阅读说明：本技术 一种磁力细胞及其制备方法和应用 (Magnetic cell and preparation method and application thereof ) 是由 马晓蕾 曹军瑞 王旭楠 谢宝龙 王勋亮 于 2020-12-28 设计创作，主要内容包括：本发明提供了一种磁力细胞及其制备方法和应用,所述磁性细胞为吸附有磁性纳米颗粒的油污降解菌；所述磁性细胞的制备方法包括：将油污降解菌与磁性纳米颗粒共孵育,在活化培养基中培养活化,得到所述磁性细胞。本发明的磁性细胞分散性好、传质性好、易于回收,油污降解能力强,制备方法简单、条件温和,在石油及石油产品污染水环境的生物修复领域具有重要的应用前景。(The invention provides a magnetic cell and a preparation method and application thereof, wherein the magnetic cell is an oil stain degrading bacterium adsorbed with magnetic nano particles; the preparation method of the magnetic cell comprises the following steps: and co-incubating the oil stain degrading bacteria and the magnetic nanoparticles, and culturing and activating in an activation culture medium to obtain the magnetic cell. The magnetic cell provided by the invention has the advantages of good dispersibility, good mass transfer, easiness in recovery, strong oil stain degradation capability, simple preparation method and mild conditions, and has an important application prospect in the field of bioremediation of petroleum and petroleum product polluted water environment.)

1. The magnetic cell is an oil stain degrading bacterium adsorbed with magnetic nanoparticles, the oil stain degrading bacterium is Rhodococcus, is named as SY095 Rhodococcus sp, is preserved in China general microbiological culture Collection center (CGMCC), has a preservation number of CGMCC No.10724, and has a preservation date of 2015, 04 months and 16 days.

2. The magnetic cell of claim 1, wherein the magnetic nanoparticles comprise any one or a combination of at least two of hydroxylated magnetic nanoparticles, carboxylated magnetic nanoparticles, or aminated magnetic nanoparticles;

preferably, the carboxylated magnetic nanoparticles comprise hydrophilic carboxylated magnetic nanoparticles and/or hydrophobic carboxylated magnetic nanoparticles;

preferably, the particle size of the magnetic nanoparticles is 10-50 nm;

preferably, the age of the oil stain degrading bacteria is 24-36 h.

3. A method for preparing a magnetic cell according to claim 1 or 2, comprising:

and co-incubating the oil stain degrading bacteria and the magnetic nanoparticles, and culturing and activating in an activation culture medium to obtain the magnetic cell.

4. The method according to claim 3, wherein the ratio of the oil-degrading bacteria to the magnetic nanoparticles is (5-10)% to (4-8) g;

preferably, the co-incubation is performed under shaking conditions;

preferably, the temperature of the co-incubation is 25-28 ℃;

preferably, the rotation speed of the co-incubation is 100-150 rpm;

preferably, the co-incubation time is 10-15 h;

preferably, the activation medium comprises 8-12 g/L tryptone, 8-12 g/L NaCl and 3-7 g/L yeast extract;

preferably, the pH value of the activation medium is 7-8.

5. The method according to claim 3 or 4, wherein the screening method of the oil stain degrading bacteria comprises:

collecting oil-containing sludge, adding sterile physiological saline water for mixing, standing, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium for culture, sucking a lower layer solution and inoculating the lower layer solution into the oil-containing liquid culture medium, and repeatedly culturing to obtain a bacterial suspension;

and inoculating the bacterial suspension into a solid culture medium for culture to obtain the oil stain degrading bacteria.

6. The preparation method according to claim 5, wherein the inoculum size of the bacterial suspension to be screened inoculated in the oil-containing liquid culture medium is 1-5%;

preferably, the oil-containing liquid culture medium comprises 1-5 g/L NaNO₃、0.1～1g/L K₂HPO₄、1～5g/L KH₂PO₄、0.01～0.1g/L CaCl₂、0.1～0.5g/L KCl、0.1～0.5g/L MgSO₄·7H₂O、0.01～0.1g/L FeSO₄·7H₂O, 0.1-0.5 g/L yeast powder, 0.1-0.5 mL/L microelement solution and 0.5-2% crude oil;

preferably, the trace element solution comprises 0.1-0.5 g/L H₃BO₃、0.1～0.5g/L CuSO₄·5H₂O、0.1～0.5g/L MnSO₄·H₂O、0.01～0.1g/L MoNa₂O₄·2H₂O and 0.5-1 g/L ZnSO₄·7H₂O；

