Bisphenol A degrading bacterium XYQ100 as well as culture method and application thereof

Abstract

The invention discloses a bisphenol A degrading bacterium XYQ100 as well as a culture method and application thereof, and relates to the technical field of microbial pollution degradation and environmental pollution, the bisphenol A degrading bacterium XYQ100 provided by the invention is classified and named as sphingobacterium sp., and is preserved in the China General Microbiological Culture Collection Center on April 18, 2025, and the preservation number of the strain is CGMCC NO.34264. The bisphenol A degrading bacterium XYQ100 provided by the invention is named as sphingobacterium sp. The strain can realize efficient removal of bisphenol A pollutants, and the degradation rate of bisphenol A within 54 hours reaches 100%.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pollution degradation and environmental pollution, and in particular to a bisphenol A-degrading bacterium XYQ100, a culture method and application thereof. Background Art

[0002] Bisphenol A, also known as BPA, is an organic compound with the molecular formula C 15 H 16 O2, BPA is almost insoluble in water, but soluble in organic solvents such as acetone and methanol. In industry, bisphenol A is used to synthesize materials such as polycarbonate (PC) and epoxy resin, which are widely used in food packaging, medical devices, etc. However, bisphenol A enters the environment during production and use. The abuse of BPA and improper disposal can lead to soil and water pollution, posing a potential threat to the ecosystem and human health. This is because BPA is also active at low levels, and the range of low-dose effects is lower than the no observed adverse effect level (NOAEL); BPA content in natural water bodies and water sources is extremely low, making detection difficult. It is easily absorbed by organisms and causes damage to individual organs.

[0003] Traditional physical and chemical methods have problems such as incomplete degradation, high cost, and possible secondary pollution when treating bisphenol A pollution. In contrast, bioremediation technology has the advantages of high degradation rate, low cost and no secondary pollution, making it a more environmentally friendly solution. In the activated sludge of sewage treatment plants, there are bacterial strains that are tolerant to and capable of degrading bisphenol A. These strains will become good materials for degrading bisphenol A. However, current research on the degradation mechanism and application of bisphenol A-degrading bacteria is still relatively limited, and there is an urgent need to further explore and develop efficient, safe, and low-cost microbial degradation technologies.

[0004] To this end, the present invention aims to provide a strain that can efficiently degrade bisphenol A and its application, so as to solve the key problems in the treatment of bisphenol A pollution. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a bisphenol A-degrading bacterium XYQ100 and its culture method and application.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A bisphenol A-degrading bacterium XYQ100, characterized in that the bisphenol A-degrading bacterium XYQ100 is classified and named Sphingobacterium sp., and was deposited in the China General Microbiological Culture Collection Center on April 18, 2025, with a culture collection number of CGMCCNO.34264.

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

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

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

[0011] Using a screening medium containing bisphenol A, bacterial strains were screened from a suspension of activated sludge samples from a sewage treatment plant, and the strains were transferred and enriched and cultured for at least three cycles using an inorganic salt medium containing bisphenol A;

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

[0013] The bacterial liquid grown with bisphenol A as the sole carbon source was streaked on an LB medium plate, and the strain was isolated and purified to obtain the bisphenol A single degradation strain XYQ100.

[0014] Preferably, in step S1, the inorganic salt culture medium needs to be added with sterile trace element stock solution and FeSO4 before use, and the volume 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 components of the inorganic salt culture medium are 1.75g / LNa2HPO4·12H2O, 0.5g / LKH2PO4, 0.5g / L(NH4)2SO4, 0.1g / LMgCl2·6H2O, and 50mg / 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] The present invention also provides the use of the 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 a fermentation broth of the bisphenol A-degrading bacteria XYQ100.

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

[0020] Sphingobacterium sp. XYQ10016S 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] The present invention isolated a bisphenol A-degrading bacterium XYQ100 from activated sludge in a sewage treatment plant. The bacterium was identified as Sphingobacterium sp. and named Sphingobacterium sp. The bisphenol A-degrading bacterium XYQ100 can effectively remove BPA pollutants and achieve a bisphenol A degradation rate of 100% within 54 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a colony plate image of the bisphenol A-degrading bacteria XYQ100 of the present invention;

[0025] Figure 2 This is a microscopic examination image of the bisphenol A-degrading bacteria XYQ100 of the present invention;

[0026] Figure 3 is the 16S rDNA phylogenetic tree of the bisphenol A-degrading bacteria XYQ100 of the present invention;

[0027] Figure 4 is a degradation curve diagram showing the degradation rate of bisphenol A in the inorganic salt culture medium over time;

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

[0029] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

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

[0031] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[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°C 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, make up to 1 L with dd H2O and sterilize by autoclave at 121°C for 20 min.

[0034] LB solid medium: 5 g / L yeast powder, 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 method for screening a bisphenol A-degrading bacterium strain XYQ100 comprises the following steps:

[0037] S1. Enrichment and acclimation of bisphenol A-tolerant bacteria:

[0038] The bacterial strains were screened from the suspension of activated sludge samples from a sewage treatment plant using an inorganic salt culture medium containing bisphenol A at an initial concentration of 50 mg / L, and were transferred and enriched and cultured for three rounds using an inorganic salt culture medium containing bisphenol A at a concentration of 50 mg / L.

