Aniline-degrading bacterial strain and its application

Abstract

The invention relates to the field of bioengineering and is applied in the technical field of microbial wastewater treatment. The invention provides an aniline-degrading bacterial strain, which is Delftia sp., and has a deposit number of CGMCC NO.27609. The invention can degrade aniline in a wide range of concentrations and has a high degradation efficiency. Aniline in a high concentration can be completely treated within 24 hours after inoculation.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and specifically relates to an aniline-degrading bacterial strain and application thereof. Background Art

[0002] Aniline is an important chemical raw material often used in the manufacture of pesticides, dyes, plastics, rubber and other industries. It is also a toxic pollutant and is listed as one of the pollutants under priority control by the state. The large-scale discharge of aniline wastewater not only pollutes the environment, but also threatens human health. The treatment methods of aniline wastewater include physical, chemical and biological. Compared with physical and chemical methods, biological degradation of aniline is highly economical and efficient, and is the most important method for treating large amounts of aniline-containing wastewater. However, the traditional biological method for aniline degradation also has some defects, such as low tolerance to aniline and poor resistance to shock loads. Therefore, it is necessary to screen high-efficiency aniline-degrading bacteria for bio-enhancement.

[0003] At present, the research on efficient aniline-degrading bacteria mainly focuses on the deep degradation of low-concentration aniline, while there are few studies on the degradation of high-concentration aniline and aniline-degrading bacteria that can adapt to different environments. Therefore, screening of efficient degradation strains that can use aniline as the only carbon source and energy source has important application value in sewage treatment and is one of the keys to solving aniline wastewater treatment.

[0004] Therefore, it is necessary to provide an aniline-degrading bacterial strain and its application to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide an aniline-degrading bacterial strain and application thereof.

[0006] The present invention achieves the above object through the following technical solutions:

[0007] One of the technical solutions is:

[0008] The invention discloses an aniline degrading bacterial strain, which is Delftia sp., with the deposit number of CGMCCNO.27609, referred to as HZ-O-031.

[0009] Furthermore, the nucleotide sequence of 16S rDNA is shown in SEQ ID No.1.

[0010] Furthermore, activated sludge from aerobic tanks in wastewater treatment systems of textile printing and dyeing enterprises was used to enrich aniline-degrading bacteria;

[0011] Enrichment method: After centrifuging the bottom mud, remove the supernatant and add the precipitate into the enrichment medium at 10% (w / v) and carry out enrichment at 35°C and 150r / min.

[0012] Furthermore, the changes of aniline in the culture medium are detected every day. For example, when the aniline in the culture medium drops to 0 mg / L, it is re-inoculated into a new culture medium at a ratio of 10%, and the aniline content is increased at the same time. After acclimation for 1-2 months, the aniline degradation efficiency of the sludge is greatly improved, and an aniline-degrading bacteria enriched solution is obtained at this time, and the enriched bacterial solution can be separated and purified by plate dilution coating and streaking method.

[0013] Further, the enriched bacterial solution was diluted to 10 -1 -10 -8 There were 8 gradients, and 100ul of each gradient was spread on the solid purification medium. Each gradient was made in parallel 5 times and cultured at 30℃ for 48h. Different colonies grown on the plate were picked for streaking purification. After 7-8 generations of streaking, a strain was finally purified and named HZ-O-031.

[0014] Further, the strain HZ-O-031 was streaked on beef extract peptone solid medium and cultured at 30°C under anoxic conditions for 24-48 hours. The colony diameter of HZ-O-031 was 0.5-1.0 mm, and the colony was white, raised, smooth and dull, round, with smooth edges, transparent, and odorless.