Preferably, the crude oil comprises heavy crude oil;

preferably, the density of the crude oil is 0.92-1 g/cm³；

Preferably, the condition of inoculating the bacterial suspension to be screened into the oil-containing liquid culture medium for culture is shake culture;

preferably, the temperature of the shake culture is 28-32 ℃;

preferably, the rotation speed of the shake culture is 150-200 r/min;

preferably, the shake culture time is 7-15 days;

preferably, the number of times of repeated culture is 5-8.

7. The production method according to any one of claims 3 to 6, wherein the magnetic nanoparticles comprise any one of or a combination of at least two of hydroxylated magnetic nanoparticles, carboxylated magnetic nanoparticles, or aminated magnetic nanoparticles;

preferably, the carboxylated magnetic nanoparticles comprise hydrophilic carboxylated magnetic nanoparticles and/or hydrophobic carboxylated magnetic nanoparticles;

preferably, the preparation method of the hydroxylated magnetic nanoparticles comprises:

mixing Fe₃O₄Respectively dissolving the nano particles and the glucan in 0.05-0.1 mol/L sodium citrate solution according to Fe₃O₄The mass ratio of the nano particles to the glucan is 1 (0.5-2) to Fe₃O₄Adding a glucan-sodium citrate solution into the nanoparticle-sodium citrate solution, carrying out ultrasonic oscillation for 30-60 min, stirring in a water bath at 85-100 ℃ for 1-3 h, carrying out magnetic separation, cleaning and vacuum drying to obtain the hydroxylated magnetic nanoparticles;

preferably, the preparation method of the hydrophilic carboxylated magnetic nanoparticles comprises the following steps:

preparing 4-6 mol/L Fe₃O₄Adding citric acid into the aqueous solution according to the molar ratio of (2-5): 1, performing ultrasonic oscillation for 20-40 min, stirring in a water bath for 1-3 h, performing magnetic separation, cleaning and vacuum drying to obtain the hydrophilic carboxylated magnetic nanoparticles;

preferably, the preparation method of the hydrophobic carboxylated magnetic nanoparticle comprises the following steps:

preparing 2-4 mol/L Fe₃O₄Adding 2-4 mol/L oleic acid into a normal hexane solution, carrying out ultrasonic oscillation for 20-40 min, stirring in a water bath at 50-68 ℃ for 0.5-2 h, carrying out magnetic separation, cleaning and vacuum drying to obtain the hydrophobic carboxylated magnetic nanoparticles;

preferably, the preparation method of the aminated magnetic nanoparticle comprises:

preparing 0.005-0.01 g/mL Fe₃O₄Adding 4-8 times of volume of ethanol into the ethanol solution, carrying out ultrasonic oscillation for 5-10 min, adding 3-aminopropyltriethoxysilane, stirring in a water bath at 50-70 ℃ for 6-8 h, carrying out magnetic separation, cleaning, and carrying out vacuum drying to obtain the aminated magnetic nanoparticles.

8. The method of any one of claims 3 to 7, comprising the steps of:

(1) collecting oil-containing sludge, adding sterile physiological saline water for mixing, standing, and taking supernatant as bacterial suspension to be screened; inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium according to the inoculation amount of 1% -5%, performing shake culture at 28-32 ℃ at 150-200 r/min for 7-15 days, absorbing the lower layer solution, inoculating into the oil-containing liquid culture medium, and performing repeated culture for 5-8 times to obtain bacterial suspension; inoculating the bacterial suspension into a solid culture medium for culture to obtain oil stain degrading bacteria;

(2) preparing any one or combination of at least two of hydroxylated magnetic nanoparticles, hydrophilic carboxylated magnetic nanoparticles, hydrophobic carboxylated magnetic nanoparticles or aminated magnetic nanoparticles;

(3) and (3) incubating the oil stain degrading bacteria and the magnetic nanoparticles for 10-15 hours at 25-28 ℃ and 100-150 rpm in a shaking manner, and culturing and activating in an activation culture medium to obtain the magnetic cells.

