Microbiome with function of efficiently degrading tetrabromobisphenol A based on cell surface hydrophobicity as well as construction method and application of microbiome

Abstract

The invention discloses a tetrabromobisphenol A efficient degradation functional microbiome based on cell surface hydrophobicity as well as a construction method and application of the tetrabromobisphenol A efficient degradation functional microbiome. Microorganisms with high cell surface hydrophobicity are introduced into a tetrabromobisphenol A degradation microflora to construct the microbiome, so that the degradation and removal efficiency of the microbiome on tetrabromobisphenol A can be effectively improved, and an engineering microbial preparation is provided for wastewater treatment and polluted water body remediation or sediment remediation; meanwhile, theoretical guidance is provided for wastewater treatment and polluted water body or sediment remediation.

Description

Technical field:

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity, and a construction method and application thereof. Background technology:

[0002] The process of microbial degradation of pollutants is first of all a mass transfer and adsorption process, that is, the process of pollutants adsorbing on the surface of microorganisms in the environmental medium. The adsorption and mass transfer efficiency of pollutants is the premise for determining whether pollutants can be quickly degraded by microorganisms. However, many pollutants in the environment have the characteristics of low concentration and high toxicity. At low concentrations, microorganisms may not be able to identify pollutants and degrade them. In particular, some organic pollutants with strong hydrophobicity have lower concentrations in the aqueous environment and their degradation efficiency is lower.

[0003] Cell surface hydrophobicity (CSH) is an important physicochemical property of microbial cells. It is an important factor that determines the nonspecific adhesion of microorganisms to various biological and non-biological surfaces and interfaces, and is also an important factor that affects the absorption and degradation of hydrophobic organic matter by bacteria. Studies have shown that microorganisms with higher cell surface hydrophobicity have higher degradation efficiency for hydrophobic pollutants. Therefore, the construction of a hydrophobic organic pollutant degradation microbiome based on microorganisms with high cell surface hydrophobicity is expected to improve the degradation and removal efficiency of this type of pollutants. Based on this, the present invention constructs a tetrabromobisphenol A degradation microbiome by introducing microorganisms with high cell surface hydrophobicity to improve the efficiency of the microbiome in the degradation and removal of tetrabromobisphenol A. Summary of the invention:

[0004] The purpose of the present invention is to utilize microorganisms with high cell surface hydrophobicity to carry out tetrabromobisphenol A microbial group and its construction method, strengthen the mass transfer process of tetrabromobisphenol A to microorganisms, and improve the efficiency of its decomposition and removal.

[0005] A method for constructing a tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity, characterized by comprising the following steps:

[0006] a. Screening strains with high cell surface hydrophobicity;

[0007] b. Using tetrabromobisphenol A as a screening condition, enriching and culturing strains or bacterial groups with tetrabromobisphenol A degradation function;

[0008] c. Combining a strain with high cell surface hydrophobicity with a strain or bacterial colony having tetrabromobisphenol A degradation function;

[0009] d. Evaluate the degradation capacity and stability of tetrabromobisphenol A by the compounded microbiome;

[0010] e. Optimize the structure of the tetrabromobisphenol A microbiome, and then compound and evaluate it to obtain a functional microbiome that efficiently degrades tetrabromobisphenol A.

[0011] Preferably, the strains screened for high cell surface hydrophobicity can be isolated or enriched from a hydrophobic environment, or can be obtained by measuring the cell surface hydrophobicity of strains in a strain library.

[0012] Preferably, the screening of strains with high cell surface hydrophobicity is performed by measuring the cell surface hydrophobicity, specifically, after the strains are cultured to the logarithmic growth phase, the measurement is performed using the hydrocarbon method.

[0013] Preferably, the enrichment culture of the strain or bacterial community having tetrabromobisphenol A degradation function is carried out by enriching the tetrabromobisphenol A degradation functional microbial group using a selective culture medium containing tetrabromobisphenol A.

[0014] Preferably, the combination of the strain with high cell surface hydrophobicity and the strain or bacterial community with tetrabromobisphenol A degradation function is carried out by adjusting the type and biomass of the strain with high cell surface hydrophobicity according to the type and biomass of the strain or bacterial community with tetrabromobisphenol A degradation function, and compounding them in a certain ratio.

