Use of a streptomyces for efficient decolorization and degradation of dyes

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a use of streptomyces in efficient decolorization and degradation of dyes; the streptomyces is MS-S2, the streptomyces MS-S2 can grow on an inorganic salt culture medium with malachite green, aniline blue, reactive blue and reactive red as the only carbon source, and has the ability of decolorization and degradation of dyes. It is verified through experiments that the streptomyces MS-S2 can quickly and efficiently decolorize and degrade malachite green, the decolorization rate is as high as 100% in a short time, and the streptomyces can adapt to a wide pH range, and the degradation product is non-toxic and non-harmful. The streptomyces can be applied to wastewater treatment in the industries of printing and dyeing, spinning, breeding and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a use of streptomyces in efficient degradation of dyes. BACKGROUND

[0002] Synthetic dyes are widely used in textile printing and dyeing, papermaking, leather, cosmetics, pharmaceutical and other industries due to their strong coloring ability, easy synthesis, stable fixing effect and other advantages. China is a major producer of dyes, producing more than 1.5 million tons of dyes per year. During the production and use of dyes, about 10% to 15% of dyes are released into the ecological environment. Dye wastewater has the characteristics of deep color, high acid and alkalinity, high content of organic pollutants, complex composition, high biological toxicity and difficult biodegradation, which seriously threatens human health and the safety of the ecological environment. It is considered as one of the most difficult industrial wastewater to be treated due to its great difficulty in treatment.

[0003] Ozone dyes, anthraquinone dyes and triphenylmethane dyes are most widely used in dyes, accounting for more than 93%. Triphenylmethane dyes are the third most used dyes after azo and anthraquinone dyes, mainly including malachite green, aniline blue, crystal violet, etc. Among them, malachite green (MG) is a common basic triphenylmethane dye, which is easily soluble in water, and its aqueous solution is blue-green. It can be used as a dye for silk, leather and paper, and also as a chemical agent for sterilization and parasiticides for aquaculture, but it has high toxicity, high residue, and carcinogenic and mutagenic hazards. Efficient and harmless treatment of dye wastewater has become a problem to be solved.

[0004] At present, the treatment methods of dye wastewater mainly include physical method, chemical method and biological method. Physical method for treating wastewater mainly uses adsorption method, membrane filtration method, permeation method, extraction technology, etc., and chemical method mainly includes electrochemical treatment method and chemical oxidation method. However, these methods have high cost and low efficiency when treating wastewater, and secondary pollution is easy to occur, which greatly limits their wide application. The biological method of using microorganisms to degrade dyes does not need the presence of catalysts and high temperature and pressure conditions, and has the advantages of low cost, green environmental protection and easy operation, which has attracted more and more attention in the field of dye wastewater treatment.

[0005] Among the three major categories of dyes studied for biodegradation, research on triarylmethane dyes is relatively limited. Reported microorganisms capable of removing malachite green include yeasts, fungi, and bacteria. Compared to fungi and yeasts, bacteria have advantages such as rapid growth, strong adaptability, and ease of genetic manipulation. These mainly include *Enterobater*, *Aeromonas*, *Pseudomonas*, *Sphingomonas*, and *Bacillus*. However, currently obtained strains exhibit relatively low dye degradation efficiency, poor broad-spectrum degradation, and sensitivity to pH and dye concentration. In practical applications, the decolorization rates of these strains cannot meet the treatment requirements of actual dye wastewater, greatly limiting the industrial application of dye decolorizing bacteria. Screening microbial strains that rapidly and efficiently degrade dyes, possess strong adaptability to pH and temperature, and produce non-toxic metabolites is of great significance for dye wastewater treatment. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an application of Streptomyces in the efficient degradation of dyes, providing an excellent resource for the application of microorganisms in dye wastewater treatment.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0008] This invention provides the use of Streptomyces in highly efficient decolorization and degradation of dyes, wherein the Streptomyces is Streptomyces sp. MS-S2.

[0009] Furthermore, the Streptomyces MS-S2 strain was isolated from the gut of wood-feeding termites. The specific isolation method can be found in the paper published by our laboratory (Blessing Danso, Sameh S Ali, Rongrong Xie*, Jianzhong Sun*, Valorisation of wheat straw and bioethanol production by a novel xylanase-and cellulase-producing Streptomyces strain isolated from the wood-feedingtermite, Microcerotermes species, Fuel, 2022, 310: 122333). This strain is preserved in the applicant's laboratory, and the applicant promises to release it to the public for verification of the technical effects of the present invention within 20 years from the date of this application.

[0010] Further, the use includes the use in decolorization and degradation of dyes in wastewater of printing and dyeing, textile, breeding and the like, or bioremediation of soil or water body polluted by dyes.

