Acinetobacter Seyfertii LY-1 and its application in degradation of 6PPDQ

Abstract

The invention discloses a Seyfert bacterium LY-1 and an application thereof in degrading 6PPDQ. The Seyfert bacterium LY-1 is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M20241742 and a preservation time of August 5, 2024. The invention obtains the Seyfert bacterium LY-1 from activated sludge of a sewage treatment plant through acclimation, separation and screening. Within 7 days, the strain is cultured under pH, 30°C temperature and good growth conditions, and the degradation rate of 0.5 mg / L of 6PPDQ reaches 35.26%. In the case of adding glucose, the degradation efficiency of 0.5 mg / L of 6PPDQ within 7 days is 17.37% higher than that of a blank group without adding an external carbon source. The Seyfert bacterium LY-1 obtained by the invention has certain application value in degrading 6PPDQ in an actual environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Acinetobacter seifertii LY-1 and application thereof in degrading 6PPDQ. Background Art

[0002] The antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is usually used as an additive for rubber tires. It is often released into the environment (atmospheric environment, surface runoff, soil sediments, etc.) due to friction between rubber tires and road surfaces, and then oxidized to N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPDQ); therefore, tire wear has become one of the main forms of 6PPDQ pollution in the environment.

[0003] Studies have found that 6PPDQ is the main cause of death of coho salmon in the Seattle area of ​​the United States. In addition to acute toxicity to salmon, 6PPDQ may also cause long-term adverse reactions such as deformities and abnormal behavior in zebrafish larvae; in addition, 6PPDQ can enter the human body through multiple routes such as inhalation and skin contact, and can be detected in human urine. Its combination with PM2.5 may induce oxidative stress and DNA damage. Therefore, 6PPDQ has a potential threat to human health, so how to safely degrade it has become one of the research directions of many researchers.

[0004] At present, the degradation of 6PPDQ in the environment mainly adopts non-biological degradation, which is cumbersome and ineffective. With the development of microbial pollution treatment technology, the degradation of 6PPDQ by microorganisms is simple and low-cost. However, due to the lack of strains that can efficiently degrade 6PPDQ, it is only in the conceptual stage. Therefore, screening strains that can efficiently degrade 6PPDQ is of great significance for the industrial application of microbial degradation of 6PPDQ. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a Seyfertia Acinetobacter LY-1 and its application in degrading 6PPDQ, which can efficiently degrade 6PPDQ.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A strain of Acinetobacter Seyferti LY-1, Acinetobacter seifertii LY-1, the strain is deposited in China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20241742, the deposit address is Wuhan University, and the deposit time is August 5, 2024.

[0008] As a preferred technical solution, the nucleotide sequence of 16S rDNA of the Acinetobacter seiferti LY-1 is shown as SEQ ID: 1.

[0009] As a preferred technical solution, Acinetobacter Seyferti LY-1 was isolated and screened from activated sludge in a sewage treatment plant. Before isolation and screening, the activated sludge was domesticated and cultured in LB liquid culture medium using gradient concentrations of 6PPDQ.

[0010] The application of the Seyfertia acinetobacter LY-1 in the degradation of 6PPDQ.

[0011] The method for degrading 6PPDQ by using the Seyfertia Acinetobacter LY-1 comprises: inoculating Seyfertia Acinetobacter LY-1 into an enriched culture medium containing 6PPDQ, and aerobically culturing the culture medium at 30° C. for 7 days to degrade 6PPDQ, wherein the degradation rate of 6PPDQ is 52.93%.

[0012] As a preferred technical solution, the enrichment medium comprises: NaCl 0.2g, K 2 HPO 4 0.5g, KH 2 PO 4 0.5g, NH 4 Cl 1g, MgSO 4 7H 2 O 0.2g, FeSO 4 7H 2 O trace, glucose 5g / L.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention obtains Seyfertia Acinetobacter LY-1 from activated sludge of a sewage treatment plant through domestication, separation and screening. The Seyfertia Acinetobacter LY-1 belongs to a new species under the genus Seyfertia and can efficiently degrade 6PPDQ. In addition, the present invention also optimizes the optimal culture medium and culture conditions for degrading 6PPDQ by Seyfertia Acinetobacter LY-1. Within 7 days, under the conditions of pH, 30 DEG C of temperature and good growth, the strain is cultured in an enriched culture medium without adding a carbon source, and the degradation rate of 0.5 mg / L of 6PPDQ reaches 35.26%; within 7 days, when the strain is cultured in an enriched culture medium with added glucose, the degradation efficiency of 0.5 mg / L of 6PPDQ is 17.37% higher than that of a blank group without adding an external carbon source, and can reach 52.93%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the streak pattern of the 6PPDQ-degrading strain LY-1. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] In the present invention:

[0018] 1. The activated sludge used for strain isolation and screening of the present invention comes from a sewage treatment plant in Deqing County, Huzhou City, Zhejiang Province.

