A Burkholderia bacteria for degrading aniline black medicine and its biochar-degrading bacteria preparation and application

Abstract

The present invention discloses a Burkholderia sp. for degrading aniline black medicine and its biochar-degrading bacterial preparation and application. Burkholderia sp. WX-6 has been deposited in the Guangdong Microbial Culture Collection Center on August 23, 2022, and its deposit number is GDMCC No: 62722. The WX-6 strain and biochar are made into a bacterial agent, and the removal rates of neutral and alkaline aniline black medicine wastewater without heavy metal ions after 96 hours are 89.7% and 88.8% respectively, and the removal rates of Cu-containing wastewater are 1.34% and 1.73% respectively. 2+ and complex heavy metal ions (Cu 2+ , Pb 2+ and Zn 2+ ) after 144 hours, the removal rates of aniline black medicine wastewater were 79.9% and 61.2%, and the removal rates of chemical oxygen demand (COD) were 79.6% and 83.3%, respectively. The WX-6 strain had a good degradation effect on No. 25 black medicine, sodium butyrate black medicine, aniline and catechol.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a Burkholderia species capable of degrading nigrospermum, and a biochar-degrading bacteria preparation and application thereof. Background Art

[0002] Froth flotation is widely used in processing ores to enrich valuable minerals. Aniline black is widely used as a good collector to separate and extract valuable metals in the mineral processing process. However, aniline black cannot be completely consumed in the flotation process, and the residual aniline black is discharged with the wastewater. Moreover, the existing technology has no way to reuse wastewater economically and on a large scale. In the end, most of these mineral processing wastewaters containing aniline black are directly discharged into natural waters without being treated. This has caused great damage to the ecological environment around the mines.

[0003] Aniline black medicine is a bio-persistent flotation collector with aniline and dithiophosphate groups. Recent studies have shown that aniline black medicine has been found in many environmental media, including wastewater, surface water, irrigation water, soil and municipal sludge. More seriously, when the residual aniline black medicine in the wastewater enters the natural water body, it is easily decomposed into aniline and dithiophosphate under the action of light, oxygen, high temperature and microorganisms. Aniline is highly toxic and can cause serious damage to the liver of animals. Dithiophosphates can cause toxicity to organisms by nonspecific inhibition of acetylcholinesterase in the nervous system. Therefore, both aniline and dithiophosphates are more toxic than aniline black medicine, and will eventually endanger human health. Therefore, it is very important to carry out research on the effective treatment of mineral processing wastewater.

[0004] At present, a variety of water treatment technologies (such as physical adsorption, chemical oxidation and biodegradation) have been developed to remove pollutants. However, so far, there have been few studies on the treatment of aniline in wastewater, especially the degradation by microbial immobilization agents. High cost and secondary pollution limit the application of physical and chemical treatment methods. Biodegradation can not only selectively eliminate aniline from wastewater, but also has a fast metabolism and reproduction rate of microorganisms, and can self-renew, thereby improving wastewater treatment efficiency and reducing maintenance costs. It is a very economical method. Degrading aniline using the biological process of the main strains that can degrade aniline is an ideal technology. However, wastewater contains not only aniline but also a large amount of heavy metals. When the content of heavy metals reaches a certain concentration, it can produce toxic and lethal effects. The complex wastewater environment affects the survival rate and degradation performance of the degradation strains. There is also a long degradation reaction time (relative to physical and chemical methods), which makes biodegradation limited in treating flotation wastewater.

[0005] Immobilized microbial technology is a technology that fixes microorganisms on the surface of a carrier to provide a shelter for microorganisms to cope with complex environments. It can also adsorb and fix pollutants, thereby providing an effective environment for microorganisms to degrade pollutants. Immobilized microbial technology can increase microbial density and activity, speed up the reaction rate, provide a stable attachment environment for microorganisms, and make microorganisms more resistant to environmental influences. In recent years, biochar has been considered an ideal carrier for microorganisms due to its specific surface volume, pore size and good absorbability. For example: Zhao et al. reported that biochar as a simultaneous shelter, adsorbent, pH buffer, and substrate of Pseudomonas citronellolis to promote biodegradation of high concentrations of phenol in wastewater (Zhao, L., Xiao, D., Liu, Y., Xu, H., Nan, H., Li, D.,...&Cao, X. (2020). Biochar assimultaneous shelter, adsorbent, pH buffer, and substrate of Pseudomonas citronellolis to promote biodegradation of high concentrations of phenol in wastewater. Water Research, 172, 115494.).

[0006] In summary, at this stage, there is an urgent need for a microorganism that can efficiently degrade aniline black medicine, and at the same time, a carrier material suitable for fixing the microorganism to improve the degradation activity of the microorganism. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a Burkholderia species for degrading nigrosin and a biochar-degrading bacteria preparation and application thereof.

[0008] The first object of the present invention is to provide a Burkholderia sp. WX-6.

[0009] The second object of the present invention is to provide the use of the Burkholderia sp. WX-6 in degrading black medicine compounds and / or preparing biological preparations for degrading black medicine compounds.

[0010] The third object of the present invention is to provide the use of the Burkholderia sp. WX-6 in degrading aniline and / or catechol, and / or preparing a biological preparation for degrading aniline and / or catechol.

[0011] The fourth object of the present invention is to provide a biological agent.

[0012] The fifth object of the present invention is to provide the use of the biological preparation in degrading black medicine compounds and / or preparing the biological preparation for degrading black medicine compounds.

[0013] The sixth object of the present invention is to provide the use of the biological agent in degrading aniline and / or catechol, and / or in preparing the biological agent for degrading aniline and / or catechol.