9. Use of the magnetic cell of claim 1 or 2 for the treatment of oily wastewater.

10. A method for treating oily sewage, which is characterized by comprising the following steps:

placing the magnetic cells of claim 1 or 2 in oil-containing wastewater, performing oil degradation, and magnetically separating and recovering the magnetic cells;

preferably, the temperature of the oil stain treatment is 25-35 ℃;

preferably, the oil stain treatment time is 3-8 days.

Technical Field

The invention belongs to the technical field of bioremediation of petroleum and petroleum product polluted water environments, and relates to a magnetic cell and a preparation method and application thereof.

Background

With the increasing exploration and exploitation of petroleum resources, the crude oil industry is rapidly developed, crude oil and products bring economic benefits to society, meanwhile, the natural environment in which human beings depend on living is also greatly damaged, and crude oil pollutants enter soil, surface water and underground water through various ways and seriously pollute the environment. After an accident of petroleum pollution occurs, most of surface oil stains are usually cleaned by physical and chemical methods, however, alkane and aromatic hydrocarbon pollutants in residual oil stains still exist in a water body, and the harm of the pollutants is not small and varied, and serious and persistent harm can be caused to the water body and human health. The bioremediation method is used as an ultimate strategy for oil stain treatment, and has the advantages of safety, thoroughness and no secondary pollution although the process is slow. The prior thallus treatment method is mainly used for immobilizing strains in the marine environment and soil water body environment polluted by oil stain wastewater to form high-concentration strains and prevent the thallus from dispersing in the marine environment and the soil water body so as to degrade pollutants.

CN101768583A discloses a method for immobilizing petroleum degrading bacteria by using reed straw as a carrier and an application thereof, which comprises the steps of firstly screening out petroleum degrading bacteria by using soil polluted by petroleum, preparing a suspension of the petroleum degrading bacteria by using the bacteria, adding sodium alginate into the suspension of the petroleum degrading bacteria, uniformly spraying the suspension of the petroleum degrading bacteria on the reed straw for 5 hours after mixing to achieve immobilization, or adding the sodium alginate into the suspension of the petroleum degrading bacteria, mixing the suspension of the petroleum degrading bacteria with the crushed reed straw, and then dropping saturated calcium chloride solution to prepare hardened particles so as to achieve immobilization. However, the method has the defects of lower purification efficiency than that of free bacteria, short service life of straws and the like.

CN105936901A discloses an immobilized microbial agent, a preparation method and an application thereof, wherein the preparation method comprises the following steps: inoculating Dietzia cerealis strain HJZX-2 into an R2A culture medium for activated culture to obtain a bacterial liquid; inoculating the bacterial liquid into an immobilized culture medium, adding modified zeolite, then placing the immobilized culture medium in a shaking table, and culturing at a preset immobilization temperature for a preset immobilization time to obtain a liquid immobilized bacterial agent. However, the method is not suitable for the on-site treatment of the oil stains in the natural environment, and the immobilization cost and the reaction cost are high.

The prior art shows the high efficiency and reliability of immobilized microorganisms in the aspect of oil stain degradation, however, the strain immobilization mostly adopts a carrier adsorption or carrier embedding mode, and the problems of low mechanical strength, poor mass transfer performance, poor dispersion performance, difficult recovery and the like exist.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a magnetic cell and a preparation method and application thereof, wherein magnetic nanoparticles are adsorbed on the surface of oil stain degrading bacteria, the magnetic cell is endowed with magnetism, the oil stain degrading magnetic cell which is good in dispersity, easy to fix and close to free bacteria in mass transfer performance is constructed, and the effect of treating oily sewage based on a magnetic separation technology is realized.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a magnetic cell, wherein the magnetic cell is an oil stain degrading bacterium adsorbed with magnetic nanoparticles, the oil stain degrading bacterium is Rhodococcus, is named as SY095 Rhodococcus sp, is preserved in China general microbiological culture Collection center (CGMCC), is addressed as No. 3 of Xilu No.1 of Beijing Inward area of Tokyo, zip code 100101, has a preservation number of CGMCC No.10724, and has a preservation date of 2015 04/16 days.

