Microbiome with highly-efficient tetrabromobisphenol a degradation function on basis of cell-surface hydrophobicity, construction method therefor and use thereof

Abstract

Disclosed in the present invention are a microbiome with a highly-efficient tetrabromobisphenol A degradation function on the basis of cell-surface hydrophobicity, a construction method therefor and the use thereof. According to the present invention, microorganisms with a high cell-surface hydrophobicity are introduced into a tetrabromobisphenol A-degrading microbial community to construct a microbiome, which can effectively improve the degradation and removal efficiency of tetrabromobisphenol A by the microbiome, can provide an engineered microbial preparation for wastewater treatment and contaminated water body remediation or sediment remediation, and can also provide theoretical guidance for the application thereof in wastewater treatment and contaminated water body remediation or sediment remediation.

Description

A four-bromine bisphenol A high-efficiency degradation functional microbial group based on cell surface hydrophobicity and a construction method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a four-bromine bisphenol A high-efficiency degradation functional microbial group based on cell surface hydrophobicity and a construction method and application thereof. BACKGROUND

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

[0003] Cell-surface hydrophobicity (CSH) is an important physicochemical property of microbial cells, and is an important factor for determining the non-specific adhesion of microorganisms to various biological and non-biological surfaces and interfaces, and is also an important factor affecting 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 microbial group based on high cell-surface hydrophobicity microorganisms is expected to improve the degradation removal efficiency of this type of pollutants. Based on this, the present application introduces high cell-surface hydrophobicity microorganisms to construct a four-bromine bisphenol A degradation microbial group, so as to improve the efficiency of the microbial group in degrading and removing four-bromine bisphenol A. SUMMARY

[0004] The purpose of the present application is to use high cell-surface hydrophobicity microorganisms to construct a four-bromine bisphenol A microbial group and a construction method, to strengthen the mass transfer process of four-bromine bisphenol A to microorganisms, and to improve the removal efficiency.

[0005] A construction method of a four-bromine bisphenol A high-efficiency degradation functional microbial group based on cell surface hydrophobicity, characterized in that it comprises the following steps:

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

[0007] b. Enriching and culturing of strains or bacterial flora with four-bromine bisphenol A degradation function by using four-bromine bisphenol A as a screening condition;

[0008] c. Combination and compounding of high cell-surface hydrophobicity strains and strains or bacterial flora with four-bromine bisphenol A degradation function;

[0009] d. evaluating the degradation ability and stability of the microorganism group after the combination;

[0010] e. optimizing the microorganism group structure of tetrabromobisphenol A, and then re-combining and evaluating, thereby obtaining the tetrabromobisphenol A high-efficiency degradation functional microorganism group.

[0011] Preferably, the strain with high cell surface hydrophobicity is obtained by separation or enrichment from a hydrophobic environment, or is obtained by selection from a strain library according to the cell surface hydrophobicity.

[0012] Preferably, the strain with high cell surface hydrophobicity is obtained by separation or enrichment from a hydrophobic environment, or is obtained by selection from a strain library according to the cell surface hydrophobicity.

[0013] Preferably, the enrichment culture of the tetrabromobisphenol A degradation functional strain or group is achieved by using a selective medium containing tetrabromobisphenol A to enrich the tetrabromobisphenol A degradation functional microorganism group.

[0014] Preferably, the combination of the strain with high cell surface hydrophobicity and the strain or group with tetrabromobisphenol A degradation function is achieved by adjusting the type and biomass of the strain with high cell surface hydrophobicity according to the type and biomass of the strain or group with tetrabromobisphenol A degradation function, and then combining them in a certain ratio.

[0015] Preferably, the evaluation of the degradation ability and stability of the microorganism group after the combination is achieved by inoculating the combined microorganism group into a system containing tetrabromobisphenol A, and then determining the content of tetrabromobisphenol A and its degradation products in the system at different times after inoculation, so as to characterize the degradation ability of the microorganism group to tetrabromobisphenol A.

[0016] The application also provides the application of the tetrabromobisphenol A high-efficiency degradation functional microorganism group based on cell surface hydrophobicity in degrading and removing tetrabromobisphenol A in contaminated water bodies, sediments or soils.

[0017] Preferably, the microorganism group is prepared into a bacterial suspension and added to the contaminated water body, sediment or soil for removing tetrabromobisphenol A, or the microorganism group is loaded on a functional carrier material and then added to the contaminated water body, sediment or soil for removing tetrabromobisphenol A.

