Azo dye degrading bacterium RL2 and application thereof
By isolating and identifying the azo dye-degrading strain RL2, the problem of difficult degradation of azo dyes is solved, efficient dye wastewater treatment is achieved, and the accumulation of toxic substances is reduced, and an ecologically friendly solution is provided.
Patent Information
- Application Number
- CN202411663654.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art is difficult to effectively degrade azo dyes, especially under oxidation conditions, which leads to the accumulation of toxic aromatic amines in dye wastewater, posing a threat to the environment and human health.
Isolated and identified an azo dye degrading strain RL2 (Kurtzmaniella quercitrusa) which was able to efficiently degrade azo dye under oxidation conditions with a decolorization rate of 99%. Azo dye decolorization preparation containing this strain was developed for use in Treat dye wastewater.
It achieves efficient degradation of azo dye, significantly improves the decolorization rate of dye wastewater, reduces the accumulation of toxic substances, and provides an eco-friendly treatment solution.
Smart Images
Figure CN119286668A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of azo dye degradation, and in particular relates to an azo dye degrading bacterium RL2 and application thereof. Background Art
[0002] Azo dyes are synthetic dyes named after the azo groups in their molecular structure. They have strong light and oxidation resistance and are widely used in textile, dyeing, papermaking, printing and other fields. Due to the stable nature of azo dyes, the dyes discharged with wastewater can remain in the water environment for a long time, thus causing serious damage to the aquatic ecosystem. In addition, dye wastewater has the characteristics of large total amount, high chromaticity and poor biodegradability, and is difficult to degrade.
[0003] Common methods for treating azo dye wastewater include physicochemical method and biological method. Currently, commonly used physicochemical methods include adsorption decolorization technology, coagulation and sedimentation technology, chemical oxidation technology, ion exchange technology, ultrafiltration membrane decolorization technology, photocatalysis technology and high-voltage pulse electrolysis technology. Compared with physicochemical method, biological treatment has low cost, eco-friendly and strong feasibility, and is an important way to treat water pollution. Activated sludge method is a traditional biological treatment method for wastewater, which has the characteristics of low cost and small secondary pollution. However, due to the poor degradability of azo dyes, it is difficult for ordinary activated sludge microorganisms to achieve decolorization, and its application is subject to certain restrictions. In recent years, researchers have discovered a variety of bacterial strains that can degrade azo dyes. There are many types of such bacteria, and the reduction degradation of azo dyes is mainly completed under anaerobic conditions using the efficient decolorization of azo reductase. However, the anaerobic degradation products of azo dyes are mostly toxic aromatic amines, which have carcinogenic, teratogenic and mutagenic effects, and are easily accumulated in the environment, thus posing a hazard to various organisms and human health.
[0004] In addition to bacteria, filamentous fungi and yeast also have good decolorization effects on azo dyes. Compared with the decolorization of bacteria under anaerobic conditions by enzyme catalysis, fungal decolorization methods are divided into adsorption decolorization and degradation decolorization. Most fungi, including filamentous fungi, first adsorb and then degrade, and some only adsorb. As a large group of unicellular fungi, yeast has the advantages of fast growth and metabolism, can produce enzymes of various fungi, easy reactor design, and high wastewater treatment efficiency. Because it is relatively tolerant to high organic loads and high-salt environments, it can treat special wastewaters including dye wastewater, and has great potential in sewage treatment. Therefore, how to provide a biological treatment method that effectively degrades azo dyes through yeast and achieves decolorization of azo dyes is a problem that technicians in this field urgently need to solve. Summary of the invention
[0005] In order to solve the above technical problems, the present invention proposes an azo dye degrading bacterium RL2 and its application. The azo dye degrading bacterium RL2 can decolorize azo dyes, and the decolorization rate reaches 99%, with significant effect.
[0006] The present invention provides the following technical solutions:
[0007] Technical solution 1: An azo dye-degrading bacterium (Kurtzmaniella quercitrusa) RL2, the azo dye-degrading bacterium RL2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 25, 2023, and the deposit number is CGMCC No.28007.
[0008] Technical solution 2: An azo dye decolorizing preparation, the active ingredient of which includes the azo dye degrading bacteria RL2.
[0009] Technical solution three: Use of the azo dye degrading bacteria RL2 or the azo dye decolorizing preparation in decolorizing azo dyes.
[0010] Technical Solution 4: Application of the azo dye-degrading bacteria RL2 or the azo dye decolorizing preparation in treating wastewater containing azo dyes or decolorizing water bodies contaminated by azo dyes.
[0011] Furthermore, the azo dye includes Reactive Brilliant Red K-2BP.
[0012] Technical Solution 5: A method for treating wastewater containing azo dyes, comprising the step of inoculating the azo dye-degrading yeast RL2 into the wastewater containing azo dyes for cultivation to degrade the azo dyes in the wastewater.
[0013] Furthermore, the inoculation amount of the azo dye degrading yeast RL2 is 8%.
[0014] Furthermore, the azo dye includes Reactive Brilliant Red K-2BP.
[0015] Compared with the prior art, the present invention has the following advantages and technical effects:
[0016] The present invention isolates a strain RL2 with azo dye degradation function from the soil. The azo dye degrading bacteria RL2 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 25, 2023, and the deposit number is CGMCC No. 28007. The strain can decolorize azo dyes, and the decolorization rate reaches more than 99%, with significant effects. The present invention provides a new solution for the biological treatment of azo dye wastewater decolorization. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] 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.
[0018] Figure 1 This is a graph showing the growth of the RL2 strain after streaking the plate;
[0019] Figure 2 This is the bacterial morphology of the RL2 strain under a microscope;
[0020] Figure 3 is the phylogenetic tree of the RL2 strain;
[0021] Figure 4 The decolorization effect diagram of the shake bottle experiment, where A and B are the effect diagrams after two parallel groups of treatment;
[0022] Figure 5 This is a diagram of the decolorization effect of the SBR reactor. DETAILED DESCRIPTION
[0023] 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.
[0024] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.
[0025] 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.
[0026] 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.
[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0028] Example 1 Isolation, activation and preservation of bacterial strains
[0029] 1. Isolation and Purification of Dye-Degrading Strain RL2
[0030] The azo dye-degrading bacteria were obtained from the soil of the fourth flowerbed of the East Campus of Yanqi Lake Campus of University of Chinese Academy of Sciences by dilution coating and streaking isolation. The culture conditions were 28-30°C for 7 days.
[0031] 1) The specific operation of dilution and coating is as follows: weigh 0.1g soil sample and add it to a test tube containing 9mL water, mix well and let it stand for separation. When separating, in the clean bench, use a pipette to suck 100μL of soil suspension into the culture dish, and spread it evenly with a disposable sterile coating stick. Cultivate at 30℃ for 7 days and observe colony growth (first place the culture medium upright, and then invert it until the surface is slightly dry).
[0032] 2) Streak isolation: Use an inoculation loop to pick up a single colony with a degradation zone on the dye selection medium, streak it on another dye selection medium, and isolate and purify the colony. Figure 1 The culture medium is separated by two streakings. The isolated single colony is round, with a smooth, protruding, moist surface, 1 to 2 mm in diameter, milky white (pink due to enriched dye), and a transparent degradation zone is formed around the colony. The colony is named RL2.
[0033] 3) Microscopic observation of dye-degrading bacteria RL2: Observe the bacterial morphology (e.g. Figure 2 The bacteria are oval, colorless and transparent, with spore structures.
[0034] 4) The formula of the culture medium used for strain isolation is: glucose 10g / L, potassium dihydrogen phosphate 1g / L, ammonium chloride 1g / L, magnesium sulfate 0.5g / L, yeast extract 0.2g / L, agar 20g, active brilliant red (K-2BP) 50mg / L (mother solution is 1000mg / L). The sterilization condition is 115℃30min. When the temperature of the culture medium drops to 50-60℃, pour 15-20mL of the culture medium into each disposable sterile culture dish and cool to room temperature to complete the preparation.
