A biochar-based degradation bacterial agent and its preparation method and application

Abstract

The present invention relates to a biochar-based degradation bacterial agent and a preparation method and application thereof, and belongs to the field of biotechnology. The present invention modifies rice husk biochar with nitric acid, and then adsorbs and immobilizes it with Rhodococcus sp.DG1 to obtain a biochar-based degradation bacterial agent. The biochar-based degradation bacterial agent prepared by the present invention can achieve efficient degradation of polycyclic aromatic hydrocarbons under the combined action of adsorption-degradation, and promotes the contact between degradation bacteria and pollutants while adsorbing and enriching polycyclic aromatic hydrocarbons in contaminated soil, so that the loaded strains can play a role in efficient degradation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a biochar-based degradation bacterial agent and a preparation method and application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds composed of two or more benzene rings arranged in a linear, angular or clustered manner. They are a typical persistent organic pollutant with high biological toxicity, accumulation, carcinogenicity, teratogenicity and mutagenicity. PAHs have a huge destructive effect on the structure and function of soil ecosystems. They are highly toxic to humans and animals and can enter the human or animal body through breathing, skin contact, diet, etc., and accumulate in the animal body through the food chain, affecting the normal function of the body and even causing cancer. Soil is an important place for PAHs to exist. PAHs have a high affinity for soil organic matter and can be absorbed by the soil, thus posing a serious threat to natural ecosystems and human health. The pollution of PAHs in my country's soil has become quite serious. Therefore, the effective remediation of PAH-contaminated soil is of great significance to improving soil environmental quality and thus protecting human and animal health.

[0003] At present, the remediation technologies for PAH-contaminated soil are roughly divided into physical and chemical remediation methods and biological remediation methods. Although physical and chemical technologies such as heating, leaching, fracturing, and multiphase extraction can effectively remove PAHs, they are costly, energy-intensive, and easily cause secondary pollution. Compared with physical and chemical methods, microbial-based bioremediation methods have attracted much attention due to their low cost, high efficiency, and environmental protection. However, microbial remediation itself has limitations such as soil environmental inhibition and competition from indigenous bacteria. Immobilized microbial technology is an effective technical means to solve the above problems. It is considered to be an effective and feasible method for bioremediation of contaminated soil because it can help microorganisms resist harsh environments and competition from indigenous microorganisms, and improve the survival rate and metabolic activity of added degradation bacteria.

[0004] Immobilized microbial technology is to use immobilized carriers as key carriers of efficient degradation bacteria and apply them to contaminated areas to achieve the effect of microbial remediation. Current immobilization methods include adsorption, embedding, and cross-linking. The adsorption method mainly uses the principles of electrostatic action, van der Waals force, hydrogen bond, ionic bond, and covalent bond to generate interaction forces between microorganisms and carriers to adsorb and combine the two. Commonly used adsorption carriers include sawdust, silica gel, diatomaceous earth, activated carbon, biochar, porous glass, cellulose, ion exchange resin, etc. The adsorption method is simple to prepare immobilized bacterial agents and has little effect on the activity of degradation bacteria, but the number and intensity of fixed microorganisms on different material carriers vary greatly, and the fixation effect is diverse.

[0005] In recent years, biochar has attracted the attention of many scholars at home and abroad due to its good soil improvement ability and high pollutant adsorption performance and has been applied to soil pollution remediation research. The porous structure, large specific surface area and rich surface functional groups of biochar are conducive to the attachment and growth of microorganisms, and can provide good protection and habitat for bacteria. Biochar and microorganisms have biostimulation and bioenhancement effects respectively. The combination of the two and the use of immobilization technology to prepare immobilized microbial agents can make the two play a synergistic role of biostimulation and enhancement, and can achieve efficient remediation of contaminated soil. However, the current technology of using biochar as an immobilized carrier has the disadvantages of weak microbial activity and poor immobilization effect during the remediation process. In addition, most of the methods for preparing modified biochar carriers have complicated steps, high energy consumption, high cost, and the risk of secondary environmental pollution. Furthermore, the method of making the agent into a ball in order to suit the characteristics of biochar greatly limits the contact effect between the degradation bacteria and the pollutants. Therefore, it is necessary to provide a biochar-based degradation agent that is conducive to the attachment and growth of microorganisms and can improve the degradation performance of the agent. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a biochar-based degradation bacterial agent and a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing a biochar-based degradation bacterial agent, comprising the following steps:

