Application and method of Tween80 combined immobilization technology enhanced microorganisms in degradation of naphthalene pollution in water

Abstract

The invention discloses an application and method of Tween 80 combined immobilization technology enhanced microorganisms in degradation of naphthalene pollution in water, the main body of the combined immobilization technology is SA / CS at Tween 80 hydrogel microspheres loaded with NM-1 flora, and the NM-1 flora is halotolerant bacteria obtained by enrichment and domestication from deep-sea sediments of a certain sea ditch. The SA / CS at Tween 80 microspheres are formed by crosslinking sodium alginate (SA), chitosan (CS) and Tween 80, hydroxyl functional groups of the Tween 80 and SA / CS are crosslinked to enhance the mechanical strength of the microspheres and regulate and control the pore structure to improve the mass transfer efficiency, hydrophobic-CH functional groups at the end positions of the Tween 80 strengthen the adsorption capacity to naphthalene, meanwhile, a co-crosslinking structure at the interface of the SA / CS at Tween 80 hydrogel microspheres forms a biochemical metabolic reaction buffer area, and the adsorption capacity to naphthalene is improved. The direct toxicity stress of the high-concentration naphthalene on the flora is effectively avoided, and the effective biological removal of the high-concentration naphthalene is realized. The SA / CS-coated Tween 80 hydrogel microspheres prepared by the invention can effectively maintain the efficient metabolic activity and degradation efficiency of the loaded NM-1 flora, and show excellent cycle stability and good engineering application potential.

Description

An application and method for enhancing microbial degradation of naphthalene contamination in water using Tween80 combined with immobilization technology. Technical Field

[0001] This invention relates to the field of biodegradation technology, and in particular to the application and method of enhancing microorganisms in degrading naphthalene pollution in water using Tween80 combined with immobilization technology. Background Technology

[0002] Naphthalene (C 10 Naphthalene (H8) is a low-ring polycyclic aromatic hydrocarbon (PAH) composed of two fused benzene rings. Naphthalene primarily originates from the incomplete combustion of fossil fuels (coal, oil, natural gas) and biomass, as well as certain industrial production activities such as coal coking, oil refining, asphalt production, and wood preservation. It can enter water bodies, soil, and the atmosphere through various pathways, including atmospheric deposition, industrial wastewater discharge, surface runoff, and oil spills. As the lowest molecular weight among the 16 priority controlled PAHs, naphthalene's volatility and water solubility facilitate migration and diffusion. Furthermore, its stable chemical structure allows it to persist in the environment, resists natural degradation, and possesses potential carcinogenic, teratogenic, and mutagenic toxicity and bioaccumulation, posing a serious threat to ecosystems and human health.

[0003] Currently, the main technologies for treating naphthalene-containing wastewater include physical, chemical, and biological methods. Physical methods primarily include adsorption and membrane separation technologies, which are simple and efficient, but suffer from problems such as easy adsorption saturation, severe membrane fouling, and high costs. Chemical methods mainly utilize advanced oxidation processes, including Fenton oxidation, ozone oxidation, or photocatalytic degradation of naphthalene, offering advantages such as fast reaction rates and high selectivity. However, they require large amounts of chemical reagents, causing secondary pollution, and are difficult to implement for continuous large-scale wastewater treatment. Biological methods, particularly those utilizing microorganisms to degrade organic pollutants, are considered one of the ideal approaches for treating PAH-contaminated wastewater containing naphthalene due to their high economic efficiency, environmental friendliness, and ability to achieve complete mineralization of pollutants. Traditional biological methods for treating naphthalene-containing wastewater often face three key problems: (1) Low bioavailability of naphthalene: Naphthalene's strong hydrophobicity makes it easily adsorbed onto solid particles or in a non-dissolved state, limiting microbial access and utilization, resulting in a slow degradation rate; (2) Microbial toxicity inhibition: High concentrations of naphthalene and its toxic metabolic intermediates inhibit microbial growth and metabolic activity; (3) Poor microbial stability: Under continuous flow or high load conditions, functional microorganisms are easily lost, leading to fluctuations in treatment effectiveness. Therefore, there is an urgent need for an excellent microbial enhancement technology to construct a novel enhanced naphthalene pollution remediation system in water, in order to solve the core problems existing in the current traditional biological methods. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems existing in the prior art and to provide an application and method for enhancing the degradation of naphthalene pollution in water using Tween80 combined with immobilization technology.