According to the invention, the oil stain degrading bacteria Rhodococcus SY095 Rhodococcus sp are obtained by separating and screening sludge from the coastal mudflat of Tianjin Dagang, the oil stain degrading bacteria and magnetic nanoparticles are vibrated under mild conditions and incubated for a period of time, the magnetic nanoparticles are adsorbed on the surface of the oil stain degrading bacteria based on the binding property of characteristic groups on the surface of the bacteria and modification groups of the magnetic nanoparticles, the bacteria are not changed or damaged, and the prepared magnetic cells have good free dispersibility and good magnetic responsiveness, are beneficial to recovery and have mass transfer performance equivalent to that of the free bacteria.

Preferably, the magnetic nanoparticles comprise any one of or a combination of at least two of hydroxylated magnetic nanoparticles, carboxylated magnetic nanoparticles, or aminated magnetic nanoparticles.

Preferably, the carboxylated magnetic nanoparticles comprise hydrophilic carboxylated magnetic nanoparticles and/or hydrophobic carboxylated magnetic nanoparticles.

Preferably, the particle size of the magnetic nanoparticles is 10-50 nm, preferably 20 nm.

In the invention, the magnetic nanoparticles with the particle size of about 20nm are adsorbed on the surface of the oil stain degrading bacteria, the volume of the oil stain degrading bacteria is not obviously influenced, and the prepared magnetic cells have good free dispersibility.

Preferably, the bacterial age of the oil stain degrading bacteria is 24-36 h, for example, 24h, 28h, 32h or 36h, preferably 28 h.

In a second aspect, the present invention provides a method for preparing a magnetic cell according to the first aspect, the method comprising:

and co-incubating the oil stain degrading bacteria and the magnetic nanoparticles, and culturing and activating in an activation culture medium to obtain the magnetic cell.

Preferably, the ratio of the quantity of the oil stain degrading bacteria to the mass of the magnetic nanoparticles is (5-10)% to (4-8).

Preferably, the co-incubation is performed under shaking conditions.

Preferably, the temperature of the co-incubation is 25-28 ℃, for example, 25 ℃, 26 ℃, 27 ℃ or 28 ℃, preferably 26 ℃.

Preferably, the rotation speed of the co-incubation is 100-150 rpm, for example, 100rpm, 110rpm, 120rpm, 130rpm, 140rpm or 150rpm, preferably 110 rpm.

Preferably, the co-incubation time is 10-15 h, for example, 10h, 11h, 12h, 13h, 14h or 15h, preferably 12 h.

Preferably, the activation medium comprises 8-12 g/L tryptone, 8-12 g/L NaCl and 3-7 g/L yeast extract.

Preferably, the pH of the activation medium is 7 to 8, for example, 7, 7.2, 7.5, 7.8 or 8, preferably 7.5.

Preferably, the screening method of the oil stain degrading bacteria comprises the following steps:

collecting oil-containing sludge, adding sterile physiological saline water for mixing, standing, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium for culture, sucking a lower layer solution and inoculating the lower layer solution into the oil-containing liquid culture medium, and repeatedly culturing to obtain a bacterial suspension;

and inoculating the bacterial suspension into a solid culture medium for culture to obtain the oil stain degrading bacteria.

Preferably, the bacterial suspension to be screened is inoculated in the oil-containing liquid culture medium in an inoculation amount of 1-5%, for example, 1%, 2%, 3%, 4% or 5%.

Preferably, the oil-containing liquid culture medium comprises 1-5 g/L NaNO₃、0.1～1g/L K₂HPO₄、1～5g/L KH₂PO₄、0.01～0.1g/L CaCl₂、0.1～0.5g/L KCl、0.1～0.5g/L MgSO₄·7H₂O、0.01～0.1g/L FeSO₄·7H₂O, 0.1-0.5 g/L yeast powder, 0.1-0.5 mL/L microelement solution and 0.5-2% crude oil, preferably 2.5g/L NaNO₃、0.5g/L K₂HPO₄、1.0g/L KH₂PO₄、0.01g/L CaCl₂、0.1g/L KCl、0.5g/L MgSO₄·7H₂O、0.01g/L FeSO₄·7H₂O, 0.1g/L yeast powder, 0.5mL/L trace element solution and 1% crude oil.