[0014] A seventh object of the present invention is to provide a method for degrading black medicine compounds and / or intermediate products of black medicine compound degradation.

[0015] In order to achieve the above object, the present invention is implemented by the following scheme:

[0016] A Burkholderia sp. WX-6, wherein the Burkholderia sp. WX-6 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, and its preservation number is GDMCC No: 62722.

[0017] The Burkholderia sp. WX-6 is used in degrading black medicine compounds and / or preparing biological preparations for degrading black medicine compounds.

[0018] Preferably, the black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

[0019] The Burkholderia sp. WX-6 is used in degrading aniline and / or catechol, and / or preparing a biological preparation for degrading aniline and / or catechol.

[0020] A biological preparation, wherein the biological preparation contains the Burkholderia sp. WX-6.

[0021] Preferably, the biological preparation also contains biochar.

[0022] Preferably, the preparation method of the biological agent is:

[0023] S1: The Burkholderia sp. WX-6 was inoculated into LB medium, and the inoculation amount OD 600nm The pH value is 0.4 to 0.8. After culturing for 23.8 to 24.2 hours, the solid and liquid are separated, the bacteria are washed and resuspended to obtain a resuspended bacterial liquid;

[0024] S2: mixing the biochar with the resuspended bacterial liquid in step S1, wherein the ratio of the biochar to the resuspended bacterial liquid is 3.8-4.2:1, to obtain a bacterial biochar mixture;

[0025] S3: The bacterial biochar mixture of step S2 is fully mixed with glutaraldehyde fixative, fixed, hydrated with ethanol, and freeze-dried to obtain a biochar-degrading bacteria preparation.

[0026] Preferably, the content of the LB culture medium in step S1 is 49.8-50.2 mL, and the bacteria are resuspended in 4.8-5.2 mL of sterile saline.

[0027] More preferably, the content of the LB culture medium in step S1 is 50 mL, and the bacteria are resuspended with 5 mL of sterile saline.

[0028] Preferably, the ratio of the glutaraldehyde fixative in step S3 to the bacterial biochar mixture in step S2 is 0.9-1.1 mL:1 g, and the volume concentration of glutaraldehyde in the glutaraldehyde fixative is 2.4-2.6%.

[0029] More preferably, the ratio of the glutaraldehyde fixative in step S3 to the bacterial biochar mixture in step S2 is 1 mL:1 g, and the volume concentration of glutaraldehyde in the glutaraldehyde fixative is 2.5%.

[0030] Preferably, the fixing condition in step S3 is standing at 3.8-4.2° C. for 4-16 hours.

[0031] More preferably, the fixing condition in step S3 is standing at 4° C. for 4 hours.

[0032] Preferably, the gradient volume concentration of ethanol in the ethanol shrinkage in step S3 is 30%, 50%, 70%, 80%, 90% and 100% respectively.

[0033] More preferably, shrinking is performed once with ethanol having a volume concentration of 30%, 50%, 70%, 80% and 90%, and shrinking is performed twice with ethanol having a volume concentration of 100%, each time standing for 14 to 16 minutes, wherein shrinking with ethanol having a volume concentration of 70% is followed by standing for 14 to 16 hours.

[0034] More preferably, each ethanol shrinkage is allowed to stand for 15 minutes.

[0035] The biological preparation is used in degrading black medicine compounds and / or preparing the biological preparation for degrading black medicine compounds.

[0036] Preferably, the black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

[0037] The biological preparation is used in degrading aniline and / or catechol, and / or in preparing the biological preparation for degrading aniline and / or catechol.

[0038] A method for degrading black medicine compounds, aniline and / or catechol, using the Burkholderia sp. WX-6 or the biological agent according to claim 5 or 6 for degradation.

[0039] Preferably, the black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention discloses a Burkholderia for degrading aniline black medicine and its biochar-degrading bacterial preparation and application. The Burkholderia sp. WX-6 of the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, and its deposit number is GDMCC No: 62722. After the Burkholderia sp. WX-6 is mixed with an immobilized carrier such as biochar to prepare a bacterial agent, the WX-6 strain grows inside and on the surface of the immobilized carrier such as biochar.

[0042] On the one hand, the bacterial agent of the present invention enriches the density of the WX-6 strain in the contaminated wastewater, reduces the adverse effects of the external environment including heavy metal ions on the functional strain, provides a stable growth environment for the WX-6 strain, is conducive to the survival of the WX-6 strain in the wastewater, improves the treatment effect of heavy metal wastewater, and enhances its degradation and removal effect on the target pollutants.

[0043] On the other hand, immobilized carriers such as biochar have a certain specific surface area, which can directionally adsorb nigrosine drug substances in heavy metal contaminated wastewater onto the surface, promote the directional migration of pollutants, enhance the interface contact between the WX-6 strain and pollutants, and improve the degradation and removal effect of pollutants.

[0044] After WX-6 strain was mixed with immobilized carriers such as biochar to prepare bacterial agents, the removal rates of neutral and alkaline aniline black drug simulated wastewater without heavy metal ions reached 89.7% and 88.8% respectively after 96 hours. 2+ ) and complex heavy metal ions (50mg / L Cu 2+ 、100mg / LPb 2+ and 50mg / L Zn 2+) after 144 hours, the removal rates of aniline black medicine wastewater were 79.9% and 61.2%, and the removal rates of chemical oxygen demand (COD) were 79.6% and 83.3%, respectively. In addition, the WX-6 strain also has a broad spectrum of substrates, and has a good degradation effect on other black medicine compounds such as No. 25 black medicine and sodium butyrate black medicine, as well as aniline and catechol, the intermediate products of aniline black medicine degradation.

