Application of a special bacterium in degrading malachite green wastewater

Abstract

The application belongs to the field of microbial degradation of dyes, and relates to application of deinococcus in degradation of malachite green wastewater. The deinococcus is Deinococcus wulumuqiensis R12. When the initial concentration of malachite green MG is 200 mg / L, the cell concentration of R12 is 8.0 g / L, the reaction is carried out at 30 DEG C and pH 6.0 for 30 min, the degradation rate is 99.80%. When the initial concentration of MG is 1000 mg / L, the degradation rate can reach more than 95% after 1.5 h of reaction. Meanwhile, the R12 has wide adaptability to pH and temperature in degrading malachite green, and can efficiently degrade malachite green MG under the conditions of pH 5.0-10.0 and 10-45 DEG C.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial degradation of dyes, and relates to application of Deinococcus in degradation of malachite green wastewater. BACKGROUND

[0002] Malachite green (MG) is also called alkali green, which is a synthetic compound with metallic luster and dark green color. The molecular formula of malachite green is C 23 H 25 C l N2, and the molecular weight is 364.911 g / mol. Malachite green is easily soluble in water and is a toxic triphenylmethane chemical. In the natural environment, the existence form of malachite green also includes leucomalachite green, and the structural formula of the two is shown as follows, wherein a is the molecular structural formula of malachite green, and b is the molecular structural formula of leucomalachite green. Leucomalachite green is hydrogenated and reduced on the basis of malachite green, which destroys the cationic chromophore group, but the structure connected with the benzene ring is not destroyed, thereby forming leucomalachite green compound.

[0003]

[0004] Malachite green is a dye and also has the effects of sterilization and insecticide. As a typical representative of triphenylmethane dyes, the central carbon atom of malachite green is surrounded by three benzene groups to form a chromophore group, which is widely used in silk, wood, cotton, leather, papermaking, ceramic and nylon industries. However, such dyes also cause environmental problems, and a large amount of industrial wastewater containing malachite green is discharged into the environment, causing serious pollution. In addition to being used as a dye, malachite green can also be used as a bactericide and an insecticide. Malachite green is effective for fish body and egg water mold diseases, and can also be used for treating external fungal and protozoan infections of fish, and therefore has been widely used in aquaculture.

[0005] Both malachite green and leucomalachite green contain aromatic structures of triphenylmethane in their structures. After such substances enter the organism, free radicals similar to aromatic amine structures are formed, and eventually produce electrophilic ions “positive nitrogen ions”. These active “positive nitrogen ions” can destroy the DNA structure and cause canceration and deformation. In addition, due to the complex aromatic molecular structure, malachite green in water is not easy to degrade, which can reduce the propagation of sunlight in the water body, thereby affecting the aquatic biological group.

[0006] Malachite green wastewater generally has deep color, is alkaline, and has a COD of 1-100 g / L and high salinity (2-4%). Since most of the compounds in malachite green wastewater have difficult-to-degrade aromatic hydrocarbons and heterocyclic parent compounds, the treatment of malachite green wastewater has always been a difficult problem in engineering and a research hotspot in the academic field.

[0007] Currently, the methods for removing malachite green from wastewater include physical-chemical methods and biological methods. Among them, physical-chemical methods include adsorption, photodegradation, electrode method and oxidation method. However, these methods have limitations such as high cost and secondary pollution. In contrast, biological methods are widely used in the treatment of urban domestic wastewater as an economical and effective wastewater treatment method. Biological methods have the characteristics of environmental protection, high efficiency and low cost. Currently, microorganisms capable of degrading malachite green have been found, including bacteria, yeasts, filamentous fungi and algae. Among them, bacteria include Bacillus, Pseudomonas, Aeromonas, Klebsiella, Exiguobacterium, Stenotrophomonas, Micrococcus, Photobacterium, Ochrobactrum and Tenacibaculum. SUMMARY

[0008] The purpose of the present application is to provide an application of Deinococcus in degrading malachite green wastewater.

[0009] The present application discloses an application of Deinococcus in degrading malachite green wastewater, wherein the Deinococcus is Deinococcus wulumuqiensis R12. Deinococcus wulumuqiensis R12 has completed biological preservation in the method for catalytically synthesizing 5-hydroxymethyl furfuryl acid by using Deinococcus wulumuqiensis R12 in Chinese patent CN109811020B, and is preserved in China Center for Type Culture Collection (CCTCC) on March 14, 2019, with the preservation number of CCTCC No: M2019142.