Preferably, the trace element solution comprises 0.1-0.5 g/L H₃BO₃、0.1～0.5g/L CuSO₄·5H₂O、0.1～0.5g/L MnSO₄·H₂O、0.01～0.1g/L MoNa₂O₄·2H₂O and 0.5-1 g/L ZnSO₄·7H₂O, preferably 0.26g/L H₃BO₃、0.5g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.06g/L MoNa₂O₄·2H₂O and 0.7g/L ZnSO₄·7H₂O。

Preferably, the crude oil comprises heavy crude oil, such as crude oil with complex components which can be processed without refining, residual oil after refining gasoline and diesel oil, and the heavy crude oil is basically characterized by high viscosity, complex components, large molecular weight, great harm to ecological environment and human health and the like.

Preferably, the density of the crude oil is 0.92-1 g/cm³。

Preferably, the condition for inoculating the bacterial suspension to be screened into the oil-containing liquid culture medium is shake culture.

Preferably, the temperature of the shake culture is 28-32 ℃, for example, 28 ℃, 29 ℃, 30 ℃, 31 ℃ or 32 ℃.

Preferably, the rotation speed of the shaking culture is 150-200 r/min, such as 150r/min, 160r/min, 170r/min, 180r/min, 190r/min or 200 r/min.

Preferably, the period of shake culture is 7-15 days, for example, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days.

Preferably, the number of times of repeated culture is 5-8.

Preferably, the magnetic nanoparticles comprise any one of or a combination of at least two of hydroxylated magnetic nanoparticles, carboxylated magnetic nanoparticles, or aminated magnetic nanoparticles.

Preferably, the carboxylated magnetic nanoparticles comprise hydrophilic carboxylated magnetic nanoparticles and/or hydrophobic carboxylated magnetic nanoparticles.

Preferably, the preparation method of the hydroxylated magnetic nanoparticles comprises:

mixing Fe₃O₄Respectively dissolving the nano particles and the glucan in 0.05-0.1 mol/L sodium citrate solution according to Fe₃O₄The mass ratio of the nano particles to the glucan is 1 (0.5-2) to Fe₃O₄And adding a glucan-sodium citrate solution into the nanoparticle-sodium citrate solution, carrying out ultrasonic oscillation for 30-60 min, stirring in a water bath at 85-100 ℃ for 1-3 h, carrying out magnetic separation, cleaning and vacuum drying to obtain the hydroxylated magnetic nanoparticles.

Preferably, the preparation method of the hydrophilic carboxylated magnetic nanoparticles comprises the following steps:

4-6 mol/L Fe is prepared₃O₄Adding citric acid into the aqueous solution according to the molar ratio of (2-5): 1, carrying out ultrasonic oscillation for 20-40 min, stirring in a water bath for 1-3 h, carrying out magnetic separation, cleaning, and vacuum drying to obtain the hydrophilic carboxylated magnetic nanoparticles.

Preferably, the preparation method of the hydrophobic carboxylated magnetic nanoparticle comprises the following steps:

preparing 2-4 mol/L Fe₃O₄Adding 2-4 mol/L oleic acid into the n-hexane solution, carrying out ultrasonic oscillation for 20-40 min, stirring in a water bath at 50-68 ℃ for 0.5-2 h, carrying out magnetic separation, cleaning and vacuum drying to obtain the hydrophobic carboxylated magnetic nanoparticles.

Preferably, the preparation method of the aminated magnetic nanoparticle comprises:

preparing 0.005-0.01 g/mL Fe₃O₄Adding 4-8 times of volume of ethanol into the ethanol solution, carrying out ultrasonic oscillation for 5-10 min, adding 3-aminopropyltriethoxysilane, stirring in a water bath at 50-70 ℃ for 6-8 h, carrying out magnetic separation, cleaning, and carrying out vacuum drying to obtain the aminated magnetic nanoparticles.

As a preferred technical scheme, the preparation method of the magnetic cell comprises the following steps:

(1) collecting oil-containing sludge, adding sterile physiological saline water for mixing, standing, and taking supernatant as bacterial suspension to be screened; inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium according to the inoculation amount of 1% -5%, performing shake culture at 28-32 ℃ at 150-200 r/min for 7-15 days, absorbing the lower layer solution, inoculating into the oil-containing liquid culture medium, and performing repeated culture for 5-8 times to obtain bacterial suspension; inoculating the bacterial suspension into a solid culture medium for culture to obtain oil stain degrading bacteria;

(2) preparing any one or combination of at least two of hydroxylated magnetic nanoparticles, hydrophilic carboxylated magnetic nanoparticles, hydrophobic carboxylated magnetic nanoparticles or aminated magnetic nanoparticles;

(3) and (3) incubating the oil stain degrading bacteria and the magnetic nanoparticles for 10-15 hours at 25-28 ℃ and 100-150 rpm in a shaking manner, and culturing and activating in an activation culture medium to obtain the magnetic cells.