[0045] The WX-6 strain was obtained by enrichment culture from activated sludge collected from mines contaminated by aniline. The WX-6 strain has high vitality, strong ability to degrade aniline, fast growth rate, simple culture method, and is not easy to mutate. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The morphological characteristics of Burkholderia sp. WX-6 are shown in Figure 1. A is a colony morphological characteristic picture of Burkholderia sp. WX-6 on an LB plate, and B is a scanning electron microscope picture of Burkholderia sp. WX-6.

[0047] Figure 2 This is the phylogenetic tree of Burkholderia sp. WX-6.

[0048] Figure 3 This is an analysis of the factors affecting the degradation of aniline by Burkholderia sp. WX-6. A is the effect of temperature on the degradation of aniline by Burkholderia sp. WX-6; B is the effect of pH on the degradation of aniline by Burkholderia sp. WX-6; C is the effect of inoculation amount on the degradation of aniline by Burkholderia sp. WX-6; D is the effect of salinity (NaCl) on the degradation of aniline by Burkholderia sp. WX-6.

[0049] Figure 4 The experimental results of the response surface method. A is the response analysis surface of the degradation rate of aniline black medicine by Burkholderia sp. WX-6 with temperature and pH, and B is the contour line of the degradation rate of aniline black medicine by Burkholderia sp. WX-6 with temperature and pH.

[0050] Figure 5 This is the degradation kinetic curve of Burkholderia sp. WX-6 on aniline black medicine with different initial concentrations.

[0051] Figure 6 The mass spectra of the degradation products of aniline black medicine are shown in Figure 1. A is the mass spectrum of aniline, and B is the mass spectrum of catechol.

[0052] Figure 7 The invention is to identify the amplified fragments of aniline dioxygenase and catechol 1,2-dioxygenase genes of Burkholderia sp. WX-6 by electrophoresis.

[0053] Figure 8 This is the pathway for Burkholderia sp. WX-6 to degrade aniline.

[0054] Fig. 9 The scanning electron micrographs of biochar before and after bacterial fixation. A is the electron micrograph of biochar before bacterial fixation, and B is the electron micrograph of biochar after Burkholderia sp. WX-6 was fixed.

[0055] Fig.10 Figure 2 is the removal efficiency of Burkholderia sp. WX-6 and biochar-degrading bacteria preparation on aniline black medicine in different types of wastewater. A is the simulated wastewater with pH 7.0, B is the simulated wastewater with pH 9.0, and C is the simulated wastewater containing Cu 2+ A is simulated wastewater (pH 7.0), D is simulated wastewater containing mixed metal ions (pH 7.0). The original simulated wastewater was used as blank control.

[0056] Fig.11 For the Cu 2+ (A) and mixed metal ion (B) simulated wastewater, COD removal capacity of different treatments. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified. The mine sludge samples are derived from soil and tailings sludge in the Lajiapo mining area and the Mangchang mining area in Nandan County, Guangxi Zhuang Autonomous Region.

[0058] Example 1 Isolation and identification of strains

[0059] 1. Experimental Methods

[0060] 1. Enrichment of domesticated strains

[0061] Add nigronilide to 100mL of sterilized MSM medium to make the final concentration of nigronilide 50mg / L, and add 5g of mining sludge sample, culture in a shaker (30℃, 150rpm) for 7d, and transfer to the same new MSM medium at 5% inoculation, transfer 5 times continuously, and the concentration of nigronilide in the medium doubles each time, and finally the concentration of nigronilide is raised to 1000mg / L. Take the final culture solution and streak on a solid LB plate to isolate a single colony, and store the single colony in a 4℃ refrigerator.

[0062] 2. Use universal primers F27 (5'-AGAGTTTGATCCTGGCTCAG-3') and R1492 (5'-ACGGHTACCTTGTTTACGAC TT-3') to amplify the 16S rDNA fragment of the isolated strain above. The PCR reaction conditions are as follows: initial preheating at 95°C for 5 min; melting at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min 15 s, 32 cycles, and finally keeping at 72°C for 10 min.

[0063] 3. The amplified product was purified and recovered for sequencing to obtain the 16S rDNA sequence of the strain, and the nucleotide sequence of the 16S rDNA is shown in SEQ ID NO: 1. The 16S rDNA sequence was submitted to the GenBank database, and BLAST was used for comparison analysis, and ClustalX software was used to perform homology analysis on related sequences with high matching degrees. The neighbor-joining method in MEGA 5.0 software was used for relationship analysis and construction of the phylogenetic tree of the strain.

[0064] 2. Experimental Results

[0065] The isolated strain was named WX-6 (hereinafter referred to as strain WX-6). The colonies of strain WX-6 cultured on LB agar for 3 days were round, transparent, convex, and brownish yellow. Figure 1 A)

[0066] The strain WX-6 was identified as Burkholderia sp. by 16S rDNA sequencing and showed to be straight or micro-campal bacteria, arranged singly or in pairs ( Figure 1 B).

[0067] Phylogenetic tree analysis showed that strain WX-6 was closely related to Burkholderia stagnalis strain LMG 28156 and belonged to the genus Burkholderia ( Figure 2 ).

[0068] The strain WX-6 was named Burkholderia sp. WX-6, and was preserved in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022. Its preservation number is: GDMCC No: 62722, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0069] Example 2 Analysis of factors affecting the degradation of aniline by Burkholderia sp. WX-6

[0070] 1. Experimental Methods

[0071] 1. Degradation conditions of aniline black medicine by Burkholderia sp. WX-6

[0072] (1) Preparation of Burkholderia sp. WX-6 bacterial suspension

[0073] A single colony of Burkholderia sp. WX-6 from Example 1 was selected and cultured in LB medium overnight (14-16 h), then centrifuged to remove the supernatant, rinsed three times with 0.9% saline and resuspended to serve as a seed suspension. 600nm The subsequent degradation experiments were carried out using a bacterial suspension of 1.0.