[0005] To achieve the above objectives, the technical solution provided by this invention is: an application and method of enhancing microbial degradation of naphthalene pollution in water using Tween80 co-immobilization technology. The Tween80 co-immobilization technology is SA / CS@Tween 80 hydrogel microspheres, which are composed of sodium alginate, chitosan, and Tween 80 crosslinked together. The crosslinking of the hydroxyl functional groups of Tween 80 with SA / CS enhances the mechanical strength of the SA / CS@Tween 80 hydrogel microspheres while regulating the pore structure to improve mass transfer efficiency. The hydrophobic -CH functional groups at the Tween 80 end enhance the adsorption capacity for naphthalene. At the same time, the co-crosslinking structure at the interface of the SA / CS@Tween 80 hydrogel microspheres forms a biochemical metabolic reaction buffer, which is used to avoid the direct toxic stress of high concentrations of naphthalene on the microbial community and to achieve effective biological removal of high concentrations of naphthalene.

[0006] The specific application method includes the following steps:

[0007] Step (1): Enrichment and screening of bacterial communities

[0008] S1. Enrichment of the microbial community: Using deep-sea sediments from a certain trench as the microbial source, a highly efficient degradative microbial community resistant to naphthalene stress was enriched through a gradient acclimation method; the enrichment and acclimation process of the microbial community was as follows:

[0009] Deep-sea sediment samples were collected, washed three times with sterile phosphate buffer solution, and then centrifuged at 8000 rpm / min for 8-12 min (preferably 10 min) to remove impurities and obtain a sediment microbial suspension; then 10 -5 5 mL of the dilution was inoculated into 2216E medium and cultured at 30℃ and 150 rpm for 5 days. This was repeated four times. The fifth-generation enrichment solution was then analyzed using a microplate reader, and the bacterial concentration exceeded 1×10⁻⁶. 9 Enrichment was terminated after the number of cells / mL was counted;

[0010] S2. Screening of the bacterial community: Using selective culture media, manganese-oxidizing strains were screened to construct a naphthalene-degrading functional bacterial community system; the screening process of the bacterial community:

[0011] A 1% v / v logarithmic growth phase bacterial group was inoculated into 50 mL of sterilized modified PYCM medium and cultured for 5 days in a shaker at 130 rpm / min and 30℃. Naphthalene concentration was measured at 24-hour intervals. The bacterial group exhibiting naphthalene-reducing properties was named NM-1. (Specifically, the residual naphthalene in the sample was determined using high-performance liquid chromatography (HPLC). HPLC conditions were: Agilent ZORBAC SB-C18 column, column temperature 30℃, wavelength 219 nm, mobile phase (acetonitrile: ultrapure water, v / v) = 7:3, flow rate 1.5 mL / min.)

[0012] Step (2), Preparation of SA / CS@Tween 80 microspheres loaded with NM-1 bacterial community

[0013] 2% SA was dissolved by heating, cooled to 25°C, and then 1% CS (preferably 0.5% acetic acid) was added in the same volume. The solution was sonicated until completely dissolved. 1%-4% Tween 80 (preferably 2% Tween 80) was added to the SA / CS mixture and stirred until uniformly dispersed. NM-1 bacterial culture was mixed with the SA / CS solution at a 1:1 ratio and stirred until uniformly dispersed. The solution was then added dropwise to a 3% CaCl2 solution using a 5 mL syringe. Crosslinking and curing were performed for 15 min, followed by rinsing three times with deionized water to obtain SA / CS@Tween 80 microspheres. (The use of sodium alginate-chitosan composite Tween 80 to encapsulate bacterial communities improves mass transfer efficiency, degradation stability, and recyclability under high naphthalene concentrations.)