[0015] The second technical solution is:

[0016] An application of an aniline-degrading bacterial strain for treating chlorobenzene wastewater comprises the following steps:

[0017] 1) Strain activation;

[0018] The strain HZ-O-031 was inoculated into the enrichment medium and cultured at 30°C, 150 r / min, with shaking for 24 h;

[0019] 2) Application:

[0020] 2-1) Activated HZ-O-031 The activated bacteria were inoculated into 100 ml of enrichment medium at 1% (v / v), and the aniline concentrations were set at 2000 mg / L (high concentration) and 200 mg / L (low concentration);

[0021] 2-2) 30°C, 150 r / min, shaking culture for 24 h;

[0022] 2-3), sample the culture medium every 8 hours to detect OD 600 (bacterial concentration) and aniline content.

[0023] Further, the enrichment medium formula is:

[0024] Aniline 50-100 μL, K2HPO4 0.5 g, KH2PO4 0.52 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.15 g, CaCl2 0.1 g, (NH4)2SO4 0.1-1 g, add distilled water to 1 L, pH 7.0-7.2. Add distilled water to 1 L, pH 7.0-7.2.

[0025] Compared with the prior art, the present invention can degrade aniline in a wider range of concentrations and has a higher degradation efficiency. Aniline in a higher concentration can be completely treated within 24 hours after inoculation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a scanning electron microscope photograph of the aniline-degrading bacterial strain of the present invention;

[0027] Figure 2 is a phylogenetic tree of the screening strain of the present invention and similar strains;

[0028] Deposit of microorganisms for patent procedures:

[0029] Date of deposit: June 12, 2023

[0030] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0031] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0032] Deposit number: CGMCC No.27609

[0033] Classification name: Delftia sp. DETAILED DESCRIPTION

[0034] Embodiment 1:

[0035] This example shows an aniline-degrading bacterial strain, which is Delftia sp., with a deposit number of CGMCC NO.27609.

[0036] The activated sludge in the aerobic pool of the wastewater treatment system of textile printing and dyeing enterprises was used to enrich aniline-degrading bacteria;

[0037] Enrichment method: After centrifuging the bottom mud, remove the supernatant and add the precipitate into the enrichment medium at 10% (w / v) and carry out enrichment at 35°C and 150r / min.

[0038] Enrichment medium formula:

[0039] Aniline 20-1000 μL, K2HPO4 0.5 g, KH2PO4 0.52 g, NaCl 0.2 g, MgSO4·7H2O0.2 g, MnSO4·H2O 0.15 g, CaCl2 0.1 g, (NH4)2SO4 1 g, add distilled water to 1 L, pH 7.0-7.2.

[0040] The changes of aniline in the culture medium are detected every day. When the aniline in the culture medium drops to 0 mg / L, it is re-inoculated into a new culture medium at a ratio of 10%, and the aniline content is increased at the same time. After acclimation for 1-2 months, the aniline degradation efficiency of the sludge is greatly improved, and an aniline-degrading bacteria enriched solution is obtained at this time. The enriched bacterial solution can be separated and purified by plate dilution coating and streak method.

[0041] The enriched bacterial solution was diluted to 10 -1 -10 -8 There were 8 gradients, and 100ul of each gradient was spread on the solid purification medium. Each gradient was made in parallel 5 times and cultured at 30℃ for 48h. Different colonies grown on the plate were picked for streaking purification. After 7-8 generations of streaking, a strain was finally purified and named HZ-O-031.

[0042] Purification medium (liquid): aniline 50-200 μL, K2HPO4 0.5 g, KH2PO4 0.52 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.15 g, CaCl2 0.1 g, (NH4)2SO4 1 g, add distilled water to 1 L, pH 7.0-7.2. Add distilled water to 1 L, pH 7.0-7.2.

[0043] Purification medium (solid): Add 1.5%-2% (w / v) agar powder to the liquid purification medium.

[0044] The strain HZ-O-031 was streaked on beef extract peptone solid medium and cultured at 30°C under anoxic conditions for 24-48 hours. The colony diameter of HZ-O-031 was 0.5-1.0 mm, and the colony was white, raised, smooth and dull, round, with smooth edges, transparent, odorless and tasteless. The microscopic morphology of the strain was observed by scanning electron microscopy, see Figure 1 , it can be seen that the bacteria is a short rod, 1-1.5μm long and 0.5-1μm wide.

[0045] The genome of strain HZ-O-031 was extracted and 16S rDNA sequencing was performed. The obtained sequence is as follows:

[0046]

[0047] The sequencing results were compared with the known sequences in the Genbank database for similarity. Figure 2 As shown, strain HZ-O-031 had the highest sequence homology with Delftia and was identified as Delftia sp.

[0048] Effects of strain HZ-O-031 on treating different concentrations of aniline:

[0049] Bacterial strain activation: Inoculate the bacterial strain HZ-O-031 into the bacterial enrichment medium and culture at 30°C, 150r / min, with shaking for 24h.

[0050] Effect verification of different concentrations of aniline on bacterial strains: The activated bacterial strains were inoculated into 100 ml of bacterial enrichment medium at 1% (v / v), and the aniline concentrations were set at 2000 mg / L (high concentration) and 200 mg / L (low concentration). The culture was shaken at 30°C, 150 r / min for 24 hours. During the culture period, samples were taken every 8 hours to test the OD value of the culture medium. 600 (bacterial concentration) and aniline content.

[0051] Bacterial enrichment medium formula:

[0052] Aniline 50-100 μL, K2HPO4 0.5 g, KH2PO4 0.52 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.15 g, CaCl2 0.1 g, (NH4)2SO4 0.1-1 g, add distilled water to 1 L, pH 7.0-7.2. Add distilled water to 1 L, pH 7.0-7.2.

[0053] The experimental results are shown in the following table:

[0054] Table 1 Treatment effects of different concentrations of aniline on HZ-O-031

[0055]

[0056] The strain HZ-O-031 entered the logarithmic phase at 8h in both the high-concentration aniline group and the low-concentration aniline group. The OD value of the high-concentration aniline group reached a maximum of 0.850 at 16h, and the aniline degradation efficiency also reached a maximum of 247.14mg / L·h at 16h. The changes in the low-concentration aniline group were consistent with those in the high-concentration aniline group, and the maximum values ​​of the bacterial concentration and aniline degradation efficiency were reached at 16h.

[0057] Only the OD of the low concentration aniline group 600The value can only reach 0.352, which may be due to the low concentration of aniline, which serves as a carbon source for the growth of the strain, resulting in insufficient carbon source and a low bacterial concentration.

[0058] Effect of strains on treating aniline wastewater

[0059] (1) Experimental method (treatment of aniline wastewater with different inoculation concentrations):

[0060] The activated HZ-O-031 strain was added into aniline wastewater at a ratio of 0% (control group), 1%, 3% and 5% (v / v), the aniline concentration in the wastewater was 700 mg / L, and the wastewater was treated at 30°C and 150 r / min, and the aniline content was tested regularly.

[0061] Aniline wastewater:

[0062] The effluent from the biochemical pool of a textile printing and dyeing enterprise's wastewater treatment system, the aniline concentration was adjusted according to the experimental requirements.

[0063] Experimental results:

[0064] The changes in aniline were detected every 12 hours, and the results are shown in the following table. When the strain inoculation ratio was 1% and 5%, the aniline concentration could be reduced to 6.79 mg / L and 3.25 mg / L in 36 hours.

[0065] When the inoculation ratio was 0.5%, the aniline concentration could only be reduced to 130.3 mg / L in 48 hours, and it would take a long time to degrade the aniline.

[0066] The degradation trends of aniline at inoculation concentrations of 1% and 5% are basically the same. From an economic point of view, 1% can be selected as the optimal inoculation ratio for subsequent experiments.

[0067] Table 2 Effect of different inoculation ratios of strains on aniline degradation

[0068]

[0069] (2) Experimental method (strain treatment of aniline wastewater with different pH values): The activated HZ-O-031 strain was added to the aniline wastewater at a ratio of 1% (v / v). The initial aniline concentration in the wastewater was 700 mg / L. The pH of the aniline wastewater was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. The wastewater was treated at 30°C and 150 r / min, and the aniline content was tested regularly.

