NL2029992B1 - Nanocomposite for microbial remediation and preparation method and use thereof - Google Patents

Abstract

The present disclosure provides a nanocomposite for microbial remediation and a preparation method and use thereof. The preparation method includes the following steps: (1) preparation of a Paracoccus aminovorans bacterial cell; (2) preparation of an 5 embedding agent graphene oxide—based nanocomposite; (3) preparation of a crosslinking agent CaCl2 solution; and (4) preparation of a nanocomposite for microbial remediation. In the present disclosure, a prepared immobilized microbial remediation agent of graphene oxide—based nanocomposites has strong 10 applicability to the environment, ensures a stable performance of a degradation function of polycyclic aromatic hydrocarbons (PAHs) degradation microorganisms, improves a degradation efficiency of traditional immobilized microbial preparations, and shortens a cycle of microbial remediation on PAHs—contaminated soil; the 15 preparation method has simple operations, low costs and desirable effects, has a prospect of large—scale industrial production, and is suitable for in—situ remediation of the PAHs—contaminated soil without secondary pollution.

Description

P856/NLpd

NANOCOMPOSITE FOR MICROBIAL REMEDIATION AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of mi- crobial remediation of contaminated soil, and specifically relates to a nanocomposite for microbial remediation and a preparation method and use thereof.

BACKGROUND ART Polycyclic Aromatic Hydrocarbons (PAHs) are organic contami- nants widely existing in the soil environment, and mainly derived from incomplete combustion of fossil fuels and biomass. In recent years, soil PAH contamination in China has become increasingly se- rious. In 2014, the Ministry of Environmental Protection and the Ministry of Land and Resources jointly issued a "National Soil Contamination Survey Communiqu", showing that PAHs-contaminated soil has an over-standard rate of 1.4%. The contamination has a large area and multi-component compounding, which seriously endan- gers the quality safety of agricultural products and the safety of human settlements. Therefore, it has become a major scientific and technological requirement to find a way to effectively control the PAHs and remedy the soil for ecological environment management and sustainable development in China.

Microbial remediation has simple operations, low costs and no secondary contamination, and has gradually become a highly- regarded green and sustainable remediation technology. At present, some microorganisms that can degrade or transform certain the PAHs have been screened and separated through techniques such as micro- bial enrichment culture. For example, Chinese patent

200810022333.9 reported a Paracoccus aminovorans HPD-2 that can degrade PAHs with a high molecular weight (such as benzo[alpyrene, pyrene or fluoranthene); Chinese patent 201410005967.9 reported a Bacillus cereus that can grow with fluoranthene as a sole carbon and energy source. However, in actual remediation projects, due to the susceptibility to indigenous microbial competition and envi- ronmental conditions, exogenous microorganisms are generally dif- ficult to function stably in in-situ soil environment to achieve ideal remediation effects.

Microbial immobilization technology can provide desirable living conditions for organisms, reduce damages of the external environment to microorganisms, and show a great potential in the field of sewage treatment and soil remediation. However, current immobilized microbial materials have poor mechanical properties and low mass transfer efficiency, and are easy to deform and break in a complex soil environment. Therefore, it is urgent to develop an immobilized microbial material with high mechanical strength and excellent mass transfer performance to ensure a stable perfor- mance of microbial degradation in the scil, thereby providing ef- ficient, economical and green microbial remediation materials for the field of contaminated soil remediation.

SUMMARY Technical problem solved: the present disclosure provides a nanocomposite for microbial remediation and a preparation method and use thereof. The present disclosure can overcome low remedia- tion efficiency by adding free microorganisms to actual contami- nated soil and low mechanical strength of original immobilized mi- crobial inoculants. The present disclosure promotes the growth and reproduction of degrading microorganisms, and improves the mechan- ical strength and mass transfer efficiency of the immobilized mi- crobial inoculants, to realize an efficient, green and economical sustainable remediation of PAHs-contaminated soil. The present disclosure has desirable potentials for development and use in the field of microbial remediation of PAHs-contaminated farmland and soil.

Technical solutions to be solved are: a nanocomposite for mi- crobial remediation is provided, where a microorganism is immobi- lized in a nano material; the nano material is a graphene oxide- based nanocomposite; and the microorganism is a Paracoccus amino- vorans HPD-2 deposited in the China General Microbiological Cul- ture Collection Center (CGMCC), with a deposit number of CGMCC No.

2568.

A preparation method of the nanocomposite for microbial reme- diation includes the following steps: (1) preparation of a Para- coccus aminovorans bacterial cell: subjecting Paracoccus amino- vorans HPD-2 to expansion culture in an LB medium to obtain a Par- acoccus aminovorans HPD-2 bacterial cell, denoted as a bacterial cell A; (2) preparation of an embedding agent graphene oxide-based nanocomposite: dissolving sodium alginate in 65-75°C deionized wa- ter, stirring to obtain a sodium alginate solution with a mass percentage of 1-3%, adding the LB medium to the sodium alginate solution, mixing uniformly, sterilizing at 0.12 MPa and 121°C for 20 min, and adding graphene oxide to obtain a mixed solution, de- noted as a solution B; (3) preparation of a crosslinking agent CaCl, solution: dissolving anhydrous calcium chloride in deionized water to obtain a CaCl, solution with a mass percentage of 1-3%, denoted as a solution C; and (4) preparation of a nano material- immobilized microbial remediation agent: adding the bacterial cell A to the solution B in a volume ratio of 1:9, stirring and mixing evenly, adding an obtained mixture dropwise to 500 mL of the solu- tion C, stirring slowly, crosslinking, filtering, and washing with a 1.0 % normal saline to obtain the nanocomposite for microbial remediation.

Preferably, in step(l), the Paracoccus aminovorans in the bacterial cell A may have a bacterial quantity of 2.8x10% cells/mL.

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

Preferably, in step (4), the crosslinking may be conducted at 4°C for 6 h.

Use of the nanocomposite for microbial remediation in remedi- ation of PAHs-contaminated soil is provided.

Use of the nanocomposite for microbial remediation in prepa- ration of a product for remediation of PAHs-contaminated soil is provided.

Beneficial effects are: in the present disclosure, the pre- pared graphene oxide-based nanocomposite for microbial remediation can enhance the mechanical properties and adsorption properties of the embedding agent calcium alginate by high specific surface ar- ea, high surface activity and high mechanical strength of the gra- phene oxide, to significantly improve the mechanical strength and mass transfer rate of the traditional calcium alginate-embedded immobilized microbial remediation agent. At a certain nutrient level (the system contains a 5-10% LB medium), the graphene oxide can significantly promote the growth and reproduction of the PAHs degradation microorganism Paracoccus aminovorans, enhance the for- mation of biofilm for the Paracoccus aminovorans, and improve the adaptability of the Paracoccus aminovorans to the environment; a synergy of the above three benefits ensure a stable performance of the degradation function of the PAHs degradation microorganism, thereby improving the degradation efficiency of traditional immo- bilized microbial preparations and shortening the cycle of micro- bial remediation for PAHs-contaminated soil. The preparation meth- od of the remediation agent has simple operations, low costs and desirable effects, has a prospect of large-scale industrial pro- duction, and is suitable for in-situ remediation of the PAHs- contaminated soil without secondary pollution.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an immobilized microbial remediation agent (where the left picture is a conventional immobilized microbial remedia- tion agent, and the right picture is a graphene oxide -based nano- composite microbial remediation agent; and the remediation agent has a diameter of about 3 mm); and FIG. 2 shows removal effects of different immobilization sys- tems on PAHs in contaminated soil (where CK represents a control treatment without remediation agent; HPD-2 represents a conven- tional immobilized HPD-2 remediation agent; HPD-2+LB represents an immobilized HPD-2 remediation agent with an LB medium added to conventional immobilized materials; HPD-2+GO represents an immobi- lized HPD-2 remediation agent of graphene oxide-based nanocompo- sites without the LB medium; and HPD-2+GO+LB represents an immobi- lized HPD-2 remediation agent of graphene oxide-based nanocompo- sites described in this standard (with the LB medium) ).

DETAILED DESCRIPTION OF THE EMBODIMENTS To better understand the present disclosure, the technical solution of the present disclosure is described in further detail below with reference to specific examples, but the present disclo- 5 sure is not limited thereto. Example 1: Removal effects of PAHs in soil using immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites A LB medium was prepared, including the ingredients and dos- age as follows: 5.0 g/L of a yeast extract, 10.0 g/L of peptone,