[0011] Further, the dyes include azo dyes, anthraquinone dyes and triarylmethane dyes; the azo dyes are reactive red, the anthraquinone dyes are reactive blue, and the triarylmethane dyes are malachite green and aniline blue, preferably malachite green.

[0012] The application further provides a method for efficiently decolorizing and degrading dyes, which is completed by using the streptomycete MS-S2; further, the pH for decolorization and degradation is 2.0-10.0, and the temperature is 25-37℃; preferably, the pH for decolorization and degradation is 6.0-10.0.

[0013] The product of the method for efficiently decolorizing and degrading dyes has an application in biocontrol, further, the biocontrol includes an application in inhibiting plant pathogenic bacteria; the plant pathogenic bacteria include but are not limited to Alternaria brassicae.

[0014] The application has the following beneficial effects:

[0015] The streptomycete MS-S2 provided by the application has a high-efficiency degradation effect on dyes, especially on malachite green dyes, and compared with other bacteria such as Enterobater, Aeromonas, Pseudomonas, Sphingomonas and Bacillus which can degrade malachite green, the degradation speed of the streptomycete MS-S2 is fast, the time required for other bacteria to reach a decolorization rate of 90% on malachite green is 4-168 hours, while the decolorization rate of the streptomycete MS-S2 on malachite green within 2 hours is as high as 100%. The culture medium has simple components and strong environmental adaptability, and has a good degradation efficiency within a pH range of 2.0-10.0, preferably 6.0-10.0, a temperature range of 25-37℃, and a dye concentration range of 50-500mg / L. The degradation metabolites are non-toxic to plants and have an inhibiting effect on plant pathogenic bacteria. In addition, the streptomycete MS-S2 has a good degradation effect on other azo dyes, anthraquinone dyes and triarylmethane dyes, and has a strong broad-spectrum effect. The bacteria can be applied to the field of environmental protection such as industrial dye wastewater, and can be used to solve the problem of difficult degradation of dyes in industry by using the high-efficiency degradation performance. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the decolorization effect of the streptomycete MS-S2 on various dyes, in which, Reactive red: reactive red; Reactive blue: reactive blue; Anihiline blue: aniline blue; Malachite green: malachite green.

[0017] Figure 2 is the decolorization efficiency of malachite green by Streptomyces MS-S2; the upper graph is the photograph of malachite green before and after the decolorization treatment by the strain, A is malachite green before the decolorization treatment by the strain, B is after the treatment by the strain; the lower graph is the decolorization efficiency within 30-120 min of the decolorization treatment by the strain.

[0018] Figure 3 is the UV / visible absorption spectrum of malachite green before and after the treatment by Streptomyces MS-S2.

[0019] Figure 4 is the influence of pH on the decolorization rate of malachite green.

[0020] Figure 5 is the influence of temperature on the decolorization rate of malachite green.

[0021] Figure 6 is the experimental result of the toxicity of malachite green and its degradation product to sorghum and mung bean, in the graph, Watertreated is the group sprayed with deionized water, Biotreated MG is the group sprayed with the product after the degradation of malachite green by the strain, Untreated MG is the group sprayed with malachite green.

[0022] Figure 7 is the antibacterial property of the metabolic product of Streptomyces MS-S2 after the degradation of malachite green to Alternaria brassicae; the right graph is the PDA culture medium without the addition of the fermentation product of Streptomyces MS-S2, and the left graph is the PDA culture medium with the addition of the fermentation product of Streptomyces MS-S2. DETAILED DESCRIPTION

[0023] In order for those skilled in the art to better understand the technical solutions of the present application, the preferred embodiments of the present application are described in detail below, but the following embodiments do not limit the protection scope of the present application.

[0024] In the embodiments of the present application, those not described in detail are completed by using conventional experimental methods, and those not described in detail in the embodiments are understood and easily realized by those skilled in the art according to the product manual or the basic knowledge in the art, so they are not described in detail.

[0025] The present application relates to Streptomyces sp. MS-S2, which is disclosed in the paper published by the present inventor (Valorisation of wheat straw and bioethanol production by a novel xylanase-and cellulase-producing Streptomyces strain isolated from the wood-feeding termite, Microcerotermes species, Danso et al, 2022), and the 16s ribosomal RNA sequence thereof has been submitted to the GeneBank database with the accession number MZ313968. The strain is currently preserved in the applicant's unit, and the applicant promises to provide the public with the technology for verifying the technical effects of the present application for 20 years from the filing date of the present application.