[0009] (1) mixing rice husk biochar with nitric acid solution for modification to obtain modified biochar material;

[0010] (2) mixing the modified biochar material with a bacterial suspension of Rhodococcus sp. DG1, vortexing and culturing, then centrifuging, collecting and drying the precipitate to obtain a biochar-based degradation bacterial agent;

[0011] The concentration of the nitric acid solution is 2-4 mol / L; the time of the shaking culture is 16-32 h.

[0012] The present invention obtains a modified biochar by acidifying rice husk biochar, which provides a space for colonization and proliferation of microorganisms with the ability to degrade polycyclic aromatic hydrocarbons; by selecting microorganisms with the ability to degrade polycyclic aromatic hydrocarbons to be fixed and adsorbed in the modified biochar, the degrading strains can successfully aggregate and form a uniform and dense bacterial layer on the surface of the modified biochar, and the microbial immobilization rate reaches 53%.

[0013] As a preferred embodiment of the method of the present invention, the Rhodococcus sp.DG1 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on November 21, 2019, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCCNO.18981. The strain is disclosed in Chinese patent CN114107116A.

[0014] Rhodococcus sp.DG1 is a microorganism with special metabolic ability. It can degrade polycyclic aromatic hydrocarbons (PAHs), which are compounds composed of two or more benzene rings connected in the form of fused rings. There are a large number of gene clusters involved in the degradation of PAHs in the genome of Rhodococcus DG1, including ring hydroxylation dioxygenases and other reductases that degrade PAHs. The dioxygenases expressed by Rhodococcus can attack the molecular structure of PAHs and open the aromatic rings by inserting oxygen atoms, making them easier to further biodegrade. Rhodococcus itself has good pH adaptability, and the optimal growth temperature is usually 25℃-35℃, which is suitable as a microbial material for biochar-based degradation agents.

[0015] As a preferred embodiment of the method of the present invention, the concentration of the nitric acid solution is 2 mol / L. The present application uses nitric acid to modify the rice husk biochar, which can effectively dissolve some insoluble impurities such as quartz and calcite in the biochar, thereby changing the surface structure of the rice husk biochar. Nitric acid has strong oxidizing properties and can also react with organic matter in rice husk biochar, thereby achieving efficient modification of the biochar. By adjusting the concentration of nitric acid, different modification effects can be achieved. Among them, the modified biochar obtained by modification with a nitric acid solution with a concentration of 2 mol / L has more large and dense internal pore structures, and the pore size is complete and not easy to collapse.

[0016] As a preferred embodiment of the method of the present invention, in step (1), the mass volume ratio of the rice husk biochar and the nitric acid solution is rice husk biochar: nitric acid solution = 1 g: 20 mL.

[0017] As a preferred embodiment of the method of the present invention, in step (1), the temperature of the modification treatment is 70°C-75°C; and the time of the modification treatment is 7-9h.

[0018] As a preferred embodiment of the method of the present invention, in step (2), the shaking culture time is 20-24 hours; preferably, the shaking culture time is 24 hours.

[0019] In step (2), the modified biochar material and the Rhodococcus are subjected to an oscillation culture experiment, and the modified biochar will adsorb the Rhodococcus, and the Rhodococcus will be fixed in the modified biochar as the oscillation culture time increases.

[0020] Under the preferred oscillating culture time, Rhodococcus DG1 can be effectively adsorbed to the surface of modified biochar, and at the same time will not overgrow on the surface of modified biochar to form a too thick biofilm, thereby affecting the immobilization effect; when the fixation time is too long, it will cause the microorganisms to accumulate in the biochar to form a biofilm, and the outer bacteria of the immobilized bacteria will fall off, reducing the number of adsorbed microorganisms. Preferably, the oscillating culture time is 20-24h, at which time the immobilization rate of microorganisms in the modified biochar continues to increase, indicating that the number of adsorbed microorganisms has increased significantly at this stage. More preferably, the oscillating culture time is 24h. When the oscillating culture is 24h, the immobilization rate of biochar on microorganisms is the highest, which is 53%.

