Omercodium fermentum strain hndzg-3 and application thereof

Abstract

The present application relates to the field of microbial technology and wastewater treatment, and specifically discloses a Kodamaea ohmeri strain with azo dye degradation function and its application. The strain was preserved in the China General Microbiological Culture Collection Center on September 30, 2022, with the preservation number of CGMCC No. 25860. The strain is isolated from the soil of Dongzhaigang Mangrove Nature Reserve in Hainan, and has decolorization and degradation effects on azo dyes such as reactive black 5, reactive brilliant red K-2G, reactive brilliant red X-3B, and methyl orange. The strain can adapt to changes in a relatively wide range of environmental factors such as salinity, pH, and temperature, and has a certain tolerance to the concentration of representative disazo dye reactive black 5. This provides a possible source of yeast strains for the biodegradation of azo dyes, and has good theoretical research value and broad application prospects in the field.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and wastewater treatment technology, and particularly relates to a strain of Kodamaeaohmeri with decolorizing and degrading functions for azo dyes Reactive Black 5, Reactive Brilliant Red K-2G, Reactive Brilliant Red X-3B, and methyl orange, and its applications. Background Technology

[0002] Currently, there are over 10,000 types of synthetic dyes used globally, with an annual production capacity of 700,000 tons. These dyes are widely used in numerous industrial sectors, including textiles, leather, detergents, pharmaceuticals, and cosmetics, and the demand for synthetic dyes has been continuously increasing in recent years. However, currently, 70% of dye wastewater is discharged directly into the natural environment without treatment, placing enormous pressure on human ecology and health. Therefore, there is an urgent need to treat industrial dye wastewater to reduce the environmental impact of the dye industry.

[0003] Industrial synthetic dyes can be classified into eight major categories based on the chemical structure of their chromophores: azo dyes, anthraquinone dyes, triphenylmethane dyes, indigo dyes, heterocyclic dyes, rose dyes, sulfur dyes, and phthalocyanine dyes. Among these, azo dyes are the most commonly used, accounting for approximately 50%-70% of all dyes. Depending on their application, they can be further divided into acid dyes, disperse dyes, and reactive dyes. Reactive dyes have become the primary category of dyes used for cotton fiber dyeing. Reactive Black 5, a typical diazo reactive dye, is widely used in industrial production. Its raw materials include many known carcinogens and mutagens, and the dye produces toxic intermediates after decomposition. Many of its components, such as aromatic amines, are banned in China and the European Union. Long-term contact with the human body may lead to redox reactions with human metabolic products, generating carcinogenic aromatic amine compounds and increasing the risk of cancer.

[0004] Traditional physicochemical treatment methods for dye wastewater include oxidation remediation, adsorption, and ion exchange. However, these methods suffer from limited effectiveness due to operational complexity, relatively low efficiency, and high energy costs. Biological treatment utilizes specialized microorganisms to directly biodegrade dye wastewater, offering high efficiency and environmental friendliness. However, the degradation efficiency of existing microorganisms, particularly azo dyes, is affected by pH and salinity. Summary of the Invention

[0005] The purpose of this invention is to find a microorganism that can not only adapt to high-salt environments but also has a certain tolerance to dye concentrations and pH values. Yeast, as a single-celled fungus, grows rapidly and has a wide range of pollutant removal capabilities (such as organic pollutants, heavy metals, and dyes). Compared with other microorganisms, yeast exhibits strong tolerance to external conditions such as heavy metals and low pH.

[0006] This invention discovers a strain of Kodamaeaohmeri HNDZG-3 with azo dye degradation function. This strain was deposited on September 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25860.

[0007] The present invention relates to Kodamaea ohmeri HNDZG-3, a strain isolated and screened from soil in the Dongzhaigang Mangrove Nature Reserve in Hainan Province. Based on the morphological and physiological-biochemical characteristics of the strain, and using the 26S rDNA method for sequencing analysis, the strain was finally identified as Kodamaea ohmeri. The full 26S rDNA sequence of Kodamaea ohmeri HNDZG-3 is shown in SEQ ID No:1.

[0008] Another object of the present invention is to provide the application of Omega yeast HNDZG-3 in the degradation of decolorized azo dyes.

[0009] In some embodiments, the azo dye may be Reactive Black 5, Reactive Brilliant Red K-2G, Reactive Brilliant Red X-3B, or Methyl Orange, etc.