[0026] Example 1: Decolorization effect of Streptomyces sp. MS-S2 on various dyes

[0027] Streptomyces sp. MS-S2 single colony was inoculated in LB medium and cultured for about 48 hours at a temperature of 30°C and a rotation speed of 180 rpm. When the absorbance of the bacterial solution at 600 nm reached about 1.0, the bacterial solution was centrifuged and washed with distilled water, and then inoculated into 50 mL liquid inorganic salt medium with a dye concentration of 50 mg / L at a inoculation amount of 5% (volume ratio). The culture was shaken and cultured at a temperature of 30°C and a rotation speed of 180 rpm for 12-48 h.

[0028] The formula of the liquid inorganic salt medium is as follows: KH2PO4 1.0 g, MgSO4 0.2 g, K2HPO4 1.0 g, (NH4)2SO4 2.0 g, dye 50 mg, and distilled water 1000 mL.

[0029] The dye used is selected from any one of the following:

[0030] Triarylmethane dyes: malachite green, aniline blue;

[0031] Anthraquinone dyes: reactive blue;

[0032] Azo dyes: reactive red.

[0033] Decolorization ability of the strain to dyes: The absorbance of the dyes before and after treatment with the strain was detected by UV-Vis spectroscopy. The dye solution was scanned within the wavelength range of 200-800 nm, and the wavelength of the maximum absorption peak for each dye was selected as the measurement wavelength for the dye absorbance value. After centrifuging the dye solution at 12000 rpm for 10 min, the supernatant was collected and the absorbance value was measured. The decolorization rate was calculated according to the following formula: Decolorization rate (%) = (Ai - Af) / Ai * 100

[0034] Where Ai: absorbance of the dye in the dye culture medium without bacterial inoculation; Af: absorbance of the dye in the dye culture medium with bacterial inoculation.

[0035] Experimental verification showed the following results: Figure 1 As shown, after culturing Streptomyces sp. MS-S2 in dyes for 12–48 hours, the decolorization rates of Reactive Red, Reactive Blue, and Aniline Blue were measured to be over 52.4%, 59.7%, and 74.9% respectively after 48 hours of culturing, while the decolorization rate of Malachite Green was as high as 100% after 12 hours of culturing.

[0036] Example 2: Decolorization efficiency of Streptomyces sp. MS-S2 on malachite green

[0037] A single colony of Streptomyces sp. MS-S2 was inoculated into LB medium and cultured at 30°C and 180 rpm for approximately 48 hours. When the absorbance at 600 nm reached approximately 1.0, the colony was centrifuged, rinsed with distilled water, and then inoculated at a rate of 5% (v / v) into 50 mL of liquid inorganic salt medium containing 50-1000 mg / L malachite green (the formulation was the same as in Example 1, with the addition of 0.08 g / L yeast extract). The culture was then incubated at 28°C, pH 8.0, and 180 rpm with shaking for 30-120 minutes. The results are as follows: Figure 2 As shown, Streptomyces MS-S2 can rapidly and efficiently decolorize malachite green. Within 30 minutes, the decolorization rate of malachite green in a 50 mg / L medium reaches 78.4%, within 90 minutes it reaches 95.4%, and within 2 hours it can reach 100%. Furthermore, Streptomyces MS-S2 maintains a high decolorization rate (60-100%) for malachite green concentrations ranging from 50 to 500 mg / L within 6 hours.

[0038] Malachite green solution with a concentration of 50 mg / L and Streptomyces MS-S2 were treated with malachite green for 2 h, and the supernatant after centrifugation was scanned using a UV-Vis spectrophotometer in the wavelength range of 200-800 nm. The results are as follows: Figure 3It can be seen that the main characteristic absorption peak of MG at 617 nm and the absorption peaks at 425 nm and 315 nm disappear after the strain treatment, and a new absorption peak appears at 360 nm, indicating that Streptomyces sp. MS-S2 can efficiently biodegrade MG.

[0039] Example 3: Decolorization efficiency of Streptomyces sp. MS-S2 on MG at different pH values

[0040] Streptomyces sp. MS-S2 single colony was inoculated in LB medium and cultured for about 48 hours. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 50 mL of liquid inorganic salt medium containing 50 mg / L MG was inoculated at a inoculation amount of 5% (volume ratio) at pH 2.0-10.0, the culture temperature was 30°C, and the rotation speed was 180 rpm. The culture was shaken for 6 hours. The decolorization efficiency of Streptomyces sp. MS-S2 on MG was calculated by the following formula: Figure 4 It can be seen that the decolorization rate of Streptomyces sp. MS-S2 on MG is relatively high (60-97%) in the pH range of 6.0-10.0, and the decolorization efficiency is the highest at pH 8.0.

