Construction method of functional microbiome for efficiently degrading bisphenol A in offshore area
By constructing a bisphenol A (BPA)-efficient degrading microbial community with a multi-site mixture as the bacterial source, the problem of poor stress resistance of single strains in nearshore waters was solved, achieving efficient BPA degradation, adapting to complex marine environments, and providing a stable pollution remediation solution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-27
AI Technical Summary
In existing technologies, the biodegradation method using a single strain has poor stress resistance and weak adaptability in nearshore waters, resulting in insufficient remediation effect and stability of bisphenol A pollution, making it difficult to apply on a large scale.
Using a mixture of nearshore water and sediment from multiple sites as the bacterial source, a highly efficient degradation microbiome was constructed through selective enrichment with bisphenol A and continuous screening over multiple generations. The microbiome was then cultured in seawater-R2A liquid medium, and highly efficient degradation strains were screened and optimized by mixing. Finally, a microbiome adapted to the complex marine environment was obtained.
The constructed microbial community can adapt to the actual environment in nearshore waters without additional domestication, achieving efficient degradation of bisphenol A with a degradation rate of over 90%. This solves the problem of the disconnect between laboratory results and application, and provides stable and efficient technical support for the remediation of pollution in nearshore waters.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a method for constructing a bisphenol A-efficient degrading microbial community in nearshore waters. Background Technology
[0002] Bisphenol A (BPA) is a typical endocrine disruptor, widely used in the production of industrial products such as polycarbonate plastics, epoxy resins, food packaging materials, and coatings. It is one of the world's largest-produced chemical raw materials. Through various pathways, including industrial emissions, leachate from domestic waste, degradation of plastic waste, and agricultural non-point source pollution, large amounts of BPA continuously enter nearshore marine ecosystems, becoming one of the most common emerging pollutants in nearshore waters. Although the concentration of BPA in nearshore waters is low, its toxicity is high. Long-term accumulation can interfere with the endocrine and reproductive development of marine organisms, disrupt ecological balance, and affect human health through bioaccumulation in seafood. Its environmental safety is of great concern.
[0003] Among existing remediation technologies, physical adsorption is prone to secondary pollution, while chemical oxidation is costly and disrupts native microbial communities, making both unsuitable for the remediation needs of nearshore marine pollution. Biodegradation is the preferred method due to its environmental friendliness, but current technologies rely on single bacterial strains, which have poor resilience and adaptability. Affected by the complex environment of nearshore waters, their remediation effects and stability are insufficient, hindering large-scale application. Functional microbiomes, leveraging synergistic effects to enhance degradation efficiency and environmental adaptability, represent a cutting-edge research area. However, their application in nearshore waters faces bottlenecks, such as insufficient colonization and stability of microbial communities in actual marine environments. Therefore, developing efficient and stable BPA-degrading functional microbiome construction methods adapted to nearshore environments is crucial for improving pollution remediation efficiency and ensuring ecological security, and is an urgent technical problem to be solved. Summary of the Invention
[0004] The purpose of this invention is to explore the potential of nearshore marine environmental microorganisms to degrade bisphenol A, construct a bisphenol A-degrading microbiome, and provide a technical means to solve the problem of bisphenol A pollution in nearshore marine areas.
[0005] The purpose of this invention is to provide a method for constructing a bisphenol A-efficient degrading microbial community in nearshore waters, which includes the following steps:
[0006] (1) Using a mixture of nearshore water and sediment from 3 to 8 sites as the bacterial source, inoculate it into bisphenol A-seawater-R2A liquid medium and culture it at 20 to 35°C and 120 to 210 r / min for 7 to 10 days to obtain the first generation culture medium system I;
[0007] (2) Inoculate the first generation culture medium system I into sterile bisphenol A-seawater-R2A liquid culture medium at an inoculation rate of 8-12% by volume;
[0008] (3) Repeat step (2) 3-6 times. After each enrichment, select a culture medium system with a bisphenol A removal efficiency ≥ 50% for the next round of inoculation.
[0009] (4) Mix the enrichment solutions obtained in step (3) with all sites and bisphenol A removal efficiency ≥90% in equal volume ratio to obtain the final nearshore marine bisphenol A efficient degradation functional microbiome.
[0010] Preferably, the bisphenol A-seawater-R2A liquid culture medium is a mixed culture medium containing 5~50 mg / L bisphenol A and seawater-R2A.
[0011] Preferably, the concentration of bisphenol A is 10 mg / L.
[0012] Preferably, the seawater-R2A mixed culture medium consists of 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, and 0.3 g sodium pyruvate per liter of nearshore natural seawater. After sterilization, the pH value is adjusted to 7.2±0.2. The nearshore natural seawater is nearshore natural seawater that has been filtered through a 0.22 μm filter membrane to remove impurities.