[0015] Preferably, the evaluation of the tetrabromobisphenol A degradation ability and stability of the compounded microbial group is to inoculate the compounded microbial group into a system containing tetrabromobisphenol A, determine the content of tetrabromobisphenol A and its degradation products in the system at different times after inoculation, and characterize the degradation ability of the microbial group on tetrabromobisphenol A.

[0016] The present invention also provides the use of a tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity in degrading and removing tetrabromobisphenol A in contaminated water, sediment or soil.

[0017] Preferably, the microbial group is prepared into a bacterial suspension and added to the contaminated water, sediment remediation or soil to remove tetrabromobisphenol A; or the microbial group is loaded on a functional carrier material and then added to the contaminated water, sediment remediation or soil treatment to remove tetrabromobisphenol A.

[0018] The present invention introduces microorganisms with high cell surface hydrophobicity into the tetrabromobisphenol A-degrading microbial community to construct a microbial group, which can effectively improve the degradation and removal efficiency of the microbial group on tetrabromobisphenol A, provide an engineered microbial preparation for wastewater treatment and contaminated water body remediation or sediment remediation, and provide theoretical guidance for the application in wastewater treatment and contaminated water body or sediment remediation. Description of the drawings:

[0019] Figure 1The cell surface hydrophobicity (Figure A) and tetrabromobisphenol A degradation ability (Figure B) of strain A18 and strain K7 in Example 1; T0 represents the tetrabromobisphenol A concentration on day 0; ck represents the abiotic control group; a, b, c represent the significant differences between groups at the 0.05 level.

[0020] Figure 2 It is the functional activity characteristic of the tetrabromobisphenol A degradation community enriched by the fourth transfer in Example 2; C4 in the figure represents the control group of the fourth transfer; S4 represents the enriched community of the fourth transfer; 0d represents the concentration of tetrabromobisphenol A on the 0th day after transfer, and 7d represents the concentration of tetrabromobisphenol A after 7 days of culture; a, b, and c represent the significant differences between groups at the 0.05 level.

[0021] Figure 3 The degradation of tetrabromobisphenol A by the enhanced microbial community of high cell surface hydrophobic strains A18 and K7 in Example 3; ck represents the control group without strain; K7 represents the addition of K7 strain; A18 represents the addition of K7 strain; H represents the addition of a higher concentration of strain; L represents the addition of a lower concentration of strain; 0d represents the concentration of tetrabromobisphenol A on the 0th day after transfer, and 7d represents the concentration of tetrabromobisphenol A after 7 days of culture; a, b, c, d, and e represent the significant differences among the groups at the 0.05 level.

[0022] Figure 4 The effect of the high cell surface hydrophobic strains A18 and K7 on the structure of tetrabromobisphenol A-degrading microorganisms in Example 3. ck represents a control group without strains; K7 represents the addition of K7 strains; A18 represents the addition of K7 strains; H represents the addition of higher concentrations of strains; and L represents the addition of lower concentrations of strains. Specific implementation method:

[0023] In conjunction with the accompanying drawings, the method for constructing a tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity of the present invention is further described in detail by way of examples. The present invention is not limited to the description of the following examples.

[0024] Example 1: Acquisition of microorganisms with high cell surface hydrophobicity and their tetrabromobisphenol A degradation characteristics

[0025] Food waste often contains more edible oil and a relatively high content of hydrophobic organic matter. Therefore, the food waste digestion system is an ideal source of microorganisms with high cell surface hydrophobicity. The microorganisms in the food waste digestion system were isolated and cultured and the cell surface hydrophobicity was measured. The determination method is the hydrocarbon method, which is summarized as follows:

[0026] The microbial strains were cultured to the logarithmic growth phase, centrifuged at 6000 rpm for 5-10 min to collect the bacterial solution, resuspended in PBS buffer, washed 3 times, and adjusted the OD 600 (OD600前 ) to 0.6-0.65, add n-hexadecane in a volume ratio of n-hexadecane: bacterial solution = 1:5, shake for 15-20s, let stand for 30min and then measure OD again 600 (OD 600后 ). The calculation formula of cell surface hydrophobicity (CSH) is: CSH (100%) = (OD 600前 -OD 600后 ) / OD 600前 ×100%