[0021] As a preferred embodiment of the method of the present invention, in step (2), the ratio of the mass of the modified biochar material to the volume of the bacterial suspension is modified biochar material: bacterial suspension = 1g: 10mL. Under the preferred ratio, the strains in the bacterial suspension are effectively colonized in the modified biochar material, and at the same time, as the microorganisms proliferate, the metabolites and nutrient substrates in the degradation system can easily and freely enter and exit, without constituting competitive inhibition, and the space for excretion and release of products will not be restricted.

[0022] As a preferred embodiment of the method of the present invention, in step (2), the temperature of the shaking culture is 28°C-32°C; the rotation speed of the shaking table is 100-120 r / min.

[0023] As a preferred embodiment of the method of the present invention, in step (2), the OD of the bacterial suspension is 600 The present invention selects the plants in the logarithmic growth phase and OD 600 =1.0 was used for subsequent immobilization. Since the strain was in the logarithmic growth phase, the structure of its cell wall and cell membrane was relatively complete, which was conducive to the combination of cells and immobilization carriers during the immobilization process. The strain in the logarithmic growth phase had a strong adaptability to environmental changes and could maintain good stability under different environmental conditions after immobilization, which could further improve the immobilization rate on the modified biochar.

[0024] As a preferred embodiment of the present invention, in step (2), the preparation of the bacterial suspension of Rhodococcus sp. DG1 comprises the following steps: inoculating Rhodococcus sp. DG1 into a microbial liquid culture medium, at 35°C and 150 r·min -1The strains were then centrifuged and enriched at 6000 r / min. -1 The cells were washed three times with phosphate buffer and redissolved in phosphate buffer to adjust the OD of the bacterial suspension. 600 The concentration was 1.0 and stored at 4°C for later use.

[0025] As a preferred embodiment of the method of the present invention, in step (1), the preparation of rice husk biochar specifically comprises the following steps: subjecting rice husk to anaerobic pyrolysis, drying, crushing, and sieving after cooling, and collecting the sieved material to obtain rice husk biochar.

[0026] Furthermore, the temperature of the anaerobic pyrolysis treatment is 450° C.-550° C., the time of the anaerobic pyrolysis treatment is 3-5 hours; the drying time is 18-26 hours; and the mesh size of the sieving is 60-100 meshes.

[0027] In a second aspect, the present invention provides a biochar-based degradation bacterial agent, which is prepared by the preparation method of the biochar-based degradation bacterial agent.

[0028] As the biochar-based degradation bacterial agent of the present invention, the strain on the biochar-based degradation bacterial agent is Rhodococcus sp.DG1; the number of Rhodococcus sp.DG1 on the biochar-based degradation bacterial agent is 0.5×10 8 CFU / g-1.0×10 8 CFU / g; preferably, the number of Rhodococcus sp.DG1 on the biochar-based degradation agent is 0.8×10 8 CFU / g. Under the optimal strain quantity, the biochar-based degradation agent can load a large number of degradation bacteria, and the enzymes secreted by the fixed degradation bacteria can penetrate into the biochar pores and react with the adsorbed polycyclic aromatic hydrocarbons to further enhance the degradation of pollutants.

[0029] In a third aspect, the present invention provides an application of a biochar-based degradation bacterial agent in soil remediation, wherein the soil is soil contaminated by polycyclic aromatic hydrocarbons.

[0030] The present invention is based on the adsorption-degradation synergy between biochar and degradation microorganisms in biochar-based immobilized degradation bacteria, and repairs soil contaminated by polycyclic aromatic hydrocarbons. On the one hand, the modified biochar in the biochar-based degradation bacteria can provide a safe habitat for Rhodococcus sp.DG1, shielding the harmful effects of adverse soil conditions and competition from indigenous microorganisms, and can also be used as a bulking agent to increase soil porosity, thereby improving the mass transfer efficiency between water, oxygen and nutrients in the soil. At the same time, the modified biochar has a developed internal pore structure and a large number of adsorption sites, and has good adsorption performance for organic pollutants in the system, thereby enriching polycyclic aromatic hydrocarbons in the soil, reducing the distance between the degradation bacteria cell and the polycyclic aromatic hydrocarbons, overcoming the mass transfer limitation, and increasing the contact between the degradation bacteria and the pollutants. At the same time, the enzymes secreted by the fixed degradation bacteria can penetrate into the biochar pores, and the adsorbed polycyclic aromatic hydrocarbons can further enhance the degradation of pollutants.

[0031] As a preferred embodiment of the application of the present invention, the biochar-based degradation bacterial agent is used in the restoration of phenanthrene-contaminated soil.

[0032] As a preferred embodiment of the application of the present invention, the addition ratio of the biochar-based degradation bacteria agent is 2%-5% of the mass of the soil to be treated; after adding the biochar-based degradation bacteria agent, the moisture content of the soil is adjusted to 50%-60% of the maximum water holding capacity of the soil; the treatment time of adding the biochar-based degradation bacteria agent is 30 days, during which it is stirred once every week; the treatment temperature of the biochar-based degradation bacteria agent is 26°C-30°C.

[0033] Preferably, the addition ratio of the biochar-based degradation agent is 4.90% of the mass of the soil to be treated; after adding the biochar-based degradation agent, the water content of the soil is adjusted to 53.50% of the maximum water holding capacity of the soil; the temperature of the biochar-based degradation agent treatment is 26°C-27°C. Under the above preferred treatment conditions, the biochar-based degradation agent has the best removal rate for polycyclic aromatic hydrocarbons, especially for phenanthrene, which can reach 80.93%.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The method for preparing the biochar-based degradation agent provided by the present invention has low preparation cost and high efficiency, and has significant economic and social benefits. Selecting agricultural waste as raw material to prepare biochar not only provides an effective way for the resource utilization of agricultural waste, but also plays a role in increasing carbon sinks and reducing emissions. The biochar after use does not need to be recycled, and there is no secondary pollution. It can be used as a soil conditioner to effectively improve the physical and chemical properties of the soil.

[0036] 2. The biochar-based degradation agent prepared by the present invention achieves efficient degradation of PAHs under the combined action of adsorption and degradation. The modified biochar can adsorb and enrich PAHs in contaminated soil, effectively promoting the contact between degradation bacteria and pollutants, allowing the loaded Rhodococcus sp.DG1 strain to play a role and efficiently degrade PAHs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a scanning electron microscope image of the degradation bacterial agent prepared in Example 1;

[0038] Figure 2 is a scanning electron microscope image of the surface morphology of the modified biochar material;

[0039] Figure 3 This is the effect of adsorption time on microbial immobilization rate;

[0040] Figure 4 This is the effect of adsorption time on the removal rate of phenanthrene degradation by microorganisms;

[0041] Figure 5 This is the response surface analysis result of the application conditions of biochar-based degradation bacterial agent. DETAILED DESCRIPTION

[0042] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The polycyclic aromatic hydrocarbons-degrading strain of the present invention is Rhodococcus sp.DG1, which is a polycyclic aromatic hydrocarbons-degrading strain domesticated and screened from petroleum-contaminated soil. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 21, 2019, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.18981. The strain is disclosed in Chinese patent CN114107116A.

[0043] In the following examples, the preparation method of the bacterial suspension of Rhodococcus sp.DG1 is as follows:

[0044] (1) The degradation strain Rhodococcus sp.DG1 was inoculated into a microbial liquid culture medium and heated at 35°C and 150 r·min -1 After culturing for 48 h until the logarithmic growth phase, the bacterial solution was heated at 6000 r / min. -1 Centrifuge for 10 min to enrich the efficient degradation bacteria, wash the bacteria three times with phosphate buffer and redissolve them in phosphate buffer, and adjust the bacterial suspension to OD 600 The concentration was 1.0 and stored at 4°C for later use.

[0045] The microbial liquid culture medium is prepared by uniformly mixing 10 g of peptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water, adjusting the pH to 7-7.2 with NaOH or hydrochloric acid, and sterilizing at 121° C. for 20 min.