[0010] The beneficial effects of the present invention are as follows: (1) The azo dye degrading bacteria of the present invention can not only adapt to high salt environment but also have a certain tolerance to dye concentration; (2) The azo dye degrading bacteria of the present invention can also degrade dyes such as Reactive Brilliant Red K-2G, Reactive Brilliant Red X-3B, and Methyl Orange, indicating that the strain has a certain decolorization spectrum for azo dyes; (3) Through the decolorization degradation experiment analysis of the representative diazo dye Reactive Black 5, it was found that it has a good decolorization effect on azo dyes and the degradation effect is significant, providing a possible source of strains for the biodegradation of azo dyes, and has broad application prospects in the industrial treatment of dyeing and printing wastewater. Attached Figure Description

[0011] Figure 1 The colony morphology of Omkoda yeast HNDZG-3 of the present invention;

[0012] Figure 2 The cell morphology of Omkoda yeast HNDZG-3 of the present invention;

[0013] Figure 3 Electron micrograph of the morphology of the Omekoda yeast strain HNDZG-3 of the present invention;

[0014] Figure 4 The phylogenetic tree of Omicosida yeast HNDZG-3 of the present invention;

[0015] Figure 5 The effect of salinity on the decolorization of Omkoda yeast HNDZG-3 in this invention;

[0016] Figure 6 The effect of pH on the decolorization of Omkoda yeast HNDZG-3 in this invention;

[0017] Figure 7 The effect of temperature on the decolorization of Omkoda yeast HNDZG-3 in this invention;

[0018] Figure 8 The effect of initial dye concentration on the decolorization of Omnikol yeast HNDZG-3 in this invention;

[0019] Figure 9 The decolorization effect of Omnikorda yeast HNDZG-3 on azo dyes is shown in the figure. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Example 1:

[0022] Isolation, purification and initial screening of strains

[0023] 1. Isolation and purification of strains

[0024] The Omega yeast in this invention was isolated and screened from soil living in the Dongzhaigang Mangrove Nature Reserve in Hainan. Samples were collected from the mangroves, and 1g of sample was added to a 10mL sterile seawater test tube and shaken on a shaker for 30 minutes. The suspension was then transferred from 10mL to 10mL of seawater. -1 Up to 10 -6 Perform serial dilutions. In a clean bench, take 100 μL of the liquid and spread it onto a YPD agar plate. Incubate at 26°C for 48 h. Select single colonies that appear to be yeast-like and purify them multiple times until no contaminants are found. After obtaining a single strain, store it on a slant at 4°C for later use.

[0025] 2. Observation of colony morphology and microscopic examination with iodine staining.

[0026] Colonies from the plates were smeared, fixed, stained with iodine, washed, dried, and examined under an oil immersion microscope. A total of 204 yeast strains were ultimately selected. These strains were inoculated into YPD liquid medium with the addition of 50% glycerol and stored at -80°C for later use.

[0027] Example 2:

[0028] Screening of strains for decolorization function

[0029] Purified yeast colonies were inoculated onto 50 mg / L Reactive Black 5 solid decolorizing medium using a spot inoculation method. After incubation at 26℃ for 3 days, the presence of decolorization zones on the plates was observed. Strains exhibiting decolorization were inoculated into 10 mL of 50 mg / L Reactive Black 5 dye medium in test tubes, with uninoculated medium serving as the control group. The culture was carried out at 26℃ and 180 rpm for 48 h on a shaker. After centrifugation at 10000 rpm for 5 min, the supernatant was collected, and the absorbance was measured at 598 nm (the wavelength of the dye's maximum absorption peak). The experiment was repeated three times, and the average value was calculated to determine the decolorization rate of the screened strains in degrading Reactive Black 5. The formula for calculating the decolorization rate is as follows:

[0030] Decolorization rate (%) = (AB) / A × 100%

[0031] Where A is the maximum absorbance value of the control group (i.e., no bacterial inoculation); B is the maximum absorbance value of the experimental group (i.e., after bacterial inoculation and decolorization for a certain period of time).

[0032] A strain with the strongest ability to degrade Active Black 5 was selected, and its strain number is HNDZG-3. Its average decolorization rate can reach more than 85%.

[0033] Example 3:

[0034] Identification of strains

[0035] 1. Colony characteristics and microscopic morphology

[0036] After culturing Omnikova yeast HNDZG-3 on YPD agar medium at 26℃ for 48 h, the morphology of the strain on the plate was observed. The colonies were nearly round, milky white, opaque, with wrinkled surfaces and incomplete edges. The results are as follows. Figure 1 The morphology of the strain is shown in electron micrograph 2. The strain has a diameter of 4-5 μm. After staining with iodine solution and observed under an oil immersion microscope, the bacterial cells are oval-shaped and reproduce by budding. The results are as follows. Figure 3 .

[0037] 2. Physiological and biochemical experiments of the strain

[0038] Physiological and biochemical identification results showed that strain HNDZG-3 could utilize various sugars such as glucose and maltose, but could not utilize arabinose, xylose, lactose, and mesotriose. Identification results using API 20C AUX test strips from bioMérieux, France, indicated that this strain was Omega-Korda yeast.

[0039] Table 1. Physiological and biochemical identification of yeast HNDZG-3

[0040]

[0041] 3. 26S rDNA sequencing analysis of the strain

[0042] The 26S rDNA gene sequence of strain HNDZG-3 was determined and compared with the NCBI database, identifying it as Kodamaea ohmeri. A phylogenetic tree of Kodamaea ohmeri HNDZG-3 was constructed, as shown below. Figure 4 As shown, the sequence of the strain is shown in SEQ ID NO:1 in the sequence listing.

