Acinetobacter LYS-5 for degrading tetrabromobisphenol A and application thereof

Abstract

The invention discloses an acinetobacter LYS-5 for degrading tetrabromobisphenol A and application thereof, relates to the field of microorganisms, and aims to solve the problem that tetrabromobisphenol A pollution in saline-alkali soil is difficult to repair by existing microorganisms. The acinetobacter is Acinetobacter pitefer LYS-5, and is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is April 22, 2024, and the preservation number is CGMCC No.30399. The invention further discloses a preparation method of the Acinetobacter pitefer LYS-5, and the acinetobacter pitefer LYS-5 has the advantages that the acinetobacter pitefer LYS-5 can be used for preparing the acinetobacter pitefer LYS-5; the method is used for degrading the pollutant tetrabromobisphenol A in a water body and saline-alkali soil.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to an Acinetobacter LYS-5 strain capable of degrading tetrabromobisphenol A and applications thereof. Background Art

[0002] Brominated flame retardants are a type of persistent pollutant widely used in various industries, including plastics, electronics, furniture, interior decoration, transportation, petroleum, and mining. Tetrabromobisphenol A (TBBPA), a brominated aromatic compound and a typical representative of brominated flame retardants, is a white powder with a melting point of 184°C and a boiling point of 316°C. It begins to decompose at around 240°C. It is soluble in organic solvents such as methanol, ethanol, and acetone, as well as in aqueous sodium hydroxide solutions, but slightly soluble in water. Due to its high persistence, environmental stability, and resistance to degradation, TBBPA readily accumulates in humans and animals and can be transferred through the food chain, toxic to organisms at higher trophic levels, ultimately posing a health risk. TBBPA has been detected in water, soil, and sediment. Due to excessive emissions in recent years, these halogenated flame retardants have entered the environment, accumulating in marine fish, mammals, and even humans, causing endocrine disruption, hepatotoxicity, neurotoxicity, and other health issues.

[0003] The degradation of brominated flame retardants in nature has attracted considerable attention both domestically and internationally. Studies have shown that various brominated flame retardants can photodegrade or biodegrade under certain conditions. Photodegradation also produces intermediates, some of which are even more toxic than tetrabromobisphenol A itself. Biodegradation is an effective method for removing organic pollutants from sediments, and microbial degradation is considered one of the most promising approaches due to its significant effectiveness, low technical costs, and lack of secondary pollution.

[0004] The microorganisms that degrade tetrabromobisphenol A mainly include fungi, bacteria and protozoa. Among them, the fungi involved in degradation are mainly white rot fungi, and the bacteria are mainly Pseudomonas and Ochrobacter. For example, An et al. published a strain of Ochrobacter that can degrade tetrabromobisphenol A. Ochrobactrum sp.T, with a degradation efficiency of 91.8% in 72 hours; Fan Zhenzhen et al. published a strain of Pseudomonas that can degrade TBBPA, achieving a TBBPA degradation efficiency of 55.2% using glucose as a co-metabolism carbon source; Chen Jie et al. published a strain of Tetrabromobisphenol A-degrading fungus, Oxycoperdon leucophylla F17, which can also degrade TBBPA, achieving a degradation rate of up to 85.5% at 20 mg / L. However, these strains are unable to tolerate the high salinity and alkalinity of saline-alkali soils. Saline-alkali soils contaminated with TBBPA exhibit high concentrations, strong aggregation, and difficulty in degradation. However, no strains have been reported that can remediate TBBPA contamination in saline-alkali soils. Summary of the Invention

[0005] The present invention aims to solve the problem that existing microorganisms are difficult to repair tetrabromobisphenol A pollution in saline-alkali soil, and provides a tetrabromobisphenol A-degrading Acinetobacter LYS-5 and its application.

[0006] The present invention provides a strain of Acinetobacter LYS-5, which is Acinetobacter pituitus ( Acinetobacter pittii )LYS-5 has been deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 22, 2024, and the deposit number is CGMCC No. 30399.

[0007] The cells of the Acinetobacter pituitus LYS-5 of the present invention are gram-negative when stained. When observed under an electron microscope, the colony morphology is milky white, round, and smooth. The bacterial body is coccus-shaped, has a capsule, no spores, and no flagella.

[0008] The Acinetobacter pituitii LYS-5 of the present invention can grow under conditions of pH 5-10 and a temperature growth range of 15-45° C. The physiological and biochemical characteristics are positive in catalase reaction, negative in oxidase reaction, positive in starch hydrolysis, positive in citric acid utilization, negative in methyl red test, negative in nitrate reduction reaction, and positive indole production test.