[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] The bacterial liquid that can grow with bisphenol A as the sole carbon source was streaked on LB medium plates, and the strain was isolated and purified to obtain the bisphenol A single degradation strain XYQ100; the morphology of the purified bisphenol A degradation strain XYQ100 on solid culture medium was as follows Figure 1 As shown in the figure, the colony is light yellow in color, in contrast to the colorless part of the culture medium. The colony is round, with a smooth surface, a relatively dense center area, and a relatively fluffy edge with neat and regular edges. Figure 2As shown, the bacteria are rod-shaped, long and narrow, with blunt ends and no obvious curvature or spherical structure. The length of the bacteria is about 2-5 μm, and the width is about 0.5-1 μm. The arrangement is mostly single dispersed or short chain arrangement, and no obvious aggregation phenomenon is observed.

[0042] Example 2 Identification Method of Bisphenol A-Degrading Bacteria XYQ100

[0043] The genomic DNA of the 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 of 16S rDNA were both universal bacterial primers.

[0044] Finally, the obtained PCR product was electrophoresed on 1% agarose gel. According to the electrophoresis results, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing to obtain its 16Sr DNA sequence. Then, the sequence was compared with the NCBI database. The comparison results showed that the bacteria belonged to the genus Sphingobacterium. Its phylogenetic tree is as follows: Figure 3 As shown. This strain was identified as Sphingobacterium sp. and named Sphingobacterium sp., and the nucleotide sequence is shown in SEQ ID NO. 1. The strain was deposited in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on April 18, 2025, with the accession number CGMCC NO. 34264.

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

[0046] In this example, bisphenol A was added to an inorganic salt medium at a final concentration of 50 mg / L and strain XYQ100 was inoculated at a 1% volume ratio to serve as an experimental group. Simultaneously, bisphenol A was added to the inorganic salt medium at a final concentration of 50 mg / L as a control. The cells were incubated in a constant temperature shaker at 30°C and 220 rpm for 54 hours. Ultra-high performance liquid chromatography was used to measure the degradation of bisphenol A by strain XYQ100, 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 Represent the concentrations of control samples and microbial treated samples, respectively.

[0049] The experimental results showed that the degradation rate of bisphenol A by strain XYQ100 reached 100% within 54 hours ( Figure 4The OD600 of the culture medium was measured by a microplate reader to characterize the growth of the bacteria. 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 above-described embodiments merely represent preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and such modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A bisphenol A-degrading bacterium strain XYQ100, characterized by: The bisphenol A-degrading bacteria XYQ100 is classified and named Sphingobacterium sp., and was deposited in the China General Microbiological Culture Collection Center on April 18, 2025, with the culture collection number CGMCC NO.34264.

2. The bisphenol A-degrading bacteria XYQ100 according to claim 1, characterized in that The nucleotide sequence of the bisphenol A-degrading bacteria XYQ100 is shown in SEQ ID NO.

1.

3. A method for culturing the bisphenol A-degrading bacteria XYQ100 according to claim 1 or 2, characterized in that: The steps include: S1. Enrichment and acclimation of bisphenol A-tolerant bacteria Using a screening medium containing bisphenol A, bacterial strains were screened from a suspension of activated sludge samples from a sewage treatment plant, and the strains were transferred and enriched and cultured for at least three cycles using an inorganic salt medium containing bisphenol A; S2. Isolation and purification of bisphenol A-degrading bacteria The bacterial liquid grown with bisphenol A as the sole carbon source was streaked on an LB medium plate, and the strain was isolated and purified to obtain the bisphenol A single degradation strain XYQ100.

4. The culture method according to claim 3, wherein In step S1, the inorganic salt culture medium needs to be added with sterile trace element stock solution and FeSO4 before use. The volume 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.

5. The culture method according to claim 3, wherein The components of the inorganic salt culture medium are 1.75g / LNa2HPO4·12H2O, 0.5g / L KH2PO4, 0.5g / L (NH4)2SO4, 0.1g / L MgCl2·6H2O, and 50mg / L CaCl2.

6. The culture method according to claim 3, wherein The sterile trace element stock solution is 500 mg / L ETA, 10 mg / L ZnSO4·7H2O, 3 mg / L MnCl2·4H2O, 30 mg / L H3BO3, 20 mg / L CoCl2·6H2O, 1 mg / LCuCl2·2H2O, 2 mg / L NiCl2·6H2O, and 3 mg / L Na2MoO4·2H2O.

7. Use of the bisphenol A-degrading bacteria XYQ100 according to any one of claims 1 to 6 in the biodegradation and removal of bisphenol A in wastewater.

8. A microbial agent for degrading bisphenol A, characterized in that: The invention comprises the bisphenol A-degrading bacteria XYQ100 according to any one of claims 1 to 6 or the fermentation liquid of the bisphenol A-degrading bacteria XYQ100.

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