阅读说明：本技术 一种利用真菌胞外聚合物制备赤铁矿纳米颗粒的方法 (Method for preparing hematite nanoparticles by using fungus extracellular polymers ) 是由 张彤 张展华 毕天雅 于 2021-10-26 设计创作，主要内容包括：本发明公开了一种利用真菌胞外聚合物制备赤铁矿纳米颗粒的方法,包括如下步骤：获取真菌的胞外聚合物溶液,将铁盐加入到胞外聚合物溶液中反应后即得。本发明利用真菌的胞外聚合物中含有的丰富的生物大分子以及其起到的分散剂的作用,进而有效保证制备得到具有一定的分散性的赤铁矿纳米颗粒；本发明方法的整体制备过程具有操作时间短、操作成本低、无二次污染等优点。(The invention discloses a method for preparing hematite nanoparticles by using fungus extracellular polymeric substances, which comprises the following steps: and (3) obtaining an extracellular polymeric substance solution of the fungus, and adding iron salt into the extracellular polymeric substance solution for reaction to obtain the product. According to the invention, abundant biomacromolecules contained in extracellular polymeric substances of fungi and the function of a dispersing agent are utilized, so that hematite nanoparticles with certain dispersibility are effectively ensured to be prepared; the whole preparation process of the method has the advantages of short operation time, low operation cost, no secondary pollution and the like.)

1. A method for preparing hematite nanoparticles by using fungal extracellular polymers is characterized by comprising the following steps:

obtaining the extracellular polymer solution of the fungus,

and adding the iron salt into the extracellular polymer solution for reaction to obtain the product.

2. The method of claim 1, wherein the final concentration of the iron salt is 1mmol/L after the iron salt is added to the extracellular polymeric substance solution.

3. The method of claim 2, wherein the iron salt is ferric nitrate.

4. A method according to any one of claims 1 to 3, wherein the fungus is a soil fungus.

5. The method according to any one of claims 1 to 4, wherein the extracellular polymer solution is obtained by:

firstly, culturing fungi to logarithmic phase by adopting a culture medium, collecting the fungi, suspending the fungi in a sodium chloride solution for secondary culture, wherein the secondary culture time is 72 hours; and after the re-culture is finished, separating and removing the fungal cells to obtain the extracellular polymer solution.

6. The method according to claim 5, wherein the concentration of the sodium chloride solution is 0.1%.

7. The method according to claim 5, wherein the culture conditions of the fungus are 28 ℃ and 180 rpm.

8. The method according to any one of claims 5 to 7, wherein the process of isolating and removing the fungal cells is: centrifuging and then filtering, wherein the filtered filtrate is the extracellular polymeric substance solution.

9. The method of claim 8, wherein the centrifugation conditions are 5000rpm, 20min at 10 ℃;

the specification of the filter membrane for filtration is 0.22 mu m aperture, and the material is polyether sulfone or polytetrafluoroethylene.

10. The process according to any one of claims 1 to 9, wherein the reaction conditions are 28 ℃, 180 rpm; the reaction time was 48 h.

Technical Field

The invention relates to the field of nanoparticle preparation, in particular to a method for preparing hematite nanoparticles by using fungus extracellular polymers.

Background

Iron element generally exists in earth crust, and oxides and sulfides of the iron element are widely distributed in natural environment. Because the iron-based nano material has low cost, high specific surface area and high reaction activity, the iron-based nano material is widely synthesized and used for removing heavy metal pollution in soil and underground water. Hematite (alpha-Fe)₂O₃) The nano particles are a common iron-based nano material, are iron minerals with the most excellent stability in all iron oxides, have strong light resistance and corrosion resistance, low biotoxicity and strong affinity to various heavy metal pollutants, have large specific surface area and strong adsorption performance, and are ideal iron-based nano materials capable of being applied to the heavy metal pollution remediation of soil and underground water.

Currently, the methods for preparing hematite nanoparticles commonly used include chemical methods such as precipitation, hydrothermal method, hydrolysis, and the like. These methods generally involve high temperatures, high pressures, toxic chemicals, etc., and require complex equipment, high energy consumption, and high cost. Specifically, for example: the precipitation method requires high-temperature calcination to obtain hematite nanoparticles; the hydrothermal method requires a closed condition at high temperature and high pressure; hydrolysis requires a boiling reflux operation. Meanwhile, hematite prepared by a chemical method usually needs to be cleaned by organic solvents such as ethanol and acetone, and then a stabilizer (such as sodium dodecyl sulfate, citric acid and the like) is added to maintain the dispersibility of particles, so that the operation is complex, and toxic and harmful chemicals are used.