[0009] The present invention Acinetobacter LYS-5 is analyzed by 16SrDNA sequence comparison with Acinetobacter Pittii ( Acinetobacter pittii ) has a homology of 99.72%. By combining the bacterial morphological characteristics, growth conditions, and physiological and biochemical identification results, it was determined that the Acinetobacter LYS-5 of the present invention is Acinetobacter Pittii ( Acinetobacter pittii ).

[0010] The present invention also provides Acinetobacter pituitus ( Acinetobacter pittii ) Application of LYS-5 in degradation of tetrabromobisphenol A in water.

[0011] The present invention also provides Acinetobacter pituitus ( Acinetobacter pittii ) Application of LYS-5 in degradation of tetrabromobisphenol A in saline-alkali soil.

[0012] Beneficial effects of the present invention: The Acinetobacter pitei LYS-5 of the present invention can survive under alkaline conditions (pH = 9-10) and utilize tetrabromobisphenol A as the sole carbon source. The Acinetobacter pitei LYS-5 of the present invention can degrade the pollutant tetrabromobisphenol A in water bodies, with a degradation rate of up to 26.45%. At the same time, Acinetobacter pitei LYS-5 can also degrade the pollutant tetrabromobisphenol A in saline-alkali soil. After the saline-alkali soil contaminated with tetrabromobisphenol A was treated with Acinetobacter pitei LYS-5 for 96 hours, the concentration of tetrabromobisphenol A was reduced from 20.86μg / kg to 15.42μg / kg. The present invention provides a new bacterial strain resource for the treatment of saline-alkali land contaminated by tetrabromobisphenol A. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a Gram staining image of Acinetobacter pituitus LYS-5 of the present invention; Figure 2 is a phylogenetic tree of Acinetobacter pituitus LYS-5 of the present invention; Figure 3 This is the degradation result of tetrabromobisphenol A in water by Acinetobacter pituitus LYS-5 of the present invention. DETAILED DESCRIPTION

[0014] The following embodiments of the present invention are described in detail. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation plans and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0015] Example 1: Acinetobacter LYS-5 of this embodiment is Acinetobacter pituitus ( Acinetobacter pittii )LYS-5 has been deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 22, 2024, and the deposit number is CGMCC No. 30399.

[0016] The method for obtaining Acinetobacter LYS-5 in this example: Soil from Heilongjiang University was mixed with saline-alkali soil from Daqing Oilfield in a volume ratio of 3:4, water was added and mixed, 0.1 mol / L sodium hydroxide solution was added to adjust the pH to about 9, and then 100 mg of tetrabromobisphenol A was added. The same amount of tetrabromobisphenol A was added every day at room temperature. After 7 days, 5% of the volume of the mixed soil was added to the acclimation medium containing tetrabromobisphenol A, and the pH was adjusted to 9. After acclimation, 5% of the inoculum was inoculated into the enrichment medium and cultured for 24 hours. Then 0.1 mL of the bacterial suspension was taken and diluted 10 -l , 10 -2 , 10 -3 , 10 -4 , 10-5 , 10 -6 , 10 -7 The strain LYS-5 was obtained by plating the strain on a solid culture medium containing tetrabromobisphenol A as the sole carbon source until a single pure colony appeared on the plate. The colony with a fast growth rate and regular edges was selected and purified.

[0017] Acclimation culture medium formula: NH4NO3 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, NaCl 1.0 g / L, MgSO4 0.15 g / L, tetrabromobisphenol A 50 mg / L.

[0018] Enrichment medium formula: beef extract 8.0~14.0g / L, NaCl 8.0~12.0g / L, peptone 4.5~5.5g / L, pH 9~10.

[0019] Acclimation solid culture medium formula: NH4NO3 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, NaCl 1.0 g / L, MgSO4 0.15 g / L, tetrabromobisphenol A 50 mg / L.

[0020] Example 2: Identification of strain LYS-5 In this example, the strain LYS-5 was subjected to physiological and biochemical identification with reference to the eighth edition of the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems: The cells of strain LYS-5 are Gram-negative. Under electron microscope, the colony morphology is milky white, round, smooth, and the bacteria are coccobacilli, with capsules, no spores, and no flagella. Figure 1 shown.

[0021] Strain LYS-5 can grow at a pH of 5-10 and a temperature range of 15-45°C. Its physiological and biochemical characteristics are positive for catalase reaction, negative for oxidase reaction, positive for starch hydrolysis, positive for citric acid utilization, negative for methyl red test, negative for nitrate reduction reaction, and positive for indole production test.