[0074] (2) Effect of temperature on the degradation of aniline by strains

[0075] In a flask containing 100 mL of sterilized MSM medium, add aniline black medicine to a concentration of 300 mg / L, and inoculate Burkholderia sp. WX-6 bacterial suspension at an inoculum of 0.6 (OD 600nm ); then place the conical flasks in a shaker at 20°C, 25°C, 30°C, 35°C and 40°C, respectively, and ensure that the speed of each shaker is 150rpm. Incubate for 3 days and determine the degradation rate of aniline black medicine.

[0076] (3) Effect of pH on the degradation of nigrosine by degradation strains

[0077] The prepared MSM medium was divided into five 250 mL culture bottles with 100 mL of MSM medium per bottle. The pH value was measured with a pH meter, and the pH value of the medium was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 with 1M HCl and 1M NaOH, respectively, and then sterilized with high temperature and high pressure. Nigrosin was added to the sterilized medium to a concentration of 300 mg / L. Burkholderia sp. WX-6 bacterial suspension was inoculated with an inoculum of 0.6 (OD 600nm After inoculation, the culture bottle was placed in a shaker for continuous shaking culture for 3 days (30°C, 150 rpm), and the degradation rate of nigronilide was determined.

[0078] (4) Effect of inoculum dose on the degradation of aniline by strain WX-6

[0079] The inoculum concentration was 0.2, 0.4, 0.6, 0.8 and 1.0 (OD 600nm ) were inoculated into 250 mL culture bottles containing 100 mL sterilized MSM medium, and the content of nigromatine was 300 mg / L. After culturing in a constant temperature shaker (30°C, 150 rpm) for 3 days, the degradation rate of nigromatine by strain WX-6 was determined.

[0080] (5) Effect of salinity on the degradation of aniline by strains

[0081] NaCl was added to a 250 mL culture bottle containing 100 mL sterilized MSM medium (pH 7.0) to adjust the salinity of the culture medium to 0.0%, 1.0%, 2.0%, 3.0%, 4.0% and 5.0% respectively; aniline black was added as a growth matrix to a final concentration of 300 mg / L; Burkholderia sp. WX-6 bacterial suspension was inoculated at an inoculum of 0.6 (OD 600nm The cells were shaken and cultured in a shaker (30°C, 150 rpm) for 3 days, and the degradation rate of nigronilide was determined.

[0082] (6) Based on the experimental results of steps (2) to (4) of this embodiment, the optimal degradation conditions for the degradation of aniline by Burkholderia sp. WX-6 were determined by designing the combination of pH, temperature and inoculum size.

[0083] The response surface method was used to optimize the effects of pH, temperature and inoculation amount on the ability of the strain to degrade AAF. pH (X1), temperature (X2) and inoculation amount (X3) were used as independent variables, and -1, 0, and 1 were used to represent the three levels of the independent variables. The degradation rate of 300 mg / L of aniline black medicine by the strain on the third day was used as the response value (Y). The response surface method was used to determine the optimal combination of conditions for the strain to degrade aniline black medicine. The coding of each factor level is shown in Table 1.

[0084] Table 1 Experimental factor levels and coding based on Box-Behnken design

[0085]

[0086] (7) Effects of heavy metal ions on strain growth

[0087] Mineral processing wastewater contains Cu 2+ , Pb 2+ and Zn 2+ Therefore, when using aniline black degrading bacteria to degrade aniline black in wastewater, it is necessary to consider the effect of coexisting heavy metal ions in the wastewater on the degradation bacteria. This experiment was to explore the effects of Burkholderia sp. WX-6 on the three heavy metal ions Cu in high concentration in mineral processing wastewater. 2+ , Pb 2+ and Zn 2+ tolerable concentration.

[0088] Experimental methods:

[0089] 1) Weigh 1.6g Pb(NO3)2, 4.3478g ZnSO4·7H2O, and 4.0g CuSO4·5H2O in a clean beaker, add appropriate amount of sterile ultrapure water to fully dissolve, transfer to a volumetric flask and dilute to 100mL to obtain CuSO4·5H2O. 2+ , Pb 2+ 、Zn 2+ The concentration of the mother solution is 10.0g / L.

[0090] 2) Use a pipette to draw 0.1mL, 0.5mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of Cu 2+ , Pb 2+ 、Zn 2+ Add the solution with ion concentration of 10.0g / L to 100mL of sterilized LB solid medium that has not solidified. After shaking, pour the plate. 2+ , Pb 2+ 、Zn 2+LB solid medium with ion concentrations of 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L and 500 mg / L. The activated bacterial culture solution (WX-6 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30°C and 150 rpm for 24 h) was diluted 10 -3 After 100 μL was inoculated on the culture medium by coating, and cultured for 72 h, and the growth of microorganisms was recorded every 12 h. Three replicates were set for each treatment in the above experiment.

[0091] 2. Experimental Results

[0092] 1. If Figure 3 As shown in A, within the range of 20-40°C, the degradation rate of aniline black medicine by Burkholderia sp. WX-6 is higher than 60%.

[0093] 2. If Figure 3 As shown in B, the pH range of the degradation of aniline by Burkholderia sp. WX-6 is relatively wide, and the degradation efficiency is maintained at more than 80% in the pH range of 6 to 9.