[0014] Step (3) Continuous and effective biological removal of naphthalene

[0015] 0.25±0.05 g of SA / CS@Tween 80 microspheres were added to high-concentration naphthalene wastewater, and the reaction time was 4-6 days (preferably 6 days). After the reaction, the microspheres were removed from the wastewater and washed three times with sterile water, and then transferred to new simulated wastewater. This process was repeated for five cycles. (The cyclic degradation of naphthalene in wastewater verifies the engineering application potential of Tween 80 combined with immobilization technology).

[0016] Preferably, the 2216E culture medium used in the enrichment and domestication process of the microbial community is: 5 g / L peptone, 1 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L NaCl, 5.98 g / L MgCl2, 1.8 g / L CaCl2, 0.0016 g / L NH4NO3, 0.55 g / L KCl, 3.24 g / L Na2SO4, 0.16 g / L Na2CO3, 0.08 g / L KBr, 0.034 g / L SrCl2, 0.022 g / L H3BO3, 0.0024 g / L NaF, 0.04 g / L Na2SiO3, and 0.008 g / L Na2HPO4, with an initial pH of 7.6 ± 0.2.

[0017] Preferably, the modified PYCM medium used in the microbial community screening process consists of: 0.50±0.05 g / L peptone, 0.30±0.05 g / L yeast extract, 0.30±0.05 g / L anhydrous glucose, 0.80±0.04 g / L ferric ammonium citrate, 0.41±0.06 g / L MgSO4, 0.22±0.01 g / L MnSO4, 0.10±0.01 g / L CaCl2, 0.10±0.01 g / L NH4NO3, 0.20±0.01 g / L NaNO3, and 0.20±0.05 g / L K2HPO4.

[0018] Preferably, the concentration of naphthalene in high-concentration naphthalene wastewater is 100-150 mg / L.

[0019] Beneficial effects of this invention:

[0020] (1) The SA / CS@Tween 80 microspheres of the present invention are highly correlated with the bacterial species, which can effectively maintain the stability of the ecosystem and effectively enhance the naphthalene degradation capacity of the NM-1 bacterial community.

[0021] (2) The bacterial community in the SA / CS@Tween 80 microspheres of the present invention has reduced the connection resistance and improved the information transmission efficiency between functional bacterial communities on the basis of high functionalization.

[0022] (3) The surface of the SA / CS@Tween 80 microspheres of the present invention has abundant hydrophobic groups -CH, which enhances the adsorption capacity of naphthalene.

[0023] (4) The SA / CS@Tween 80 microspheres of the present invention have a three-dimensional network structure and a stable internal environment, which effectively alleviates the toxic stress of high concentration of naphthalene and maintains the high metabolic activity and stable degradation efficiency of NM-1 bacteria. Attached Figure Description

[0024] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0025] Figure 1 shows the effect of different Tween 80 concentrations on (a) bacterial community growth and (b) naphthalene degradation in this invention;

[0026] Figure 2 shows the effect of different Tween 80 concentrations on (a) EPS content and (b) CSH content in this invention;

[0027] Figure 3 shows the relative abundance of NM-1 bacterial phyla at different Tween 80 concentrations in this invention;

[0028] Figure 4 shows the relative abundance of NM-1 bacterial genus at different Tween 80 concentrations in this invention;

[0029] Figure 5 shows the particle size distribution of the microspheres in this invention: (a) SA / CS, (b) SA / CS@Tween 80, (c) SA / CS@Tween 80+NM-1; and SEM images of the microspheres: (d) SA / CS, (e) SA / CS@Tween 80+NM-1.

[0030] een 80 (f) SA / CS@Tween 80+NM-1;

[0031] Figure 6 shows a comparison of the mass transfer efficiency of SA / CS and SA / CS@Tween 80 microspheres in this invention;

[0032] Figure 7 compares the degradation efficiencies of NM-1, SA / CS@Tween 80, and SA / CS@Tween 80+NM-1 in this invention;

[0033] Figure 8 shows the reusability of the SA / CS@Tween 80 microspheres in this invention. Detailed Implementation

[0034] This section will describe in detail specific embodiments of the present invention. Preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the drawings is to supplement the textual description with graphics, so that people can intuitively and vividly understand each technical feature and overall technical solution of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0035] This invention addresses the technical bottleneck of conventional biological methods, which are limited by the low bioavailability, toxicity inhibition, and poor microbial community stability of naphthalene, making it difficult to achieve efficient naphthalene degradation. It provides a method and application of Tween 80 combined with immobilization technology to enhance the application of microorganisms in degrading naphthalene pollution in water. By combining the stability advantages of the nonionic surfactant Tween 80 and immobilized microorganism technology, SA / CS@Tween 80 hydrogel microspheres are constructed. The microspheres have abundant hydrophobic groups that enhance the adsorption capacity of naphthalene, while their stable structure effectively alleviates the toxic stress of high concentrations of naphthalene and improves microbial community stability.

[0036] Example 1: Effect of different concentrations of Tween 80 on the naphthalene-reducing performance of bacterial communities

[0037] One mL of bacterial culture in the logarithmic growth phase was inoculated into modified PYCM medium containing 0.0%, 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% (v / v) Tween 80, respectively. The initial naphthalene concentration was 50 mg / L. The culture conditions were 130 rpm / min, 30℃, and incubation in a shaker for 5 days. The OD of the bacterial community was measured every 24 h. 600 The degradation efficiency of naphthalene was similar, as shown in Figure 1. All concentrations of Tween 80 significantly promoted bacterial growth (Figure 1(a)), with the promotion effect becoming more pronounced as the Tween 80 concentration gradually increased (0–2%). The optimal 2% concentration of Tween 80 increased the growth level of the NM-1 bacterial colony by 4.2 times. The degradation efficiency of naphthalene also showed a similar trend, as shown in Figure 1(b) (the degradation rate of naphthalene during the culture process was observed). Within the first 24 hours after the initial addition of Tween 80, the degradation rate of naphthalene did not increase significantly, but gradually increased with the increase in bacterial biomass. All concentrations of Tween 80 significantly improved the degradation rate of naphthalene. At a Tween 80 concentration of 2%, the degradation rate of naphthalene was the highest, reaching 186.6%, compared to the control group. Furthermore, the naphthalene degradation rate under the 2% Tween 80 condition was the highest, at 100%. These results indicate that Tween 80 can effectively promote bacterial growth and the utilization of intermediate products, further accelerating the degradation of naphthalene.

[0038] Example 2: Effect of different Tween 80 concentrations on EPS of NM-1 bacteria.

[0039] Figure 2(a) shows the peak EPS levels and their component contents under different treatment groups. The EPS content secreted by the NM-1 bacterial community increased with increasing Tween 80 concentration and then decreased. The EPS content extracted without Tween 80 was 123.7 mg / L. When the Tween 80 concentration was increased to 2%, the EPS yield reached a maximum of 338.5 mg / L, which was 2.7 times higher than the control group. The increase in EPS can form a dense protective layer to reduce the toxicity of persistent pollutants in the environment. Due to the increased EPS content, it can form a hydrophobic film on the cell surface, changing the cell's affinity for water and ultimately affecting CSH. Figure 2(b) shows the changes in CSH in cells at different Tween 80 concentrations. We found that the initial CSH of the blank control was low at 21.5%, indicating that most of the bacteria in this experiment were low-hydrophobic bacteria (CSH < 36%). After adding Tween 80, the CSH value was significantly increased, with a doubling to 43% at a dosage of 2%. The increase in CSH can enhance the adsorption of naphthalene by microorganisms, improve the bioavailability of organic pollutants, and promote biodegradation. These results indicate that Tween 80 can improve the degradation performance of NM-1 bacteria, reduce the toxicity of pollutants in the environment, and increase the level of CSH on the cell surface, thereby improving hydrophobicity and effectively improving the treatment efficiency of PAHs.

[0040] Example 3: Effects of different concentrations of Tween 80 on the phylum and genus levels of the NM-1 bacterial community.