[0022] The total DNA of strain LYS-5 was extracted. Its 16S rDNA was amplified using universal primers. The 16S rDNA sequence was compared with the gene sequences registered in Genbank. It was found that strain LYS-5 had the same genus as Acinetobacter Pittii ( Acinetobacter pittii) with a homology of 99.72%. The G+C% value of the screened strain LYS-5 was 38.90%, and the G+C% values of homologous bacterial genera ranged from 38.70% to 38.90%, with little difference. The sequence number was submitted to the GenBank database and obtained the accession number PP463025. The Blast tool and MEGA7.0 and other related software provided by NCBI found homologous sequences in the GenBank database and established a phylogenetic tree (such as Figure 2 By combining the results of bacterial physiological and biochemical identification with molecular identification, it was determined that strain LYS-5 was Acinetobacter pituitarius ( Acinetobacter pittii The 16S rDNA sequence of Acinetobacter pituitus LYS-5 is shown in SEQ ID NO: 1 in the sequence listing.

[0023] Example 3: Degradation of Tetrabromobisphenol A in Water by Acinetobacter pitei LYS-5 Prepare 100 mL of liquid acclimation medium and place it in a 250 mL shake flask. Sterilize at 121°C for 30 min, then remove the medium and aseptically add tetrabromobisphenol A. Acclimation medium formula: 1.0 g / L NH₄NO₃, 0.8 g / L KH₂PO₄, 1.5 g / L K₂HPO₄, 1.0 g / L NaCl, 0.35 g / L MgSO₄.

[0024] 1. Acinetobacter pituitii ( Acinetobacter pittii) LYS-5 was inoculated into a solid culture medium and activated at 30° C. for 48 hours. The solid culture medium formula was as follows: beef extract 14.0 g / L, NaCl 12.0 g / L, peptone 5.5 g / L, and agar 20 g / L.

[0025] 2. The activated Acinetobacter Pittii ( Acinetobacter pittii) LYS-5 was inoculated into liquid culture medium for fermentation at 36°C until the bacterial count per ml of fermentation liquid was about 10 12 The liquid culture medium formula is: beef extract 14.0 g / L, NaCl 12.0 g / L, and peptone 5.5 g / L.

[0026] 3. The bacterial liquid was inoculated into a liquid acclimation medium containing 20 mg / L tetrabromobisphenol A, with an inoculation amount of 10%, the pH was adjusted to 9, and the culture was carried out at 35°C. The degradation rate was measured after 96 hours of degradation. The results were as follows: Figure 3 shown.

[0027] from Figure 3 It can be seen that the Acinetobacter Pittii strain screened by the present invention ( Acinetobacter pittii) LYS-5 has a strong ability to degrade tetrabromobisphenol A, with the highest degradation rate being 26.45%.

[0028] Example 4: Degradation of Tetrabromobisphenol A in Saline-Alkaline Soil by Acinetobacter Pitt LYS-5 Activated Acinetobacter pitei LYS-5 was inoculated into enrichment medium and cultured at 30°C and 150 rpm for 24 hours. The culture was centrifuged at 12,000 rpm for 10 minutes, and the cells were harvested and washed three times with sterile water to prepare a bacterial suspension (OD600 = 1.5). The bacterial suspension was mixed with saline-alkali soil contaminated with tetrabromobisphenol A (TBBA) (pH = 9) at a volume ratio of 1:10. The TBA concentration in the saline-alkali soil was 20.86 μg / kg. The culture was incubated at 35°C with ventilation for 96 hours, and the residual TBA content was determined. The enrichment medium formulation included 14.0 g / L beef extract, 12.0 g / L peptone, 5.5 g / L NaCl, and a pH of 9.

[0029] After 96 hours of treatment, the residual TBA in the soil was 15.42 μg / kg. This experiment demonstrates that TBA-degrading bacteria can repair TBA-contaminated saline-alkali soil without the addition of other chemicals. This provides a microbial agent resource for in situ bioremediation of TBA-contaminated saline-alkali soil, demonstrating broad application prospects.

Claims

1. A strain of Acinetobacter LYS-5 that degrades tetrabromobisphenol A, characterized in that: This Acinetobacter LYS-5 is a member of the Acinetobacter Pittii family ( Acinetobacterpittii )LYS-5 has been deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is April 22, 2024, and the deposit number is CGMCC No. 30399.

2. Use of Acinetobacter pituitus LYS-5 as claimed in claim 1 in degrading tetrabromobisphenol A in water.

3. Use of the Acinetobacter pitei LYS-5 as claimed in claim 1 in degrading tetrabromobisphenol A in saline-alkali soil.

4. A bacterial agent for degrading tetrabromobisphenol A in water, characterized in that: The bacterial agent includes the Acinetobacter pituitus LYS-5 as described in claim 1.

5. A bacterial agent for degrading tetrabromobisphenol A in saline-alkali soil, characterized in that: The bacterial agent includes the Acinetobacter pituitus LYS-5 as described in claim 1.

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