In a third aspect, the present invention provides the use of the magnetic cell of the first aspect for the treatment of oily wastewater.

In a fourth aspect, the present invention provides a method for treating oily sewage, comprising:

and (2) putting the magnetic cells in the first aspect into oil-containing sewage, degrading the oil stain, and magnetically separating and recovering the magnetic cells.

Preferably, the temperature of the oil stain treatment is 25 to 35 ℃, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃, preferably 30 ℃.

Preferably, the oil stain treatment time is 3 to 8 days, for example, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days, preferably 5 days.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the nanometer magnetic particles smaller than single cells of the oil stain degrading bacteria are adsorbed on the cell walls of the oil stain degrading bacteria, so that the biological activity of microorganisms is well kept, the influence of materials on the cell surface mass transfer performance is reduced to the greatest extent, the problem that the traditional strain immobilization method is difficult to disperse once immobilized is solved, and the uniformity of dispersive degradation and the convenience of magnetic separation and centralized recovery are both considered;

(2) the preparation method of the magnetic cell is simple and mild in condition, the prepared magnetic cell has high oil stain degradation activity and cell capacity, is environment-friendly and recyclable, can meet different requirements of dispersion and centralized use, and has the oil stain degradation rate of over 65% in 72 hours;

(3) the magnetic cells can be integrated on a large-scale magnetic carrier, are uniformly dispersed in a water body, are used for in-situ treatment and sewage treatment of an oil pollution environment, and have remarkable advantages in large-scale popularization and application.

Detailed Description

To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

The preparation of the magnetic cell with the oil stain degradation function comprises the following steps:

(1) collecting oil-containing sludge of an oilfield in Tianjin Hongkong, adding sterile normal saline to immerse the sludge and fully mixing, standing for settling the sludge, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium (2.5g/L NaNO) according to the inoculation amount of 5 percent₃、0.5g/L K₂HPO₄、1g/L KH₂PO₄、0.01g/L CaCl₂、0.1g/L KCl、0.5g/L MgSO₄·7H₂O、0.01g/L FeSO₄·7H₂O, 0.1g/L yeast powder, 0.5mL/L trace element solution and 1% crude oil, wherein the trace element solution comprises 0.26g/L H₃BO₃、0.5g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.06g/L MoNa₂O₄·2H₂O and 0.7g/L ZnSO₄·7H₂O), shaking table cultivation at 30 ℃ and 160r/min for 7 days, and selecting materials with obviously reduced oil stains on the surface layer,The lower layer solution is sucked and inoculated in a new oil-containing liquid culture medium with the same volume, and the culture is continued according to the conditions; repeating for 5 times, selecting an experimental group with the least residual oil, dipping the lower layer solution, and carrying out flat plate scribing to obtain oil stain degrading bacteria;

(2) mixing Fe₃O₄The nanoparticles and dextran were dissolved separately in 0.075mol/L sodium citrate solution, as per Fe₃O₄The mass ratio of the nano particles to the glucan is 1:2 to Fe₃O₄Adding dextran-sodium citrate solution into the nanoparticle-sodium citrate solution, ultrasonically oscillating for 30min, stirring in 85 deg.C water bath for 3h, magnetically separating, washing with water for 4 times, and vacuum drying to obtain hydroxylated magnetic nanoparticles with particle diameter of 20 nm;

(3) culturing the screened oil stain degrading bacteria in an LB culture medium for 28 hours, centrifugally collecting bacteria, resuspending the bacteria with normal saline, and repeating for 3 times to remove culture medium components;

and (2) resuspending the oil stain degrading bacteria in 100mL of physiological saline to ensure that the concentration of the oil stain degrading bacteria is 5%, adding 4g of hydroxylated magnetic nanoparticles, carrying out shake co-incubation for 10h at 28 ℃ and 150rpm, and carrying out culture activation in an activation medium (10g/L tryptone, 10g/L NaCl and 5g/L yeast extract, pH7.5) to obtain the magnetic cell.