[0041] Example 4: Decolorization efficiency of Streptomyces sp. MS-S2 on MG at different temperatures

[0042] Streptomyces sp. MS-S2 single colony was inoculated in LB medium and cultured for about 48 hours. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 50 mL of liquid inorganic salt medium containing 50 mg / L MG was inoculated at a inoculation amount of 5% (volume ratio) at pH 8.0, the culture temperature was 25-40°C, and the rotation speed was 180 rpm. The culture was shaken for 6 hours. The decolorization efficiency of Streptomyces sp. MS-S2 on MG was calculated by the following formula: Figure 5 It can be seen that the decolorization rate of Streptomyces sp. MS-S2 on MG is relatively high (61-92%) at 25-37°C, and the decolorization efficiency is the highest at 28°C.

[0043] Example 5: Toxicity detection of products after Streptomyces sp. MS-S2 degrades dyes

[0044] Sorghum Vulgare pers and Vigna radiata seeds (both are conventional market) were used as materials for plant toxicity detection.

[0045] The seeds were soaked in deionized water for 8 h, and 30 expanded seeds were selected and placed on the seedling paper in 3 culture dishes. The seeds were sprayed with 100 mg / L malachite green, 100 mg / L supernatant of the product after the degradation of malachite green by the strain MS-S2 for 2 h, and deionized water (control) at 1 mL each time for 3-5 times per day, and the seeds were allowed to germinate and grow at 25°C for 5 days. The germination and growth of the seeds were observed and recorded. Figure 6 It can be seen that malachite green significantly inhibited the germination and growth of the seeds, and the germination and growth of the seeds treated with the supernatant after the strain treatment and deionized water were good, i.e., the Streptomyces MS-S2 had good detoxification effect on malachite green.

[0046] Example 6: Antibacterial property of the metabolic product of Streptomyces sp. MS-S2

[0047] The Streptomyces sp. MS-S2 single colony was inoculated in LB medium and cultured for about 48 h. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 100 mL of liquid inorganic salt medium containing 100 mg / L malachite green was inoculated with the bacterial solution at 5% (volume ratio) under the conditions of pH 8.0, culture temperature of 28°C, and rotation speed of 180 rpm. The culture was shaken for 7 days. The metabolic product of the Streptomyces MS-S2 was centrifuged at 10,000 r / min for 10 min, and the supernatant was filtered with a 0.22 μm filter. The filtered supernatant was added to sterilized PDA medium at 10% (volume ratio) to prepare PDA solid plates.

[0048] The formula of the PDA medium was as follows: potato powder 6 g / L, glucose 20 g / L, and agar 20 g / L.

[0049] The Alternaria brassicae (purchased from the market) was punched into 5 mm diameter plugs with a sterile plate puncher and placed on the PDA solid medium containing the fermentation product of the Streptomyces MS-S2 for 5 days. Meanwhile, the same size plugs were placed on the PDA medium without the fermentation product of the Streptomyces MS-S2 as a control. The antibacterial property of the metabolic product of the Streptomyces MS-S2 after the degradation of malachite green was observed. As shown in Fig. 6, the plugs on the right side of the figure grew on the PDA medium without the fermentation product of the Streptomyces MS-S2, while the plugs on the left side of the figure were obviously inhibited on the PDA medium containing the fermentation product of the Streptomyces MS-S2, indicating that the metabolic product of the Streptomyces MS-S2 after the degradation of malachite green had a certain antibacterial effect on the Alternaria brassicae. Figure 7 As can be seen, the Alternaria brassicae grew on the PDA medium without the fermentation product of the Streptomyces MS-S2 on the right side, while the growth of the Alternaria brassicae was obviously inhibited on the PDA medium containing the fermentation product of the Streptomyces MS-S2 within the same time, indicating that the metabolic product of the Streptomyces MS-S2 after the degradation of malachite green had a certain antibacterial effect on the Alternaria brassicae.

Claims

1. A method for efficient decolorization and degradation of dyes, characterized by, The method uses Streptomyces ( Streptomyces (sp.) MS-S2 completed; The decolorization degradation pH is 8.0, and the temperature is 28℃; The culture is shaken at a rotation speed of 180 rpm for 120 min; The dye is malachite green; Streptomyces ( Streptomyces MS-S2 was inoculated at a volume ratio of 5%; the dye content was 50-1000 mg / L; 0.08 g / L of yeast powder is added in the dye system.

2. The method of claim 1, wherein, The application environment of the high-efficiency decolorization degradation dye includes: dye decolorization degradation in printing and dyeing, textile, breeding industry wastewater, or bioremediation of dye-polluted soil or water.

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