[0018] The application can effectively improve the degradation and removal efficiency of the microorganism group to tetrabromobisphenol A by introducing the microorganism with high cell surface hydrophobicity into the tetrabromobisphenol A degradation microbial community to construct the microorganism group, thereby providing an engineering microbial preparation for wastewater treatment and contaminated water body or sediment remediation, and providing a theoretical guidance for the application in wastewater treatment and contaminated water body or sediment remediation. BRIEF DESCRIPTION OF DRAWINGS:

[0019] Figure 1 is the cell surface hydrophobicity (A) and tetrabromobisphenol A degradation ability (B) of strains A18 and K7 in Example 1; T0 represents the tetrabromobisphenol A concentration on day 0; ck represents the non-biological control group; a, b, c represent the significance of difference between groups at the 0.05 level.

[0020] Figure 2 is the functional activity characteristics 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 tetrabromobisphenol A concentration on day 0 after transfer, 7d represents the tetrabromobisphenol A concentration after 7 days of culture; a, b, c represent the significance of difference between groups at the 0.05 level.

[0021] Figure 3 is the degradation of tetrabromobisphenol A by the high cell surface hydrophobicity strains A18 and K7 to strengthen the microbial community in Example 3; ck represents the control group without adding strains; K7 represents adding K7 strain; A18 represents adding K7 strain; H represents adding a higher concentration of strains; L represents adding a lower concentration of strains; 0d represents the tetrabromobisphenol A concentration on day 0 after transfer, 7d represents the tetrabromobisphenol A concentration after 7 days of culture; a, b, c, d, e represent the significance of difference between groups at the 0.05 level.

[0022] Figure 4 is the effect of high cell surface hydrophobicity strains A18 and K7 on the microbial structure for tetrabromobisphenol A degradation in Example 3; ck represents the control group without adding strains; K7 represents adding K7 strain; A18 represents adding K7 strain; H represents adding a higher concentration of strains; L represents adding a lower concentration of strains. DETAILED DESCRIPTION

[0023] The application will be further described in detail below by examples in combination with the accompanying drawings. The application is not limited to the description of the following examples.

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

[0025] Kitchen waste often contains a lot of edible oil, and the content of hydrophobic organic matter is relatively high, so the kitchen waste digestion system is an ideal source place for high cell surface hydrophobicity microorganisms. The microorganisms in the kitchen waste digestion system were isolated and cultured, and the cell surface hydrophobicity was determined. The determination method is hydrocarbon method, and the method 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 with PBS buffer, washed 3 times, and adjusted OD 600 (OD 600前) to 0.6-0.65, n-hexadecane was added in the ratio of n-hexadecane: bacterial solution = 1:5 by volume, oscillated for 15-20 s, and after 30 min of standing, the OD was measured again 600 (OD 600后 ). The formula for calculating the cell surface hydrophobicity (CSH) is: CSH (100%) = (OD 600前 - OD 600后 ) / OD 600前 x 100%

[0027] It was determined that the cell surface hydrophobicity CSH values of the high cell surface hydrophobicity strains K7 and A18 were 72.5%-87.9% and 81.6%-94.5%, respectively (Figure 1A). Using R2A medium, the bacterial cells of strains K7 and A18 were collected in the logarithmic growth phase, and inoculated into a system containing 10 mg / L tetrabromobisphenol A as the sole carbon source [4 ml of medium (KH2PO41.5 g / L, Na2HPO4·12H2O 1.5 g / L, (NH4)2SO42 g / L, MgSO4·7H2O 0.22 g / L, CaCl2·2H2O 0.01 g / L, FeSO4·7H2O 0.001 g / L, and yeast extract 0.1 g / L)] at a final concentration of OD 600 0.2, and compared with a control group without inoculation, to determine the degradation effect of the strains on tetrabromobisphenol A.

[0028] The results showed that the tetrabromobisphenol A degradation efficiency of strain K7 was relatively high, and about 11.5 mg / L of tetrabromobisphenol A was degraded to about 9.1 mg / L within 7 days (Figure 1B), with a degradation rate close to 21%, which was significantly different from the control group (p < 0.001). The tetrabromobisphenol A degradation rate of strain A18 was relatively low, and there was no significant difference from the control group (Figure 1B).

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

[0030] Active sludge or sediment was taken from a landfill leachate biochemical treatment system, a municipal sewage treatment system, and a river, and mixed to obtain a mixture, which was used as an inoculum for the passage enrichment of tetrabromobisphenol A degrading microorganisms. The enrichment medium was an inorganic salt medium (1.5 g / L KH2PO4, 1.5 g / L Na2HPO4·12H2O, 2 g / L (NH4)2SO4, 0.22 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O), and tetrabromobisphenol A was added as the sole carbon source. The concentration of tetrabromobisphenol A was started at 5 mg / L, and increased by 5 mg / 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. After 7 days of culture at 30°C and 180 rpm, the degradation rate was determined, and then 20% was transferred to 100 mL enrichment medium containing 10 mg / L tetrabromobisphenol A. After 7 days of culture at 30°C and 180 rpm, the degradation rate was determined, and the enrichment was continued.