Example 2

The preparation of the magnetic cell with the oil stain degradation function comprises the following steps:

(1) collecting oil-containing sludge of an oilfield in Tianjin Hongkong, adding sterile normal saline to immerse the sludge and fully mixing, standing for settling the sludge, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium (2.5g/L NaNO) according to the inoculation amount of 2 percent₃、0.5g/L K₂HPO₄、1g/L KH₂PO₄、0.01g/L CaCl₂、0.1g/L KCl、0.5g/L MgSO₄·7H₂O、0.01g/L FeSO₄·7H₂O, 0.1g/L yeast powder, 0.5mL/L trace element solution and 1% crude oil, wherein the trace element solution comprises 0.26g/L H₃BO₃、0.5g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.06g/L MoNa₂O₄·2H₂O and 0.7g/L ZnSO₄·7H₂O), shaking and shaking the table for 7 days at 28 ℃ and 150r/min, selecting an experimental group with obviously reduced surface oil stains and obviously turbid lower layer solution, absorbing the lower layer solution, inoculating the lower layer solution into a new oil-containing liquid culture medium with the same volume, and continuously culturing according to the conditions; repeating for 6 times, selecting an experimental group with the least residual oil, dipping the lower layer solution, and carrying out flat plate scribing to obtain oil stain degrading bacteria;

(2) mixing Fe₃O₄Preparing 5mol/L Fe by nano particles₃O₄Performing ultrasonic oscillation on the aqueous solution for 10min, adding citric acid according to the molar ratio of 5:1, performing ultrasonic oscillation for 30min, stirring in a water bath for 2h, performing magnetic separation, washing with water for 4 times, and performing vacuum drying to obtain hydrophilic carboxylated magnetic nanoparticles with the particle size of 20 nm;

(3) culturing the screened oil stain degrading bacteria in an LB culture medium for 32h, centrifugally collecting bacteria, resuspending the bacteria with normal saline, and repeating for 3 times to remove culture medium components;

the oil stain degrading bacteria are resuspended in 100mL of physiological saline to make the concentration of the oil stain degrading bacteria be 10%, 8g of hydroxylated magnetic nanoparticles are added, the mixture is incubated for 10 hours at 28 ℃ and 150rpm in a shaking way, and the mixture is cultured and activated in an activation culture medium (10g/L tryptone, 10g/L NaCl and 5g/L yeast extract, pH7.5) to obtain the magnetic cell.

Example 3

The preparation of the magnetic cell with the oil stain degradation function comprises the following steps:

(1) collecting oil-containing sludge of an oilfield in Tianjin Hongkong, adding sterile normal saline to immerse the sludge and fully mixing, standing for settling the sludge, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium (2.5g/L NaNO) according to the inoculation amount of 5 percent₃、0.5g/L K₂HPO₄、1g/L KH₂PO₄、0.01g/L CaCl₂、0.1g/L KCl、0.5g/L MgSO₄·7H₂O、0.01g/L FeSO₄·7H₂O, 0.1g/L yeast powder, 0.5mL/L trace element solution and 1% crude oil, wherein the trace element solution comprises 0.26g/L H₃BO₃、0.5g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.06g/L MoNa₂O₄·2H₂O and 0.7g/L ZnSO₄·7H₂O), shaking and shaking the table for 10 days at 28 ℃ and 150r/min, selecting an experimental group with obviously reduced surface oil stains and obviously turbid lower layer solution, absorbing the lower layer solution, inoculating the lower layer solution into a new oil-containing liquid culture medium with the same volume, and continuously culturing according to the conditions; repeating for 6 times, selecting an experimental group with the least residual oil, dipping the lower layer solution, and carrying out flat plate scribing to obtain oil stain degrading bacteria;

(2) mixing Fe₃O₄Dispersing the nano particles in n-hexane to prepare 3mol/L Fe₃O₄Carrying out ultrasonic oscillation on an n-hexane solution for 10min, adding 3mol/L oleic acid, carrying out ultrasonic oscillation for 20min, stirring in a water bath at 50 ℃ for 2h, carrying out magnetic separation, cleaning with ethanol, and carrying out vacuum drying to obtain hydrophobic carboxylated magnetic nanoparticles with the particle size of 10 nm;