The hematite nanoparticle can also be prepared by adopting a method of synthesizing microorganisms (such as fungi, bacteria, actinomycetes and the like), and is paid more and more attention due to the characteristics of low energy consumption, mild reaction conditions, no secondary pollution and the like. For example, Bharde et al successfully synthesized magnetite nanoparticles extracellularly using the soil fungi Fusarium oxysporum and Verticillium sp (Bharde et al, Small,2006,2(1): 135-141). However, the iron oxide nanoparticles prepared by the microbial synthesis method using fungi, bacteria, actinomycetes and the like are mostly synthesized in microbial cells, only a small part of the iron oxide nanoparticles can be synthesized extracellularly, and the problem of separation of the nanoparticles from the microbial cells is also solved, so that complicated separation operation steps are additionally required.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect of complicated separation operation in the method for synthesizing hematite nanoparticles by using microorganisms in the prior art, and provides a method for preparing hematite nanoparticles by using extracellular polymers of fungi, which solves the problem.

A method for preparing hematite nanoparticles using fungal extracellular polymers, comprising the steps of:

obtaining the extracellular polymer solution of the fungus,

and adding the iron salt into the extracellular polymer solution for reaction to obtain the product.

After the iron salt is added into the extracellular polymeric substance solution, the final concentration of the iron salt is 1 mmol/L.

The iron salt is ferric nitrate.

The fungi are soil fungi.

The extracellular polymer solution is obtained by the following steps:

firstly, culturing fungi to logarithmic phase by adopting a culture medium, collecting the fungi, suspending the fungi in a sodium chloride solution for secondary culture, wherein the secondary culture time is 72 hours; and after the re-culture is finished, separating and removing the fungal cells to obtain the extracellular polymer solution.

The concentration of the sodium chloride solution is 0.1%.

The culture condition of the fungus is 28 ℃, and the rotating speed is 180 rpm.

The process for separating and removing the fungal cells comprises the following steps: centrifuging and then filtering, wherein the filtered filtrate is the extracellular polymeric substance solution.

The centrifugation condition is 5000rpm, the centrifugation is 20min, and the temperature is 10 ℃;

the specification of the filter membrane for filtration is 0.22 mu m aperture, and the material is polyether sulfone or polytetrafluoroethylene.

The reaction conditions are 28 ℃, and the rotating speed is 180 rpm; the reaction time was 48 h.

The technical scheme of the invention has the following advantages:

1. the method for preparing the hematite nano-particles provided by the invention can be prepared by reacting extracellular polymers of fungi with iron salts, the raw materials are easy to obtain, and the equipment and the operation are simple. In particular, fungi continue to produce and release during their cultivation a viscous colloidal substance consisting of biomacromolecules and their degradation products, namely Extracellular Polymeric Substances (EPS). The extracellular polymeric substance of the fungus is a compound which takes saccharides as a main chain structure and is attached with proteins, namely, the extracellular polymeric substance of the fungus mainly comprises biomacromolecules such as polysaccharide, protein, lipid and the like, wherein the saccharides can account for more than half of the total amount of the extracellular polymeric substance. The extracellular polymeric substance of the fungus contains rich functional groups (such as hydroxyl, carboxyl, amino and the like) and has stronger affinity to metal elements; the macromolecular substances can play a role of a dispersing agent to ensure that the prepared hematite nanoparticles have certain dispersibility; therefore, the hematite nanoparticle can be successfully prepared by reacting the extracellular polymer with iron salt.

2. The fungi in the method of the present invention are preferably soil fungi, and particularly, in soil microorganisms, the fungi can be used as one of ideal bioreactors for synthesizing iron-based nanoparticles due to the characteristics of large biomass, good tolerance and the like. In addition, the extracellular polymeric substances extracted from common soil fungi are not only easy to obtain raw materials, but also high in yield of the extracellular polymeric substances of the fungi, and the yield can be further improved.

3. In the method, the extracellular polymeric substance solution is prepared only by centrifuging and filtering the culture solution and only by centrifuging after the extracellular polymeric substance solution reacts with the ferric salt, so that the whole preparation process does not need to add other equipment besides conventional culture, centrifugation and filtering equipment, does not need to adopt other chemical reagents except the culture medium, sodium chloride and ferric nitrate, and has low preparation cost.

4. The whole preparation process of the method has the advantages of short operation time, low operation cost, no secondary pollution and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a scanning electron micrograph of Fusarium oxysporum hyphae of the present invention, at a scale of 10 μm;

FIG. 2 is a graph showing the growth of Fusarium oxysporum hyphae in the present invention;

FIG. 3 is a TEM image of hematite nanoparticles prepared by the present invention, at a scale bar of 100 nm;

FIG. 4 is a TEM image of hematite nanoparticles prepared according to the present invention, at a scale bar of 20 nm;

FIG. 5 is a statistical distribution of the particle size of hematite nanoparticles prepared according to the present invention;

FIG. 6 is an energy dispersive X-ray spectrum of hematite nanoparticles prepared in accordance with the present invention;

FIG. 7 is a plot of the selected region electron diffraction of hematite nanoparticles prepared in accordance with the present invention;

FIG. 8 is a high resolution TEM image of hematite nanoparticles prepared according to the present invention;

FIG. 9 is a second high resolution transmission electron micrograph of hematite nanoparticles prepared in accordance with the present invention.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1

The fungus adopted in the embodiment is soil fungus, and specifically the existing Fusarium oxysporum (CGMCC 3.6787) is adopted. The formula of the culture solution is a common culture medium for separating and culturing yeast and mould, namely a potato glucose broth PDB culture medium (4.0 g/L of potato extract, 20.0g/L of glucose and 5.6 +/-0.2 of pH).