[0010] Preferably, the Deinococcus degrades malachite green wastewater under the condition of pH 5.0-10.0. The Deinococcus degrades malachite green wastewater under the condition of 10-45℃. The degradation reaction time is 0.2-5h (preferably 0.5-1.5h).

[0011] Further, the Deinococcus degrades wastewater with an initial concentration of malachite green of 100-2000mg / L under the condition of 10-45℃ and pH 5.0-10.0. The pH is further preferably 5.0-9.0.

[0012] In a preferred embodiment, the initial concentration of malachite green is 100-800mg / L.

[0013] In a preferred embodiment, Deinococcus wulumuqiensis R12 is added to malachite green wastewater, and the concentration of Deinococcus wulumuqiensis R12 in the malachite green wastewater is 6-10 g / L (preferably 8 g / L).

[0014] Compared with the prior art, the degradation rate of the application is 99.80% when the initial concentration of MG is 200 mg / L, the concentration of R12 cells is 8.0 g / L, the reaction is carried out at 30°C and pH 6.0 for 30 min. When the initial concentration of MG is 1000 mg / L, the degradation rate can reach more than 95% after 1.5 h of reaction. Meanwhile, R12 has a wide pH adaptability and temperature adaptability for degrading malachite green, and can efficiently degrade MG at pH 5.0-10.0 and 10-45°C. The qualitative analysis result shows that the decolorized product of R12 degrading MG includes 4-(dimethylamino) benzophenone, etc., and two metabolic pathways are speculated, and the initial substrate malachite green is mainly degraded by continuous loss of the substituted methyl group of "bis(dimethylamino)" in the molecular structure. The antibacterial circle experiment of microbial cells shows that the degradation product does not show inhibition to microbial cells and plant cells. In summary, Deinococcus wulumuqiensis R12 in the application can effectively degrade malachite green MG, and the degradation product is significantly different from another strain of Deinococcus MG degrading bacteria Deinococcus radiodurans R1 reported. Meanwhile, the metabolic pathway of Deinococcus microorganism degrading malachite green is predicted for the first time. Deinococcus wulumuqiensis R12 has potential application prospect in the bioremediation of MG pollution. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Effect of different pH on R12 degrading malachite green;

[0016] Figure 2 Effect of different initial concentrations of MG on the degradation rate;

[0017] Figure 3 Effect of different temperatures on R12 degrading malachite green;

[0018] Figure 4 Full wavelength (200-900 nm) scanning of R12 degrading malachite green products at different time periods;

[0019] Figure 5 FTIR analysis of R12 degrading malachite green products, A: malachite green, B: malachite green degradation product at 12 h of degradation;

[0020] Figure 6 GC-MS analysis of the product after MG decolorization

[0021] Figure 7 LC-MS analysis of the product after MG decolorization, A, m / z 329, malachite green (retention time: 4.72 min); B, m / z 315, desmethylmalachite green (retention time: 4.23 min); C, m / z 301, desdimethylmalachite green (retention time: 8.10 min); D, m / z 226, 4-(dimethylamino)benzophenone (retention time: 6.45 min); E, m / z 212, (4-(methylamino)phenyl)(phenyl)methanone (retention time: 5.65 min);

[0022] Figure 8 Analysis of possible pathways of R12 to degrade malachite green

[0023] Figure 9 Toxicity test of malachite green degradation products on microbial cells, (A) Micrococcus luteus, (B) Escherichia coli, (C) Bacillus subtilis, (D) Staphylococcus aureus, a is the degradation product of 200 mg / L malachite green, b, c, d are 200 mg / L, 100 mg / L, 50 mg / L malachite green, respectively. DETAILED DESCRIPTION

[0024] The present application will be further described below in conjunction with the accompanying drawings and specific examples.