(3) culturing the screened oil stain degrading bacteria in an LB culture medium for 24 hours, centrifugally collecting bacteria, resuspending the bacteria with normal saline, and repeating for 3 times to remove culture medium components;

the oil stain degrading bacteria are suspended in 100mL of physiological saline to ensure that the concentration of the oil stain degrading bacteria is 7%, 8g of hydroxylated magnetic nanoparticles are added, the mixture is incubated for 15 hours at 25 ℃ and 150rpm in a shaking way, and the mixture is cultured and activated in an activation culture medium (8g/L tryptone, 8g/L NaCl and 3g/L yeast extract, pH7) to obtain the magnetic cells.

Example 4

The preparation of the magnetic cell with the oil stain degradation function comprises the following steps:

(1) collecting oil-containing sludge of an oilfield in Tianjin Hongkong, adding sterile normal saline to immerse the sludge and fully mixing, standing for settling the sludge, and taking supernatant as bacterial suspension to be screened;

inoculating the bacterial suspension to be screened into an oil-containing liquid culture medium (2.5g/L NaNO) according to the inoculation amount of 1 percent₃、0.5g/L K₂HPO₄、1g/L KH₂PO₄、0.01g/L CaCl₂、0.1g/L KCl、0.5g/L MgSO₄·7H₂O、0.01g/L FeSO₄·7H₂O, 0.1g/L yeast powder, 0.5mL/L trace element solution and 1% crude oil, wherein the trace element solution comprises 0.26g/L H₃BO₃、0.5g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.06g/L MoNa₂O₄·2H₂O and 0.7g/L ZnSO₄·7H₂O), shaking and shaking the table for 15 days at 32 ℃ and 200r/min, selecting an experimental group with obviously reduced surface oil stains and obviously turbid lower layer solution, absorbing the lower layer solution, inoculating the lower layer solution into a new oil-containing liquid culture medium with the same volume, and continuously culturing according to the conditions; repeating for 8 times, selecting an experimental group with the least residual oil, dipping the lower layer solution for flat plate scribing, and obtaining oil stain degrading bacteria;

(2) mixing 1g of Fe₃O₄Dispersing the nanoparticles in 100mL of ethanol, ultrasonically oscillating for 10min, adding 800mL of ethanol and 10mL of water, continuously ultrasonically oscillating for 10min, adding 10mL of 3-Aminopropyltriethoxysilane (APTES), continuously stirring for 6h in a water bath at 70 ℃, carrying out magnetic separation, cleaning with ethanol, and vacuum drying to obtain aminated magnetic nanoparticles with the particle size of 50 nm;

(3) culturing the screened oil stain degrading bacteria in an LB culture medium for 36h, centrifugally collecting bacteria, resuspending the bacteria with normal saline, and repeating for 3 times to remove culture medium components;

and (2) resuspending the oil stain degrading bacteria in 100mL of physiological saline to ensure that the concentration of the oil stain degrading bacteria is 5%, adding 4g of hydroxylated magnetic nanoparticles, performing shake co-incubation for 15h at 25 ℃ and 100rpm, and performing culture activation in an activation medium (12g/L tryptone, 12g/L NaCl and 7g/L yeast extract, pH8) to obtain the magnetic cell.

Oily sewage treatment

The magnetic cells prepared in examples 1 to 4 were placed in oil-containing wastewater, exposed at 30 ℃ for 5 days, and after the treatment, the magnetic cells were recovered by a magnetic separation apparatus and stored under refrigeration.

The results of treating the same samples with the magnetic cells prepared in examples 1 to 4 are shown in table 1, and the black oil stain (main component is petroleum alkane) in the sewage sample is significantly reduced.

TABLE 1

Numbering	Oil content before treatment	Degradation rate of oil stain
			Example 1	27.6％	68.3％
Example 2	27.6％	74.8％
			Example 3	27.6％	72.4％
Example 4	27.6％	65.5％

In conclusion, the magnetic cells adsorb the magnetic nanoparticles on the oil stain degrading bacteria, so that the magnetic cells have the advantages of good dispersibility, good mass transfer, easy magnetic recovery and strong oil stain degrading capability, can be integrated on a large-scale magnetic carrier, and have remarkable advantages in large-scale popularization and application.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.