[0094] 3. If Figure 3 As shown in C, the degradation rate of nigrosin in MSM medium by WX-6 with different inoculation amounts was 81.56-86.52% within 3 days. 600nm ) can reach a high point in the degradation rate of aniline black medicine, so the optimal inoculation size is 0.6.

[0095] 4. If Figure 3 As shown in D, Burkholderia sp. WX-6 tolerates a certain salinity and maintains a degradation rate of more than 80% when the salinity is lower than 2%, but when the salinity is higher than 3%, its degradation efficiency drops rapidly and is lower than 20%.

[0096] 5. Through Box-Behnken design, the experimental design of degradation conditions of Burkholderia sp. WX-6 was obtained and the experimental results were obtained after the experiment (see Table 2).

[0097] Table 2 Box-Behnken design experiment and results

[0098]

[0099]

[0100] Note: X1 in the table is the code value of pH; X2 is the code value of temperature; X3 is the code value of inoculation amount. The above results are the mean ± standard error of 3 replicates. The same letter after the data in the same column in the table indicates that the difference is not significant at the 5% level.

[0101] The model equation of the degradation rate (Yi) of aniline black medicine was fitted, and the reliability of the model was evaluated by model significance and variance analysis. Finally, the degradation strain was cultured according to the theoretical value of the optimal degradation conditions, and the degradation rate was used as the evaluation index to verify the reliability of the model conclusion (Table 3). The model equation is as follows:

[0102]

[0103] Table 3 Model equation significance and variance analysis

[0104]

[0105]

[0106] R 2 =0.9947 (corrected R2=0.9879), coefficient of variation (CV)=0.78%; df refers to degrees of freedom; SS refers to mean square; P<0.05 indicates that the investigated factor has a significant effect; P<0.01 indicates a very significant effect.

[0107] The statistical analysis results showed that the coefficient of determination (R 2 ) was 0.9947, indicating that there was a high correlation between the experimental measured value and the predicted value, and the model equation was suitable for the theoretical prediction of the degradation of aniline black drug by Burkholderia sp. WX-6; the coefficient of variation (CV) was 0.78%, indicating that the model equation had a high degree of fit. The model variance analysis showed that the model was highly significant (P<0.0001). The variance analysis showed that pH (X1), temperature (X2) and its quadratic coefficient X1 2 and X2 2 The linear effect on the degradation rate of nigrosine by Burkholderia sp. WX-6 was extremely significant (P<0.01).

[0108] In order to effectively and intuitively reflect the various factors and their interactions, the response surface was drawn using Design-Expert 8.0 software. Figure 4 According to the equation, the fixed inoculum amount is 0.6 (OD 600nm ), analyze the effect of temperature and pH on the degradation rate of aniline black drug by strains, and make a three-dimensional response surface ( Figure 4 A) and contour map ( Figure 4B), and the optimal degradation conditions of Burkholderia sp. WX-6 for degradation of nigromatine were obtained: temperature 33.34°C, pH 7.60, and inoculum amount 0.6 (OD600nm). At this time, the degradation rate of nigromatine predicted by the model was 89.255%.

[0109] The optimal degradation conditions (temperature 33°C, pH 7.6, inoculation amount 0.6 (OD 600nm )) Three repeated validation tests were conducted to test the accuracy of the prediction. The degradation strain WX-6 was cultured under the theoretical optimal degradation conditions. After 3 days of treatment, the degradation rates of 300 mg / L aniline black medicine in three repeated experiments were 90.74%, 90.23%, and 88.89%, respectively, which were all higher than the maximum degradation rate predicted by theory, indicating that the model fits the actual situation and the optimized conditions are available.

[0110] In summary, the optimal conditions for the degradation of aniline by Burkholderia sp. WX-6 were 33°C, pH 7.60, and inoculum concentration of 0.6 (OD 600nm )Burkholderia sp. WX6 has a wide adaptability to pH and temperature ranges during the degradation of aniline.

[0111] 6. Heavy metal ions such as Cu ions have a significant inhibitory effect on the degradation of aniline by Burkholderia sp. WX-6 at a concentration ≥100 mg / L (Table 4).

[0112] Table 4 Minimum inhibitory concentration of different heavy metal ions

[0113]

[0114] Example 3 Degradation Kinetics of Aniline Black Drug by Burkholderia sp. WX-6

[0115] 1. Experimental Methods

[0116] Different amounts of nigronilide were added to 100 mL of sterilized MSM medium to make the final concentrations of nigronilide 100, 300, 600 and 1000 mg / L, respectively, and the Burkholderia sp. WX-6 bacterial suspension of Example 2 was inoculated at an inoculum size of OD 600nm , under the optimal conditions determined in Example 2 (temperature 33°C, pH 7.60, inoculation amount 0.6 (OD 600nm)) The culture was shaken in a shaker (150 rpm), and samples were taken at 0, 12, 18, 24, 30, 48, 72 and 96 h, and the residual amount of nigrosine in each bacterial solution sample was determined by ultraviolet light, and the degradation kinetic curve of nigrosine at different concentrations was drawn. The control was non-inoculated, and 3 replicates were set for each treatment.

[0117] 2. Experimental Results

[0118] Depend on Figure 5 It can be seen that in the MSM medium, when the final concentration of nigromatine is within the range of 100-1000 mg / L, Burkholderia sp. WX-6 fully utilizes nigromatine as the sole carbon source and energy source for growth. This indicates that Burkholderia sp. WX-6 can also tolerate a higher concentration of nigromatine. Overall, the degradation rate of nigromatine by Burkholderia sp. WX-6 decreases with the increase of the initial concentration of nigromatine, indicating that high concentrations of nigromatine have an inhibitory effect on the growth of strain WX6.

