Modified fulvic acid-charcoal immobilized microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of environmental restoration, and particularly relates to a modified fulvic acid-charcoal immobilized microbial agent as well as a preparation method and application thereof. The preparation method comprises the following steps: adding a bacterial liquid, modified fulvic acid and biochar into a culture medium for co-culture to obtain the modified fulvic acid-biochar immobilized bacterial agent. The preparation method is simple, convenient, easy to control and environmentally friendly, the cost is reduced, meanwhile, the prepared modified fulvic acid-biochar immobilized microbial agent still shows high microbial strain activity, petroleum hydrocarbon and polycyclic aromatic hydrocarbon in combined pollution are efficiently degraded and can be maintained for a long time, and good industrial application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental restoration, and specifically relates to a modified fulvic acid-biochar immobilized bacterial agent and a preparation method and application thereof. Background Art

[0002] Petroleum refers to a mixture of gaseous, liquid and solid hydrocarbons, known as the "blood of industry". With the rapid development of industrialization, people's demand for petroleum products is increasing. In 2023, the global demand for crude oil has reached 102.21 million barrels per day, involving multiple fields such as energy, pharmaceuticals, cosmetics, textiles and transportation. With the continuous increase in global oil production every year, the problem of oil pollution of soil, water bodies and other environments is becoming more and more serious. In the process of oil use and transportation, the discharge of oily wastewater, sewage irrigation, volatilization of various petroleum products, and the falling of incomplete combustion products will cause petroleum substances to pollute the environment. In the process of oil production, refining and processing, there will also be oil spills and emissions due to accidents, abnormal operations and maintenance. According to relevant data, the world consumes about 3 billion tons of oil each year, of which 7% (including crude oil and products) flows back to the ground in different ways, causing serious pollution to groundwater and soil. The entry of oil into the soil will increase the density of the soil, significantly reduce the quality of the soil, and reduce the water retention capacity, oxygen content and nutrients. In addition, the toxic and mutagenic properties of petroleum compounds can cause great damage to microorganisms and higher organisms, and the pollution of soil and water by petroleum products poses serious ecological and health risks to humans and the environment.

[0003] Due to their hydrophobicity, organic pollutants usually adhere to soil particles, atmospheric particles and water sediments, and eventually enter the soil environment. Therefore, soil treatment becomes the key to the remediation of organic complex pollution. At present, the commonly used soil remediation methods at home and abroad involve physical, chemical, biological and other fields. Commonly used methods include bioremediation, adsorption, gas phase extraction, incineration, chemical oxidation, ultrasonic method, microwave heating, etc. Microbial remediation technology has the advantages of low cost, high efficiency, green environmental protection and easy application. It is considered to be the preferred method for soil remediation. However, petroleum hydrocarbons and polycyclic aromatic hydrocarbons in the soil are dispersed in the complex soil aggregate structure and are difficult to be directly used by microorganisms.

[0004] Carrier-immobilized microbial technology is to fix microorganisms on carrier materials by physical or chemical methods, thereby maintaining microbial activity and increasing microbial density. It is a promising method for remediation of petroleum-contaminated soil, with the advantages of high treatment efficiency, stable effect, and no secondary pollution. Carrier-immobilized microorganisms have a good remediation effect on petroleum hydrocarbon pollution, which is attributed to the synergistic effect of biodegradation of immobilized microorganisms and carrier adsorption. Biochar is a type of carbon-rich solid product generated by high-temperature cracking of biological organic materials under highly anoxic or anaerobic conditions. Due to its special pore structure, large specific surface area and strong cation exchange capacity, it has great advantages and potential economic and ecological value in the remediation of petroleum-contaminated soil. Fulvic acid comes from soil humus and is environmentally friendly and economical. It can not only combine with various types of ecotoxic substances to reduce the damage of toxic substances to microorganisms, but also form compounds with various types of organic and inorganic substances as a food source for microorganisms and be transformed and utilized by microbial communities.