[0070] Experimental results:

[0071] The changes of aniline were detected every 12 hours, and the results are shown in the following table. It can be seen from the table that strain HZ-O-031 has the ability to degrade aniline in the pH range of 4-10. Between pH 5.0-9.0, within 48 hours, strain HZ-O-031 can reduce the aniline concentration to below 15 mg / L, and the degradation rate reaches 98%. When the pH is 6-9, the degradation rate can exceed 99%, indicating that strain HZ-O-031 can treat aniline wastewater in a wide pH range, with high degradation efficiency, and can adapt to wastewater with different pH.

[0072] Table 3 Effect of strains on treating aniline wastewater with different pH values

[0073]

[0074] From the above results, it can be concluded that strain HZ-O-031 can utilize aniline in a wide range of concentrations and has a high degradation efficiency. It can completely process high concentrations of aniline within 24 hours after inoculation.

[0075] Embodiment 2:

[0076] The ammonia nitrogen content in the effluent of a textile printing and dyeing company in Dalian exceeded the standard. After testing, it was found that aniline was present in both the inlet and outlet water. Aniline affected the activity of nitrifying bacteria in the nitrification system, thereby affecting the efficiency of ammonia nitrogen degradation, resulting in excessive ammonia nitrogen in the effluent water. 3 , the inlet and outlet water quality is as follows:

[0077] Table 4. Water quality parameters of inlet and outlet wastewater from a printing and dyeing enterprise in Dalian

[0078] project pH <![CDATA[Aniline (mg·L -1 )]]> <![CDATA[Ammonia nitrogen (mg·L -1 )]]> Mixed water 8-9.5 300-500 300-500 Water 7-9 15-20 100-200

[0079] Aniline-degrading bacteria HZ-O-031 was added to the front end of the aerobic pool of the plant at a ratio of 1‰. After 48 hours, the aniline content in the effluent was 0 mg / L. After 72 hours, the ammonia nitrogen in the effluent dropped to below 10 mg / L, meeting the sewage discharge standard. After one month of continuous operation, the effluent indicators are shown in Table 5. Ammonia nitrogen was maintained between 0-5 mg / L, and the effluent indicators were stable for a long time, meeting the discharge standards.

[0080] Table 5 Effluent indicators of a printing and dyeing enterprise in Dalian after using bacterial agents in production wastewater

[0081] project pH <![CDATA[Aniline (mg·L -1 )]]> <![CDATA[Ammonia nitrogen (mg·L -1 )]]> Water 7-8 0-0.5 0-5

[0082] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An aniline-degrading bacterial strain, characterized in that: Delftia Delftia sp ), the deposit number is CGMCCNO.27609, and the code is HZ-O-031.

2. An application of an aniline-degrading bacterial strain, wherein the aniline-degrading bacterial strain according to claim 1 is applied to chlorobenzene wastewater treatment, characterized in that: include: 1) Strain activation; The strain HZ-O-031 was inoculated into the enrichment medium and cultured at 30°C, 150 r / min, with shaking for 24 h; 2) Application: 2-1) Activated HZ-O-031 The activated bacteria were inoculated into 100 ml of enrichment medium at 1% (v / v), and the aniline concentrations were set at 2000 mg / L and 200 mg / L respectively; 2-2) 30°C, 150 r / min, shaking culture for 24 h; 2-3), sample the culture medium every 8 hours to detect OD 600 and aniline content.

3. The use of an aniline-degrading bacterial strain according to claim 2, characterized in that: Bacterial enrichment medium formula: Aniline 50-100 μL, K2HPO4 0.5 g, KH2PO4 0.52 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.15 g, CaCl2 0.1 g, (NH4)2SO4 0.1-1 g, add distilled water to 1 L, pH 7.0-7.2.

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