[0013] Preferably, the 3 to 8 sites are six sites: Lovers' Road in Zhuhai, Dapeng Bay in Shenzhen, Huangpu Port in Guangzhou, Kaozhouyang in Huizhou, Dong'ao Bay in Zhuhai, and Jiaoyi Bay in Dongguan.
[0014] Preferably, the 3 to 8 sites are four sites: Lovers' Road in Zhuhai, Huangpu Port in Guangzhou, Kaozhouyang in Huizhou, and Jiaoyi Bay in Dongguan.
[0015] Preferably, the 20~35℃ and 120~210 r / min are 30℃ and 180 r / min.
[0016] Preferably, the volume ratio of 8-12% is 10% in volume.
[0017] Preferably, the specific steps are as follows:
[0018] (1) Dissolve bisphenol A in acetone to prepare a stock solution of 1000~5000 mg / L, and sterilize the stock solution by filtration through a 0.22 μm organic phase filter membrane;
[0019] (2) Prepare seawater-R2A mixed culture medium. Add 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, and 0.3 g sodium pyruvate to every 1 liter of nearshore natural seawater. After sterilization, adjust the pH value to 7.2±0.2. The nearshore natural seawater is nearshore natural seawater that has been filtered through a 0.22 μm filter membrane to remove impurities.
[0020] (3) Add the bisphenol A stock solution from step (1) to the seawater-R2A mixed culture medium from step (2) to prepare bisphenol A-seawater-R2A liquid culture medium, with a bisphenol A concentration of 5~50 mg / L;
[0021] (4) Using 3 to 8 sites of nearshore marine water and sediment mixture as bacterial sources, inoculate into the bisphenol A-seawater-R2A liquid culture medium of step (3), and culture in a constant temperature shaker at 20 to 35°C and 120 to 210 r / min for one week to obtain the first generation culture medium system I, and determine the degradation rate of bisphenol A in each enrichment system;
[0022] (5) Inoculate the first generation culture medium system I from step (4) into sterilized bisphenol A-seawater-R2A liquid culture medium at an inoculation volume ratio of 10%, and culture it for one week under the constant temperature shaker conditions of step (4) to obtain the second generation culture medium system II, and determine the degradation rate of bisphenol A in each enrichment system.
[0023] (6) Repeat step (5) 3-6 times. After each enrichment, the culture medium system with a bisphenol A removal efficiency ≥ 50% is selected for the next round of inoculation. The third, fourth, fifth and sixth generation enrichment solutions are obtained respectively, and the degradation rate of bisphenol A in each enrichment system is measured.
[0024] (7) Mix all the enrichment solutions obtained in step (6) with a bisphenol A removal efficiency ≥90% in equal volume ratio to obtain the final nearshore marine bisphenol A efficient degradation functional microbiome.
[0025] This invention uses a mixture of nearshore water and sediment from multiple sites as the bacterial source, employs a seawater-R2A mixed culture medium, and obtains the target microbial community through selective enrichment of bisphenol A, multi-generational continuous screening, and optimization of bacterial culture mixture. This provides effective technical support for the bioremediation of BPA pollution in nearshore waters.
[0026] This invention focuses on screening highly efficient bacterial communities that are tolerant of complex environments based on "adaptability to nearshore waters". The constructed microbiome can adapt to the actual marine environment without additional domestication, solving the key problem of "disconnect between laboratory results and application" and providing a new approach to the problem of bisphenol A (BPA) pollution control in nearshore waters. Attached Figure Description
[0027] Figure 1 This study describes the degradation effect of fourth-generation bacteria enriched from six nearshore marine samples on bisphenol A (BPA). The values represent the BPA degradation rate of the fourth-generation culture system IV from the six nearshore marine samples in Example 1 after 7 days of incubation in a medium containing 10 mg / L BPA. Specifically, QL represents the sample from the Lovers' Road section of Zhuhai; DY represents the sample from Dapeng Bay in Shenzhen; HP represents the sample from Huangpu Port in Guangzhou; KZ represents the sample from Kaozhou Bay in Huizhou; DA represents the sample from Dong'ao Bay in Zhuhai; and JY represents the sample from Jiaoyi Bay in Dongguan.
[0028] Figure 2 The degradation curves of bisphenol A (BPA) at different concentrations (10 mg / L, 20 mg / L, 50 mg / L) are shown in the figure. Detailed Implementation
[0029] The following detailed description, with reference to the accompanying drawings, further illustrates a method for constructing a highly efficient bisphenol A-degrading functional microbiome according to the present invention through embodiments. The present invention is not limited to the descriptions in the following embodiments.
