Methylobacterium sp. capable of degrading tetrabromobisphenol a and application thereof

Abstract

The application provides a strain of Stenotrophomonas maltophilia capable of degrading tetrabromobisphenol A and an application thereof, and provides an application of the Stenotrophomonas maltophilia in treatment of tetrabromobisphenol A (TBBPA) contaminated soil or water. The strain provided by the application has relatively strong TBBPA removal performance and can be used for biological remediation of TBBPA contaminated water and soil.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to the application of Stenotrophomonas maltophilia in the degradation of tetrabromobisphenol A. Background Technology

[0002] Tetrabromobisphenol A (TBBPA) is a brominated aromatic compound synthesized by the bromination of bisphenol A (BPA). It is a grayish-white powder and a typical representative of brominated flame retardants. Flame retardants are widely used as additives in materials such as electrical appliances, furniture, and textiles. During the production, use, and waste recycling of products containing TBBPA, TBBPA can escape and volatilize into the surrounding environment. It exhibits thermal stability and strong lipophilicity at room temperature, with a melting point of 184℃ and a boiling point of 316℃. It begins to decompose at around 240℃. It is soluble in organic solvents such as methanol, ethanol, and acetone, and also soluble in sodium hydroxide aqueous solution. However, it is slightly soluble in water. Due to its low water solubility and high lipophilicity, it readily accumulates in environmental samples such as soil, river sediment, and sludge.

[0003] TBBPA has been widely detected in various environmental media, organisms, and humans worldwide. Related toxicological studies have shown that TBBPA is toxic to plant and animal cells, particularly exhibiting a wide range of toxic effects in animals, including thyroid hormone interference, neurotoxicity, liver and kidney toxicity, immunotoxicity, and sex-interference.

[0004] TBBPA possesses hydrophobic and persistent properties, and currently, environmental remediation of pollutants can be achieved through both abiotic and biological methods. Biodegradation is also an important pathway for removing organic pollutants from the natural environment. It utilizes the absorption, metabolism, and degradation functions of organisms to accelerate the removal of pollutants from the environment. Due to its advantages such as low energy consumption, high efficiency, simple process, and no secondary pollution, it has been extensively and deeply studied, becoming a promising method for environmental remediation. Summary of the Invention

[0005] This application provides the application of Stenotrophomonas maltophilia in the remediation of tetrabromobisphenol A contaminated soil or water.

[0006] The strain provided by this invention exhibits strong performance in removing low concentrations of TBBPA and can be used for the bioremediation of TBBPA-contaminated water and soil. Attached Figure Description

[0007] Figure 1 It is Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia. Morphological diagram of the specimen on MHA medium.

[0008] Figure 2It is Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia. Morphological diagram of the product on an inorganic salt culture medium.

[0009] Figure 3 It is Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia. The morphology of the image under a microscope (10*100).

[0010] Figure 4 A phylogenetic tree of the 16S rDNA sequence of strain MPEB0011984 is shown.

[0011] Figure 5 The OD values ​​of the strains cultured for 16 h at different pH values ​​are shown.

[0012] Figure 6 The effect of bacterial cell addition on TBBPA degradation rate is shown.

[0013] Figure 7 The effect of the initial concentration of TBBPA on the TBBPA degradation rate is shown.

[0014] The TBBPA-removing bacteria provided by this invention belongs to the Xanthomonaceae family of the Xanthomonaceae order, and is Stenotrophomonas maltophilia MPEB0011984, with the taxonomic name Stenotrophomonas maltophilia. It was deposited on June 21, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 62558; the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0015] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0016] This invention provides a strain of Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia. The bacterium was isolated from soil surrounding an electronics factory in Qingyuan, Guangdong.

[0017] MHB medium: 17.5g casein hydrolysate, 5.0g beef extract powder, 1.5g starch, sterile water to a final volume of 1L, pH adjusted to 7.2. Solid medium (MHA) is based on MHB with the addition of 15g agar.

[0018] The inorganic salt liquid culture medium consists of: glucose 3g; NaCl 0.2g; CaCl2 0.015g; (NH4)SO4 1g; MgSO4 4.0.2g 7H2O; 0.01g FeSO4; 0.01g MnSO4; 3g K2HPO4; 1.5g KH2PO4; add water to 1L. Inorganic salt solid culture medium: add 15-20g agar per liter of liquid culture medium.

[0019] First, the sediment was enriched using inorganic salt medium (MSL). The method for isolating Stenotrophomonas maltophilia of the present invention includes the following steps:

[0020] Add 5g of soil to a 50mL centrifuge tube containing 30mL of inorganic salt medium, add TBBPA to a concentration of 10mg / L, and incubate on a shaker at 25℃ and 120rpm for 7 days. Then, transfer the culture to a new 30mg / L inorganic salt medium at a volume ratio of 10%, incubate for 7 days, then transfer to a 50mg / L TBBPA inorganic salt medium, incubate for 7 days, and then transfer again to a higher concentration of TBBPA solution until the TBBPA concentration reaches 100mg / L, obtaining a stable mud-free TBBPA degradation mixed bacterial system. Take 1mL of the above mud-free TCEP degradation mixed bacterial system, dilute it 10-fold, and perform serial dilutions with inorganic salt medium. Spread the diluted solution onto an MSL plate containing 100mg / L TBBPA, and incubate the plate at 25℃. After colonies grow, pick single bacteria. The purified single colonies were cultured in MHB solution overnight at 25°C. The bacterial culture was then mixed with a 50% glycerol aqueous solution at a ratio of 1:1 and stored at -80°C.