[0005] The present invention studies the preparation of a microbial immobilization carrier by combining fulvic acid with biochar, in order to solve the defects of existing immobilized microbial technology in terms of efficiency, stability and ecological compatibility. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a modified fulvic acid-biochar immobilized bacterial agent and a preparation method and application thereof. The present invention provides a novel preparation method of a modified fulvic acid-biochar immobilized bacterial agent, which is simple to operate, easy to control, has no stringent requirements on preparation conditions, environment and equipment, is pollution-free, energy-saving and environmentally friendly. While simplifying the preparation process and reducing the preparation cost, the prepared modified fulvic acid-biochar immobilized bacterial agent can still significantly improve the activity of microbial strains, efficiently degrade petroleum hydrocarbons and polycyclic aromatic hydrocarbons in complex pollution, and can maintain it for a long time, and still has a high level of fixed bacteria and strain activity after long-term storage.

[0007] The technical scheme of the present invention is: a method for preparing a modified fulvic acid-biochar immobilized bacterial agent, the steps comprising: adding bacterial liquid, modified fulvic acid and biochar into a culture medium for co-cultivation to obtain the modified fulvic acid-biochar immobilized bacterial agent.

[0008] Furthermore, the modified fulvic acid and biochar are mixed before being added to the culture medium, and the mass ratio of the modified fulvic acid to the biochar is 0.8-1.2:1, preferably 1:1.

[0009] The volume ratio of the bacterial liquid to the culture medium is 0.6-1.2:100, preferably 1:100; the culture medium is a liquid culture medium, including but not limited to LB culture medium, Martin culture medium, modified Martin culture medium, Sabouraud dextrose liquid culture medium, etc., preferably LB culture medium.

[0010] The total amount ratio of the modified fulvic acid and biochar to the bacterial solution is 2.5-3.5 g:1 mL, preferably 3 g:1 mL; the mass ratio of the modified fulvic acid and biochar is 0.8-1.2:1, preferably 1:1.

[0011] The bacterial solution is OD 600 = 1, the strains in the bacterial liquid are strains having degradation activity, and / or repair activity, and / or improvement activity on pollution in soil and / or water, and further are strains having degradation activity, and / or repair activity, and / or improvement activity on petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons in soil and / or water, including but not limited to Pseudomonas, Bacillus, Micrococcus, Lactobacillus, Candida, Alcaligenes, Corynebacterium variants, Acinetobacter, Staphylococcus, Flavobacterium, Achromobacter, Micrococcus, Mycobacterium, etc., specifically Acinetobacter (Acinetobacter sp.) TX3.

[0012] The co-culture temperature is 37±5°C, preferably 37°C, and the pH value is 6.5-7.5, preferably pH 7.

[0013] The preparation of the bacterial solution includes: placing the strain in a culture medium, activating the bacterial solution in a constant temperature shaker at 37±5°C until the OD of the bacterial solution is 600 =1-1.2; further, the bacterial solution was activated in a constant temperature shaker at 37°C and 180 rpm until the OD 600 =1.

[0014] The modified fulvic acid is obtained by esterification reaction of fulvic acid and saturated fatty acid, and its preparation comprises: esterification reaction of fulvic acid and saturated fatty alcohol at 100±5° C. under the catalysis of methanesulfonic acid.

[0015] Furthermore, an excess of saturated fatty alcohol is added. The saturated fatty alcohol is a primary alcohol, including but not limited to n-octanol, n-nonanol, n-decanol, n-dodecyl alcohol, etc., preferably n-decanol or n-dodecyl alcohol.

[0016] The mass ratio of fulvic acid to methanesulfonic acid is 2-3:1, preferably 2.1-2.5:1.

[0017] The esterification reaction product is ground and passed through a 200-mesh sieve.

[0018] The biochar is a product of carbonization of biomass raw materials, and its preparation includes: burning the biomass raw materials at a carbonization temperature, wherein the carbonization temperature is 500° C.-600° C. The burned product is ground and passed through a 200-mesh sieve.

[0019] Compared with the bacterial agents prepared by the adsorption method and the cross-linking method, the modified fulvic acid-biochar immobilized bacterial agent prepared by the preparation method of the present invention has excellent activity in terms of solid bacteria quantity, petroleum hydrocarbon degradation and polycyclic aromatic hydrocarbon degradation, and can be maintained for a long time. In addition, the preparation is convenient, simple, easy to control, has no strict requirements, is environmentally friendly, and is suitable for industrial application.

