Method for bioremediation of soil by using crude oil degradation fungi

Abstract

The invention discloses a method for biologically repairing soil by using crude oil degrading fungi, and belongs to the technical field of microorganisms and biology. The method comprises the following steps: step 1, directionally screening strains of the crude oil degrading fungi; step 2, function enhancement of the screened strains; 3, preparing a repair carrier; and 4, carrying out gradient process and composite flora synergistic remediation. According to the method, indigenous fungi are screened and separated in a crude oil pollution area, the screened strains are subjected to adaptive laboratory evolution, the degradation capacity and the environmental endurance capacity are enhanced, a preparation process of composite microspheres is combined, adsorption-degradation efficient remediation of crude oil pollution is achieved, and a gradient process and other biological methods are used for cooperating remediation measures; the remediation effect is further enhanced, the natural ecological environment is stably restored, secondary pollution in the remediation process is avoided, and the risk of ecological pollution is reduced.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biotechnology, and in particular is a method for bioremediation of soil using crude oil-degrading fungi. Background Art

[0002] Throughout the entire petroleum and petrochemical industry chain—from exploration and extraction to transportation, processing, storage, and sales—poor management and accidents can lead to oil spills, causing serious environmental damage. According to statistics, approximately 8 million tons of crude oil enter the environment worldwide each year, contaminating soil, groundwater, rivers, and oceans. The main sources of oil contamination in soil include crude oil leaks and spills, the accumulation of oily slag, sludge, and garbage, wastewater irrigation, air pollution, vehicle exhaust emissions, and chemical contamination.

[0003] In the field of remediation of crude oil-contaminated soil, there are currently a variety of treatment methods, mainly including physical methods, chemical methods and biological methods. However, these existing methods all have defects to varying degrees. Physical methods, such as the use of oil spill treatment agents (such as surface cleaning agents and chemical oil collection agents), can be degraded by microorganisms, but will temporarily slow down the degradation rate of petroleum hydrocarbons, especially for high molecular weight polycyclic aromatic hydrocarbons (PAHs), which have high residual toxicity. When faced with high-concentration contaminated soil, off-site thermal desorption is required, which consumes a lot of energy and may damage the soil structure.

[0004] Chemical methods also face many problems. For example, the chemical oxidation process may produce secondary pollutants, such as chloroform, which may cause new harm to the environment.

[0005] Although biological methods have great development potential, they also have some limitations at this stage. On the one hand, plant roots will be inhibited in high-concentration oil pollution (>2%), resulting in a significant decrease in the remediation effect. They are only applicable to moderately contaminated soils, and the remediation depth is also limited by the root distribution. On the other hand, natural bacterial strains can usually only degrade a few pollutants and it is difficult to effectively deal with complex pollution situations, such as the pollution scene where petroleum hydrocarbons and PAHs coexist. If they rely on genetically engineered strains, there will be ecological risks of environmental release, as shown in patent document CN111808770B.

[0006] Therefore, it is necessary to propose a method for bioremediation of soil with crude oil-degrading fungi that can utilize indigenous strains combined with chemical microparticle preparation, combine biological and chemical treatment methods to efficiently repair crude oil-contaminated soil, avoid the generation of secondary pollutants, and reduce the risk of ecological pollution. Summary of the Invention

[0007] In order to solve the above problems, the purpose of the present invention is to provide a method for bioremediation of soil with crude oil-degrading fungi. By screening and isolating indigenous fungi in crude oil-contaminated areas, adaptive laboratory evolution of the screened strains is carried out to enhance degradation ability and environmental tolerance. Combined with the preparation process of composite microspheres, efficient adsorption-degradation remediation of crude oil pollution is achieved. Gradient process and other biological methods are used to coordinate remediation measures to further enhance the remediation effect, stably restore the natural ecological environment, avoid secondary pollution during the remediation process, and reduce the risk of ecological pollution.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows: A method for bioremediation of soil by crude oil-degrading fungi comprises the following steps:

[0009] Step 1: Directed screening of crude oil degrading fungal strains: isolate indigenous fungi from crude oil contaminated areas, screen the isolated indigenous fungi based on salt and low temperature tolerance, and give priority to those with C 10 -C 40 Strains with broad-spectrum degradation capabilities for alkanes and 3-5-ring PAHs were selected through transcriptomics to screen strains with high expression of laccase and lignin peroxidase, and inducers were used to enhance enzyme activity;

[0010] Step 2: Enhance the function of the selected strains. Through adaptive laboratory evolution technology, the tolerance of the selected strains to high-concentration oil pollution and extreme pH is improved to ensure their activity and stability in complex environments. A composite bacterial community containing core degradation bacteria, auxiliary bacteria and growth-promoting bacteria is constructed.

[0011] Step 3: Preparation of a remediation carrier, preparing biochar particles, using chitosan to embed fungal spores of a composite bacterial community to form chitosan pellets, using biochar to adsorb and load the chitosan pellets in an adsorption preparation device to form composite microspheres, and modifying the surface of the composite microspheres with hydrophobic groups to target the adsorption of crude oil pollutants, and the composite microspheres have a pH-responsive release function;

[0012] Step 4: Gradient technology and composite bacterial community synergistic remediation: For high-concentration pollution areas, that is, areas with crude oil content greater than 2%, fungi-biochar combination is used, and fungi-plant combination is used for medium and low-concentration pollution areas, and remediation parameters are adjusted through dynamic monitoring.

[0013] Furthermore, the salt tolerance condition in step 1 refers to a salt concentration greater than 5%, and a low temperature tolerance range of 4-15°C.

[0014] Furthermore, the inducer in step one includes resveratrol, which increases enzyme activity and is used to enhance the oxidative degradation efficiency of PAHs by the indigenous strain.

[0015] Furthermore, the high-concentration oil pollution in step 2 refers to a concentration greater than 2% and an extreme pH range of 3-10.

[0016] Furthermore, the composite bacterial community in step 2 includes core degradation bacteria, auxiliary bacteria and growth-promoting bacteria.

[0017] Furthermore, the core degradation bacteria in step 2 are strains with the highest crude oil degradation efficiency, the auxiliary bacteria are Trichoderma, which secrete cellulase to enhance pollutant dissolution, and the growth-promoting bacteria are arbuscular mycorrhizal fungi, which coexist with plants to promote ecological restoration.

[0018] Furthermore, the hydrophobic group in step three is an octadecyl group or a phenyl group.

[0019] Furthermore, the pH-responsive release function in step three is to slowly release spores when the soil pH is less than 6, and to accelerate spore germination when the pH is 7-8.

[0020] Furthermore, the fungus-biochar combination in step four includes laying a biochar adsorption layer on the surface of the contaminated soil to form a degradation buffer zone, and then spraying fungal microspheres.

[0021] Furthermore, the fungus-plant combination in step four includes planting ryegrass in the later stage of restoration and enhancing plant tolerance through the secretion of indoleacetic acid by fungal hyphae, and the restoration depth is extended to less than 1.5m.

[0022] Furthermore, the dynamic monitoring and adjustment of remediation parameters in step four include implanting microbial electrochemical sensors during the remediation phase to monitor the concentrations of phenanthrene and pyrene in the soil in real time, and regulating the frequency of nutrient solution injection through Internet of Things technology.

[0023] The principle of the basic scheme is: in the targeted screening stage of crude oil degrading fungal strains, indigenous fungi are isolated from crude oil contaminated areas, and salt resistance and low temperature resistance are used as key screening conditions, with priority given to fungi with specific C 10 -C 40 Strains with broad-spectrum degradation capabilities for alkanes and 3-5-ring PAHs. Transcriptomics was used to screen strains with high expression of laccase and lignin peroxidase, and resveratrol was used as an inducer to increase enzyme activity, thereby enhancing the oxidative degradation efficiency of PAHs. The principle is to focus on screening fungal strains that can survive stably in harsh environmental conditions (such as high salt and low temperature) and have the ability to efficiently degrade specific crude oil components. This lays a solid foundation for subsequent remediation work and ensures that the selected strains can accurately and effectively degrade crude oil pollutants.

[0024] In the functional enhancement stage of the screening strains, adaptive laboratory evolution technology is used to improve the tolerance of the screening strains to high-concentration oil pollution and extreme pH, and to ensure the activity stability of the strains in complex environments. At the same time, a composite microbial community containing core degrading bacteria, auxiliary bacteria and growth-promoting bacteria is constructed. This principle reflects the deep mining and optimization of the performance of the strains. Through simulation of the complex pollution environment that may be encountered, the strains are strengthened so that they can better adapt to the changing soil conditions in the actual remediation process. The construction of a composite microbial community is to take advantage of the complementary effects of different strains. The core degrading bacteria focus on efficient decomposition of the main components of crude oil, the auxiliary bacteria enhance the dissolution of pollutants by secreting cellulase and other substances, break the close contact between pollutants and soil particles, and the growth-promoting bacteria are symbiotic with plants to promote ecological recovery. This way of cooperative combat can comprehensively improve the efficiency and quality of remediation.

[0025] In the preparation of the remediation carrier, biocarbon microparticles are prepared, chitosan is used to embed the fungal spores of the composite microbial community to form chitosan bacterial spheres, and then biocarbon is used to adsorb and load the chitosan bacterial spheres to form composite microspheres. The surface of the composite microspheres is modified with hydrophobic groups to target adsorb oil pollutants, and the composite microspheres are given a pH-responsive release function. The principle is to create a high-efficiency remediation carrier that combines adsorption and degradation. Biocarbon has a rich pore structure and a large specific surface area, which can effectively adsorb oil pollutants and facilitate subsequent fungal degradation. Chitosan-embedded fungal spores can protect the spores from inactivation under adverse external factors and also control the release rate of the spores. The surface-modified hydrophobic groups can precisely target and adsorb the hydrophobic pollutants in the oil, achieving efficient enrichment of the pollutants. The pH-responsive release function is an adaptive design that adjusts the release rate of the spores according to the change in soil pH, ensuring that the fungal spores can be activated and put into degradation work at the most suitable time, avoiding the inhibition of high-concentration oil pollution on the activity of the spores in the early stage, and improving the accuracy and effectiveness of remediation.

[0026] Finally, in the gradient process and the remediation stage of the combined microbial population, for the high concentration pollution area (crude oil content > 2%), the fungus-biochar combination is used, and for the medium and low concentration pollution area, the fungus-plant combination is used, and the remediation parameters are adjusted through dynamic monitoring. The core of this principle is to adopt different remediation strategies according to local conditions and time. For the high concentration pollution area, due to the high content of pollutants and high risk, the fungus-biochar combination is used to take advantage of the strong adsorption capacity of biochar and the high degradation capacity of fungi to quickly adsorb and decompose pollutants and prevent further spread of pollutants. For the medium and low concentration pollution area, combined with the remediation capacity of plants, through the synergistic effect of fungi and plants, the absorption and enrichment function of plant roots and the degradation function of fungi are used to achieve deep purification and ecological restoration of the soil. At the same time, through dynamic monitoring of the concentration changes of pollutants in the soil and other key parameters, and with the help of Internet of Things technology, the remediation parameters such as nutrient solution injection frequency are adjusted in real time, which can ensure that the remediation process is always in the best state, and the remediation scheme can be adjusted in time according to the actual situation to ensure the efficiency and adaptability of the remediation work, and achieve a multiplier effect of remediation.

[0027] The beneficial effects of the basic scheme are: 1. The remediation efficiency is greatly improved. Through directional screening, high-efficiency fungal strains with salt tolerance (adaptation to > 5% salinity), low-temperature tolerance (4-15℃ activity retention) and broad-spectrum degradation ability for C 10 -C 40 Alkanes and 3-5 ring PAHs, precise degradation of complex components in crude oil, compared with traditional remediation methods, the degradation rate of difficult-to-degrade high molecular weight polycyclic aromatic hydrocarbons and other pollutants is significantly accelerated, which can effectively reduce the concentration of soil pollutants in a shorter time and shorten the remediation period, which is of great significance for timely governance of crude oil pollution and restoration of land ecological function.

[0028] 2. Strong environmental adaptability. The selected fungal strains are strengthened by adaptive laboratory evolution technology, and the tolerance to high-concentration oil pollution (> 2%) and extreme pH (3-10) is significantly enhanced, ensuring that in different regions, different pollution levels of soil environment, especially in harsh environmental conditions, it can still maintain stable activity and degradation ability. This strong environmental adaptability enables the method to be widely applied to various crude oil contaminated soil scenes, whether it is a high-salt coastal area or a cold northern region, or a soil environment with strong acidity or alkalinity, it can be effectively implemented, greatly expanding its application range and solving the problem of greatly reduced remediation effect of traditional methods in complex environments.

[0029] 3. Low ecological risk. Different from the repair method of genetically engineered strains, the present invention gives priority to the use of natural indigenous fungal strains to construct a composite bacterial community, avoiding the ecological risks that may be brought by exogenous genetically engineered bacteria. These indigenous strains originally existed in the crude oil contaminated area and have adapted to the local ecological environment in the long-term natural selection process. They will not damage the original ecological balance in the soil. At the same time, by rationally matching degradation core bacteria, auxiliary bacteria and growth-promoting bacteria, a functionally complementary ecological restoration system is formed. While degrading crude oil pollutants, it can also promote the restoration and reconstruction of soil ecology, which is beneficial to the optimization of soil microbial community structure and the improvement of soil fertility, achieving true green restoration and being friendly to the ecological environment.

[0030] 4. Balancing the depth and breadth of remediation. On the one hand, in the remediation process, a fungus-biochar combination is used for high-concentration pollution areas. A biochar adsorption layer is laid on the surface of the contaminated soil to form a degradation buffer zone, and then fungal microspheres are sprayed. This can effectively treat crude oil pollution in the surface and shallow layers of the soil. On the other hand, in the later stages of remediation, a fungus-plant combination is used for low- and medium-concentration pollution areas. Plants such as ryegrass are planted, and fungal hyphae secrete indoleacetic acid to enhance plant tolerance. This extends the remediation depth to below 1.5 meters, achieving the treatment of deep-seated pollutants in the soil. This breaks through the limitation of traditional plant remediation, where the depth is limited by root distribution, ensuring the remediation effect of the entire soil layer, making the remediation more thorough and comprehensive.

[0031] 5. High degree of intelligence. By implanting microbial electrochemical sensors during the remediation phase, the concentration changes of key pollutants such as phenanthrene and pyrene in the soil are monitored in real time. