阅读说明：本技术 纳米材料固定化微生物修复剂及其制备方法和应用 (Nano material immobilized microorganism repairing agent and preparation method and application thereof ) 是由 任文杰 滕应 毛婷玉 骆永明 于 2020-06-05 设计创作，主要内容包括：纳米材料固定化微生物修复剂及其制备方法和应用,包括如下步骤：(1)噬氨副球菌菌体的制备；(2)包埋剂氧化石墨烯基纳米复合材料的制备；(3)交联剂CaCl<Sub>2</Sub>溶液的制备；(4)纳米材料固定化微生物修复剂制备。本发明制备的氧化石墨烯基纳米材料固定化微生物修复剂对环境的适用性强,保证了多环芳烃降解微生物降解功能的稳定发挥,提高了传统固定化微生物制剂的降解效率,缩短了多环芳烃污染土壤微生物修复的周期；制备方法操作简单、成本低、效果好,具有大规模工业化生产的前景,适合多环芳烃污染土壤的原位修复且无二次污染。(The preparation method and the application of the nano material immobilized microorganism repairing agent comprise the following steps: (1) preparing paracoccus aminovorans thallus; (2) preparing an embedding agent graphene oxide-based nano composite material; (3) crosslinking agent CaCl 2 Preparing a solution; (4) preparing the nano material immobilized microorganism repairing agent. The graphene oxide-based nanomaterial immobilized microorganism repairing agent prepared by the invention has strong applicability to the environment, and guarantees the degradation of polycyclic aromatic hydrocarbonThe biodegradation function is stably exerted, the degradation efficiency of the traditional immobilized microbial preparation is improved, and the microbial remediation period of the soil polluted by the polycyclic aromatic hydrocarbon is shortened; the preparation method has the advantages of simple operation, low cost and good effect, has the prospect of large-scale industrial production, is suitable for in-situ remediation of polycyclic aromatic hydrocarbon polluted soil, and has no secondary pollution.)

1. The nano material immobilized microorganism repairing agent is characterized in that microorganisms are immobilized in a nano material, and the nano material is a graphene oxide-based nano composite material; the microorganism is Paracoccus aminovorans (A)Paracoccus aminovorans) HPD-2 is preserved by China general microbiological culture Collection center with the preservation number of CGMCCNo.2568.

2. The method for preparing the nano-material immobilized microbial remediation agent of claim 1, characterized by comprising the steps of: (1) preparation of Paracoccus aminovorans cells: paracoccus aminovorans (A)Paracoccus aminovorans) HPD-2 is enlarged cultured in LB culture medium to obtain Paracoccus aminovoransParacoccus aminovorans) HPD-2 thallus, marked as thallus A; (2) preparing an embedding agent graphene oxide-based nano composite material: dissolving sodium alginate in deionized water at 60-80 ℃, stirring to obtain a solution with the sodium alginate mass percentage content of 2.5%, adding an LB culture medium into the solution, uniformly mixing, sterilizing for 20 min under the conditions of 0.12 MPa and 121 ℃, and then adding graphene oxide, and marking as a solution B; (3) crosslinking agent CaCl₂Solutions ofThe preparation of (1): dissolving anhydrous calcium chloride in deionized water to obtain CaCl with the mass percentage of 3%₂Solution, denoted as solution C; (4) preparing a nano material immobilized microorganism repairing agent: adding the thallus A into the solution B according to the volume ratio of 1:9, stirring and mixing uniformly, then dripping into 500 mL of solution C, slowly stirring, performing crosslinking treatment, filtering, and washing with 1.0% of normal saline to obtain the nano-material immobilized microorganism repairing agent.

3. The method for preparing a nanomaterial-immobilized microbe remediation agent according to claim 2, wherein the number of Paracoccus aminovorans in the thallus A in step (1) is 2.8 x 10 ⁸one/mL.

4. The method for preparing the nano-material immobilized microbial remediation agent of claim 2, wherein the volume percentage of the LB medium in the solution B in the step (2) is 5% to 10% (v/v), and the mass percentage of the graphene oxide is 0.01%.