[0020] The present invention provides a modified fulvic acid-biochar immobilized bacterial agent prepared by the above-mentioned modified fulvic acid-biochar immobilized bacterial agent preparation method, comprising a strain, modified fulvic acid and biochar, wherein the modified fulvic acid and biochar are connected to form a composite material, and the strain is attached to the inside of the modified fulvic acid and biochar composite material.

[0021] The total amount of the modified fulvic acid and the biochar is in a ratio of 2.5-3.5 g:1 mL to the bacterial solution of the strain, preferably 3 g:1 mL; the mass ratio of the modified fulvic acid and the biochar is 0.8-1.2:1, preferably 1:1.

[0022] The bacterial solution is OD 600 =1-1.2 bacterial liquid, wherein the strains in the bacterial liquid are strains having degradation activity, and / or repair activity, and / or improvement activity on pollution in soil and / or water, and further are strains having degradation activity, and / or repair activity, and / or improvement activity on petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons in soil and / or water, including but not limited to Pseudomonas, Bacillus, Micrococcus, Lactobacillus, Candida, Alcaligenes, Corynebacterium variants, Acinetobacter, Staphylococcus, Flavobacterium, Achromobacter, Micrococcus, Mycobacterium, etc., specifically Acinetobacter (Acinetobacter sp.) TX3.

[0023] The fixed bacteria amount of the modified fulvic acid-biochar immobilized bacteria agent provided by the present invention is as high as 4.95×10 10 CFU / g, and after 90 days of storage, the solid bacteria count still remained at 9.45×10 9 CFU / g. After 60 days, the degradation rate of diesel in diesel-benzo[a]pyrene composite contaminated soil reached 39.30%, and the degradation rate of benzo[a]pyrene reached 64.87%. The degradation and remediation of petroleum hydrocarbons and polycyclic aromatic hydrocarbons pollution showed good stability and efficiency.

[0024] The modified fulvic acid-biochar immobilized bacterial agent provided by the present invention is used to prepare products for use in petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons. The product has degradation activity on petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons, and has repair activity and / or improvement activity on the pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons. The product can be applied to environmental systems such as soil and water bodies.

[0025] The polycyclic aromatic hydrocarbons refer to aromatic compounds containing two or more benzene rings, which are divided into non-condensed ring type and condensed ring type, such as biphenyl, bipolyphenyl, naphthalene, anthracene, phenanthrene, benzo[a]pyrene, etc., preferably benzo[a]pyrene.

[0026] The petroleum hydrocarbons are organic compounds composed of carbon and hydrogen, mainly including alkanes, olefins, aromatic hydrocarbons and cycloalkanes, etc., alkanes such as methane and ethane, olefins such as ethylene and propylene, aromatic hydrocarbons such as benzene, cycloalkanes such as cyclohexane, etc.

[0027] Pollution caused by PAHs and / or petroleum hydrocarbons includes but is not limited to pollution caused by oil, gasoline, diesel, kerosene, paint, coatings, plastics, rubber, ink, lubricants, asphalt, petroleum coke, liquefied gas, pesticides, fertilizers, detergents, fibers, etc.

[0028] The above product of the present invention can be in liquid, solid or semi-solid state, and can be prepared into a desired state as required.

[0029] The present invention also provides a product for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, wherein the product contains the modified fulvic acid-biochar immobilized bacterial agent provided by the present invention. The product has degradation activity for petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons, and has repair activity and / or improvement activity for the pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons. The product can be applied to environmental systems such as soil and water bodies.