[0119] As shown in Table 5, the degradation kinetic equation and half-life results of Burkholderia sp. WX-6 for different initial concentrations of aniline black medicine were obtained according to the degradation kinetic fitting model and the half-life model. The half-life of aniline black medicine degradation by Burkholderia sp. WX-6 was positively correlated with the substrate concentration, and the half-lives corresponding to 100, 300, 600 and 1000 mg / L aniline black medicine were 16.39h, 18.73h, 31.11h and 35.55h, respectively. The initial concentration range of aniline black medicine degradation by Burkholderia sp. WX-6 was wide, the degradation half-life was short, and the tolerance and degradation efficiency of aniline black medicine were high.

[0120] Table 5 Degradation kinetic parameters of aniline black medicine at different initial concentrations

[0121]

[0122] Example 4 Determination of the substrate broad spectrum of Burkholderia sp. WX-6

[0123] 1. Experimental Methods

[0124] The test substrates are No. 25 black medicine, sodium butyrate black medicine, aniline and catechol, among which No. 25 black medicine and sodium butyrate black medicine belong to black medicine compounds, and aniline and catechol are intermediate products that may be produced during the biodegradation of aniline black medicine.

[0125] These test substrates were added to the sterilized basic MSM medium to a final concentration of 300 mg / L, and fresh Burkholderia sp. WX-6 bacterial suspension of Example 2 was inoculated. The optimal degradation conditions (temperature 33°C, pH 7.6, inoculation amount 0.6 (OD 600nm )) was cultured under shaking for 3 days, and the OD of the Burkholderia sp. WX-6 culture solution in the culture solution was measured by a spectrophotometer. 600nm To determine whether Burkholderia sp. WX-6 can use these substrates for growth. The experiment used MSM medium without inoculated suspension plus the same concentration of test substrate as the control, and each group of treatments was set up with 3 replicates.

[0126] 2. Experimental Results

[0127] Table 6 shows that Burkholderia sp. WX-6 makes good use of No. 25 black medicine and sodium butane black medicine as the only carbon source and energy source, and also uses aniline black medicine to degrade intermediates aniline and catechol, indicating that Burkholderia sp. WX-6 has a wide range of substrate utilization capabilities and can further degrade aniline black medicine for use in repairing environments contaminated by aniline black medicine.

[0128] Table 6 The substrate spectrum of Burkholderia sp. WX-6

[0129]

[0130] Example 5 Identification of Nigella sativa Degradation Products and Analysis of Degradation Pathways

[0131] 1. Experimental Methods

[0132] The Burkholderia sp. WX-6 bacterial suspension of Example 2 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterilized liquid MSM medium, and nigrosine was added to a final concentration of 300 ppm. The optimal degradation conditions determined in Example 2 (temperature 33° C., pH 7.6, inoculation amount 0.6 (OD 600nm )) Oscillating culture, regular sampling for pre-treatment, the same culture medium without bacterial suspension but with the same concentration of aniline black drug was used as control, and each treatment was repeated 3 times.

[0133] Analysis of degradation pathway: The degradation products of aniline black medicine at different time periods were determined by LC-MS. Combined with the chromatographic analysis and substrate utilization, the degradation pathway of aniline black medicine by Burkholderia sp. WX-6 was inferred.

[0134] Referring to the nucleotide sequence of the aniline dioxygenase gene reported in NCBI (Accession number. D86080), a pair of degenerate primers were designed to perform PCR amplification on the aniline dioxygenase gene of Burkholderia sp. WX-6. The PCR reaction system is as follows:

[0135] Target bacterial solution: 2 μL, primers 1 μL each, Taq enzyme 12.5 μL, add sterile deionized water to make up to 50 μL. PCR amplification program: 95℃4min; 95℃ melting for 30s; 55℃ annealing for 30s; 72℃ extension for 1min; 29 cycles in total; 72℃ extension for 10min, 4℃ storage.

[0136] In order to clarify the degradation pathway of nigrosin by Burkholderia sp. WX-6, the intermediate products in the test solution were identified and analyzed by LC-TOF-MS / MS.

[0137] 2. Experimental Results

[0138] From the secondary mass spectrum, we can see that the product of aniline black medicine degradation by Burkholderia sp. WX-6 contains aniline ( Figure 6 A) Two substances: catechol ( Figure 6 B).

[0139] The aniline dioxygenase gene ( Figure 7 ), comprehensive analysis speculated that the degradation pathway of aniline black drug by Burkholderia sp. WX-6 is as follows Figure 8 As shown, aniline is first degraded into aniline, then generates catechol under the action of aniline dioxygenase, and finally enters the tricarboxylic acid cycle after catechol 1,2-dioxygenase opens the ring.

[0140] Example 6 Preparation of Biochar-Degrading Bacteria Preparation

[0141] 1. Experimental Methods

[0142] A single colony of Burkholderia sp. WX-6 from Example 1 was selected and inoculated into 50 mL of LB medium at an inoculum size of 0.6 (OD 600nm), after culturing on a shaking table for 24 h, the supernatant was removed, and the bacteria were washed with 0.9% sterile saline, and the bacteria were resuspended in 5 mL of saline after centrifugation again.

[0143] Weigh 20 g of biochar into a sterilized Erlenmeyer flask, pour in the resuspended bacteria, mix well, and use as a sample for subsequent experiments.

[0144] Take 1g of the above experimental sample, rinse it twice with physiological saline, add 1mL of 2.5% glutaraldehyde fixative and mix thoroughly, and let it stand at 4°C for 4h (extend the time as much as possible or overnight (14-16h)) for fixation; after washing with physiological saline for 3 times, perform gradient ethanol shrinkage, that is, add 30%, 50%, 70%, 80% and 90% (v / v) ethanol once, 100% ethanol twice, let it stand for 15min each time, centrifuge to remove the supernatant (after dehydration with 70% ethanol, leave it overnight (14-16h), and continue gradient dehydration on the next day), freeze-dry to obtain the biochar-degrading bacteria preparation, and observe it with a scanning electron microscope.

[0145] 2. Experimental Results

[0146] like Fig. 9 As shown, before solidification ( Fig. 9 A), the surface of biochar is not smooth, with irregular wrinkles and gaps; after solidification ( Fig. 9 B), there was no obvious change in the surface morphology of biochar, and Burkholderia sp. WX-6 was captured in the wrinkles and cracks on the surface of biochar; under a high-power electron microscope, the morphology of Burkholderia sp. WX-6 on the surface of biochar was clearly visible, without any obvious changes and rich in content, indicating that the biochar had a significant ability to fix bacteria.

[0147] Example 7 Actual repair effect of the degradation bacteria preparation

[0148] 1. Experimental Methods

[0149] The concentration of aniline black in wastewater is roughly in the range of 150-350 mg / L, and wastewater is usually alkaline. Experiments were carried out according to the actual situation. In subsequent experiments, the simulated wastewater was treated with immobilized microorganisms. The basic data was determined through simulation experiments, laying a scientific foundation for the application in actual occasions.

[0150] Weigh 1.6 g Pb(NO3)2, 4.3478 g ZnSO4·7H2O and 4.0 g CuSO4·5H2O in a clean beaker, add appropriate amount of sterile ultrapure water to fully dissolve, transfer to a volumetric flask and dilute to 100 mL to obtain CuSO4·5H2O. 2+ , Pb 2+ and Zn 2+ The concentration of the mother solution is 10.0g / L.

[0151] The formula of each liter of original simulated wastewater is as follows: 300 mg of aniline black, 1000 mg of KH2PO4, 1000 mg of K2HPO4, 1000 mg of NaCl and 10 mL of trace element aqueous solution (500 mg / LMgSO4·7H2O, 17 mg / L MnSO4·H2O, 11.6 mg / LH3BO3, 11.5 mg / LZnSO4·7H2O and 3 mg / LFeSO4·7H2O), and the solvent is water.

[0152] The pH and metal ion content of the original simulated wastewater were adjusted to obtain four types of wastewater:

[0153] 1. The pH value of wastewater is 7.

[0154] 2. The pH value of wastewater is 9.

[0155] 3. Adding Cu to simulated wastewater 2+ , Pb 2+ and Zn 2+ ions to make their final concentrations of 50, 100 and 50 mg / L respectively, and the concentration of nigrosine added was 300 mg / L. The initial pH value of the wastewater was adjusted to 7. After leaving it in the dark overnight (14-16 h), samples were taken to detect nigrosine and chemical oxygen demand (COD).

[0156] 4. Add 100 mg / L Cu ions to the simulated wastewater. Other conditions are the same as those of the third wastewater in this example.

[0157] Three experimental groups were set up with the above four types of wastewater to compare the treatment effects of biochar, Burkholderia sp. WX-6 of Example 2 and the biochar-degrading bacteria preparation of Example 6 on aniline black medicine.

[0158] Group 1: Add 2g of biochar to adsorb aniline black medicine;

[0159] Group 2: 1 mL of the Burkholderia sp. WX-6 suspension of Example 2 was added, and the inoculation amount was 0.6 (OD 600nm ), degrading aniline black medicine;

[0160] The third group: 2 g of the biochar-degrading bacteria preparation of Example 6 was added to degrade nigrosin.

[0161] Each group was placed in a constant temperature shaking incubator set at 33°C, samples were taken at regular intervals, the samples were filtered, the remaining nigrosine in the solution was determined by an ultraviolet spectrophotometer, and the chemical oxygen demand (COD) in the water was determined by the acid potassium permanganate method.

[0162] 2. Experimental Results

[0163] 1. By Fig.10 A shows that: from 0 to 24 hours, the degradation efficiency of aniline black medicine is from high to low: biochar>biochar-degrading bacteria preparation>single bacteria (Burkholderia sp. WX-6 in Example 2)>CK; after 24 hours, the biochar-degrading bacteria preparation and single bacteria successively surpassed the biochar, and the adsorption and fixation of biochar after 48 hours reached saturation. During the entire experimental period, the biochar-degrading bacteria preparation had the strongest degradation ability, followed by the single bacteria. The removal rates of aniline black medicine in neutral wastewater (pH 7.0) without heavy metal ions by the single bacteria after 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours and 96 hours were 1.8%, 9.69%, 15.76%, 66.41%, 74.01%, 80.33% and 83.67%, respectively. The removal rate of nigrosin in neutral wastewater (pH 7.0) without heavy metal ions by the biochar-degrading bacteria preparation reached 89.7% after 96 hours of treatment.

[0164] Depend on Fig.10 B It can be seen that under the influence of the strong alkaline environment, the degradation rate of aniline black medicine by the biochar-degrading bacteria preparation and the single bacteria within 48 hours is compared with the neutral environment, but it is not significant. The Burkholderia sp. WX-6 of Example 2 has a strong alkaline tolerance, which is the reason why the strong alkaline environment has little effect on the degradation of aniline black medicine by the bacterial suspension. The removal rate of aniline black medicine in alkaline wastewater (pH 9.0) without heavy metal ions by the single bacteria after 6h, 12h, 24h, 36h, 48h, 72h and 96h treatment is 0.43%, 8.95%, 7.42%, 54.67%, 66.41%, 74.01% and 77.99% respectively. The removal rate of aniline black medicine in alkaline wastewater (pH 9.0) without heavy metal ions by the biochar-degrading bacteria preparation after 96h treatment is 88.8%.