10.0 g/L of NaCl, and deionized water as a balance; the LB medium was adjusted to a pH value of 7.0, and sterilized at 0.12 MPa and 121°C for 20 min for later use. A Paracoccus aminovorans strain stored at -80°C was taken out, 100 pL of a bacterial solution was drawn and inoculated in 10 mL of a liquid LB medium for rejuvena- tion culture at 30°C and 150 r/min for 8 h, and inoculated into the liquid LB medium at a volume ratio of 10% to continue cultur- ing for 16 h. After centrifugation at 6,000 r/min for 5 min, cells in a logarithmic growth phase of the above-mentioned bacteria cell were obtained; after washing twice with phosphate-buffered saline (PBS), the cells were resuspended, a bacterial suspension was ad- Justed to an ODgy of 1.0, and 100 mL of the bacterial suspension was concentrated by centrifugation to obtain a Paracoccus amino- vorans bacterial cell for PAHs degradation; sodium alginate was dissolved in deionized water at 70°C and stirred quickly to obtain a sodium alginate solution with a mass percentage of 3%, an LB me- dium was added to the sodium alginate solution at a volume ratio of 10%, mixed evenly, sterilized at 0.12 MPa and 121°C for 20 min, graphene oxide was added after the above solution was cooled, to obtain a mixed solution containing graphene oxide with a mass per- centage of 0.01%, as an embedding agent solution; anhydrous calci- um chloride was dissolved in deionized water to obtain a CaCl, so- lution with a mass percentage of 3% as a crosslinking agent solu- tion; the bacteria cell was added to the embedding agent solution in a volume ratio of 1:9, stirred and mixed evenly, an obtained mixture was added dropwise to the crosslinking agent solution, crosslinked at 4°C for 6 h, filtered, and washed with a 1.0% nor- mal saline, to obtain an immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites.

Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites was added to the soil at a dosing ratio of 3 wt.%. It can be seen from FIG. 2 that after 35 days of remediation, a removal rate of PAHs in the soil by the immobilized HPD-2 remediation agent of graphene oxide-based nano- composites is 70.31%, which is 12.57% higher than that of the con- ventional immobilized HPD-2 remediation agent. The immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites significantly promotes the removal of PAHs in the soil, and also significantly improves the removal effect of PAHs in the soil. Es- pecially for high molecular weight of PAHs in the soil, the remov- al effect is more obviously promoted (Table 1). The reason is that the graphene oxide can promote the growth and reproduction of HPD- 2 in the presence of LB medium nutrition and accelerate the for- mation of HPD-2 biofilm, to improve the adaptability of microor- ganisms to the environment as well as improve the mass transfer performance and mechanical strength of the conventional immobi- lized microbial remediation agents, thereby ultimately promoting the degradation of PAHs by microorganisms.

Table 1 Removal effect (%) of different immobilization sys- tems on PAHs in contaminated soil Immobilizg Immobilized | Immobilized HPD-2 remg HPD-2 remedi HPD-2 remedig Convention diation tion agent d tion agent of immobilizg agent wit graphene ox‘ graphene oxid HPD-2 remd LB medium Treatment ide-based based nanocon diation added to nanocomposit posites de- agent (HPI conventio without LB scribed in th 2) immobilize medium (HPD- standard (HPQ materials 2+G0O) 2+GO+LB) (HPD-2+LB)

Benzol[alanthracene 69.11 Example 2: Removal effects of PAHs in soil using immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites Compared with Example 1, in this example, an LB medium was added to a sodium alginate solution at a volume ratio of 5%, and other steps and parameters were the same as in Example 1. An immo- bilized HPD-2 remediation agent of graphene oxide-based nanocompo- sites was obtained. Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites was added to the soil at a dosing ratio of 3 wt.%. It can be seen from FIG. 2 that after 35 days of remediation, a removal rate of PAHs in the soil by the immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites is 68.24%, which is not significantly different from the immobilized remediation agent with 10% LB medi- um and is 10.5% higher than that of the conventional immobilized HPD-2 remediation agent. The immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites significantly promotes the removal of PAHs in the soil, and also significantly improves the removal effect of PAHs in the soil. Example 3: Removal effects of PAHs in soil using immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites Compared with Examples 1 and 2, in this example, an LB medium was added to a sodium alginate solution at a volume ratio of 8%, and other steps and parameters were the same as in Examples 1 and

2. An immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites was obtained. Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared immobilized HPD-2 remediation agent of graphene oxide-based nano- composites was added to the soil at a dosing ratio of 3 wt.%. It can be seen from FIG. 2 that after 35 days of remediation, a re- moval rate of PAHs in the soil by the immobilized HPD-2 remedia-

tion agent of graphene oxide-based nanocomposites is 71.35%, which is not significantly different from the immobilized remediation agent with 10% LB medium and is 13.61% higher than that of the conventional immobilized HPD-2 remediation agent.

The immobilized HPD-2 remediation agent of graphene oxide-based nanocomposites significantly promotes the removal of PAHs in the soil, and also significantly improves the removal effect of PAHs in the soil.