[0019] 2. Each culture medium:

[0020] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0021] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 10 g / L.

[0022] Inorganic salt liquid medium (enriched medium without carbon source): NaCl 0.2g, K 2 HPO 4 0.5g, KH 2 PO 4 0.5g, NH 4 Cl 1g, MgSO 4 7H 2 O 0.2g, FeSO 4 7H 2 HO and dissolve it in 1 L of deionized water and adjust the medium pH to 7.0.

[0023] Enrichment medium: NaCl 0.2g, K 2 HPO 4 0.5g, KH 2 PO 4 0.5g, NH 4 Cl 1g, MgSO 4 7H 2 O 0.2g, FeSO 4 7H 2 O trace, glucose 5g / L.

[0024] All culture media were sterilized in an autoclave at 121°C for 30 min before use.

[0025] 3. Main reagents and instruments:

[0026] N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPDQ), dichloromethane (HPLC), acetone (HPLC), methanol (HPLC), ultra-high performance liquid chromatography (Agilent Technologies Co., Ltd.), high-pressure steam sterilizer (Shidukai Instrument Equipment Co., Ltd.), constant temperature shaking incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), multi-tube vortex shaker (Wuxi Laipu Instrument Equipment Co., Ltd.), rotary evaporator (Aika Instrument Equipment Co., Ltd.).

[0027] Example 1: Isolation and purification of 6PPDQ-degrading bacteria

[0028] The isolation and purification of 6PPDQ-degrading bacteria includes the following steps:

[0029] Step 1: domestication:

[0030] Add 10 mL of activated sludge to a 250 mL conical flask containing 100 mL of LB liquid culture medium, and place it in a constant temperature shaking incubator at 30°C and 180 r / min for culture. Enrichment culture for 24 h, then take 1 mL of the enriched culture solution and add it to 100 mL of inorganic salt liquid culture medium containing 0.1 mg / L 6PPDQ, and place it in a constant temperature shaking incubator at 30°C and 180 r / min for culture. After 7 days, take 1 mL of the culture solution and transfer it to a fresh inorganic salt liquid culture medium containing 0.2 mg / L 6PPDQ, and continue to culture in a constant temperature incubator at 30°C and 180 r / min for 7 days, thereby gradually increasing the 6PPDQ concentration to 0.5 mg / L with a concentration gradient of 0.1 mg / L, and culture for 7 days under the same culture conditions.

[0031] Step 2, separation and purification:

[0032] Take 1 mL of the last cultured bacterial solution and press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 After dilution, take 50 μL of diluted bacterial solution and drop it into LB solid culture medium, use a coating rod to spread it evenly, and when the bacterial solution is almost dry, seal the culture dish with a sealing film, then invert the plate, place it in a 30°C constant temperature incubator for static culture, and when obvious single colonies are formed in the culture medium, select single colonies of different colors and shapes for plate streaking to purify the strains, purify the obtained single colonies 3-5 times, and obtain 4 strains after separation and purification, named LY-1, LY-2, LY-3, and LY-4. Take the four single strains purified for the last time and store them in a -80°C refrigerator with 30% glycerol for standby use.

[0033] Example 2: Screening of 6PPDQ-degrading bacteria

[0034] The screening of 6PPDQ-degrading bacteria includes the following steps:

[0035] Four frozen strains LY-1, LY-2, LY-3, and LY-4 were screened and streaked onto LB solid culture medium and cultured in a 30°C constant temperature incubator. When single colonies grew out, they were inoculated into LB liquid culture medium and cultured in a 30°C, 180 r / min shaking incubator for bacterial amplification. After 24 hours, the enriched bacterial liquid was centrifuged in an 8000 rpm high-speed centrifuge for 5 minutes, the upper culture medium liquid was discarded, the bacteria were washed 3 times with sterile water and resuspended, and OD600 was adjusted to 1.0. 5% of LY-1, LY-2, LY-3, and LY-4 bacterial liquid were inoculated in 50 mL conical flasks containing 10 mL of inorganic salt liquid culture medium, and 0.5 mg / L of 6PPDQ was added. The culture was incubated at 30°C, 180 r / min constant temperature shaking incubator, wherein a blank group without bacterial solution was set up, each in triplicate, after 7 days, the samples were subjected to liquid-liquid extraction to extract 6PPDQ in the culture solution, and the residual content of 6PPDQ in each culture medium was detected by ultra-high performance liquid chromatography, and the degradation rate of 6PPDQ was calculated. The results are shown in Table 1;