[0030] Compared with the prior art, the modified fulvic acid-biochar carrier immobilized petroleum degrading bacteria (MFA-BC-B) of the present invention has the following significant beneficial effects in the field of soil and / or water remediation:

[0031] (1) Significantly improve the efficiency and stability of microbial immobilization

[0032] Compared with conventional carriers (such as activated carbon, clay, etc.), the loading capacity of microorganisms is not high, the long-term survival rate is low, and it is easily inactivated by environmental factors. The present invention enhances its hydrophobicity and carrier affinity by modifying fulvic acid (esterification of n-decyl alcohol / n-dodecyl alcohol), and combines the high specific surface area and pore structure of biochar to form a synergistic immobilization system. The composite structure of modified fulvic acid and biochar provides a physical protection barrier and a nutrient slow-release environment for microorganisms, reducing the damage to the bacteria caused by environmental stress. Experimental data show that the modified fulvic acid-biochar carrier immobilized microorganisms prepared by the present invention still maintain 9.45×10 9 The high number of viable bacteria in CFU / g far exceeds that of traditional carriers (usually ≤10 8 CFU / g).

[0033] (2) Synergistic pollution degradation capabilities

[0034] The degradation efficiency of single carriers or free bacteria for composite pollutants (such as petroleum hydrocarbons + polycyclic aromatic hydrocarbons) is limited, and they are easily inhibited by the toxicity of pollutants. The fulvic acid-biochar composite carrier has both adsorption and bioactivation functions: biochar adsorbs pollutants (such as diesel and benzopyrene), shortening the contact distance between microorganisms and pollutants; fulvic acid combines with pollutants to reduce the damage of pollutants to microorganisms. Experimental verification shows that in composite contaminated soil (diesel 7.5g / kg + benzopyrene 6mg / kg), after 60 days of remediation: the diesel degradation rate is 39.30%, and the benzo[a]pyrene degradation rate is 64.87%, which is significantly higher than that of single carriers or free bacteria.

[0035] 3. Systematic restoration of soil ecological functions

[0036] The present invention can improve the physical and chemical properties of soil: biochar increases soil porosity and water holding capacity, fulvic acid supplements humic acid organic matter and promotes soil aggregate formation. In addition, the composite carrier can provide an ecological niche for indigenous microorganisms, forming a synergistic community with degradation bacteria (TX3) as the core, and enhancing the soil self-repair potential. In addition, the present invention can also avoid secondary pollution of chemical repair agents, which is in line with the concept of green and sustainable repair.

[0037] 4. Long-term effectiveness and universal application

[0038] The stable composite structure of modified fulvic acid and biochar delays the decomposition of the carrier, providing continuous nutrient release and pollutant adsorption capacity. Experiments have shown that the immobilized bacterial agent remains highly active after long-term storage, which is suitable for the delayed remediation needs of sudden pollution scenarios. It can be adapted to a variety of petroleum-degrading strains and expanded to other organic pollution (such as pesticides and plasticizers) remediation fields. The raw materials (fulvic acid, biochar) are widely available and can be prepared based on agricultural / forestry waste (such as straw and sawdust), which is in line with the trend of resource recycling. The carrier can be made into granular or powder dosage forms, which are convenient for mechanical spreading or in-situ injection, and are suitable for the remediation of large-scale contaminated sites.

[0039] This invention breaks through the bottlenecks of traditional immobilized microbial technology in efficiency, stability and ecological compatibility through the triple mechanism of carrier modification, microbial synergy and ecological regulation, and provides an efficient, long-term and low-cost remediation solution for soil contaminated by petroleum hydrocarbons and polycyclic aromatic hydrocarbons. It has significant scientific research value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the humic acid esterification reaction.

[0041] Figure 2 Infrared spectra of FA and MFA.

[0042] Figure 3 Scanning electron micrographs of MFA-BC (a) and MFA-BC-B (b).

[0043] Figure 4 Preservation properties of the immobilized carrier MFA-BC-B prepared for co-culture (stored at 4°C for 90 days).

[0044] Figure 5 The remediation effect of diesel and benzo[a]pyrene co-contaminated soil under different treatments (60d). DETAILED DESCRIPTION

[0045] 1. Preparation of MFA-BC-B

[0046] 1. Preparation of MFA-BC-B Co-culture

[0047] (1) Fulvic acid modification: Fulvic acid (7 g) was esterified with excess n-decanol in an oil bath at 100°C under the catalysis of methanesulfonic acid (3 g) in a reaction vessel. Figure 1 ), the reaction time is not less than 24h. After the modification, the fulvic acid and biochar (BC) are ground and passed through a 200-mesh sieve, cooled and dried, and mixed in a ratio of 1:1 (MFA-BC) for standby use.

[0048] (2) Co-cultivation: A petroleum-degrading bacterium TX3 (Acinetobacter sp., with a deposit number of CGMCC No. 30200 and deposited by China National Microbiological Culture Collection Center) was selected for activation treatment, and the bacteria in the solid preservation medium of the inclined tube were selected and placed in LB medium, and activated in a constant temperature shaker at 37°C and 180 rpm until the bacterial solution OD600 = 1. Then 1 mL of the bacterial solution was inoculated in 100 mL of LB medium, and 3 g of a mixture of modified fulvic acid and biochar was added, and co-cultivated at 37°C and pH = 7 for 12 h.

[0049] Each liter of LB medium includes: Tryptone 10g, yeast extract

[0050] 5g, sodium chloride (NaCl) 10g.

[0051] (3) Preparation of carrier-immobilized microorganisms: After the carrier and the strain were co-cultured, the modified fulvic acid-biochar immobilized petroleum-degrading bacteria (MFA-BC-B) were obtained after centrifugation and drying.

[0052] 2. Preparation of MFA-BC-B by cross-linking method

[0053] Take 100mL OD 600 = 1 TX3 petroleum degradation bacteria liquid was centrifuged and mixed with 3g of a mixture of modified fulvic acid and biochar (1:1) to form the carrier bacterial agent; the carrier bacterial agent was mixed with 10.0g·L-1 Sodium alginate (SA) solution was mixed at a ratio of 2:1 (w / v), the resulting mixture was sucked with a plastic pipette, and 50.0 g·L -1 The samples were fixed in a CaCl2 solution for 4 h and then the excess CaCl2 solution was washed away with distilled water to obtain MFA-BC-B prepared by the cross-linking method.

[0054] 3. Preparation of MFA-BC-B by adsorption method

[0055] Take the mixture of modified fulvic acid and biochar (1:1) and OD 600 =1, the ratio of the mixture of modified fulvic acid and biochar to the bacterial liquid was 1:10 (w / v), and the adsorption was carried out at 37°C and 180 rpm for 12 hours. The free bacteria were removed by filtration to obtain MFA-BC-B prepared by the adsorption method.

[0056] 2. Characterization of MFA-BC-B prepared by co-culture method

[0057] 1. Infrared spectroscopy

[0058] Fulvic acid (FA) and modified fulvic acid (MFA) were characterized by infrared spectroscopy. FA has a wavelength of 3600 to 3300 cm -1 Contains a very broad absorption region, which is caused by OH stretching vibration. -1 and 1320~1210cm -1 The peaks of 3000-2850cm-1 correspond to the absorption of carboxyl C=O and the expansion of CO. -1 ) and esters (1750~1735cm -1 1210~1163cm -1 ) together confirmed the occurrence of esterification reaction.

[0059] 2. Scanning electron microscopy

[0060] MFA-BC has a smooth surface, relatively flat, and wrinkles at the joints ( Figure 3 a), providing suitable shelter and protection for the strain; in addition, TX3 was successfully immobilized and evenly distributed inside the MFA-BC composite ( Figure 3 b). The attachment of strain TX3 to MFA-BC can protect them from direct pollution of diesel and benzo[a]pyrene and provide them with a good habitat for growth and colonization, which is beneficial to the degradation of the combined pollution by the strain.