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

[0033] After four generations of enrichment, a tetrabromobisphenol A degrading microbial enrichment population with stable degradation rate was obtained. The population could degrade about 22 mg / L of tetrabromobisphenol A to 14.8 mg / L within 7 days (Figure 2), with a removal rate of about 32%.

[0034] Example 3: Microbial community construction based on cell surface hydrophobicity and its tetrabromobisphenol A degradation ability

[0035] Based on Examples 1 and 2, the high cell surface hydrophobicity strains K7 and A18 in Example 1 were added to the tetrabromobisphenol A degrading enrichment community in different amounts (1% and 5% of the total protein amount of the enrichment community), and a treatment group without addition was used as a control. The effect of adding different high cell surface hydrophobicity strains on the efficiency of the microbial community in degrading tetrabromobisphenol A was determined.

[0036] The results showed that the addition of both strains significantly improved the removal effect of tetrabromobisphenol A. The control group without any strain had a removal rate of about 25%, 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). Increasing the biomass did not have a consistent pattern on the removal of tetrabromobisphenol A. Increasing K7 only slightly increased the removal rate of tetrabromobisphenol A, while increasing A18 significantly decreased the removal rate. This may be due to the fact that K7 has a certain tetrabromobisphenol A degradation ability, while A18 does not have a tetrabromobisphenol A degradation ability. Therefore, the addition of cell surface hydrophobicity strains needs to be determined according to different strains.

[0037] The analysis of the structure of the microbiome revealed that the addition of cell surface hydrophobic strains caused significant changes in the microbial community. Compared with the control group without the addition of bacteria, the relative abundance of some genera continued to rise or remained stable during the enrichment process (Figure 4), and most of these microorganisms have been reported to have the ability to degrade tetrabromobisphenol A or aromatic hydrocarbons.

Claims

1. A method for constructing a high-efficiency degrading functional microbial group of tetrabromobisphenol A based on cell surface hydrophobicity, characterized in that, The method comprises the following steps: a. screening strains with high cell surface hydrophobicity; b. enriching and culturing strains or groups with tetrabromobisphenol A degradation function using tetrabromobisphenol A as a screening condition; c. combining and compounding strains with high cell surface hydrophobicity and strains or groups with tetrabromobisphenol A degradation function; d. evaluating the tetrabromobisphenol A degradation capacity and stability of the microbial group after compounding; e. optimizing the structure of the tetrabromobisphenol A microbial group, and then compounding and evaluating, thereby obtaining a tetrabromobisphenol A high-efficiency degradation functional microbial group.

2. The construction method of claim 1, wherein, The strains with high cell surface hydrophobicity are obtained by isolation or enrichment from a hydrophobic environment, or are obtained by selection from a strain library through determination of the cell surface hydrophobicity of the strains.

3. The construction method of claim 1, wherein, The strains with high cell surface hydrophobicity are determined by cell surface hydrophobicity determination, preferably by using a hydrocarbon method after the strains are cultured to the logarithmic growth phase.

4. The construction method of claim 1, wherein, The strains or groups with tetrabromobisphenol A degradation function are enriched by using a selective culture medium containing tetrabromobisphenol A.

5. The construction method of claim 1, wherein, The strains with high cell surface hydrophobicity are combined and compounded with the strains or groups with tetrabromobisphenol A degradation function according to the types and biomass of the strains or groups with tetrabromobisphenol A degradation function, the types and biomass of the strains with high cell surface hydrophobicity are adjusted, and the compounding is performed at a certain ratio.

6. The construction method of claim 1, wherein, The tetrabromobisphenol A degradation capacity and stability of the microbial group after compounding are evaluated by inoculating the compounded microbial group into a system containing tetrabromobisphenol A, determining the content of tetrabromobisphenol A and its degradation products in the system at different times after inoculation, and characterizing the tetrabromobisphenol A degradation capacity of the microbial group.

7. A tetrabromobisphenol A high-efficiency degradation functional microbial group based on cell surface hydrophobicity, which is constructed by the method according to any one of claims 1-6.

8. The use of the tetrabromobisphenol A high-efficiency degradation functional microbial group based on cell surface hydrophobicity according to claim 7 in degrading and removing tetrabromobisphenol A in contaminated water bodies, sediments or soil.

9. Use according to claim 8, characterized in that, The microbial group is prepared into a bacterial suspension and added to the contaminated water bodies, sediments or soil to remove tetrabromobisphenol A.

10. Use according to claim 8, characterized in that, The microbial group is loaded on a functional carrier material and then added to the contaminated water bodies, sediments or soil to remove tetrabromobisphenol A. The microbial group is loaded on a functional carrier material and then added to the contaminated water bodies, sediments or soil to remove tetrabromobisphenol A.

Images