[0165] Depend on Fig.10 C and Fig.10 D shows that high concentrations of single Cu ions and mixed heavy metal ions (Cu 2+ , Pb 2+ and Zn 2+ ) is toxic to the strain, significantly reducing the strain's degradation efficiency of aniline black medicine. The use of biochar to fix the strain (biochar-degrading bacteria preparation) is used to protect the strain and improve the degradation rate. Due to the presence of heavy metals, the adsorption and removal efficiency of biochar on aniline black medicine is also significantly reduced. This is because heavy metal ions can also be adsorbed by biochar. When the adsorption capacity of biochar reaches saturation, the aniline black medicine it adsorbs is lower than when there is no heavy metal. Fig.10D It can be further seen that the adsorption capacity of biochar is nearly saturated at 72h, but at this time, the efficiency of biochar-degrading bacteria preparation in degrading aniline black medicine has been significantly improved (compared with single bacteria). This reflects the fixation effect of biochar on Burkholderia sp. WX-6 in Example 2, especially in a complex environment with high concentrations of mixed heavy metals. The removal rates of aniline black medicine in wastewater containing Cu ions (pH 7.0) by a single bacterium after 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, and 144h were 7.3%, 13.66%, 12.55%, 26.78%, 35.00%, 41.46%, 43.27%, 46.23% and 52.58%, respectively. The removal rate of single bacteria on wastewater containing mixed heavy metal ions (Cu 2+ , Pb 2+ and Zn 2+ The removal rates of nigrosine in wastewater (pH 7.0) were 6.27%, 8.80%, 4.12%, 2.51%, 8.81%, 16.82%, 24.52% and 30.41% after treatment for 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h and 144h, respectively. The biochar-degradation bacteria preparation had an obvious effect on the removal of nigrosine in wastewater containing Cu ions and mixed heavy metal ions (Cu 2+ , Pb 2+ and Zn 2+ ) in the wastewater (pH 7.0) after 144 hours of treatment, the removal rates of aniline black were 79.9% and 61.2% respectively.

[0166] 2. If Fig.11 As shown in A, in wastewater containing a single Cu ion, within 60 hours, there was no significant difference in the removal efficiency of wastewater COD by single bacteria, biochar-degrading bacteria preparation and biochar, but the biochar-degrading bacteria preparation maintained a high COD removal rate during the 84-hour experimental time. On the contrary, the removal rates of single bacteria and biochar decreased successively at 60 hours and 72 hours. After 96 hours, the removal efficiency of each pair of wastewater COD from high to low was: biochar-degrading bacteria preparation>biochar>single bacteria; Fig.11 As shown in B, in the presence of high concentrations of mixed heavy metal ions, the COD removal capacity of biochar-degrading bacteria preparations and single bacteria was weakened, among which the removal capacity of single bacteria decreased most significantly, and the capacity of biochar-degrading bacteria preparations decreased less, so the COD removal capacity of biochar-degrading bacteria preparations was highlighted. 2+ , Pb 2+ and Zn 2+) wastewater (pH 7.0) was treated for 144 hours, and the COD removal rates of nigrosine were 79.6% and 83.3% respectively.

[0167] In summary, the inoculation of Burkholderia sp. WX-6 alone, the addition of biochar alone, and the addition of biochar-degrading bacteria preparations all promoted the removal of aniline black medicine and COD in the simulated wastewater to varying degrees, among which the addition of biochar-degrading bacteria preparations had the best effect.

[0168] In view of this, the removal mechanism of aniline by biochar-degrading bacteria preparation is that biochar first combines with a part of aniline and heavy metal ions in the solution through adsorption, thereby reducing the concentration of aniline in the solution and reducing the toxicity of heavy metal ions, thereby indirectly improving the resistance of Burkholderia sp. WX-6 to toxic heavy metal ions, allowing Burkholderia sp. WX-6 to grow and reproduce, and then through the degradation of aniline by Burkholderia sp. WX-6 in the solution, aniline is further removed.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Burkholderia sp. WX-6, characterized in that The Burkholderia sp. WX-6 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, and its preservation number is GDMCC No: 62722.

2. The use of Burkholderia sp. WX-6 as claimed in claim 1 in degrading black medicine compounds and / or preparing biological preparations for degrading black medicine compounds, characterized in that: The black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

3. Use of the Burkholderia sp. WX-6 described in claim 1 in degrading aniline and / or catechol, and / or in preparing a biological agent for degrading aniline and / or catechol.

4. A biological agent, characterized in that: The biological preparation contains the Burkholderia sp. WX-6 described in claim 1.

5. The biological agent according to claim 4, characterized in that The biological preparation also contains biochar.

6. Use of the biological agent according to claim 4 or 5 in degrading black medicine compounds and / or preparing a biological agent for degrading black medicine compounds, characterized in that: The black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

7. Use of the biological agent according to claim 4 or 5 in degrading aniline and / or catechol, and / or in preparing a biological agent for degrading aniline and / or catechol.

8. A method for degrading black medicine compounds, aniline and / or catechol, characterized in that: The degradation is carried out using the Burkholderia sp. WX-6 described in claim 1 or the biological preparation described in claim 4 or 5, wherein the black medicine compound is one or more of aniline black medicine, No. 25 black medicine and sodium butadiene black medicine.

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