[0046] The phosphate buffer is prepared by uniformly mixing 21.75 g of K2HPO4H2O, 33.4 g of Na2HPO4·12H2O, 8.7 g of KH2PO4, 5.0 g of NH4Cl and 1000 mL of distilled water, adjusting the pH to about 7 with NaOH or hydrochloric acid, and sterilizing at 121° C. for 20 min.

[0047] Modified biochar material 1 (BC-H2), the specific preparation method is as follows:

[0048] (1) The raw material biomass rice husk is cleaned and dried at 60°C, then anaerobic pyrolysis is performed at 500°C for 4 h, cooled, placed in an oven at 60°C for 24 h, crushed, passed through a 60-mesh sieve, and rice husk biochar is obtained, which is then sealed and dried for storage;

[0049] (2) The biochar material prepared in step (1) was mixed with a nitric acid solution at a ratio of 1:20 (w / v), and the concentration of the nitric acid solution was 2 mol·L -1 ;

[0050] (3) The mixed solution of step (2) was placed in a magnetic stirrer and heated to 75°C and stirred for 8 h. After cooling, it was filtered and washed with deionized water until the filtrate was neutral. It was then dried in an oven at 60°C for 12 h. After cooling, it was placed in a sample bottle and dried to store the modified biochar material, which was named BC-H2.

[0051] Modified biochar material 2 (BC-H4)

[0052] The difference between modified biochar material 2 (BC-H4) and modified biochar material BC-H2 is that the concentration of modified biochar material 2 (BC-H4) is 4 mol·L -1 of nitric acid solution for modification.

[0053] Modified biochar material 3 (BC-H1)

[0054] The difference between modified biochar material 3 (BC-H1) and modified biochar material BC-H2 is that the concentration of modified biochar material 3 (BC-H1) is 1 mol·L -1 of nitric acid solution for modification.

[0055] Modified biochar material 4 (BC-H8)

[0056] The difference between modified biochar material 4 (BC-H8) and modified biochar material BC-H2 is that the concentration of modified biochar material 4 (BC-H8) is 8 mol·L -1of nitric acid solution for modification.

[0057] Modified biochar material 5 (BC-H16)

[0058] The difference between modified biochar material 5 (BC-H16) and modified biochar material BC-H2 is that the concentration of modified biochar material 5 (BC-H16) is 16 mol·L -1 of nitric acid solution for modification.

[0059] Modified biochar material results and surface morphology analysis

[0060] The modified biochar materials 1-5 and the unmodified rice husk biochar were sieved through a 100-mesh sieve and dried in an oven at 60°C for 12 h. About 10 mg of the sample was glued to the sample stage, and then the structure and surface morphology characteristics were observed using a scanning electron microscope (SEM) at an operating acceleration voltage of 20.0 kV and room temperature. The modified biochar material results and the surface morphology of the SEM results are shown in Figure 2. Figure 2 Reference Figure 2 The fine debris that appears on the surface of the unmodified original biochar BC is the ash produced by the pyrolysis of biomass to prepare biochar, which blocks the surface pores.

[0061] After being modified with different concentrations of nitric acid, its surface structure changed to varying degrees: the scanning electron microscopy results of modified biochar material 3 (BC-H1) showed that after being modified with 1 mol / L nitric acid, the ash content on the surface of the biochar was slightly reduced, but the pore structure of the prepared modified biochar material was not complete; the scanning electron microscopy results of modified biochar material 1 (BC-H2) showed that after being modified with 2 mol / L nitric acid, the pore size of the biochar was clear and complete, with more large and dense internal pore structures. The pore structure of modified biochar material 1 (BC-H2) is uniform and complete, which can provide bacteria with more sufficient growth space and can effectively accommodate and fix more microbial cells. At the same time, the more developed the pore structure of the biochar material, the better its mass transfer and permeability performance, the metabolites and nutrient substrates in the degradation system can easily and freely enter and exit, and the space for excretion and release of products will not be restricted. The scanning electron microscopy results of modified biochar material 2 (BC-H4) showed that after being modified with 4 mol / L nitric acid, the pore size of the biochar remained generally clear and complete. This indicates that when preparing modified biochar materials, materials with good structural properties can be prepared when the nitric acid concentration is in the range of 2M-4M.