5. The method for preparing a nano-material immobilized microbial remediation agent according to claim 2, wherein the crosslinking treatment in step (4) is crosslinking at 4 ℃ for 6 hours.

6. The application of the nanomaterial-immobilized microorganism remediation agent of claim 1 to remediation of polycyclic aromatic hydrocarbon-contaminated soil.

7. The application of the nanomaterial-immobilized microorganism repairing agent of claim 1 in preparation of a product for repairing polycyclic aromatic hydrocarbon-polluted soil.

Technical Field

The invention belongs to the technical field of microbial remediation of contaminated soil, and particularly relates to a nano-material immobilized microbial remediation agent as well as a preparation method and application thereof.

Background

Polycyclic Aromatic Hydrocarbons (PAHs) are a class of organic pollutants widely present in soil environments, mainly derived from incomplete combustion of fossil fuels and biomass. In recent years, PAHs pollution in soil of China is increasingly serious, and the national soil pollution condition survey bulletin issued by the environmental protection department and the national soil resource department in 2014 shows that the point standard exceeding rate of PAHs polluted soil is 1.4%, the pollution is large in area, multiple components are compounded and the like, and the quality safety of agricultural products and the safety of human living environment are seriously endangered. Therefore, how to effectively control and repair PAHs in soil has become a great scientific and technological demand for ecological environment management and sustainable development in China.

The microbial remediation has the advantages of simple operation, low cost, no secondary pollution and the like, and gradually becomes a highly advocated green sustainable remediation technology. At present, some microorganisms capable of degrading or transforming certain PAHs have been screened and separated by technologies such as microorganism enrichment culture, for example, Chinese patent 200810022333.9 reports a Paracoccus aminovorans HPD-2 capable of degrading high molecular weight PAHs (benzo [ a ] pyrene, pyrene or fluoranthene); chinese patent 201410005967.9 reports a bacillus cereus that can grow with fluoranthene as the sole carbon and energy source. However, in the actual repair engineering, since it is easily restricted by the competition of indigenous microorganisms and environmental conditions, exogenous microorganisms are often difficult to stably play a role in the in-situ soil environment, so that an ideal repair effect cannot be obtained.

The microorganism immobilization technology can provide good living conditions for organisms, reduce the damage of the external environment to microorganisms, and show great application potential in the fields of sewage treatment and soil remediation. However, the existing immobilized microbial material has the defects of poor mechanical property, low mass transfer efficiency and the like, and is easy to deform and crack in a complex soil environment. Therefore, the research and development of immobilized microbial materials with high mechanical strength and excellent mass transfer performance are urgently needed, and the stable exertion of the microbial degradation function in the soil is ensured, so that the efficient, economic and green microbial remediation material is provided for the field of contaminated soil remediation.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a nano-material immobilized microorganism repairing agent and a preparation method and application thereof, which can overcome the problems of low repairing efficiency of free microorganisms added into actual polluted soil, low mechanical strength of the original immobilized microorganism agent and the like, are beneficial to promoting the growth and propagation of degraded microorganisms, improving the mechanical strength and mass transfer efficiency of the immobilized microorganism agent, realizing efficient, green and economic sustainable repair of PAHs polluted soil, and have good development and application potential in the field of PAHs polluted farmland and field soil bioremediation.

The technical scheme is as follows: a nano-material immobilized microorganism repairing agent, wherein microorganisms are immobilized in a nano-material, and the nano-material is a graphene oxide-based nano-composite material; the microorganism is Paracoccus aminovorans (Paracoccus aminovorans) HPD-2, and is preserved by the common microorganism center of China Committee for culture Collection of microorganisms with the strain preservation number of CGMCCNo.2568.