[0061] 3. Study on the amount of solid bacteria in the carrier

[0062] (1) This study compared the degradation efficiency of diesel and benzo[a]pyrene using three microbial immobilization techniques (adsorption, crosslinking, and co-cultivation). MFA-BC-B prepared by the adsorption method, MFA-BC-B prepared by the crosslinking method, and MFA-BC-B prepared by the co-cultivation method were added to a composite contaminated MS culture medium containing 7.5 g / L diesel and 6 mg / L benzo[a]pyrene at a concentration of 4% (w / w), and cultured at 37°C and 220 r / min for 7 days. The degradation rate of benzo[a]pyrene was determined by HPLC, and the degradation rate of diesel was determined by dry weight method. In terms of the amount of fixed bacteria, the amount of fixed bacteria by the co-cultivation method (4.95×10 10 CFU / g was 34.9 times that of the adsorption method and 93.2 times that of the cross-linking method. The high fixed bacteria content provided a higher degradation efficiency, indicating that the ability of carriers to immobilize microorganisms is the key to technical optimization. The cross-linking method had the lowest fixed bacteria content (5.31×10 8 CFU / g), which may be related to the damage of chemical cross-linking agents to the cell membrane or mass transfer limitation, resulting in decreased bacterial activity. The co-culture method significantly improved the degradation efficiency of diesel and benzo[a]pyrene due to its high solid bacteria content. The degradation rates of diesel and benzo[a]pyrene were 40.63% and 60.33%, respectively, and it is suitable for high pollution load scenarios.

[0063]

[0064] (2) The results showed that MFA-BC was slightly better than BC in terms of specific surface area, total pore volume and bacterial consolidation. Compared with BC, the specific surface area increased by 13.31%, the total pore volume increased by 21.50%, and the bacterial consolidation amount increased by 13.06 times. This was due to the mutual influence and joint action of MFA and BC during the carrier preparation process. The stable composite structure formed by MFA and BC provided a good habitat for microorganisms to grow and colonize.

[0065]

[0066] (3) The number of immobilized microorganisms in the co-cultured MFA-BC-B slowly decreased with the increase of storage time, and the degradation performance was reduced. After being stored at 4°C for 90 days, the number of immobilized microorganisms was 9.45*10 9 CFU / g, the degradation rates of diesel and benzo[a]pyrene after activation were 24.01% and 46.99%, respectively. The stable composite structure of modified fulvic acid and biochar can delay the decomposition of the carrier and provide continuous nutrient release and protection for microorganisms. Experiments have shown that the MFA-BC-B prepared by co-cultivation still maintains high activity and performance after long-term storage.

[0067] 4. Remediation of diesel-benzo[a]pyrene composite contaminated soil by FA-BC-B prepared by co-culture

[0068] (1) Configuration of diesel-benzo[a]pyrene composite contaminated soil

[0069] Spray 75g diesel evenly into 10kg clean soil, add it in 3 times, each time with an interval of 10min, stir it manually with a stainless steel shovel during the period, and then transfer it to a mechanical stirrer and mix it at 30rpm for 30min. Then add benzo[a]pyrene-n-hexane mother liquor (10mL) drop by drop into the diesel-contaminated soil, and stir it synchronously (40rpm, 1h) until the solvent is completely evaporated. Finally, transfer the contaminated soil to a brown glass jar and seal it with a sealing film (with ventilation holes reserved). Place it in a temperature and humidity controlled incubator, and set the conditions: temperature 25±1℃, humidity 60%±5%, and avoid light. Turn the soil once a day for 14 days.

[0070] (2) Soil degradation experiment

[0071] There were 7 treatment groups (n=3): CK (blank control): no restoration materials were added; BC: biochar (4% w / w); MFA: modified fulvic acid (4% w / w); TX3: free bacterial agent (4% v / w, OD 600 =1.0±0.05); BC-B: biochar immobilized petroleum-degrading bacteria (4% w / w); MFA-B: modified fulvic acid immobilized petroleum-degrading bacteria TX3 (4% w / w); MFA-BC-B: modified composite carrier immobilized petroleum-degrading bacteria TX3 (4% w / w). 500g soil sample (dry weight) was placed in a 1L culture bottle for each treatment group. The soil moisture content was adjusted to 60% of the field water holding capacity. The culture was kept at a constant temperature of 25±1℃, and the water was weighed and replenished every 3 days.

[0072] Sampling at 0, 15, 30, 45, and 60 days: 50 g of soil samples were collected by aseptic operation and divided into sterile centrifuge tubes; stored at 4°C for microbiological analysis and -80°C for molecular biology detection. The remaining samples were air-dried and tested for physical and chemical indicators.