[0062] When the concentration of nitric acid continued to increase, the surface structure of biochar collapsed more obviously. The scanning electron microscopy results of modified biochar material 4 (BC-H8) showed that after modification with 8 mol / L nitric acid, most of the pore structures of the modified biochar materials collapsed; after modification with 16 mol / L nitric acid, the pore structure of modified biochar material 5 (BC-H16) basically disappeared.

[0063] After the modified biochar material 1 (BC-H2) and the unmodified biochar (BC) were vacuum degassed at 100 °C for 12 h, the specific surface area and pore size distribution data of the biochar were measured by the low-temperature nitrogen adsorption method at 77 K using a fully automatic Brunaer-Emmett-Teller measuring instrument, as shown in the following table:

[0064] Table 1 Structural data of modified biochar and unmodified biochar

[0065]

[0066] From the data in Table 1, we can see that using 2 mol·L -1 The specific surface area of ​​the modified biochar material 1 (BC-H2) modified by nitric acid solution is increased by 158% compared with the unmodified rice husk biochar, and the pore volume is also much greater than the pore volume of the unmodified biochar, which is increased by 171%. Therefore, by using 2 mol / L nitric acid concentration for modification, a modified biochar material 1 (BC-H2) with a large specific surface area and pore volume can be prepared. The increase in specific surface area and pore volume helps to increase the metabolic efficiency of the microbial degradation system, thereby improving the degradation effect. Therefore, the modified biochar material 1 (BC-H2) is used in the subsequent embodiments.

[0067] Examples 1-4 and Comparative Examples 1-3 provide a biochar-based degradation bacterial agent, which is as follows:

[0068] Example 1

[0069] (1) The modified biochar material 1 (BC-H2) was mixed with a suspension of Rhodococcus sp. DG1 at a ratio of 1 g:10 mL (w / v) and heated at 30 °C for 120 r / min. -1 The adsorption was carried out by shaking culture, and the shaking culture time was 24h.

[0070] (2) The mixed solution obtained in step (1) was heated at 6000 r / min -1 Centrifuge for 10 min, discard the supernatant, and freeze-dry the lower precipitate to obtain the biochar-based degradation agent. The number of DG1 strain cells attached to the biochar was determined by the traditional plate count method to be about 0.8×10 8 CFU·g -1The scanning electron microscopy image of the prepared degradation agent is as follows Figure 1 shown.

[0071] Example 2

[0072] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 16 hours in step (2).

[0073] Example 3

[0074] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 20 hours in step (2).

[0075] Example 4

[0076] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 32 hours in step (2).

[0077] Comparative Example 1

[0078] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 36 hours in step (2).

[0079] Comparative Example 2

[0080] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 40 hours in step (2).

[0081] Comparative Example 3

[0082] The biochar-based degradation bacterial agent prepared by steps (1)-(2) of Example 1 is different in that the modified biochar material and the Rhodococcus sp. DG1 bacterial suspension are shaken and cultured for adsorption and fixation for 48 hours in step (2).

[0083] Optimization of preparation conditions for biochar-based degradation agents

[0084] After preparing the modified biochar materials of Examples 1-4 and Comparative Examples 1-3, the modified biochar materials were mixed with the bacterial suspension at a ratio of 1 g:10 mL (w / v), and the mixture was cultured for adsorption at 120 r / min at 30° C., and samples were taken at regular intervals. After centrifugation at 6000 r / min for 10 min, the photometric value (OD) of the supernatant at a wavelength of 600 nm was immediately measured. 600 The original OD0 (1.0) was subtracted from the measured OD of the supernatant. 600 The obtained value represents the amount of biochar adsorbed on the bacterial agent, recorded as OD1. The immobilization rate of strain DG1 on biochar was calculated as follows: immobilization rate (%) = OD1 / OD0 × 100. The immobilization rate and phenanthrene degradation removal rate of Rhodococcus DG1 on modified biochar with different fixation (adsorption) time are shown in the following figure. Figure 3 shown.