[0041] ① The Tween 80 concentration gradient exhibited a significant dose-response effect on bacterial community structure. As shown in Figure 3, at the phylum level, in the control group (N1, 0% Tween 80), Firmicutes (52.4±3.2%), Proteobacteria (28.0±2.1%), and Actinobacteriota (22.4±0.2%) constituted the core bacterial community, accounting for a cumulative 96.7%, with Firmicutes playing a dominant role. However, as the Tween 80 concentration increased to 2% (N3 group), the community structure changed significantly: the abundance of Proteobacteria increased to 62.2±4.5%, becoming dominant, while Firmicutes and Actinobacteriota decreased to 20.7±1.8% and 18.8±1.5%, respectively, but still played important roles in the community. Proteobacteria, Firmicutes, and Actinobacteria are key phyla involved in the conversion of polycyclic aromatic hydrocarbons (PAHs), with Proteobacteria being the dominant phylum in PAH degradation. The data above indicate that screening under Tween 80 stimulation enhanced the growth of Proteobacteria in the experimental aquatic environment, leading to a more specialized community structure and function. Therefore, the increased abundance of Proteobacteria is a key reason for the improved NM-1 naphthalene degradation efficiency under the influence of Tween 80.

[0042] ② As shown in Figure 4, at the genus level, Tween 80 induced the targeted enrichment of functional bacterial genera. The abundance of shingomonas (7.2%→27.7%), Pseudomonas (4.1%→13.2%), Candidimonas (4.9%→7.0%), Rhodococcus (2.0%→7.8%), and Phenylobacterium (1.8%→6.5%) increased significantly with increasing Tween 80 concentration. In all treatment groups, the abundance of Bacillus was 43.5%, 35.7%, and 24.6%, respectively. These key genera have been previously reported to be polycyclic aromatic hydrocarbon degrading bacteria.

[0043] In summary, the screening effect of Tween 80 significantly altered the community structure, particularly increasing the abundance of key bacterial genera, which greatly enhanced the microbial degradation performance.

[0044] Example 4: Improved mass transfer performance of SA / CS@Tween 80 microspheres

[0045] The morphology of microspheres prepared with different components was characterized. Figure 5(ac) shows camera images and particle size distributions of SA / CS, SA / CS@Tween 80, and SA / CS@Tween 80 microspheres loaded with NM-1 bacterial colony. It can be clearly seen that the SA / CS microspheres are transparent, while the SA / CS@Tween 80 microspheres are translucent. This is mainly due to the uniform distribution of Tween 80 within the SA / CS microspheres, while the SA / CS@Tween 80 microspheres loaded with NM-1 bacterial colony are translucent yellow due to the addition of NM-1 bacterial suspension. The particle size distribution of the SA / CS, SA / CS@Tween 80, and SA / CS@Tween 80 microspheres loaded with NM-1 bacterial colony was determined using ImageJ professional image analysis software. The microspheres with added Tween 80 had a significantly larger particle size than the SA / CS microspheres, which had a size of 3.91±0.33 mm. The differences between the SA / CS@Tween 80 and SA / CS@Tween 80 microspheres loaded with the NM-1 microbial community were minimal, at 4.18±0.26 mm and 4.22±0.27 mm, respectively. This is mainly because Tween 80 acts as an emulsifier, making the solution more stable and reducing droplet shrinkage during the solidification process, resulting in a larger final microsphere particle size. Further analysis of the SEM images (Figure 5(df)) reveals that the SA / CS microspheres have a fractured network structure inside. Numerous pores and irregular cracks indicate low mechanical strength, making them susceptible to breakage under external forces. The addition of Tween 80 significantly alters the microsphere structure. Figure 5e shows that the internal pores of the microspheres become more uniform and dense, forming a cross-shaped three-dimensional network structure, which improves the mechanical strength of the microspheres. These dense pores provide a suitable growth environment for the bacteria and also verify the phenomenon that Tween 80 makes the microspheres less prone to shrinkage. After encapsulating the NM-1 bacterial community in SA / CS@Tween 80, a large number of bacteria adhered to the inside of the microspheres, indicating that the microspheres are an excellent carrier with a porous structure, facilitating microbial loading.