[0021] After isolation, purification, and screening, the strain was obtained, with the strain number MPEB0011984. The characteristics of strain MPEB0011984 are described below:

[0022] Morphology: When this strain was cultured on MHA (beef meal 6 g / L, soluble starch 1.5 g / L, acid-hydrolyzed casein 17.5 g / L, agar 17 g / L, pH 7.3) plates at 35°C for 24 h, the colonies were observed to be pale yellow, with a smooth and glossy surface. Figure 1 The strain, when incubated on an inorganic salt plate at 35°C for 24 hours, exhibited transparent, smooth, and matte colonies. Figure 2 The strains are rod-shaped with blunt, rounded ends, and are irregularly dispersed. Figure 3 Gram staining of the strain revealed it to be a Gram-negative bacterium.

[0023] 16S analysis: Genomic DNA was extracted from the pure culture of the strain described in this invention, amplified and sequenced using general software 27F and 1492r, and a phylogenetic tree was further constructed using MEGA software. Figure 4 The results showed that the bacterium was Stenotrophomonas maltophilia (Stenotrophomonas maltophilia). Stenotrophomonas maltophilia. ).

[0024] Antibiotic resistance of the strain (Table 1) showed that the strain was sensitive to chloramphenicol, ceftazidime, tigecycline, ciprofloxacin, and levofloxacin.

[0025] Table 1

[0026] antibiotic resistance results Chloramphenicol S Ceftazidime S Tigecycline S Ciprofloxacin S Levofloxacin S

[0027] The effect of pH on bacterial growth: Bacteria activated for 16 hours were added at a 6% inoculum to MHB solutions with pH 3-11. All samples were divided into three aliquots and incubated at 150 RPM in a shaker for 16 hours. Spectrophotometry was used for measurement, and the culture medium was zeroed. Bacterial growth was relatively small between pH 5 and 9, and bacteria could not grow below pH 4. Figure 5 As shown.

[0028] Degradation effect of strains on TBBPA

[0029] Preparation of bacterial suspension: One loopful of the glycerol-preserved strain was streaked onto MHA medium and cultured for 1 day. Single colonies were picked and incubated in MHB solution at 35℃ and 160 rpm for 16 hours to prepare the seed culture. The MPEB0011984 seed culture was inoculated into MHB medium at a 2% inoculum and cultured at 35℃ and 150 RPM in a constant temperature shaker for 18 hours. The bacterial cells were collected by centrifugation at 8000 rpm for 10 minutes, washed three times with ultrapure water, and the wet bacterial cells were used as a biosorbent.

[0030] Adsorption capacity calculation of strain: Method for calculating the degradation rate R of TBBPA by the strain:

[0031]

[0032] Where R is the degradation rate of TBBPA, C0 is the initial concentration of TBBPA (mg / L), and Ce is the concentration of TBBPA in the solution at equilibrium (mg / L).

[0033] Prepare the TBBPA solution to the concentrations shown in the table below. Add wet bacterial cells to the inorganic salt solution containing TBBPA at a volume ratio of 2%. Incubate at 35°C and 150 RPM in a constant temperature shaker for 5 days. Centrifuge and collect the supernatant to determine the TBBPA degradation rate. The detection method is HPLC-1260 high-performance liquid chromatography (Agilent Technologies, UV-Vis detector). The instrument conditions for HPLC measurement of TBBPA concentration are shown in Table 2 below:

[0034] Table 2

[0035]

[0036] Effect of bacterial cell addition amount: In inorganic salt solution, the strain achieved a 100% degradation rate of TBBPA at an initial concentration of 20 mg / L. Furthermore, the removal efficiency was 100% at addition amounts of 2% and 4%. Figure 6 As shown. Therefore, an addition amount of 2% by volume was chosen.

[0037] Effect of initial concentration: The strain achieved a 100% degradation rate of TBBPA at an initial concentration of 20 mg / L. At a TBBPA concentration of 60 mg / L, the degradation rate also reached 64%, demonstrating good degradation performance. The strain exhibited the highest degradation efficiency for TBBPA at concentrations below 20 mg / L.

[0038] Therefore, the strain provided by this invention can achieve a 100% degradation rate of TBBPA at concentrations of 20 mg / L and below when cultured in a constant temperature shaker at 35°C and 150 RPM for 5 days. Even when the concentration of TBBPA reaches 60 mg / L, its degradation rate of TBBPA also reaches more than 60%.

[0039] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. Application of Stenotrophomonas maltophilia in the remediation of tetrabromobisphenol A contaminated soil or water, among which, The Stenotrophomonas maltophiliae is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 62558.

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