[0085] As the adsorption time increases, OD1 shows a trend of first increasing and then decreasing, indicating that the adsorption time has a significant effect on the biomass of degrading bacteria fixed on the biochar. Starting from a culture time of 8 hours, the OD1 value of biochar continues to increase, reaching a peak of 0.53 at 24 hours, indicating that the number of adsorbed microorganisms has increased significantly during this stage. After the adsorption time exceeds 24 hours, the OD1 value begins to decrease, and the number of adsorbed microorganisms decreases. When the fixation time is too long, microorganisms will accumulate in the biochar to form a biofilm, and the outer layer of bacteria of the immobilized agent will fall off, reducing the number of adsorbed microorganisms. When the oscillation culture time is 24 hours, the immobilization rate of biochar on microorganisms is the highest, at 53%.

[0086] The bacterial agents prepared in Examples 1-4 and Comparative Examples 1-3 at different fixed (i.e., shaking culture) times were added to 300 mL of 50 mg L -1 In phenanthrene solution, at 35℃, 150r min -1 After constant temperature shaking culture for 3 days, the samples were extracted and purified, and the content of polycyclic aromatic hydrocarbons in the system was determined by high performance liquid chromatography. The removal rate of phenanthrene by the bacterial agent at different fixed time was calculated. The results are as follows Figure 4As shown. Among them, when the fixing time exceeds 16h (Example 2), the degradation and removal rate of phenanthrene in the solution by the biochar-based degradation bacteria agent exceeds 80%; as the fixing time increases, when it reaches 20h in Example 3, the degradation and removal rate of phenanthrene in the solution by the biochar-based degradation bacteria agent exceeds 90%; when the fixing time is 24h, the degradation and removal rate of phenanthrene in the solution by the biochar-based degradation bacteria agent is the highest, which is 92.33%; as time continues to increase, after the fixing time reaches 32h in Example 4, the degradation rate of phenanthrene decreases slightly, but still remains at 80-90%. When the fixing time exceeds 32h, reaching 36-48h in Comparative Examples 1-3, the removal rate of phenanthrene is less than 80%. The above results show that an oscillation culture time of 24h is the preferred preparation condition for the biochar-based degradation bacteria agent.

[0087] The following Examples 5-6 provide an application of a biochar-based degradation bacterial agent in the remediation of polycyclic aromatic hydrocarbons in soil, as follows:

[0088] Example 5

[0089] The biochar-based degradation agent prepared in Example 1 was used for the remediation of polycyclic aromatic hydrocarbons. The response surface methodology was first used to design the agent dosage of 2%, 4% and 6%, the water holding capacity of 20%, 50% and 80%, the temperature of 20°C, 25°C and 30°C, and the contaminated soil was remediated for 30 days. The phenanthrene concentration in the contaminated soil was 55.39 mg kg -1 After adding the biochar-based degradation agent, the soil was stirred once every week, and polycyclic aromatic hydrocarbons were extracted and purified after sampling. The content of polycyclic aromatic hydrocarbons remaining in the soil was determined by high performance liquid chromatography, and the removal rate of phenanthrene under different conditions was calculated.

[0090] Example 6

[0091] Weigh 100g of contaminated soil, where the concentration of phenanthrene in the contaminated soil is 55.39mg kg -1 , add 5g of the biochar-based degradation bacterial agent prepared in Example 1, adjust the moisture content of the soil mixture after adding the bacterial agent to 50%-60% of the maximum water holding capacity of the soil, culture at a temperature of 26°C-30°C for 30 days, stir once every week, and add ultrapure water regularly.

[0092] Comparative Example 4

[0093] The contaminated soil was remediated under the conditions of Example 6, except that 5% of free Rhodococcus sp. DG1 bacterial suspension was added. The residual rate of phenanthrene in the contaminated soil was measured regularly, and the degradation removal rate was calculated.

[0094] Comparative Example 5

[0095] The original contaminated soil was treated without adding any microorganisms. The soil moisture content was adjusted to 50%-60% of the maximum water holding capacity. It was cultured at a temperature of 26℃-30℃ for 30 days, stirred once every week, and ultrapure water was added regularly.