[0036] Table 1 Degradation rate data table

[0037]

[0038] From the data in Table 1, it can be seen that strain YL-1 has the best effect in degrading 6PPDQ.

[0039] Example 3: Identification of strain YL-1

[0040] The identification process of strain YL-1 is as follows:

[0041] 1) After activation, LY-1 was cultured in LB solid medium in a 30°C constant temperature incubator for 24 hours. Its morphological characteristics are: the colonies of the strain are round with a diameter of 1.0-2.0 mm, light yellow in color, opaque, with obvious protrusions in the middle, smooth and moist surface, and identified as Gram-positive bacteria after Gram staining. The colonies grown on the plate are as follows: Figure 1 shown.

[0042] 2) The isolated and screened strain LY-1 was sequenced and identified by 16S rDNA sequencing. The 16S rDNA base sequence is shown in SEQ ID: 1.

[0043]

[0044] The strain LY-1 was identified as belonging to the genus Acinetobacter Seifertii ( Acinetobacter seifertii ), so it was named Seyfert rod LY-1 ( Acinetobacter seifertii LY-1).

[0045] Example 4: Optimization of conditions for degradation of 6PPDQ by Acinetobacter Seyfertii LY-1

[0046] Condition Optimization Test Method

[0047] Acinetobacter Seyfertii LY-1 was inoculated into enriched medium containing 0.5 mg / L 6PPDQ and inorganic salt liquid medium containing 0.5 mg / L 6PPDQ at an inoculum size of 5%, respectively, and cultured in a constant temperature shaking incubator at 30℃ and 180 r / min, in triplicate. After 7 days, the samples were subjected to liquid-liquid extraction to extract 6PPDQ from the culture medium. The residual content of 6PPDQ in each culture medium was detected by ultra-high performance liquid chromatography, and the degradation rate of 6PPDQ was calculated. The results showed that under the same culture conditions, the degradation rate of 6PPDQ was 35.26% when Acinetobacter Seyfertii LY-1 was cultured in inorganic salt liquid medium (without carbon source), and the degradation rate of 6PPDQ reached 52.93% when cultured in enriched medium (with carbon source), which was 17.37% higher than that in inorganic salt liquid medium.

[0048] Therefore, it can be seen that the optimal culture conditions for the degradation of 6PPDQ by using Seyfert bacterium LY-1 are: using enriched culture medium (containing carbon source) in a constant temperature shaking incubator at 30°C and 180 r / min for 7 days.

[0049] In summary, the Acinetobacter Seyferti LY-1 isolated and screened by the present invention can be used as a 6PPDQ-degrading bacterium for 6PPDQ degradation. The present invention provides excellent microbial strain resources for microbial remediation of 6PPDQ pollution and provides a possible way to degrade 6PPDQ.

[0050] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A Seifertia bacteria ( Acinetobacter seifertii )LY-1, characterized in that The strain is deposited in China Center for Type Culture Collection with the accession number CCTCC NO: M 20241742.

2. Acinetobacter Seifertii as claimed in claim 1 ( Acinetobacter seifertii )Application of LY-1 in the degradation of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone.

3. Using the Acinetobacter Seifertii as claimed in claim 1 ( Acinetobacter seifertii ) A method for degrading N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone by LY-1, characterized in that: Acinetobacter seifertii LY-1 is inoculated into a fermentation medium containing N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone, and cultured aerobically at 30°C for 7 days to degrade N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone; wherein the fermentation medium adopts an enriched medium.

4. Acinetobacter Seifertii according to claim 3 ( Acinetobacter seifertii ) A method for degrading N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone by LY-1, characterized in that: The enrichment medium includes: NaCl 0.2 g / L, K2HPO4 0.5 g / L, KH2PO4 0.5 g / L, NH4Cl 1 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O trace and glucose 5 g / L.

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