[0043] Based on the above results, yeast HNDZG-3 was identified as Kodamaea ohmeri.

[0044] Example 4:

[0045] Effect of salinity on decolorization

[0046] Industrial dye wastewater typically contains a certain concentration of salt ions. The decolorization efficiency of strain HNDZG-3 on Reactive Black 5 under different salinity conditions was investigated by adjusting the NaCl concentration in the decolorization medium. Figure 5 The results showed that strain HNDZG-3 could maintain a decolorization rate of over 70% for 12 hours when the salt content was 0-70 g / L, and could still effectively decolorize Reactive Black 5 when the salt content was 110 g / L, with a decolorization rate of over 80% after 48 hours.

[0047] Example 5:

[0048] The effect of pH on decolorization

[0049] The decolorization efficiency of strain HNDZG-3 on Active Black 5 under different pH conditions was investigated by adjusting the pH of the decolorization medium. The results are shown in [Figure number missing]. Figure 6 The strain HNDZG-3 has a wide pH range of adaptability. When the pH is 3, the decolorization rate of strain HNDZG-3 can reach 95.38% in 12 hours, which is the best and fastest decolorization effect. When the pH is 2-8, strain HNDZG-3 can grow and maintain a decolorization rate of more than 75%. This shows that strain HNDZG-3 has a good pH range in practical applications.

[0050] Example 6:

[0051] Effect of temperature on decolorization

[0052] The decolorization efficiency of strain HNDZG-3 on Active Black 5 was investigated by culturing the decolorization medium at different temperatures. The results are shown in [Figure number missing]. Figure 7 The optimal temperature for degradation by strain HNDZG-3 is 26-38℃. Within this temperature range, the average degradation rate of Active Black 5 reaches over 90% within 24 hours. Excessively high culture temperature will affect the decolorization rate, while excessively low culture temperature will affect the decolorization efficiency in the early stages. However, as the culture time increases, the decolorization rate gradually increases, but it takes a long time. When the temperature is 32℃, strain HNDZG-3 has the fastest decolorization speed and can maintain a high decolorization rate.

[0053] Example 7:

[0054] Effect of dye concentration on decolorization

[0055] Different concentrations of Reactive Black 5 dye were added to the decolorizing medium to investigate the decolorizing ability of strain HNDZG-3 at different concentrations of Reactive Black 5. Dye was added to the decolorizing medium at final concentrations of 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, and 600 mg / L, respectively. The results are shown in [Figure number missing]. Figure 8 The strain HNDZG-3 has a high tolerance to dyes. It can achieve a decolorization rate of over 90% for 100mg-500mg / L Reactive Black 5 in 24h and over 90% for 600mg / L in 48h. Moreover, the bacterial cells are all white after decolorization, which indicates that even if the dye concentration increases, the strain will undergo biodegradation decolorization rather than decolorization through its own adsorption.

[0056] Example 8:

[0057] Effects of strain HNDZG-3 on decolorization of other dyes

[0058] To investigate the decolorization spectrum of strain HNDZG-3, azo dyes Reactive Brilliant Red K-2G, Reactive Brilliant Red X-3B, Methyl Orange, and Congo Red, triphenylmethane dye Malachite Green, and anthraquinone dyes Reactive Brilliant Blue KN-R and Reactive Brilliant Blue X-BR were added to the decolorization medium at a final concentration of 50 mg / L. Results are shown below. Figure 9The strain HNDZG-3 achieved a maximum decolorization rate of 79.49% for Reactive Brilliant Red K-2G, 81.76% for Reactive Brilliant Red X-3B, and 83.14% for methyl orange. It showed no significant decolorization effect on Congo Red and triphenylmethane malachite green. The highest decolorization rates for the anthraquinone dyes Reactive Brilliant Blue KN-R and Reactive Brilliant Blue X-BR were 41.49% and 36.01%, respectively. This indicates that strain HNDZG-3 exhibits a certain degree of decolorization spectrum for azo dyes, while its decolorization effect on triphenylmethane and anthraquinone dyes remains to be investigated. The above are merely some embodiments of the present invention. The present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. An Omicosac yeast strain with azo dye degradation function ( Kodamaea ohmeri HNDZG-3, characterized in that, The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 30, 2022, with accession number CGMCC No. 25860.

2. The Omnikorda yeast as described in claim 1 ( Kodamaea ohmeri HNDZG-3, characterized in that The source was soil from the Dongzhaigang Mangrove Nature Reserve in Hainan.

3. The Omnikorda yeast according to claim 1 or 2 ( Kodamaea ohmeri Application of HNDZG-3 in the degradation of azo dyes Reactive Black 5, Reactive Brilliant Red K-2G, Reactive Brilliant Red X-3B or Methyl Orange.

Images