[0096] Optimization of application conditions for biochar-based degradation agents to remediate soil polycyclic aromatic hydrocarbons

[0097] Example 5 The response surface methodology was used to design the repair of contaminated soil (phenanthrene concentration was 55.39 mg kg) with the dosage of bacterial agent being 2%, 4% and 6%, the water holding capacity being 20%, 50% and 80%, and the temperature being 20°C, 25°C and 30°C. -1 )30d, stirred once every week, extracted and purified PAHs after sampling, and determined the content of PAHs remaining in the soil by HPLC, and calculated the removal rate of phenanthrene under different conditions. The data are shown in Table 2 below:

[0098] Table 2 Data of the bacterial repair test group in Example 5

[0099]

[0100]

[0101] According to Table 2, the best remediation conditions in the test group are 4% dosage of bacterial agent, 50% water holding capacity, and 25°C temperature, at which the removal rate of phenanthrene is 80.93%. Response surface analysis was further performed based on the results of Table 2. The results of response surface analysis are shown in Figure 5 As shown, according to the results of the test group, the best remediation conditions are predicted to be a dosage of bacteria of 4.942%, a specific retention of 53.030%, and a temperature of 27.148°C, at which the removal rate of phenanthrene is 82.319%.

[0102] For further verification, Example 6 weighed 100g of contaminated soil, added 5g of degradation bacteria, adjusted the soil moisture content after addition to 50%-60% of the maximum water holding capacity, cultured at a temperature of 26℃-30℃ for 30d, stirred once every week, and regularly added ultrapure water to set up three parallel experiments; at the same time, set up the same conditions, the free degradation bacteria experimental group added in Comparative Example 4 and the blank control group of Comparative Example 5. The residual rate of phenanthrene in the contaminated soil was regularly measured, and the degradation removal rate was calculated. Among them, within 30 days, the degradation removal rate of phenanthrene in the soil by the biochar-based degradation bacteria in Example 6 was 80.15%, while the degradation removal rate of phenanthrene in the soil by adding the free Rhodococcus group in Comparative Example 4 was only 41.37%. Compared with Comparative Example 4, under preferred conditions, the degradation capacity of the biochar-based degradation bacteria was increased by 93.7%. It can be seen that the biochar bacteria prepared by the present invention has a high degradation and removal efficiency for polycyclic aromatic hydrocarbons in the soil. At the same time, the preparation process of the bacterial agent is simple and the degradation bacteria fixation effect is stable. It has the advantages of improving soil environmental quality and avoiding secondary pollution. It is an environmentally friendly soil remediation material.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a biochar-based degradation bacterial agent, characterized in that: The following steps are involved: (1) mixing rice husk biochar with nitric acid solution for modification to obtain modified biochar material; (2) mixing the modified biochar material with a bacterial suspension of Rhodococcus sp. DG1, vortexing and culturing, then centrifuging, collecting and drying the precipitate to obtain a biochar-based degradation bacterial agent; The concentration of the nitric acid solution is 2-4 mol / L; the shaking culture time is 20-24 h; the ratio of the mass of the modified biochar material to the volume of the bacterial suspension is modified biochar material: bacterial suspension = 1 g: 10 mL; the OD of the bacterial suspension is 600 is 1.

0.

2. The preparation method according to claim 1, characterized in that In step (1), the concentration of the nitric acid solution is 2 mol / L.

3. The preparation method according to claim 2, characterized in that: In step (1), the temperature of the modification treatment is 70°C-75°C; the time of the modification treatment is 7-9h.

4. The preparation method according to claim 1, characterized in that: In step (2), the shaking culture time is 24 hours.

5. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the shaking culture is 28°C-32°C; during the shaking culture, the rotation speed of the shaker is 100-120r / min.

6. The preparation method according to claim 1, characterized in that: In step (1), the preparation of rice husk biochar specifically includes the following steps: taking rice husk for anaerobic pyrolysis, drying, crushing, and sieving after cooling, and collecting the sieved material to obtain rice husk biochar.

7. A biochar-based degradation bacterial agent, characterized in that: The biochar-based degradation bacterial agent is prepared by the preparation method of any one of claims 1 to 6.

8. The use of a biochar-based degradation bacterial agent in soil remediation according to claim 7, characterized in that: The soil is polycyclic aromatic hydrocarbons contaminated soil.

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