[0025] In the early stage, researchers isolated a strain of extremely radiation-tolerant microorganism Deinococcus wulumuqiensis R12 from radiation-polluted soil in Xinjiang (see the literature: W. Wang, J. Mao, Z. Zhang, Q. Tang, Y. Xie, J. Zhu, L. Zhang, Z. Liu, Y. Shi, M. Goodfellow, Deinococcus wulumuqiensis sp. nov., and Deinococcus xibeiensis sp. nov., isolated from radiation-polluted soil, International Journal of Systematic and Evolutionary Microbiology 60(9) (2010) 2006-2010. and Chinese patent CN109811020B). R12 is a red spherical gram-positive bacterium. The bacterium has strong resistance to ionizing radiation and ultraviolet radiation, and also shows high tolerance to other DNA damaging agents.

[0026] 1) Strains, media and solutions

[0027] Strain: Deinococcus wulumuqiensis R12 was isolated from radiation area of Xinjiang. The strain was cultivated at 28°C using TGY medium.

[0028] TGY liquid medium: weigh 10 g of peptone, 3 g of yeast powder, 1 g of glucose, add distilled water to 1000 mL, adjust pH to 7.2, sterilize at 121°C, 0.1 MPa for 30 minutes.

[0029] 2) Determination method

[0030] a. Determination of decolorization rate:

[0031] Strain R12 was grown in TGY liquid medium in a shaker flask to the late logarithmic phase, centrifuged at 10000 rpm for 10 min, the supernatant was removed to obtain the bacterial slurry, and a cell suspension was prepared. The cell suspension was transferred to a 50 mL centrifuge tube containing fresh TGY medium and MG, so that the cell concentration was 8.0 g / L. The system without R12 cells was used as a control experiment. The decolorization reaction was carried out at 200 rpm. After the reaction was completed, centrifugation was carried out at 12000 rpm for 10 minutes, the supernatant was diluted to an appropriate multiple, and the absorbance of the supernatant was determined using a spectrophotometer at a wavelength of 618 nm. The decolorization rate (%) was calculated according to the following formula:

[0032]

[0033] In the formula: d - decolorization rate, %;

[0034] A0 - absorbance of control tube;

[0035] A - absorbance of sample tube

[0036] b. Effect of different factors on R12 degradation of malachite green:

[0037] In order to analyze the effect of different environmental factors on the decolorization efficiency of MG, batch experiments were carried out in TGY medium at different initial MG concentrations (100-2000 mg / L), temperatures (10-45°C) and initial pH values (pH 5.0-12.0). Each experiment was repeated three times.

[0038] c. Ultraviolet (UV) full wavelength scan analysis:

[0039] For qualitative analysis of the reaction products, physiological saline was used instead of TGY medium for the decolorization. The decolorization conditions were: 30°C, pH 6.0, 200 rpm, initial MG concentration of 200 mg / L, and initial cell concentration of 4.0 g / L. That is, the degradation system only included MG, physiological saline, and R12 cells. At specific time points, the reaction was collected, and after centrifugation at 10,000 rpm for 10 min, the supernatant was taken and measured in the wavelength range of 200-800 nm.

[0040] d. Fourier transform infrared absorption spectroscopy (FTIR) analysis:

[0041] To prepare samples for FTIR measurement, at specific time points, the reaction was collected, and after centrifugation at 12,000 rpm for 10 min, the supernatant was taken and passed through a 0.22 μm aqueous membrane. 6 mL of filtrate was collected and extracted with chloroform (3 x 2 mL). The extract was dried over anhydrous sodium sulfate, and the supernatant was taken. The sample was concentrated by nitrogen blowing. 20 μL of the supernatant was spread on a potassium bromide window sheet and dried under light for 10 min. Then the dried sample was analyzed on an infrared spectrometer, and the FTIR spectrum of 400-4000 cm -1 was collected.

[0042] e. Gas chromatography-mass spectrometry (GC-MS) analysis:

[0043] After decolorization for a certain period of time, centrifugation was performed at 10,000 rpm for 10 min. The supernatant was collected, passed through a 0.22 μm aqueous membrane, extracted with an equal amount of ethyl acetate three times, the extract was combined, concentrated by nitrogen blowing, dried over anhydrous sodium sulfate, and used for GC / MS analysis after membrane filtration.

[0044] GC / MS analysis was performed using a Thermo GC (TRACE GC ULTRA) coupled with a Thermo MS (TSQ QUANTUM GC); EI mode (70 eV) was used. The analysis column was a DB-5MS capillary column (30 m x 0.25 mm ID, 0.25 μm membrane thickness). The column oven temperature was initially 50°C for 5 min, then increased to 280°C at a rate of 15°C / min, and maintained at 280°C for 5 min. The carrier gas was He, the injection amount was 1 uL, the splitless mode was used, and the solvent delay was 6.1 min. The injection port temperature was 280°C, the ion source and transfer tube temperature were both 280°C, and the GC / MS system was operated in full scan mode (m / z 50-900).

[0045] f. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) analysis:

[0046] After the reaction time, the reaction mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was collected, passed through a 0.22 μm water phase membrane, and 6 mL of the filtrate was collected and extracted with chloroform (3 x 2 mL). The extracts were combined, concentrated under nitrogen, and dissolved in methanol for LC / MS analysis.

[0047] UPLC Method: 12 min, 190-400 nm (PDA detection), A (0.1% formic acid water), B (acetonitrile), C18 column.

[0048] Table 1. Ultra Performance Liquid Chromatography Conditions

[0049]

[0050]

[0051] Table 2. Ultra Performance Liquid Chromatography Elution Program

[0052]

[0053] Mass Spectrometry Conditions: Electrospray ion source (ESI) in positive ion mode, m / z scan range 100-1000, desolvation gas flow: 800.0 L / h, desolvation gas temperature: 400 °C, cone gas flow: 30.0 L / h, ion source temperature: 120 °C, cone energy: 40 V. Capillary voltage 3.5 kV.

[0054] g. Toxicity analysis of decolorized product:

[0055] Microbial toxicity test: Based on the principle of the tube method in the Chinese Pharmacopoeia, E. coli ATCC25922, P. aeruginosa ATCC15442, S. aureus CMCC(B)26003, and B. subtilis ATCC6633 were used as test bacteria to test the toxicity of MG degradation products. A culture dish with a diameter of about 90 mm was injected with 20 mL of melted PCA medium and evenly spread on the bottom of the dish, placed on a water platform to solidify as the bottom layer. Another appropriate amount of medium was heated and cooled to 48-50 °C, and the above bacterial suspension was added, 5 mL was added to evenly spread on the bottom layer as the bacterial layer. Four stainless steel tubes (inner diameter 6.0 mm ± 0.1 mm, height 10.0 mm ± 0.1 mm, outer diameter 7.8 mm ± 0.1 mm) were evenly placed at equal distances, and an equal amount of MG and MG degradation products were added to the stainless steel tubes, and a tile round cover was used to cover them. Place at 37 °C and incubate overnight.

[0056] Example 1. Effect of different pH on R12 degradation of malachite green

[0057] To understand the effect of different pH values ​​on the degradation of malachite green by R12, degradation studies were conducted within the pH range of 5-12. In this experiment, the initial concentration of MG was 200 mg / L, the concentration of R12 cells was 8.0 g / L (wet weight), the reaction time was 30 min, the temperature was 30℃, and the rotation speed was 200 rpm.

[0058] The experimental results are shown in Figure 1 As shown, R12 exhibits a wide pH adaptability in degrading malachite green. The degradation rate remains above 99% within the pH range of 5.0–9.0. At pH 10, the degradation rate decreases slightly to 95.04%. Significant decreases occur at pH 11 and 12, with degradation rates of 88.40% and 89.55%, respectively. It is noteworthy that spontaneous decolorization of MG occurred in the control system without R12 cells. The decolorization rate increased from 20.54% to 70.74% when the pH range was 7.0–12.0. Furthermore, in the degradation system with R12, the pH range at the end of decolorization did not change significantly when the initial pH was 5.0–10.0. However, when the initial pH was 11.0 and 12.0, the final pH decreased to 10.35 and 10.87, respectively. To reduce the influence of solution pH on decolorization, a pH of 6 was determined for subsequent MG decolorization studies.

[0059] Compared to its genus *Deinococcus radiodurans* R1, R1 has an optimal initial pH range of 6.0–8.0. R1 exhibits decolorization rates of approximately 55%, 85%, 90%, 90%, and 90% in environments with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. In contrast, R12 maintains a degradation rate exceeding 99% across a pH range of 5.0–9.0. Therefore, R12 demonstrates highly efficient degradation of malachite green across a wider pH range. Compared to most reported strains, R12 can efficiently degrade MG within a pH range of 5.0–10.0. Since dye wastewater is generally highly alkaline, the use of R12 for treating dyeing plant wastewater across a wider pH range has potential value.