[0035] 2. Activation of strains
[0036] After continuous streaking separation for more than 2 times, pick a single colony on a loop of culture medium with an inoculation loop, inoculate it into a liquid culture medium containing 10 mg / L Reactive Brilliant Red, and culture at 30°C and 180 rpm until the culture solution turns milky white.
[0037] The formula of the liquid culture medium used for activation is: glucose 10g / L, potassium dihydrogen phosphate 1g / L, ammonium chloride 1g / L, magnesium sulfate 0.5g / L, yeast extract powder 0.2g / L, active brilliant red (K-2BP) 10mg / L (mother liquor is 1000mg / L). The sterilization conditions are 115℃, 30min.
[0038] 3. Preservation of strains
[0039] Prepare 50% glycerol by volume and sterilize (115°C for 30 min), mix the secondary activated bacterial solution and sterilized glycerol in a cryopreservation tube at a volume ratio of 1:1, and store at -80°C.
[0040] 4. Molecular Identification of Strains
[0041] After RL2 gene extraction and PCR amplification, 18S rDNA sequencing was performed. A phylogenetic tree was established, such as Figure 3 shown.
[0042] The PCR process is as follows: extract genomic DNA and use universal primers NS1 and NS8 for PCR amplification.
[0043] Primer NS1: 5'-GTAGTCATATGCTTGTCTC-3' (SEQ ID NO. 1);
[0044] Primer NS8: 5'-TCCGCAGGTTCACCTACGGA-3' (SEQ ID NO. 2).
[0045] PCR amplification is divided into three stages, and the conditions are as follows: the first stage is 94℃ pre-denaturation for 5min, and 1 cycle; the second stage is 94℃ denaturation for 30s, 55℃ annealing for 30s, and 72℃ extension for 1min45s, for a total of 35 cycles; the third stage is 72℃ extension for 7min, for 1 cycle, and PCR is completed. The amplified DNA is to be sequenced and stored at 4℃. The sequencing method is the Sanger method, and the sequencer model is ABI 3730x. After RL2 gene extraction and PCR amplification, 18S rDNA sequencing was performed, and the 18SrDNA sequence is as follows:
[0046]
[0047] The sequencing results were compared with the ITS sequence homology in the online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of NCBI (https: / / www.ncbi.nlm.nih.gov / ). The sequence of the strain RL2 was 99.82% homologous to the Kurtzmaniella quercitrusa strain, and was classified and named Kurtzmaniellaquercitrusa. The azo dye degrading bacteria RL2 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit date of July 25, 2023 and a deposit number of CGMCC No.28007. The detection on July 25, 2023 proved that the bacteria were alive.
[0048] Example 2 Decolorization experiment
[0049] 1. Chroma measurement and decolorization rate calculation
[0050] The colorimetric detection uses an ultraviolet spectrophotometer to measure the absorbance of the supernatant at the maximum absorption peak of the dye. Reactive brilliant red (K-2BP) has a maximum absorption peak at λ = 534nm. The decolorized solution is centrifuged at 8000rpm for 3min, and the supernatant is taken to measure the absorbance OD at λ = 534nm. Decolorization rate (%) = (AB) / A*100, where: A is the OD value of the test solution before decolorization, and B is the OD value of the supernatant after decolorization and centrifugation.
[0051] 2. Shake flask decolorization experiment
[0052] After the dye-degrading strain RL2 was streaked for more than 2 times, a single colony on a loop of culture medium was picked with an inoculation loop and inoculated into a liquid culture medium containing 50 mg / L Reactive Brilliant Red (two parallel experiments: RL2-1 and RL2-2), and cultured at 30°C and 160 rpm for 7 days. Using a sterile liquid culture medium without Reactive Brilliant Red as a control, it was observed that the culture medium was milky white and the decolorization effect was good (such as Figure 4 The colorimetric determination and decolorization rate calculation were performed, wherein the results of the shake flask experiment on dye degradation and decolorization are shown in Table 1. The results show that the decolorization rate is more than 95%.