[0073] (3) Degradation of diesel and benzo[a]pyrene by TX3

[0074] The degradation rate of petroleum hydrocarbons was calculated by dry weight method. After the degradation was completed, the remaining petroleum hydrocarbons in the culture medium were extracted with dichloromethane, and then the dichloromethane was completely volatilized in a fume hood to obtain the remaining petroleum hydrocarbons after degradation.

[0075] The residual amount of benzo(a)pyrene in the system was determined by high performance liquid chromatography. The remaining benzo(a)pyrene content after the reaction system was extracted with dichloromethane, and then the dichloromethane was evaporated using a rotary evaporator, and 1 mL of acetonitrile (HPLC purity) was added to redissolve the benzo(a)pyrene, and filtered through a 0.22 μm organic phase filter membrane to obtain 1 mL of the sample to be tested.

[0076] High performance liquid chromatography instrument model: Shimadzu LC-20AT, high performance liquid chromatography detection conditions are as follows: chromatographic column: reverse phase chromatographic column (AQ-C18, 5um, 4.6×250mm); column temperature: 30°C; detector: ultraviolet spectrophotometer (254nm); mobile phase: acetonitrile: water = 4:1; flow rate: 1mL / min; injection volume: 1μL.

[0077] As the remediation time increases, the degradation rate of diesel and benzo[a]pyrene in each treatment group shows a gradually decreasing trend. After 60 days of remediation, in the soil contaminated with diesel (7.5g / kg) and benzo[a]pyrene (6mg / kg), the degradation rates of diesel and benzo[a]pyrene by MFA-BC-B were 39.30% and 64.87%, respectively. Compared with the degradation effect of modified fulvic acid or biochar alone, the immobilized microorganisms have been significantly improved.

[0078] It should be pointed out that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a modified fulvic acid-biochar immobilized bacterial agent, characterized in that: The steps include: The bacterial liquid, modified fulvic acid and biochar are added into the culture medium for co-cultivation to obtain the modified fulvic acid-biochar immobilized bacterial agent.

2. The preparation method according to claim 1, characterized in that: The modified fulvic acid and biochar were mixed before adding to the culture medium.

3. The preparation method according to claim 1 or 2, characterized in that: The volume ratio of the bacterial liquid to the culture medium is 0.6-1.2:100, the total amount ratio of the modified fulvic acid to the biochar to the bacterial liquid is 2.5-3.5 g:1 mL, and the mass ratio of the modified fulvic acid to the biochar is 0.8-1.2:

1.

4. The preparation method according to claim 3, characterized in that: The OD of the bacterial solution 600 =1-1.2, the strains in the bacterial liquid are strains that have degradation activity, and / or repair activity, and / or improvement activity on pollution in soil and / or water.

5. The preparation method according to claim 1, characterized in that: The co-culture temperature is 37±5°C and the pH value is 6.5-7.

5.

6. The preparation method according to claim 1, characterized in that: The modified fulvic acid is the modified fulvic acid obtained by esterification reaction of fulvic acid and saturated fatty acid.

7. The modified fulvic acid-biochar immobilized bacterial agent prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The invention comprises bacterial strains, modified fulvic acid and biochar. The modified fulvic acid and biochar are connected to form a composite material, and the bacterial strains are attached to the inside of the composite material of modified fulvic acid and biochar.

8. The modified fulvic acid-biochar immobilized bacterial agent according to claim 7, characterized in that: The total amount of the modified fulvic acid and the biochar is in a ratio of 2.5-3.5 g:1 mL to the bacterial solution containing the strain, and the mass ratio of the modified fulvic acid to the biochar is 0.8-1.2:

1.

9. The modified fulvic acid-biochar immobilized bacterial agent prepared by the preparation method according to any one of claims 1 to 6 or the modified fulvic acid-biochar immobilized bacterial agent according to claim 7 or 8 is used to prepare products for use in petroleum hydrocarbons and / or polycyclic aromatic hydrocarbons.

10. A product for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, characterized in that: Contains the modified fulvic acid-biochar immobilized bacterial agent prepared by the preparation method according to any one of claims 1 to 6 or the modified fulvic acid-biochar immobilized bacterial agent according to claim 7 or 8.

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