[0060] In this invention, pH affects microorganisms, malachite green, and other substances in the reaction system. At lower pH levels (pH 3 and pH 4), significant precipitation occurs in the degradation system; therefore, this invention selects a pH range of 5-12 for experiments. At higher pH levels (pH 7-12), the self-decolorization of malachite green becomes more pronounced. This is because malachite green and decolorized malachite green undergo interconversion, and their equilibrium depends on pH. At higher pH levels, colored malachite green molecules transform into colorless decolorized malachite green. Therefore, the high decolorization efficiency of malachite green under strongly alkaline conditions is partly attributed to the production of the colorless form of malachite green, a result of chemical transformation rather than biodegradation. pH affects microorganisms in several ways; for example, pH influences cell membrane permeability, membrane structure stability, and the rate of enzymatic reactions; the solubility or ionization of substances differs in different pH environments; and pH can also alter the absorption of nutrients by microorganisms by changing the surface charge of the microorganisms. Studies have shown that at lower pH levels, microbial surfaces carry a positive charge, preventing positively charged MG molecules from approaching and resulting in a decreased observed decolorization efficiency. In this invention, the above phenomenon was not observed, possibly because R12 degrades malachite green very efficiently. Furthermore, it is known that the pH of the culture medium dominates the transport of dye molecules across the cell membrane, which is considered the rate-limiting step in the decolorization process.

[0061] Example 2: Effect of different initial concentrations on the degradation of malachite green by R12

[0062] To investigate the effect of initial malachite green concentration on the R12 degradation system, MG was used as the substrate at an initial concentration of 100-2000 mg / L. Degradation was carried out at 30℃, pH 6.0, R12 cell concentration of 8.0 g / L (wet weight), and a rotation speed of 200 rpm. Samples were taken at 0.5 h, 1.5 h, and 2 h to determine the degradation rate. The results are shown below. Figure 2 .like Figure 2As shown, after 30 minutes of reaction, the degradation rate of MG with initial concentrations of 100-800 mg / L was above 95%. When the initial MG concentration increased from 1000 mg / L to 2000 mg / L, the degradation rate decreased from 75.58% to 35.40% after 30 minutes of reaction. The degradation of MG by R12 showed an increasing trend with increasing reaction time. After 1 hour of reaction, the degradation rate was greater than 90% for reaction systems with initial MG concentrations less than 1200 mg / L; for reaction systems with initial MG concentrations of 1400-2000 mg / L, the degradation rate increased to 68.39%-42.18%, respectively. After continuing the reaction for another half hour, the degradation rate in the reaction system with an MG concentration of 2000 mg / L was 53.14%. With increasing dye concentration, the decolorization rate gradually decreased. This decrease may be related to the toxicity of the dye to bacterial cells or the incorrect binding of dye molecules to enzyme active sites.

[0063] The typical concentration of dyes found in wastewater discharged from the textile industry is between 10-200 mg / L; therefore, the degradation rate within this concentration range is a key focus of this invention. Under the above conditions, for an initial concentration of 200 mg / L of MG, the degradation rate of MG after treatment with R12 for 30 min reached as high as 99.80%. When the initial concentration of MG was 1000 mg / L, the degradation rate reached over 95% after 1.5 h of reaction. Compared with other microorganisms capable of degrading MG (all of which have been disclosed in existing literature, and the corresponding degradation time and degradation effect are data disclosed in existing literature), the microorganism Deinococcus wulumuqiensis R12 degrades MG faster and with a higher degradation rate, as detailed in Table 3.

[0064] Table 3 Degradation efficiency of malachite green by microorganisms

[0065]

[0066] Example 3: Effect of different temperatures on the degradation of malachite green by R12

[0067] Under conditions of 200 rpm rotation speed and pH 6.0, the initial concentration of MG in the degradation system was controlled at 200 mg / L, and the concentration of R12 wet cells was controlled at 8.0 g / L. The degradation rate was measured after 30 min within the range of 10-45℃ to study the effect of temperature on the above degradation system. The experimental results are shown in [Figure number missing]. Figure 3 .Depend on Figure 3 It can be seen that R12 exhibits high degradation efficiency for malachite green within the temperature range of 10-45℃, with degradation rates all exceeding 98%. The enzyme system related to MG degradation in R12 has good thermal stability and maintains high activity even at low temperatures.

[0068] Generally, suitable temperature is conducive to the rapid growth of the strain, thereby conducive to the degradation of malachite green. The optimal degradation temperature of bacteria to malachite green is generally 30-37℃, which is basically consistent with the optimal growth temperature, for example, the optimal temperature for Pseudomonas plecoglossicide MG2 and Pseudomonas veronii to degrade malachite green is 30-35℃ and 30℃, respectively. In the decolorization system with Deinococcus radiodurans R1 cells as catalyst, when the temperature decreases from 45℃ to 25℃, the decolorization rate decreases from 97.2% to 88.0%. In the present application, R12 has good MG degradation capacity in a wider temperature range, which may be related to the higher catalytic efficiency of the related enzyme and the high activity of the related enzyme in a wider pH range. In general, R12 can perform biological remediation of MG in a wider temperature range. The high degradation rate of R12 in the low temperature range is remarkable, which has a positive significance for reducing energy consumption in industrial applications and efficient degradation in low temperature weather.

[0069] Example 4 Analysis of decolorization products

[0070] The decolorization conditions are: 30℃, pH 6.0, 200 rpm, initial MG concentration of 200 mg / L, and initial cell concentration of 4.0 g / L (wet weight). Physiological saline is used instead of TGY medium for decolorization. Then, the decolorization products are analyzed by ultraviolet (UV) full wavelength scanning, Fourier transform infrared absorption spectroscopy (FTIR) analysis, gas chromatography-mass spectrometry (GC-MS) analysis, and liquid chromatography-mass spectrometry (LC-MS) analysis.

[0071] 1) Ultraviolet (UV) full wavelength scanning analysis:

[0072] In order to understand the product production of R12 degrading malachite green, the system before and after decolorization of malachite green is analyzed by UV, FTIR, GC-MS, and LC-MS. In order to reduce the influence of other substances in the degradation system on the analysis results, physiological saline is used instead of TGY medium for decolorization reaction. In addition, considering that R12 has high and fast effect on analyzing malachite green, in order to better capture the related conditions of possible intermediate products, 4.0 g / L of R12 is used for decolorization reaction.

[0073] The results of ultraviolet full wavelength scanning are as follows: Figure 4As shown. After incubation with strain R12 for 30 min, the characteristic peak of malachite green at 617 nm (peak 4) completely disappeared, as did the peaks at 316 nm (peak 2) and 425 nm (peak 3). Conversely, the slight peak 5 at 254 nm gradually increased, and peak 1 at 203 nm showed a slight red shift over time. Simultaneously, a significant absorbance was measured at 368 nm (peak 6).

[0074] If the decolorization of malachite green by microorganisms is based on adsorption, then the absorption peaks should decrease proportionally after the reaction. If the decolorization of malachite green by microorganisms is based on chemical degradation, then the main visible light absorption peaks of the reaction substrate should disappear, and new absorption peaks should appear. Therefore, based on the comparison of spectra before and after the reaction, in this invention, the decolorization of malachite green by R12 is mainly due to chemical degradation. Furthermore, since the wavelength of the auxochrome in the conjugated polycyclic aromatic compounds is longer than that of the auxochrome on the monobenzene derivative, it is speculated that the absorption peaks at 203 nm (peak 1) and 252 nm (peak 5) are caused by monobenzene vibrations, and the peak at 368 nm (peak 6) can be attributed to vibrations of the conjugated polycyclic aromatic hydrocarbon structure. 4-(dimethylamino)benzophenone is one of the main products by attacking the central carbon of malachite green and has a significant absorbance at 360 nm; therefore, it is speculated that the peak at 368 nm represents 4-(dimethylamino)benzophenone.

[0075] 2) Fourier transform infrared absorption spectroscopy (FTIR) analysis:

[0076] To further understand the product formation of R12 degrading malachite green, the 0 min ( Figure 5 (A) in the middle, 30 min, 1 h, 12 h ( Figure 5 FTIR analysis was performed on degradation products treated at different time points (B) in the figure. The results are shown in [Figure B]. Figure 5 The FTIR spectra of the degradation products at 30 min, 1 h, and 12 h are similar (not shown in the figure).