Comparative Example 1: Removal effects of PAHs in soil using conventional immobilized HPD-2 remediation agent A Paracoccus aminovorans strain stored at -80°C was taken out, 100 pL of a bacterial solution was drawn and inoculated in 10 mL of a liquid LB medium for rejuvenation culture at 30°C and 150 r/min for 8 h, and inoculated into the liquid LB medium at a vol- ume ratio of 10% to continue culturing for 16 h.

After centrifuga- tion at 6,000 r/min for 5 min, cells in a logarithmic growth phase of the above-mentioned bacteria cell were obtained; after washing twice with PBS, the cells were resuspended, a bacterial suspension was adjusted to an ODg of 1.0, and 100 mL of the bacterial sus- pension was concentrated by centrifugation to obtain a Paracoccus aminovorans bacterial cell for PAHs degradation; sodium alginate was dissolved in deionized water at 60°C and stirred quickly to obtain a sodium alginate solution with a mass percentage of 2.5%, and the sodium alginate solution was sterilized at 0.12 MPa and 121°C for 20 min as an embedding agent solution; anhydrous calcium chloride was dissolved in deionized water to obtain a CaCl; solu- tion with a mass percentage of 3% as a crosslinking agent solu- tion; the bacteria cell was added to the embedding agent solution, stirred and mixed evenly, an obtained mixture was added dropwise to the crosslinking agent solution, crosslinked at 4°C for 6 h, filtered, and washed with a 1.0% normal saline, to obtain a con- ventional immobilized HPD-2 remediation agent.

Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared conventional immobilized HPD- 2 remediation agent was added to the soil at a dosing ratio of 3 wt.%. It can be seen from FIG. 2 that after 35 days of remedia- tion, a removal rate of PAHs in the soil by the conventional immo- bilized HPD-2 remediation agent is 57.74%.

Comparative Example 2 Removal effects of PAHs in soil using immobilized HPD-2 remediation agent with LB medium added to con- ventional immobilized materials A Paracoccus aminovorans strain stored at -80°C was taken out, 100 uL of a bacterial solution was drawn and inoculated in 10 mL of a liquid LB medium for rejuvenation culture at 30°C and 150 r/min for 8 h, and inoculated into the liquid LB medium at a vol- ume ratio of 10% to continue culturing for 16 h. After centrifuga- tion at 6,000 r/min for 5 min, cells in a logarithmic growth phase of the above-mentioned bacteria cell were obtained; after washing twice with PBS, the cells were resuspended, a bacterial suspension was adjusted to an ODgy of 1.0, and 100 mL of the bacterial sus- pension was concentrated by centrifugation to obtain a Paracoccus aminovorans bacterial cell for PAHs degradation; sodium alginate was dissolved in deionized water at 60°C and stirred quickly to obtain a sodium alginate solution with a mass percentage of 2.5%, an LB medium was added to the sodium alginate solution at a volume ratio of 10%, mixed evenly, sterilized at 0.12 MPa and 121°C for 20 min to obtain a mixed solution as an embedding agent solution; anhydrous calcium chloride was dissolved in deionized water to ob- tain a CaCl, solution with a mass percentage of 3% as a crosslink- ing agent solution; the bacteria cell was added to the embedding agent solution, stirred and mixed evenly, an obtained mixture was added dropwise to the crosslinking agent solution, crosslinked at 4°C for 6 h, filtered, and washed with a 1.0% normal saline, to obtain an immobilized HPD-2 remediation agent with an LB medium added to immobilized materials (calcium alginate embedding agent).

Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared immobilized HPD-2 remediation agent with an LB medium added to immobilized materials was added to the soil at a dosing ratio of 3 wt.%. It can be seen from FIG.

2 that after 35 days of remediation, a removal rate of PAHs in the soil by the immobilized HPD-2 remediation agent with an LB medium added to immobilized materials is 50.92%, which is even lower than that of the conventional immobilized HPD-2 remediation agent.

The reason may be that after adding the LB medium, the HPD-2 mainly grows with the LB medium as a carbon source, which reduces the degradation of PAHs.

Comparative Example 3: Removal effects of PAHs in soil using immobilized HPD-2 remediation agent of graphene oxide-based nano- composites without LB medium A Paracoccus aminovorans strain stored at -80°C was taken out, 100 pL of a bacterial solution was drawn and inoculated in 10 mL of a liquid LB medium for rejuvenation culture at 30°C and 150 r/min for 8 h, and inoculated into the liquid LB medium at a vol- ume ratio of 10% to continue culturing for 16 h.

After centrifuga- tion at 6,000 r/min for 5 min, cells in a logarithmic growth phase of the above-mentioned bacteria cell were obtained; after washing twice with PBS, the cells were resuspended, a bacterial suspension was adjusted to an ODgy of 1.0, and 100 mL of the bacterial sus- pension was concentrated by centrifugation to obtain a Paracoccus aminovorans bacterial cell for PAHs degradation; sodium alginate was dissolved in deionized water at 60°C and stirred quickly to obtain a sodium alginate solution with a mass percentage of 2.5%, and the sodium alginate solution was sterilized at 0.12 MPa and 121°C for 20 min, graphene oxide was added after the above solu- tion was cooled, to obtain a mixed solution containing the gra- phene oxide with a mass percentage of 0.01%, as an embedding agent solution; anhydrous calcium chloride was dissolved in deionized water to obtain a CaCl: solution with a mass percentage of 3% as a crosslinking agent solution; the bacteria cell was added to the embedding agent solution, stirred and mixed evenly, an obtained mixture was added dropwise to the crosslinking agent solution, crosslinked at 4°C for 6 h, filtered, and washed with a 1.0% nor- mal saline, to obtain an immobilized HPD-2 remediation agent with graphene oxide added to immobilized materials.

Soil collected from a contaminated site of a coking plant in Nanjing was used as test soil, where a content of PAHs in the soil was 344.48 mg/kg; the above-prepared immobilized HPD-2 remediation agent with graphene oxide added to immobilized materials was added to the soil at a desing ratio of 3 wt.%. It can be seen from FIG. 2 that after 35 days of remediation, a removal rate of PAHs in the soil by the immobilized HPD-2 remediation agent with graphene ox- ide added to immobilized materials is 58.20%, which is 1.19% high- er than that of the conventional immobilized HPD-2 remediation agent, with no obvious promotion effect.

The above examples are preferred embodiments of the present disclosure.

However, the embodiments of the present disclosure are not limited by the above examples.

Any change, modification, sub- stitution, combination and simplification made without departing from the spiritual essence and principle of the present disclosure should be an equivalent replacement manner, and all are included in a protection scope of the present disclosure.

Claims (7)

CONCLUSIONS 1. Nanocomposite for microbial remediation, where a microorganism is immobilized in a nanomaterial; wherein the nanomaterial is a graphene oxide based nanocomposite; and the microorganism is a Paracoccus aminovorans HPD-2 deposited in the China General Microbiological Culture Collection Center (CGMCC), with a deposit number of CGMCC No. 2568. A method for preparing the nanocomposite for microbial remediation according to claim 1, comprising the following steps: (1) preparation of a Paracoccus aminovorans bacterial cell: subjecting Paracoccus aminovorans HPD-2 to expansion culture in an LB medium to obtain a Paracoccus aminovorans HPD-2 bacterial cell, designated as a bacterial cell A; (2) preparation of a graphene oxide based nanocomposite embedding agent: dissolving sodium alginate in 65-75°C deionized water, stirring to obtain a sodium alginate solution with a mass percentage of 1-3%, adding the LB- medium to the sodium alginate solution, mix evenly, sterilize at 0.12 MPa and 121°C for 20 min and add graphene oxide to obtain a solution designated as solution B; (3) preparation of a cross-linking agent CaCl: solution: dissolving anhydrous calcium chloride in deionized water to obtain a CaCl: solution having a mass percentage of 1-3 +, referred to as a solution C; and (4) preparation of a nanomaterial-immobilized microbial remediation agent: adding the bacterial cell A to the solution B in a volume ratio of 1:9, stirring and mixing uniformly, adding a resulting mixture dropwise to 500 ml of the solution C , stir slowly, crosslink, filter and wash with a 1.0% normal saline solution to obtain the nanocomposite for microbial remediation. The method for preparing the nanocomposite for microbial remediation according to claim 2, wherein in step (1), the Paracoccus aminovorans in the bacterial cell A has a bacterial amount of 2.8 x 10% cells/ml. The method for preparing the nanc composite for microbial remediation according to claim 2, wherein in step (2) the LB medium in the solution B has a volume percentage of 5-10% (v/v) and the graphene oxide has a mass percentage of 0.01%. The method for preparing the nanocomposite for microbial remediation according to claim 2, wherein in step (4), the cross-linking is performed at 4°C for 6 hours. Use of the nanocomposite for microbial remediation according to claim 1 in the remediation of soil contaminated with polycyclic aromatic hydrocarbons (PAHs). Use of the nanocomposite for microbial remediation according to claim 1 for preparing a product for the remediation of soil contaminated with PAHs.