[0027] After measurement, the cell surface hydrophobicity CSH values ​​of the strains K7 and A18 with high cell surface hydrophobicity were 72.5%-87.9% and 81.6%-94.5%, respectively. Figure 1 A) Using R2A medium strains K7 and A18, the cells were collected in the logarithmic growth phase and the final concentration of the bacterial solution was OD 600 The strain was inoculated with 0.2% Bacillus subtilisin into a sole carbon source system [4ml culture medium (1.5g / L potassium dihydrogen phosphate, 0.9g / L sodium hydrogen phosphate, 2g / L ammonium sulfate, 0.2g / L magnesium sulfate heptahydrate, 0.01g / L calcium chloride dihydrate, 0.001g / L ferrous phosphate and 0.1g / L yeast powder)] containing 10mg / L tetrabromobisphenol A, and compared with the uninoculated control group, the degradation effect of the strain on tetrabromobisphenol A was determined.

[0028] The results showed that strain K7 had a high degradation efficiency of tetrabromobisphenol A, and was able to degrade about 11.5 mg / L of tetrabromobisphenol A to about 9.1 mg / L ( Figure 1 B), the degradation rate was close to 21%, which was significantly different from the control group (p<0.001). However, the degradation rate of tetrabromobisphenol A by strain A18 was lower, with no significant difference from the control group ( Figure 1 B).

[0029] Example 2: Enrichment of tetrabromobisphenol A-degrading microbial flora

[0030] Activated sludge or sediments were taken from the biochemical treatment system of landfill leachate, municipal sewage treatment system and river channel, mixed, and the mixture was used as an inoculum source to enrich the degradation microorganisms of tetrabromobisphenol A. The enrichment medium was an inorganic salt medium (1.5g / L KH2PO4, 1.5g / L Na2HPO4·12H2O, 2g / L (NH4)2SO4, 0.22g / LMgSO4·7H2O, 0.01g / L CaCl2·2H2O, 0.001g / L FeSO4·7H2O), tetrabromobisphenol A was added as the sole carbon source, and the concentration of tetrabromobisphenol A started from 5mg / L and increased by 5mg / L each time during the enrichment process.

[0031] The mixed inoculum was inoculated into 100 mL enrichment medium containing 5 mg / L tetrabromobisphenol A, and a sterile control group was set up. After culturing at 30°C and 180 rpm for 7 days, the degradation rate was determined, and the culture was transferred to 100 mL enrichment medium containing 10 mg / L tetrabromobisphenol A at a rate of 20%. After culturing at 30°C and 180 rpm for 7 days, the degradation rate was determined, and the enrichment was continued by subculturing.

[0032] Determination of tetrabromobisphenol A degradation rate: 5 mL of enriched sample was taken to a 20 mL brown sample bottle for freeze-drying. After removing the water, 5 mL of 5:5 methanol:water solution was used for dissolution and extraction. The extract was filtered through a 0.22 μm filter membrane into a 2 mL brown chromatographic injection bottle. HPLC was used with a C18 column as the separation column and 9:1 acetonitrile:water as the mobile phase at a flow rate of 1.0 mL / min. The concentration of tetrabromobisphenol A was determined according to the standard curve at 290 nm and 206 nm.

[0033] After four generations of enrichment, a tetrabromobisphenol A-degrading microbial community with a relatively stable degradation rate was obtained. The community was able to degrade about 22 mg / L of tetrabromobisphenol A to 14.8 mg / L ( Figure 2 ), the removal rate is about 32%.

[0034] Example 3: Construction of microbial community based on cell surface hydrophobicity and its tetrabromobisphenol A degradation ability

[0035] Following Example 1 and Example 2, the high cell surface hydrophobic strains K7 and A18 in Example 1 were added to the tetrabromobisphenol A degradation enrichment community according to different biomass (1% and 5% of the total protein amount of the enriched community), and the treatment group without addition was used as the control to determine the effect of the addition of different high cell surface hydrophobic strains on the efficiency of the microbial group in degrading tetrabromobisphenol A.