[0077] Regarding malachite green itself, 1585cm -1 The peak at 1363 cm⁻¹ corresponds to the C=C stretching vibration of the benzene ring. -1 The peak at 1168 cm is caused by CC extension. -1 The peak at 2918 cm⁻¹ corresponds to the CN stretching vibration of aromatic compounds. -1 and 2860cm -1 The peak at 3430 cm⁻¹ is due to an asymmetric CH₃ group. -1 The peak at that point corresponds to the antisymmetric stretching of the amide.

[0078] For malachite green degradation solution, 3428 cm -1The new peaks at 2922 cm -1 and 2852 cm -1 represent the stretching of the asymmetric CH2group -CH2-. This indicates the production of methylene-substituted metabolites. The slight peak at 1722 cm -1 corresponds to the C=0 stretching of ketone. The peaks at 1633 cm -1 and 1385 cm -1 are caused by the -NH or -CN stretching vibration in amine I, II and III groups. Therefore, the FTIR analysis results show that the apparently exposed chemical groups in the MG decolorization product include C=0 and -NH2, which confirms the degradation of MG by strain R12. In addition, the following absorption peaks are characteristic peaks of aromatic compounds, and the fewer the absorption peaks, the more the aromatic compounds are broken down. -1 The following absorption peaks are characteristic peaks of aromatic compounds, and the fewer the absorption peaks, the more the aromatic compounds are broken down.

[0079] 3) Gas chromatography-mass spectrometry (GC-MS) analysis:

[0080] GC-MS, i.e. gas-liquid chromatography-mass spectrometry, has the characteristics of high resolution, high sensitivity and simple and rapid analysis process, and is one of the powerful tools for separating and detecting complex compounds. In order to identify the decolorization product obtained after the degradation of MG, the degradation products at different time periods of 0 min, 30 min, 1 h and 12 h were treated and subjected to GC-MS analysis. The generated intermediate at a retention time of 16.71 min was identified as 4-(dimethylamino) benzophenone (CAS No. 530-44-9, m / z 225). R12 can effectively degrade MG by gradually removing the benzene rings in the complex aromatic structure, which is an important step in degrading recalcitrant compounds. The intermediate detected by GC / MS may be the result of the cleavage of the entire conjugated chromophore structure, thereby producing amino benzene derivatives.

[0081] In addition, although they belong to the same Deinococcus, the degradation product of R12 in the present application is different from that of Deinococcus radiodurans R1 reported. The two intermediate products of R1 degrading malachite green are 4-(dimethylamino) phenol (m / z 136) and 4-(dimethylamino) benzophenone (m / z 225). During the incubation period of 1-12 hours, 4-(dimethylamino) phenol and 4-(dimethylamino) benzophenone are not further degraded. However, in the present application, the presence of 4-(dimethylamino) phenol is not detected, which indicates that different strategies leading to different chemical bond breaking tendencies will result in different accumulation of compounds.

[0082] 4) Liquid chromatography-mass spectrometry (LC-MS) analysis:

[0083] The degradation products of the model compound malachite green were analyzed by using electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) in positive ion scanning mode (+ESI-MS), and the structures of the degradation products were determined according to the information provided by the obtained mass spectrum as shown in Figure 7 . It is presumed that the detected related substances include malachite green (m / z 329), desmethyl malachite green (m / z 315), desdimethyl malachite green (m / z 301), 4-(dimethylamino) benzophenone (m / z 226), (4-(methylamino) phenyl) (phenyl) methanone (m / z 212). Among them, the ion peak (m / z 329) is produced by ionization of the initial substrate malachite green electrospray mass spectrometry, and the latter four substances are presumed to be degradation products and intermediates. MG (m / z 329) with a retention time of 4.72 min gradually degrades into a small amount, which indicates that the high-efficiency decolorization of MG in the reaction system is caused by R12 biodegradation.

[0084] 5) Analysis of the possible metabolic pathway of R12 degrading malachite green:

[0085] According to the reports of other researchers, the MG degradation process is relatively complex, because OH free radicals attack multiple sites of MG and its by-products in aqueous solution. The initial degradation of malachite green usually includes the following paths: hydroxylation reaction of the central carbon of MG; MG generates leucomalachite green under the action of MG reductase; and demethylation reaction of MG, which may also be caused by the attack of hydroxyl radicals.