[0046] This invention employs the methylene blue method to evaluate the mass transfer efficiency of different immobilized microspheres. 0.25 ± 0.05 g of blank SA / CS and SA / CS@Tween 80 microspheres were prepared and added to 50 mL of a 20 mg / L methylene blue solution. The mixture was magnetically stirred at 150 rpm, and samples were taken at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The absorbance of the samples at 664 nm was measured using a UV-Vis spectrophotometer, and the methylene blue absorbance was calculated using the following formula:

[0047]

[0048] Where OD0 represents the initial absorbance of the solution, OD t The absorbance of the solution at the time of sampling.

[0049] In studies on the degradation of polycyclic aromatic hydrocarbons (PAHs) by immobilized microorganisms, the diffusion efficiency of pollutants from the outside to the inside of the carrier is a key factor affecting the bioavailability of pollutants and the metabolic activity of microorganisms. As shown in Figure 6, SA / CS@Tween 80 microspheres exhibit superior mass transfer performance compared to SA / CS microspheres, indicating that SA / CS@Tween 80 microspheres can provide an efficient substrate transport channel for the continuous metabolic activities of organisms and provide a good mass transfer basis for the subsequent efficient degradation of high-concentration naphthalene by the microbial community.

[0050] Example 5: Blank SA / CS@Tween 80 microspheres and SA / CS@Tween 80 microspheres loaded with bacterial communities were added to simulated wastewater containing naphthalene at concentrations of 25–125 mg / L. The results are shown in Figure 7. Under naphthalene concentrations of 25–125 mg / L, the SA / CS@Tween 80 microspheres loaded with NM-1 showed a greater ability to degrade naphthalene than the free bacterial community NM-1. When the naphthalene concentration was ≤50 mg / L, the degradation efficiency of phenol by the free bacterial community NM-1 and the immobilized NM-1 microspheres was not significantly different, both being around 100%. When the naphthalene concentration increased to 75 mg / L and above, the difference between the two began to emerge. Immobilized NM-1 microspheres achieved degradation efficiencies of 99.3%, 96.5%, and 92.6% for naphthalene at concentrations of 75 mg / L, 100 mg / L, and 125 mg / L, respectively, while free NM-1 microspheres showed degradation efficiencies of 83.5%, 72.6%, and 60.7%. These results indicate that the encapsulation of SA / CS@Tween 80 microspheres can reduce the initial toxicity stress on the NM-1 microspheres and improve their degradation performance on naphthalene.

[0051] Example 6: SA / CS@Tween 80 microspheres loaded with NM-1 were added to a high-concentration wastewater containing 125 mg / L naphthalene for five consecutive cycles. The results are shown in Figure 8. After five cycles, the degradation performance of SA / CS@Tween 80 microspheres for naphthalene did not show a significant decrease and remained at a high level. The degradation rates reached 92.6%, 91.7%, 90.3%, 89.2%, and 88.5% in the five cycles, respectively. This result fully verifies that SA / CS@Tween 80 microspheres loaded with NM-1 possess both excellent naphthalene degradation efficiency and cycle stability.

[0052] Without causing conflict, those skilled in the art can freely combine and use the above-mentioned additional technical features.

[0053] The above description is only a preferred embodiment of the present invention. Any technical solution that achieves the purpose of the present invention by essentially the same means is within the protection scope of the present invention.