[0036] The results showed that the addition of both strains significantly improved the removal of tetrabromobisphenol A. The control group without any strain had only about 25% removal efficiency, the treatment group with K7 had a degradation rate of 39-43%, and the treatment group with A18 had a degradation rate of 46-52% ( Figure 3 ). There is no consistent rule for the removal of tetrabromobisphenol A by increasing biomass. Increasing K7 only slightly increases the removal rate of tetrabromobisphenol A, while increasing A18 significantly reduces the removal rate. This may be due to the fact that K7 has a certain ability to degrade tetrabromobisphenol A, while A18 does not have the ability to degrade tetrabromobisphenol A. Therefore, the amount of cell surface hydrophobic strains added needs to be determined according to different strains.

[0037] Analysis of the structure of the microbiome revealed that the addition of hydrophobic strains on the cell surface led to significant changes in the microbial community. Compared with the control group without bacteria, the relative abundance of some bacterial genera continued to increase or remain stable during the enrichment process ( Figure 4 ), most of these microorganisms have been reported to have the ability to degrade tetrabromobisphenol A or aromatic compounds.

Claims

1. A method for constructing a tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity, characterized in that: The following steps are involved: a. Screening strains with high cell surface hydrophobicity; b. Using tetrabromobisphenol A as a screening condition, enriching and culturing strains or bacterial groups with tetrabromobisphenol A degradation function; c. Combining a strain with high cell surface hydrophobicity with a strain or bacterial colony having tetrabromobisphenol A degradation function; d. Evaluate the degradation capacity and stability of tetrabromobisphenol A by the compounded microbiome; e. Optimize the structure of the tetrabromobisphenol A microbiome, and then compound and evaluate it to obtain a functional microbiome that efficiently degrades tetrabromobisphenol A.

2. The construction method according to claim 1, characterized in that: The strains screened for high cell surface hydrophobicity are separated or enriched from a hydrophobic environment, or are selected by measuring the cell surface hydrophobicity of strains in a bacterial strain library.

3. The construction method according to claim 1, characterized in that: The screening of strains with high cell surface hydrophobicity is carried out by measuring the cell surface hydrophobicity, preferably by culturing the strains to the logarithmic growth phase and then measuring using the hydrocarbon method.

4. The construction method according to claim 1, characterized in that: The enrichment culture of the tetrabromobisphenol A-degrading functional strain or bacterial community is carried out by enriching the tetrabromobisphenol A-degrading functional microbial group using a selective culture medium containing tetrabromobisphenol A.

5. The construction method according to claim 1, characterized in that: The combination and compounding of the strain with high cell surface hydrophobicity and the strain or bacterial community with tetrabromobisphenol A degradation function is carried out according to the type and biomass of the strain or bacterial community with tetrabromobisphenol A degradation function, and the type and biomass of the strain with high cell surface hydrophobicity are adjusted, and the compounding is carried out in a certain ratio.

6. The construction method according to claim 1, characterized in that: The method for evaluating the degradation ability and stability of tetrabromobisphenol A of the compounded microbial group is to inoculate the compounded microbial group into a system containing tetrabromobisphenol A, measure the content of tetrabromobisphenol A and its degradation products in the system at different times after inoculation, and characterize the degradation ability of the microbial group on tetrabromobisphenol A.

7. A tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity, constructed according to any construction method of claims 1-6.

8. Use of the tetrabromobisphenol A efficient degradation functional microbial group based on cell surface hydrophobicity as claimed in claim 7 in degrading and removing tetrabromobisphenol A in contaminated water, sediment or soil.

9. The use according to claim 8, characterized in that: The microbial group is prepared into a bacterial suspension and added to contaminated water, sediment remediation or soil to remove tetrabromobisphenol A.

10. The use according to claim 8, characterized in that: The microbial community is loaded onto a functional carrier material and then added to contaminated water bodies, sediment remediation or soil treatment to remove tetrabromobisphenol A.

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