[0086] According to the determination of the degradation products by UV, FTIR, GC-MS and LC-MS, no decolorized MG and MG hydroxylated products are detected in the present application. As shown in Figure 8 , the degradation pathway is inferred, and the continuous loss of the substituted methyl groups of the "bis(dimethylamino)" in the molecular structure of the initial substrate malachite green is mainly performed during the degradation, and the degradation is performed through two reaction channels. The first degradation reaction channel is that malachite green undergoes Csp3-N bond cleavage, and after the removal of the methyl group, the product m / z 315 is generated, and the latter is further oxidized, and again undergoes Csp3-N cleavage, and after the removal of another methyl group, the product m / z 301 is obtained, or the product m / z 226 is obtained by further oxidation and removal of the phenyl group, and the product m / z 212 is obtained by further removal of the methyl group through Csp3-N bond cleavage. The second degradation reaction channel is that malachite green is oxidized to remove N,N-dimethylaniline to obtain the product m / z 226, and the product m / z 212 is obtained by further removing the ethyl group through Csp3-N bond cleavage. The above products are further degraded, the benzene ring is cracked to generate small molecular acid, amine compounds, and finally degraded to generate H2O, CO2, NH4 + , NO3 - , CO3 2-and so on. In general, R12 can achieve high efficiency of degrading MG by removing benzene rings in complex aromatic structure step by step.

[0087] Example 5 Toxicity study of decolorized product

[0088] The decolorization conditions are: 30°C, pH 6.0, 200 rpm, initial MG concentration of 200 mg / L, and initial cell concentration of 8.0 g / L (wet weight). The degradation product (i.e. decolorized product) is obtained. The toxicity analysis of the decolorized product of MG is carried out using the existing strains in the laboratory, and the results are shown in Table 2. Figure 9 The toxicity study of microorganisms shows that the medium containing 50, 100 and 200 mg / L MG shows obvious inhibition zone, indicating that MG has toxicity to the tested Staphylococcus aureus, Micrococcus luteus, Escherichia coli and Bacillus subtilis. The degradation product of 200 mg / L MG does not show any growth inhibition to the above microorganisms. Therefore, the wastewater containing MG treated by the strain R12 will not cause fatal impact on the microbial community.

[0089] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the above examples do not limit the present application in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present application.

Claims

1. The use of Deinococcus in the degradation of malachite green wastewater, characterized by, The Deinococcus wulumuqiensis R12.

2. The use of Deinococcus in the degradation of malachite green wastewater according to claim 1, characterized in that, The Deinococcus wulumuqiensis R12 degrades malachite green wastewater under the condition of pH 5.0-10.

0.

3. The use of Deinococcus in the degradation of malachite green wastewater according to claim 1, characterized in that, The Deinococcus wulumuqiensis R12 degrades malachite green wastewater under the condition of 10-45℃.

4. The use of Deinococcus in the degradation of malachite green wastewater according to claim 1, characterized in that, The degradation reaction time is 0.2-5h.

5. The use of Deinococcus in the degradation of malachite green wastewater according to claim 1, characterized in that, The Deinococcus wulumuqiensis R12 degrades malachite green wastewater under the condition of 10-45℃ and pH 5.0-10.0, and the initial concentration of malachite green is 100-2000mg / L.

6. The use of Deinococcus in the degradation of malachite green wastewater according to claim 5, characterized in that, The initial concentration of malachite green is 100-800mg / L.

7. The use of Deinococcus in the degradation of malachite green wastewater according to claim 2, characterized in that, The pH is 5.0-9.

0.

8. The use of Deinococcus in the degradation of malachite green wastewater according to claim 4, characterized in that, The degradation reaction time is 0.5-1.5h.

9. The use of Deinococcus in the degradation of malachite green wastewater according to claim 1, characterized in that, The Deinococcus wulumuqiensis R12 is added into malachite green wastewater, and the concentration of Deinococcus wulumuqiensis R12 in the malachite green wastewater is 6-10g / L.

10. The use of Deinococcus in the degradation of malachite green wastewater according to claim 9, characterized in that, The concentration of Deinococcus wulumuqiensis R12 in the malachite green wastewater is 8g / L.

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