Claims

1. An application and method for enhancing microbial degradation of naphthalene pollution in water using Tween80 combined with immobilization technology, characterized in that: Tween 80 co-immobilization technology produces SA / CS@Tween 80 hydrogel microspheres. These microspheres are composed of sodium alginate, chitosan, and Tween 80 cross-linked together. The hydroxyl functional groups of Tween 80 cross-link with SA / CS enhance the mechanical strength of the SA / CS@Tween 80 hydrogel microspheres while regulating the pore structure to improve mass transfer efficiency. The hydrophobic -CH functional groups at the Tween 80 terminals strengthen the adsorption capacity for naphthalene. Simultaneously, SA / CS@Tween... The co-crosslinked structure of the 80 hydrogel microsphere interface forms a biochemical metabolic reaction buffer, which is used to avoid the direct toxic stress of high concentration naphthalene on the microbial community and to achieve the effective biological removal of high concentration naphthalene; the specific application method includes the following steps: Step (1), enrichment and screening of microbial community S1, enrichment of microbial community: using deep-sea sediments of a certain trench as the source of bacteria, the highly efficient degradation microbial community resistant to naphthalene stress is enriched by gradient domestication method; the enrichment and domestication process of microbial community is as follows: after collecting deep-sea sediment samples and washing them 3 times with sterile phosphate buffer solution, centrifuge them at 8000 rpm / min for 8-12 min to remove impurities and obtain bottom sediment bacterial suspension; then 10 -5 5 mL of the dilution was inoculated into 2216E medium and cultured at 30℃ and 150 rpm for 5 days. This was repeated four times. The fifth-generation enrichment solution was then analyzed using a microplate reader, and the bacterial concentration exceeded 1×10⁻⁶. 9 Enrichment ends after 1 / mL; S2, Screening of bacterial communities: Manganese-oxidizing strains are screened in combination with selective culture medium to construct a naphthalene-degrading bacterial community system; Screening process of bacterial communities: 1% v / v logarithmic growth phase bacterial communities are inoculated into 50 mL of sterilized modified PYCM medium and cultured in a shaker at 130 rpm / min and 30℃ for 5 days. The naphthalene concentration is measured by taking samples at 24 h intervals; and the bacterial community with naphthalene-reducing properties is named NM-1; Step (2), Preparation of SA / CS@Tween 80 microspheres loaded with NM-1 bacterial community: 2% SA is heated to dissolve, cooled to 25℃ and then 1% CS of the same volume is added. CS is dissolved in 0.3%-0.7% acetic acid and ultrasonically treated until completely dissolved; 1%-4% Tween 80 is added to the SA / CS mixed solution and stirred until uniformly dispersed. Mix NM-1 bacterial solution with SA / CS solution at a ratio of 1:1, stir until uniformly dispersed, and then use a 5 mL syringe to dropwise add the solution into 3% CaCl2 solution. Crosslink and solidify for 15 min and rinse three times with deionized water to obtain SA / CS@Tween 80 microspheres; Step (3): Continuous and effective biological removal of naphthalene. Add 0.25±0.05 g of SA / CS@Tween 80 microspheres into high concentration naphthalene wastewater. The reaction time is 4-6 days. After the reaction is completed, remove the microspheres from the wastewater and wash them three times with sterile water. Then transfer them to new simulated wastewater and repeat five cycles.

2. The application and method of enhancing microbial degradation of naphthalene pollution in water using Tween80 combined immobilization technology according to claim 1, characterized in that: The 2216E medium used for the enrichment and domestication of the microbial community consisted of: 5 g / L peptone, 1 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L NaCl, 5.98 g / L MgCl2, 1.8 g / L CaCl2, 0.0016 g / L NH4NO3, 0.55 g / L KCl, 3.24 g / L Na2SO4, 0.16 g / L Na2CO3, 0.08 g / L KBr, 0.034 g / L SrCl2, 0.022 g / L H3BO3, 0.0024 g / L NaF, 0.04 g / L Na2SiO3, and 0.008 g / L Na2HPO4, with an initial pH of 7.6 ± 0.

2.

3. The application and method of enhancing microbial degradation of naphthalene pollution in water using Tween80 combined immobilization technology according to claim 1, characterized in that: The modified PYCM medium used in the microbial community screening process consisted of: 0.50±0.05 g / L peptone, 0.30±0.05 g / L yeast extract, 0.30±0.05 g / L anhydrous glucose, 0.80±0.04 g / L ferric ammonium citrate, 0.41±0.06 g / L MgSO4, 0.22±0.01 g / L MnSO4, 0.10±0.01 g / L CaCl2, 0.10±0.01 g / L NH4NO3, 0.20±0.01 g / L NaNO3, and 0.20±0.05 g / L K2HPO4.

4. The application and method of enhancing microbial degradation of naphthalene pollution in water using Tween80 combined immobilization technology according to claim 1, characterized in that: The concentration of naphthalene in high-concentration naphthalene wastewater is 100-150 mg / L.

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