Bisphenol a degrading bacterium xyq100, culture method and application thereof

Abstract

This invention discloses a bisphenol A (BPA)-degrading bacterium, XYQ100, its cultivation method, and its applications, relating to the fields of microbial contamination degradation and environmental pollution technology. The BPA-degrading bacterium XYQ100 provided by this invention is classified as *Sphingobacterium sp.*, and was deposited at the China General Microbiological Culture Collection Center on April 18, 2025, with accession number CGMCC NO.34264. This strain can efficiently remove BPA contaminants, achieving a 100% degradation rate of BPA within 54 hours.

Description

Technical Field

[0001] This invention relates to the fields of microbial pollution degradation and environmental pollution technology, specifically to a bisphenol A degrading bacterium XYQ100, its culture method, and its application. Background Technology

[0002] Bisphenol A, also known as BPA, is an organic compound with the molecular formula C6H2O. 15 H 16 O2, BPA is almost insoluble in water but soluble in organic solvents such as acetone and methanol. Industrially, bisphenol A is used to synthesize materials such as polycarbonate (PC) and epoxy resins, and is widely used in food packaging and medical devices. However, bisphenol A enters the environment during production and use. The misuse and improper handling of BPA can lead to soil and water pollution, posing a potential threat to ecosystems and human health. This is because BPA is active even at low levels, producing low-dose effects below the no-observable-adverse-effect level (NOAEL); BPA is present in extremely low concentrations in natural water bodies and source water, making detection difficult; it is easily absorbed by organisms and can damage their organs.

[0003] Traditional physicochemical methods for treating bisphenol A (BPA) pollution suffer from problems such as incomplete degradation, high costs, and potential secondary pollution. In contrast, bioremediation technology offers advantages such as high degradation rates, low costs, and no secondary pollution, making it a more environmentally friendly solution. BPA-tolerant and degrading bacterial strains exist in activated sludge from wastewater treatment plants, making them excellent materials for BPA degradation. However, current research on the degradation mechanisms and applications of BPA-degrading bacteria remains limited, necessitating further exploration and development of efficient, safe, and low-cost microbial degradation technologies.

[0004] Therefore, the present invention aims to provide a strain that efficiently degrades bisphenol A and its application, in order to solve key problems in the treatment of bisphenol A pollution. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a bisphenol A degrading bacterium, XYQ100, its culture method, and its applications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A bisphenol A degrading bacterium, XYQ100, is characterized in that the bisphenol A degrading bacterium XYQ100 is classified and named Sphingobacterium sp., and was deposited at the China General Microbiological Culture Collection Center on April 18, 2025, with the culture accession number CGMCC NO.34264.

[0008] Preferably, the nucleotide sequence of the bisphenol A degrading bacteria XYQ100 is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for culturing the above-mentioned bisphenol A degrading bacteria XYQ100, comprising the following steps:

[0010] S1. Enrichment and domestication of bisphenol A tolerant bacteria

[0011] Bacterial strains were screened from the suspension of activated sludge samples from a wastewater treatment plant using a screening medium containing bisphenol A, and then transferred and enriched in an inorganic salt medium containing bisphenol A for at least three rounds of acclimatization.

[0012] S2. Isolation and purification of bisphenol A-degrading bacteria

[0013] The bacterial culture grown with bisphenol A as the sole carbon source was streaked on LB agar plates to isolate and purify the strain, obtaining the bisphenol A-degrading strain XYQ100.

[0014] Preferably, in step S1, sterile trace element stock solution and FeSO4 are added to the inorganic salt culture medium before use, and the volume-to-mass ratio of the inorganic salt culture medium, sterile trace element stock solution and FeSO4 is 1L:0.8~1.2mL:0.15~0.25mg.

[0015] Preferably, the inorganic salt culture medium consists of 1.75 g / L Na2HPO4·12H2O, 0.5 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.1 g / L MgCl2·6H2O, and 50 mg / L CaCl2.

[0016] Preferably, the sterile trace element stock solution is 500 mg / L EDTA, 10 mg / L ZnSO4·7H2O, 3 mg / L MnCl2·4H2O, 30 mg / L H3BO3, 20 mg / L CoCl2·6H2O, 1 mg / L CuCl2·2H2O, 2 mg / L NiCl2·6H2O, and 3 mg / L Na2MoO4·2H2O.

[0017] This invention also provides the application of the aforementioned bisphenol A degrading bacteria XYQ100 in the biodegradation and removal of bisphenol A in wastewater.

[0018] The present invention also provides a microbial agent for degrading bisphenol A, comprising the above-mentioned bisphenol A degrading bacteria XYQ100 or the fermentation broth of bisphenol A degrading bacteria XYQ100.

[0019] The sequence involved in this invention is as follows:

[0020] Bisphenol A degrading bacteria XYQ100 (Sphingobacterium sp.) 16S rRNA gene sequence (SEQ ID NO.1):

[0021]

[0022] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0023] This invention isolates a bisphenol A (BPA) degrading bacterium, XYQ100, from activated sludge in a wastewater treatment plant. It is identified as belonging to the genus Sphingobacterium and named Sphingobacterium sp. This BPA-degrading bacterium, XYQ100, can efficiently remove BPA pollutants and achieves a 100% degradation rate of BPA within 54 hours. Attached Figure Description

[0024] Figure 1 This is a colony plate image of the bisphenol A degrading bacterium XYQ100 used in this invention;

[0025] Figure 2 This is a microscopic image of the bisphenol A degrading bacterium XYQ100 used in this invention.

[0026] Figure 3 This is a phylogenetic tree of the 16S rDNA of the bisphenol A degrading bacterium XYQ100 in this invention;

[0027] Figure 4 This is a degradation curve showing the change of bisphenol A degradation rate over time in the inorganic salt culture medium of the present invention.

[0028] Figure 5 This is a graph showing the OD value changes during the growth of bisphenol A in an inorganic salt culture medium according to the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Inorganic salt culture medium (liquid): 1.75 g Na2HPO4·12H2O, 0.5 g KH2PO4, 0.5 g (NH4)2SO4, 0.1 g MgCl2·6H2O, 50 mg CaCl2, diluted to 1 L with dd H2O, sterilized at 121℃ for 20 min, and cooled to room temperature.

[0033] Trace element stock solution: 500 mg EDTA, 10 mg ZnSO4·7H2O, 3 mg MnCl2·4H2O, 30 mg H3BO3, 20 mg CoCl2·6H2O, 1 mg CuCl2·2H2O, 2 mg NiCl2·6H2O, 3 mg Na2MoO4·2H2O, diluted to 1 L with dd H2O, autoclaved at 121˚C for 20 min.

[0034] LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 1.5 g agar, sterilized at 121°C for 20 min, and cooled to room temperature.

[0035] Example 1: Isolation and Screening of Bisphenol A Degrading Bacteria

[0036] A screening method for a bisphenol A degrading bacterium, XYQ100, includes the following steps:

[0037] S1. Enrichment and domestication of bisphenol A-resistant bacteria:

[0038] Bacterial strains were screened from the suspension of activated sludge samples from a wastewater treatment plant using an inorganic salt medium containing an initial concentration of 50 mg / L bisphenol A, and then subjected to three rounds of transfer, enrichment, and acclimatization culture using an inorganic salt medium containing 50 mg / L bisphenol A.

[0039] Before use, add 1 mL / L of sterile trace element stock solution and 0.2 mg / L of FeSO4 to the inorganic salt culture medium.

[0040] S2. Isolation and purification of bisphenol A degrading bacteria:

[0041] Bacterial suspensions capable of growth using bisphenol A as the sole carbon source were streaked onto LB agar plates for bacterial isolation and purification, yielding the bisphenol A-degrading strain XYQ100. The morphology of the purified bisphenol A-degrading strain XYQ100 on solid culture medium is as follows: Figure 1 As shown, the colonies are generally light yellow, contrasting with the colorless portion of the culture medium. The colonies are round, with a smooth surface, relatively dense in the center, and looser at the edges, which are neat and regular. The morphology observed under a microscope is as follows: Figure 2As shown, the bacteria are rod-shaped, elongated, with blunt, rounded ends, and no obvious curved or spherical structures were observed. The bacteria are approximately 2-5 μm long and 0.5-1 μm wide, and are arranged mostly as single, dispersed or in short chains, with no obvious aggregation observed.

[0042] Example 2: Identification method of bisphenol A degrading bacteria XYQ100

[0043] Genomic DNA of bisphenol A degrading bacteria strain XYQ100 was extracted using a bacterial genome extraction kit, and its 16S rDNA gene was amplified by PCR. The upstream and downstream primers for the 16S rDNA were universal primers for bacteria.

[0044] Finally, the obtained PCR products were subjected to electrophoresis on a 1% agarose gel. Based on the electrophoresis results, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing to obtain their 16S rDNA sequences. The sequences were then compared using the NCBI database. The comparison results showed that this bacterium belongs to the genus *Sphingomonas*, and its phylogenetic tree is as follows: Figure 3 As shown in the figure, this strain was identified as *Sphingobacterium* sp., and named *Sphingobacterium*. The 16S rRNA gene sequence of the bisphenol A degrading bacterium XYQ100 is shown in SEQ ID NO. 1. The strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on April 18, 2025, with accession number CGMCC NO. 34264.

[0045] Example 3: Degradation effect of strain XYQ100 on bisphenol A in inorganic salt culture medium

[0046] In this embodiment, bisphenol A (BPA) was added to an inorganic salt culture medium at a final concentration of 50 mg / L, and strain XYQ100 was inoculated at a 1% (v / v) ratio as the experimental group. Simultaneously, an inorganic salt culture medium with a final concentration of 50 mg / L BPA was added as a control. The cultures were incubated at 30°C and 220 rpm for 54 h in a constant temperature shaker. The degradation of BPA by strain XYQ100 was detected by ultra-high performance liquid chromatography (UHPLC), and the degradation rate was calculated using the following formula:

[0047] Degradation rate (%) = (C0) - C X ) / C0×100%;

[0048] Among them, C0 and C X These represent the concentrations of the control sample and the microbial-treated sample, respectively.

[0049] Experimental results showed that strain XYQ100 achieved a 100% degradation rate of bisphenol A within 54 hours. Figure 4 The OD600 of the culture medium was measured using a microplate reader to characterize bacterial growth. XYQ100 began to grow significantly after 24 hours of culture, reached the logarithmic growth phase at 30 hours, and ended the logarithmic growth phase at 48 hours. Figure 5 ).

[0050] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A bisphenol A degrading bacterium, XYQ100, characterized in that, The bisphenol A degrading bacteria, classified as Sphingobacterium sp. by XYQ100, was deposited at the China General Microbiological Culture Collection Center on April 18, 2025, with the accession number CGMCC NO.34264.

2. The bisphenol A degrading bacterium XYQ100 according to claim 1, characterized in that, The 16S rRNA gene sequence of the bisphenol A degrading bacteria XYQ100 is shown in SEQ ID NO.

1.

3. The application of the bisphenol A degrading bacteria XYQ100 as described in claim 1 or 2 in the biodegradation and removal of bisphenol A in wastewater.

4. A microbial agent for degrading bisphenol A, characterized in that, Includes the bisphenol A degrading bacteria XYQ100 as described in claim 1 or 2.

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