A strain of Burkholderia vietnamese and its application in treating dye wastewater containing 3,4-dichlorophenylthiourea

Abstract

A strain of Burkholderia vietnamense and its application in treating dye wastewater containing 3,4-dichlorophenylthiourea, the strain can remove 3,4-dichlorophenylthiourea in wastewater by biosorption, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.26144, the strain can be applied to the treatment of wastewater containing dye intermediate 3,4-dichlorophenylthiourea in dye industry, when treating the wastewater containing simulated 3,4-dichlorophenylthiourea, 5~10 g / L glucose is added as an additional carbon source, and the constant temperature shock culture is 2 d, and the content of 3,4-dichlorophenylthiourea in the simulated dye wastewater after treatment is greatly reduced. Compared with the traditional wastewater treatment method, the strain described in the present invention and its application method have high treatment efficiency, wide adaptability, simple operation, easy operation and control, low requirements for process equipment, greatly reduced treatment cost, and solve the problems generated in the treatment process of other methods.

Description

Technical Field

[0001] The invention relates to the technical field of industrial wastewater treatment, and in particular to a strain of Burkholderia vietnamese and application thereof in treating dye wastewater containing 3,4-dichlorophenylthiourea. Background Art

[0002] With the increasing application of various dyes, about 10~15% of dyes and dye intermediates will be discharged into the environment during production or use. Most dyes and dye intermediates are relatively stable and highly toxic. They are difficult to degrade naturally in the soil or water environment, which will have a great impact on the environment and human body.

[0003] Adsorption and degradation are two important processes of environmental behavior and migration transformation of dyes, pesticides and other substances. Among them, the effect of biosorption on biodegradation has gradually been paid attention to. Biosorption refers to the physical and chemical process of adsorbing chemical substances (metals, compounds, etc.) onto the surface of organisms or entering their interior, including physical and chemical adsorption, electrostatic action, ion exchange, complexation, chelation and trace precipitation. There have been a lot of reports on the biosorption of heavy metals, dyes, pesticides, etc. by microbial materials, but there are no reports on the treatment of wastewater containing 3,4-dichlorophenylthiourea dye by microbial adsorption. Summary of the invention

[0004] Technical problem solved: In view of the above technical problems, the present invention provides a strain of Vietnamese Burkholderia and its application in treating dye wastewater containing 3,4-dichlorophenylthiourea. The Vietnamese Burkholderia PMD-2 provided by the present invention can remove the dye intermediate 3,4-dichlorophenylthiourea through biological adsorption, especially after adding glucose as an external carbon source, it can provide sufficient starting carbon source for PMD-2, can proliferate in large quantities and maintain high activity, can reduce the toxicity and inhibitory effect of wastewater on microorganisms, thereby greatly reducing the content of 3,4-dichlorophenylthiourea in the water environment to achieve the removal effect, and has the advantages of short time consumption, significant and stable effect, low treatment cost, etc., the specific application method and reaction conditions are mild, the strain has a wide adaptability range, the process is simple, the operation is easy, and it is efficient and reliable.

[0005] Technical solution: A strain of Burkholderia vietnamese ( Burkholderia vietnamiensis )PMD-2, whose deposit number is CGMCC No.26144.

[0006] PMD-2 colony characteristics: The colony is single, light yellow and translucent, regular and round, with a relatively moist and shiny surface, a bulge in the middle, and easy to pick up. Gram staining is negative, and the bacterial body is short and blunt at both ends.

[0007] The above-mentioned Vietnamese Burkholderia PMD-2 is used to treat wastewater containing 3,4-dichlorophenylthiourea dye intermediates.

[0008] As a preferred embodiment, the specific application steps are as follows:

[0009] Step 1. Activate and culture Burkholderia vietnamese PMD-2, transfer the activated culture seed solution into the fermentation medium at a volume ratio of 1-10%, and ferment and culture until the late logarithmic growth period to prepare the bacterial suspension;

[0010] Step 2. Add the bacterial suspension prepared in step 1 to the wastewater to be tested containing 3,4-dichlorophenylthiourea dye intermediate, add glucose as an external carbon source, and culture it aerobically in a shaker at a temperature of 30-37 ° C and a shaker speed of 120-200 r / min for 2-7 days;

[0011] Step 3. After adding the bacterial suspension prepared in step 1 in step 2, samples are taken at intervals of 1 day, and the changes in the residual amount of pollutants in the wastewater to be tested before and after the addition of the strain are determined by HPLC. At the same time, the full wavelength scan of the solution before and after treatment is performed by ultraviolet spectrophotometer.