The preparation method of the nano material immobilized microorganism repairing agent comprises the following steps: (1) preparation of Paracoccus aminovorans cells: carrying out amplification culture on HPD-2 of Paracoccus aminovorans (Paracoccus aminovorans) in an LB culture medium to obtain a Paracoccus aminovorans (Paracoccus aminovorans) HPD-2 thallus which is marked as a thallus A; (2) preparing an embedding agent graphene oxide-based nano composite material: dissolving sodium alginate in deionized water at 60-80 ℃, stirring to obtain a solution with the sodium alginate mass percentage content of 2.5%, adding an LB culture medium into the solution, uniformly mixing, sterilizing for 20min under the conditions of 0.12MPa and 121 ℃, and then adding graphene oxide, and marking as a solution B; (3) crosslinking agent CaCl₂Preparation of the solution: dissolving anhydrous calcium chloride in deionized water to obtain CaCl with the mass percentage of 3%₂Solution, denoted as solution C; (4) preparing a nano material immobilized microorganism repairing agent: adding the thallus A into the mixture according to the volume ratio of 1:9 Adding the solution B into the solution B, stirring and mixing uniformly, then dropwise adding the solution B into 500mL of the solution C, slowly stirring, performing crosslinking treatment, filtering, and washing with 1.0% of normal saline to obtain the nano material immobilized microorganism repairing agent.

Preferably, the number of Paracoccus aminovorans in the cell A in the step (1) is 2.8X 10⁸one/mL.

Preferably, the volume percentage of the LB medium in the solution B in the step (2) is 5% to 10% (v/v), and the mass percentage of the graphene oxide is 0.01%.

Preferably, the crosslinking treatment in the step (4) is crosslinking at 4 ℃ for 6 h.

The application of the nano-material immobilized microorganism repairing agent in repairing polycyclic aromatic hydrocarbon polluted soil.

The application of the nano-material immobilized microorganism repairing agent in preparation of a product for repairing polycyclic aromatic hydrocarbon contaminated soil.

Has the advantages that: the graphene oxide-based nanomaterial immobilized microorganism repairing agent prepared by the invention can utilize the excellent characteristics of high specific surface area, high surface activity, high mechanical strength and the like of graphene oxide, enhance the mechanical characteristics and the adsorption characteristics of the calcium alginate embedding agent, and obviously improve the mechanical strength and the mass transfer rate of the traditional calcium alginate embedded immobilized microorganism repairing agent; under a certain nutritional level (a system contains 5-10% of LB culture medium), the graphene oxide can remarkably promote the growth and the propagation of PAHs degrading microorganisms Paracoccus aminovorans, enhance the formation of a biological membrane of the microorganisms, improve the adaptability of the microorganisms to the environment, and ensure the stable exertion of the degrading function of the PAHs degrading microorganisms due to the synergistic effect of the three components, thereby improving the degrading efficiency of the traditional immobilized microbial preparation and shortening the period of repairing the microorganisms in the PAHs polluted soil; the preparation method of the repairing agent is simple to operate, low in cost, good in effect, has a prospect of large-scale industrial production, is suitable for in-situ repairing of PAHs polluted soil, and does not have secondary pollution.

Drawings

FIG. 1 shows an immobilized microorganism repairing agent (the left is a conventional immobilized microorganism repairing agent, the right is a graphene-based nanocomposite immobilized microorganism repairing agent; the diameter of the repairing agent is about 3 mm);

FIG. 2 shows the effect of different immobilization systems on removal of PAHs from contaminated soil (CK represents control treatment without addition of remediation agent; HPD-2 represents conventional immobilized HPD-2 remediation agent; HPD-2+ LB represents immobilized HPD-2 remediation agent with LB medium added to conventional immobilized material; HPD-2+ GO represents graphene oxide-based nanocomposite immobilized HPD-2 remediation agent without LB medium added; and HPD-2+ GO + LB represents graphene oxide-based nanocomposite immobilized HPD-2 remediation agent (with LB medium added) of the present standard).

Detailed Description

The invention will be further described with reference to the following examples for better understanding, but the scope of the invention as claimed is not limited to the examples.