The specific process is as follows:

inoculating a spore suspension of F.oxysporum into a 250mL triangular flask in a sterile super clean bench by using a disposable sterile syringe according to an inoculation ratio of 1:20 by volume, wherein the flask contains 100mL of sterilized PDB culture solution, and the concentration of the spore suspension is 2.13X 10⁶One per ml. . And (5) plugging the sterilized breathable silica gel plug, and then putting the sterilized breathable silica gel plug into a constant-temperature shaking table for cultivation. The shaker was set at 28 ℃ and a rotation speed of 180 rpm.

Since the f.oxysporum bacterial suspension is a uniform red bacterial suspension, the growth curve can be measured by measuring the optical density OD value. Samples are taken every 12h after inoculation and the absorbance value (OD) at a wavelength of 600nm is determined using an ultraviolet-visible spectrophotometer or microplate reader (e.g. Spark 10M, Tecan, Switzerland)₆₀₀) And drawing a growth curve to monitor the growth condition of the fungi. Wherein, the scanning electron microscope image of the F.oxysporum hypha is shown in figure 1, and the growth curve is shown in figure 2.

After the fungus grows to a stationary phase (60h), subpackaging the bacterial liquid into centrifuge tubes, centrifuging for 20min at 5000rpm, wherein the temperature during centrifugation is 10 ℃, removing the upper culture solution after centrifugation, cleaning with 0.1% NaCl solution, and suspending to 100mL of original volume.

Continuously culturing in a constant temperature shaking table for about 72h, subpackaging the bacterial liquid into centrifuge tubes, centrifuging at 10 ℃ and 5000rpm for 20min, centrifuging to obtain a supernatant, removing the lower layer of thallus, removing residual fungi from the supernatant through a polyether sulfone (PES) filter membrane with the aperture of 0.22 mu m to obtain a filtrate, namely an extracellular polymer solution, and temporarily storing the filtrate in a refrigerator at 4 ℃ for later use.

Mixing ferric nitrate (ferric nitrate nonahydrate Fe (NO))₃ ^.9H₂O, purchased from Aladdin, cat # F100208-100g) was added to the extracellular polymer solution such that the final concentration of Fe (III) was 1 mmol/L. Placing the mixture into a constant temperature shaking table to react for 48 hours, and obtaining the turbidity visible to the naked eye, namely the hematite nano particle aggregate.

The hematite nanoparticles in the extracellular polymeric substance solution can be collected by a centrifugation mode, specifically, the hematite nanoparticles can be collected by centrifugation for 20min at 5000rpm, and the precipitate is the hematite nanoparticles obtained by collection.

In this example, the prepared suspension containing hematite nanoparticles was dropped onto a copper grid covered with an ultra-thin carbon support film, and after the liquid was volatilized, a Transmission Electron Microscope (TEM) (JEM-2010FEF, japan JEOL) was used to perform a test, so as to obtain the morphology of hematite nanoparticles, and the test results are shown in fig. 3 and 4.

As can be seen from fig. 3 and 4, the hematite nanoparticles obtained are spherical particles with relatively uniform dispersion. The geometric diameters of the hematite particles in the TEM images were counted using Nano Measurer software and statistically analyzed, resulting in an average particle size of 5.45nm as shown in fig. 5. The elemental composition of the nanoparticles was characterized by energy dispersive X-ray spectroscopy (EDX) equipped with a transmission electron microscope, and the results are shown in fig. 6, showing that the nanoparticles contain Fe and O elements.

To confirm that the iron-containing nanoparticles formed were hematite nanoparticles, diffraction spots and diffraction rings of the particle crystals were obtained by selective electron diffraction (SAED), and as a result, as shown in fig. 7, it was found by measurement that hematite (α -Fe) was predominant₂O₃) The (024), (110), (116), (113), and (214) planes of (a) indicate that hematite nanoparticles were produced.

In this example, the lattice fringes of the individual nanoparticles were observed by a high-resolution transmission electron microscope (HR-TEM), and the results were determined as hematite (α -Fe) by measuring the intervals of the lattice fringes as shown in fig. 8 and 9₂O₃) The (104) and (110) crystal planes of the hematite nanoparticle are further verified to be obtained by the preparation of the embodiment.

Therefore, the detection results show that the hematite nanoparticle with the size of 5-6 nm can be effectively synthesized by using the fusarium oxysporum (f.oxysporum) extracellular polymer.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.