[0012] Preferably, the activation culture process in step 1 is as follows: prepare the components of the activation culture medium according to the following concentrations: yeast extract powder 10-100 g / L, peptone 10-100 g / L, NaCl 10-100 g / L, natural pH, sterilize at 121°C for 20 minutes, cool after sterilization, inoculate slant seeds Vietnamese Burkholderia PMD-2, and culture at 30-37°C and a shaking speed of 120-200 r / min for 12-18 h to serve as the activation culture seed solution.

[0013] Preferably, the preparation process of the bacterial suspension in step 1 is as follows: the strain PMD-2 is cultured in a fermentation medium to the late logarithmic growth stage, centrifuged at 6000-8000 r / min for 8 min to collect the bacterial cells at the bottom of the centrifuge tube, an equal volume of sterile basic inorganic salt medium is added to resuspend and wash the bacterial cells twice, and the basic inorganic salt medium is used to resuspend the bacterial cells and adjust the OD to 600 =1.0, as bacterial suspension.

[0014] Preferably, in the step 2, the volume ratio of the bacterial suspension added to the wastewater to be tested is 1-10%, and the amount of glucose added is 5-10 g / L.

[0015] Preferably, the HPLC determination conditions in the step three are: using a disposable syringe equipped with a 0.22 µm organic filter membrane to filter into a liquid sample bottle, and placing it at room temperature for use; chromatographic conditions: Agilent 1260 infinityⅡ high performance liquid chromatograph, Waters® SPHERISORB® C18 (4.6 mm×250 mm, 5 µm) chromatographic column, mobile phase acetonitrile: ultrapure water = 60:40 (volume ratio); flow rate: 0.8 mL / min; column temperature: 35 °C; detection wavelength: 240 nm; injection volume: 10 µL, and 3,4-dichlorophenylthiourea is quantified by the external standard method.

[0016] Beneficial effect: The present invention obtains Burkholderia vietnamiensis (Burkholderia vietnamese) PMD-2 is a Gram-negative bacterium. After the bacterial suspension was prepared with sterilized basic inorganic salt medium and added to the simulated wastewater with high content of 3,4-dichlorophenylthiourea for treatment, the concentration of 3,4-dichlorophenylthiourea in the wastewater was reduced from 50 mg / L before treatment to below 1.15 mg / L. Especially when the inoculation amount was 1~10% and the amount of glucose added was 5~10 g / L, the removal rate of 3,4-dichlorophenylthiourea in the wastewater reached more than 98% after two days of treatment. Inoculating the wastewater with a bacterial suspension of 1~10% by volume and adding 5~10g / L of glucose can treat the wastewater by itself, which is low-cost and has low requirements for equipment. It is an effective new method for treating difficult organic wastewater in industry.

[0017] The bacteria that adsorb the dye intermediate 3,4-dichlorophenylthiourea can also act on a variety of dye intermediates with similar structures.

[0018] Deposit information: The strain of the present invention was deposited on November 14, 2022, with the deposit number: CGMCC No.26144, and the classification name is Burkholderia vietnamiensis (Burkholderia vietnamese) PMD-2, the name of the depository is the General Microbiology Center of China Culture Collection Administration, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony diagram of pure PMD-2 obtained by streaking separation in Example 1 of the present invention;

[0020] Figure 2 The microscopic morphology of PMD-2 after Gram staining according to Example 1 of the present invention;

[0021] Figure 3It is the phylogenetic tree diagram of PMD-2 of the present invention;

[0022] Figure 4 This is a full-wavelength ultraviolet scanning diagram of the culture fluid before and after PMD-2 treatment after adding glucose in Example 4 of the present invention;

[0023] Figure 5 The present invention provides a comparison chart of the removal effect of strain PMD-2 on 3,4-dichlorophenylthiourea after adding glucose and without adding glucose. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments. It should be noted that the present invention is not limited to the following specific embodiments. A person skilled in the art can adopt other various specific embodiments to implement the present invention based on the contents disclosed in the present invention, or any design structure and idea of ​​the present invention with simple changes or modifications, all fall within the protection scope of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0025] Example 1

[0026] The domestication and separation process of the present invention is as follows:

[0027] 1) Samples: Samples were provided by Zhejiang Wanfeng Chemical Co., Ltd.

[0028] 2) Culture medium

[0029] LB medium: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, natural pH.

[0030] Basic inorganic salt culture medium: NaCl 1 g / L, K2HPO4 1 g / L, (NH4)2SO4 1 g / L, MgSO4 0.2 g / L, buffered salt solution 1 mL. When preparing the basic inorganic salt culture medium, first weigh 0.1 g NaCl and 0.13 g K2HPO4 in a conical flask, add 100 mL of distilled water, and sterilize by high pressure. After sterilization, cool to room temperature, and add the remaining culture medium components: buffered salt solution, 10% MgSO4 solution, and 10% (NH4)2SO4 solution to the culture medium at a volume ratio of 1% respectively in a clean bench.

[0031] 3) Strain domestication, separation and purification

[0032] Acclimation: Accurately weigh 0.05 g of 3,4-dichlorophenylthiourea, add 10 mL of anhydrous ethanol, and sonicate until all dissolved to prepare a 5 g / L organic pollutant 3,4-dichlorophenylthiourea mother solution. Take 1 mL of the organic pollutant 3,4-dichlorophenylthiourea mother solution and add it to 100 mL of basic inorganic salt culture medium to make the concentration of 3,4-dichlorophenylthiourea in the culture medium 50 mg / L. Inoculate the wastewater into the culture medium at a volume ratio of 1-10%, and culture at 30-37 °C and 100-200 r / min for 15 days. After the culture medium becomes turbid, transfer it to a new basic inorganic salt culture medium. Each time it is transferred, the concentration of 3,4-dichlorophenylthiourea in the culture medium increases by 10 mg / L until the final concentration is 100 mg / L. This process is acclimation, which improves the strain's tolerance to 3,4-dichlorophenylthiourea.

[0033] Isolation and purification: dilute the last turbid culture solution with sterile water in a concentration gradient, spread it on LB solid medium, culture it at 30-37 °C for 48-72 h, pick colonies with good growth and different morphological characteristics, and perform several streaking separations for purification. The purified strains are stored in glycerol freezer.

[0034] 4) Screening out the strain PMD-2 that can efficiently treat 3,4-dichlorophenylthiourea

[0035] After each purified strain was activated in LB medium, it was transferred to fermentation medium for cultivation and OD was prepared. 600 =1.0 were inoculated into a basic inorganic salt culture medium containing 50 mg / L 3,4-dichlorophenylthiourea at an inoculum size of 1-10%, and samples were taken at intervals of 1 d for HPLC detection. By comparing the results, it was found that the treatment effects of other strains were minimal, and only PMD-2 had a significant effect. Therefore, subsequent research was carried out based on the PMD-2 strain provided by the invention.

[0036] 5) Classification and molecular biological identification of PMD-2

[0037] See the picture of pure PMD-2 colonies obtained by streaking isolation for details. Figure 1 For the microscopic morphology of PMD-2 after Gram staining, see Figure 2 The strains obtained above were classified and identified molecularly, and the results are as follows:

[0038] PMD-2 was identified as Burkholderia (Burkholderia spp.), presumably Burkholderia vietnamiensis (Burkholderia vietnamese). Burkholderia vietnamiensisColony characteristics of (Vietnamese Burkholderia) PMD-2: The colony is single, light yellow and translucent, regular and round, with a relatively moist and shiny surface, a raised middle, and easy to pick up. Gram staining is negative, and the bacteria are short rod-shaped with blunt ends.

[0039] The sequence information of PMD-2 strain is as follows (SEQ ID NO: 1):

[0040]

[0041] Phylogenetic tree of PMD-2 strains is shown in Figure 3 .

[0042] Example 2

[0043] Preparation of bacterial suspension:

[0044] The components of the activation culture medium were prepared according to the following concentrations: yeast extract powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, natural pH, sterilized at 121°C for 20 minutes, cooled after sterilization, inoculated with 1% by volume of the PMD-2 frozen bacterial solution prepared in Example 1, and cultured at 30-37°C and a shaking speed of 100-200 r / min for 12-18 h to serve as the activation culture seed solution.

[0045] The components of the fermentation medium were prepared according to the following concentrations: sucrose 20 g / L, peptone 10 g / L, NaCl 10 g / L, pH = 7.0, sterilized at 121 ° C for 20 minutes, cooled after sterilization, inoculated with 1-10% activated culture seed solution, cultured at 30-37 ° C, shaker speed 100-200 r / min for 12-24 h, and PMD-2 fermentation culture medium was obtained. The fermentation culture medium was centrifuged at 6000-8000 r / min for 8 min, the supernatant was discarded, the bottom bacteria were collected, and the bacteria were resuspended and washed twice with sterilized basic inorganic salt medium, the supernatant was discarded, and finally the bacteria were resuspended with basic inorganic salt medium and the OD was adjusted. 600 =1.0 and stored at 4 ℃ as bacterial suspension for later use.

[0046] Example 3

[0047] The removal effect of PMD-2 on 3,4-dichlorophenylthiourea:

[0048] PMD-2 was inoculated into a basic inorganic salt medium containing 50 mg / L 3,4-dichlorophenylthiourea at a concentration of 1-10% with a culture period of 7 days. The residual amount of 3,4-dichlorophenylthiourea was detected by HPLC.

[0049] The results showed that without adding glucose, after 7 days of cultivation, the content of 3,4-dichlorophenylthiourea in the solution decreased from 50 mg / L to 12 mg / L, and the adsorption removal rate was 76%.

[0050] Example 4

[0051] The removal effect of PMD-2 on 3,4-dichlorophenylthiourea after adding glucose:

[0052] PMD-2 was inoculated into a basic inorganic salt medium containing 50 mg / L 3,4-dichlorophenylthiourea at a concentration of 1-10% at a bacterial inoculation rate, and 5-10 g / L glucose was added as an external carbon source. After culturing for 2 days, the residual amount of 3,4-dichlorophenylthiourea was detected by HPLC and by UV full wavelength scanning (200-500 nm).

[0053] HPLC results showed that after adding glucose, the content of 3,4-dichlorophenylthiourea in the solution decreased from 50 mg / L to 1.15 mg / L after 2 days of cultivation, and the adsorption removal rate reached as high as 99%.

[0054] The UV full wavelength scanning results of the culture medium solution before and after PMD-2 treatment after adding glucose are as follows Figure 4 See the figure for the comparison of the removal effect of strain PMD-2 on 3,4-dichlorophenylthiourea after and without glucose addition. Figure 5 .

[0055] Example 5

[0056] Effect of different substrate concentrations on the adsorption and removal of PMD-2:

[0057] The scheme is as follows: prepare basal inorganic salt culture medium with concentrations of 3,4-dichlorophenylthiourea of ​​25 mg / L, 50 mg / L, 80 mg / L, and 100 mg / L (which deviates from the actual detection concentration), add 5-10 g / L glucose, inoculate at 1-10%, and culture at constant temperature and shaking for 2 days under the same conditions. Detect the changes in the residual amount of 3,4-dichlorophenylthiourea before and after treatment by HPLC. The results are shown in the following table.

[0058]

[0059] In addition, the culture conditions and bioconversion process of the strain of the present invention can be used to treat wastewater containing other dye intermediates with similar structures in addition to 3,4-dichlorophenylthiourea.

[0060] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A Burkholderia vietnamiensis PMD-2, characterized in that: Its deposit number is CGMCC No.26144.

2. Use of the Vietnamese Burkholderia described in claim 1 in adsorption treatment of wastewater containing 3,4-dichlorophenylthiourea dye intermediate.

3. The use according to claim 2, characterized in that: The specific application steps are as follows: Step 1. Activate and culture Vietnamese Burkholderia PMD-2, transfer the activated culture seed liquid into the fermentation medium at a volume ratio of 1~10%, ferment and culture to the late logarithmic growth stage, and prepare the bacterial suspension; Step 2. Add the bacterial suspension prepared in step 1 to the wastewater to be tested containing 3,4-dichlorophenylthiourea dye intermediates, add glucose as an external carbon source, and place it in a shaker for aerobic culture at a temperature of 30~37°C and a shaker speed of 120~200 r / min for 2~7 days; Step 3. After adding the bacterial suspension prepared in step 1 in step 2, take samples at intervals of 1 day, and determine the changes in the residual amount of pollutants in the wastewater to be tested before and after the addition of the strain by HPLC, and at the same time, use an ultraviolet spectrophotometer to perform a full-wavelength scan of the solution before and after treatment.

4. The use according to claim 3, characterized in that The activation culture process in step 1 is as follows: prepare the components of the activation culture medium according to the following concentrations: yeast extract powder 10-100 g / L, peptone 10-100 g / L, NaCl 10-100 g / L, natural pH, sterilize at 121°C for 20 minutes, cool after sterilization, inoculate slant seeds Vietnamese Burkholderia PMD-2, and culture at 30-37°C and a shaking speed of 120-200 r / min for 12-18 hours to use as the activation culture seed solution.

5. The use according to claim 3, characterized in that: The preparation process of the bacterial suspension in step 1 is as follows: the strain PMD-2 is cultured in a fermentation medium to the late logarithmic growth stage, the bacteria at the bottom of the centrifuge tube are collected by centrifugation at 6000-8000 r / min for 8 min, an equal volume of sterile basic inorganic salt culture medium is added to resuspend and wash the bacteria twice, and the basic inorganic salt culture medium is used to resuspend the bacteria and adjust to OD600=1.0 as a bacterial suspension.

6. The use according to claim 3, characterized in that In the step 2, the volume ratio of the bacterial suspension added to the wastewater to be tested is 1-10%, and the amount of glucose added is 5-10 g / L.

7. The use according to claim 3, characterized in that: The HPLC determination conditions in the step three are: use a disposable syringe equipped with a 0.22 µm organic filter membrane to filter into a liquid sample bottle, and place it at room temperature for use; chromatographic conditions: C18 chromatographic column, 4.6 mm×250 mm, 5 µm; the mobile phase is acetonitrile: ultrapure water = 60:40, volume ratio; flow rate: 0.8 mL / min; column temperature: 35°C; detection wavelength: 240 nm; injection volume: 10 µL, and 3,4-dichlorophenylthiourea is quantified according to the external standard method.

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