[0053] Table 1 Shake flask experiment dye degradation and decolorization table
[0054] Experimental groups Absorbance Decolorization rate RL2-1 0.021 95.28％ RL2-2 0.017 96.16％ Comparison 1.065 8.62％
[0055] The formula of the culture medium used in the shake flask decolorization experiment is: glucose 10g / L, potassium dihydrogen phosphate 1g / L, ammonium chloride 1g / L, magnesium sulfate 0.5g / L, yeast extract powder 0.2g / L, active brilliant red (K-2BP) 50mg / L (mother liquor is 1000mg / L). The sterilization conditions are 115℃, 30min.
[0056] 3. SBR reactor decolorization experiment
[0057] The sequencing batch reactor activated sludge process refers to a method for treating sewage with activated sludge, which consists of five basic processes, namely, water intake, aeration, sedimentation, drainage and standby, in the same reactor (vessel) in chronological order. It is referred to as the SBR process. In this embodiment, strain RL2 was added without adding activated sludge, and water intake, aeration and drainage were carried out in sequence in the reactor to verify the decolorization and degradation effect of the strain RL2 to be tested on the dye liquor.
[0058] Specifically, 1L of dye solution to be decolorized is prepared with the following formula: 10g / L glucose, 1g / L potassium dihydrogen phosphate, 1g / L ammonium chloride, 0.5g / L magnesium sulfate, 0.2g / L yeast extract powder, 30mg / L reactive brilliant red (K-2BP) (mother liquor is 1000mg / L). It does not need to be sterilized and is directly introduced into the SBR reactor.
[0059] RL2 activated by azo dye Reactive Brilliant Red (OD reached 1.3-1.5) was added to the SBR reactor at 8% (v / v). The aeration flow rate was 0.5Nl / min, and the dissolved oxygen DO was maintained at 4-5mg / L. After running at 25℃ for 16h, the color was significantly decolored (the decolorization effect of the SBR reactor is shown in Figure 5 The colorimetric determination and decolorization rate calculation were performed, wherein the results of dye degradation and decolorization in the SBR reactor experiment are shown in Table 2. The results show that the decolorization rate is more than 99%.
[0060] Table 2 SBR reactor experimental dye degradation and decolorization table
[0061] Experimental groups Absorbance Decolorization rate SBR water inlet 0.622 / SBR outlet 0.005 99.20％
[0062] 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. An azo dye degrading bacterium (Kurtzmaniella quercitrusa) RL2, characterized in that The azo dye-degrading bacteria RL2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 25, 2023, and the deposit number is CGMCC No.28007.
2. An azo dye decolorizing preparation, characterized in that: The active ingredient comprises the azo dye degrading bacteria RL2 described in claim 1.
3. Use of the azo dye degrading bacteria RL2 according to claim 1 or the azo dye decolorizing preparation according to claim 2 in decolorizing azo dyes.
4. Use of the azo dye degrading bacteria RL2 according to claim 1 or the azo dye decolorizing preparation according to claim 2 in treating wastewater containing azo dyes or decolorizing water bodies contaminated by azo dyes.
5. The use according to claim 3 or 4, characterized in that: The azo dye includes Reactive Brilliant Red K-2BP.
6. A method for treating wastewater containing azo dyes, characterized in that: The method comprises the steps of inoculating the azo dye degrading yeast RL2 according to claim 1 into wastewater containing azo dyes for culturing so as to degrade the azo dyes in the wastewater.
7. The method according to claim 6, characterized in that The inoculation amount of the azo dye degrading yeast RL2 is 8%.
8. The method according to claim 6, characterized in that The azo dye includes Reactive Brilliant Red K-2BP.