[0030] Example 1: Enrichment and Degradation Efficiency of Bisphenol A Degrading Microbial Community
[0031] (1) Bisphenol A was dissolved in acetone to prepare a stock solution of 2000 mg / L, and the stock solution was sterilized by filtration through a 0.22 μm organic phase filter membrane;
[0032] (2) Prepare seawater-R2A mixed culture medium, wherein for every 1 liter of nearshore natural seawater, add 0.5 g of yeast extract powder, 0.5 g of peptone, 0.5 g of casein hydrolysate, 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.05 g of magnesium sulfate (heptahydrate), and 0.3 g of sodium pyruvate. After sterilization, the pH value should be adjusted to 7.2±0.2. The nearshore natural seawater is nearshore natural seawater that has been filtered through a 0.22 μm filter membrane to remove impurities and is collected from Zhuhai Lovers Road, Shenzhen Dapeng Bay, Guangzhou Huangpu Port, Huizhou Kaozhou Bay, Zhuhai Dong'ao Bay, and Dongguan Jiaoyi Bay.
[0033] (3) Add the bisphenol A stock solution from step (1) to the seawater-R2A mixed culture medium from step (2) to prepare bisphenol A-seawater-R2A liquid culture medium with a bisphenol A concentration of 10 mg / L;
[0034] (4) Using the mixture of nearshore waters and sediments from six sites (QL, Lovers' Road, Zhuhai; DY, Dapeng Bay, Shenzhen; HP, Huangpu Port, Guangzhou; KZ, Kaozhouyang, Huizhou; DA, Dong'ao Bay, Zhuhai; and JY, Jiaoyi Bay, Dongguan) as the bacterial source, the mixture was inoculated into 60 mL of bisphenol A-seawater-R2A liquid culture medium in step (3) at a volume ratio of 10%. The mixture was cultured for one week in a constant temperature shaker at 30℃ and 180 r / min to obtain the first generation culture medium system I. The degradation rate of bisphenol A in each enrichment system was determined by high performance liquid chromatography.
[0035] (5) Inoculate the first generation culture medium system I from step (4) into sterilized bisphenol A-seawater-R2A liquid culture medium at an inoculation amount of 10% (v / v), and culture it for one week under the constant temperature shaker conditions of step (4) to obtain the second generation culture medium system II, and determine the degradation rate of bisphenol A in each enrichment system.
[0036] (6) Repeat step (5) 6 times. After each enrichment, the culture medium system with a bisphenol A removal efficiency ≥ 50% is selected for the next round of inoculation. The third, fourth, fifth and sixth generation enrichment solutions are obtained respectively, and the degradation rate of bisphenol A in each enrichment system is measured.
[0037] (7) The enrichment solutions obtained in step (6) with bisphenol A removal efficiency ≥90% (QL Lovers Road, Zhuhai; HP, Huangpu Port, Guangzhou; KZ, Kaozhouyang, Huizhou; JY, Jiaoyi Bay, Dongguan) are mixed in equal volume ratio to obtain the final nearshore marine bisphenol A efficient degradation functional microbiome.
[0038] (8) The surface seawater of Zhuhai Lovers' Road, Guangzhou Huangpu Port, Huizhou Kaozhou Bay and Dongguan Jiaoyi Bay were mixed in a volume ratio of 1:1:1:1. The microbial community was inoculated into 80 mL of the mixed seawater. The initial OD was adjusted to 0.3. The initial concentrations of bisphenol A were set to 10 mg / L, 20 mg / L and 50 mg / L respectively. The samples were cultured at 30℃ and 180 rpm. Samples were taken on days 0, 2, 4 and 6 respectively to determine the content of bisphenol A. The determination method is as follows: 5 mL of sample was taken, freeze-dried and 5 mL of methanol was added. The mixture was shaken for 20 minutes. Then, 1 mL of extract was taken with a syringe and filtered through a 0.22 μm organic filter membrane. The extract was determined by high performance liquid chromatography. The chromatographic column was an Agilent C18 reversed phase column. The mobile phase was acetonitrile: ultrapure water = 65:35 (V / V). The elution was isogradient, the flow rate was 1.0 mL / min, and the ultraviolet light detection wavelength was 278 nm.
[0039] Using samples from six nearshore marine sites as bacterial sources, the bacteria were inoculated into bisphenol A-seawater-R2A liquid medium. After four subcultures in a constant-temperature shaker at 30℃ and 180 rpm, bisphenol A-degrading and enriching bacterial communities were obtained in all systems using samples from each site as bacterial sources, with degradation rates exceeding 50%. Among these, the bacterial communities obtained from some sites achieved a 100% degradation rate of bisphenol A. Figure 1 Further mixing of microbial communities with degradation rates exceeding 90% yielded the final nearshore marine bisphenol A (BPA) highly efficient degrading microbiome. Its removal efficiency for different concentrations of BPA was measured. The microbiome completely degraded 10 mg / L and 20 mg / L BPA within 4 days, and completely degraded 50 mg / L BPA within 6 days. Figure 2 ).
Claims
1. A method for constructing a functional microbial community for efficient degradation of bisphenol A in nearshore waters, characterized in that, Includes the following steps: (1) Using a mixture of nearshore water and sediment from 3 to 8 sites as the bacterial source, inoculate it into bisphenol A-seawater-R2A liquid medium and culture it at 20 to 35°C and 120 to 210 r / min for 7 to 10 days to obtain the first generation culture medium system I; (2) Inoculate the first generation culture medium system I into sterile bisphenol A-seawater-R2A liquid culture medium at an inoculation rate of 8-12% by volume; (3) Repeat step (2) 3-6 times. After each enrichment, select a culture medium system with a bisphenol A removal efficiency ≥ 50% for the next round of inoculation. (4) Mix the enrichment solutions obtained in step (3) with all sites and bisphenol A removal efficiency ≥90% in equal volume ratio to obtain the final nearshore marine bisphenol A efficient degradation functional microbiome.
2. The method according to claim 1, characterized in that, The bisphenol A-seawater-R2A liquid culture medium is a mixed culture medium containing 5~50 mg / L bisphenol A and seawater-R2A.
3. The method according to claim 2, characterized in that, The concentration of bisphenol A is 10 mg / L.
4. The method according to claim 2, characterized in that, The seawater-R2A mixed culture medium consists of 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, and 0.3 g sodium pyruvate per liter of nearshore natural seawater. After sterilization, the pH value is adjusted to 7.2±0.
2. The nearshore natural seawater is nearshore natural seawater that has been filtered through a 0.22 μm filter membrane to remove impurities.
5. The method according to claim 1, characterized in that, The 3 to 8 sites are six sites: Lovers' Road in Zhuhai, Dapeng Bay in Shenzhen, Huangpu Port in Guangzhou, Kaozhouyang in Huizhou, Dong'ao Bay in Zhuhai, and Jiaoyi Bay in Dongguan.
6. The method according to claim 5, characterized in that, The 3 to 8 sites are the four sites of Lovers' Road in Zhuhai, Huangpu Port in Guangzhou, Kaozhouyang in Huizhou, and Jiaoyi Bay in Dongguan.
7. The method according to claim 1, characterized in that, The 20~35℃ and 120~210 r / min are 30℃ and 180 r / min.
8. The method according to claim 1, characterized in that, The volume ratio of 8-12% is equivalent to a volume ratio of 10%.
9. The method according to claim 1, characterized in that, The specific steps are as follows: (1) Dissolve bisphenol A in acetone to prepare a stock solution of 1000~5000 mg / L, and sterilize the stock solution by filtration through a 0.22 μm organic phase filter membrane; (2) Prepare seawater-R2A mixed culture medium. Add 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, and 0.3 g sodium pyruvate to every 1 liter of nearshore natural seawater. After sterilization, adjust the pH value to 7.2±0.
2. The nearshore natural seawater is nearshore natural seawater that has been filtered through a 0.22 μm filter membrane to remove impurities. (3) Add the bisphenol A stock solution from step (1) to the seawater-R2A mixed culture medium from step (2) to prepare bisphenol A-seawater-R2A liquid culture medium, with a bisphenol A concentration of 5~50 mg / L; (4) Using 3 to 8 sites of nearshore marine water and sediment mixture as bacterial sources, inoculate into the bisphenol A-seawater-R2A liquid culture medium of step (3), and culture in a constant temperature shaker at 20 to 35°C and 120 to 210 r / min for one week to obtain the first generation culture medium system I, and determine the degradation rate of bisphenol A in each enrichment system; (5) Inoculate the first generation culture medium system I from step (4) into sterilized bisphenol A-seawater-R2A liquid culture medium at an inoculation volume ratio of 10%, and culture it for one week under the constant temperature shaker conditions of step (4) to obtain the second generation culture medium system II, and determine the degradation rate of bisphenol A in each enrichment system. (6) Repeat step (5) 3-6 times. After each enrichment, the culture medium system with a bisphenol A removal efficiency ≥ 50% is selected for the next round of inoculation. The third, fourth, fifth and sixth generation enrichment solutions are obtained respectively, and the degradation rate of bisphenol A in each enrichment system is measured. (7) Mix all the enrichment solutions obtained in step (6) with a bisphenol A removal efficiency ≥90% in equal volume ratio to obtain the final nearshore marine bisphenol A efficient degradation functional microbiome.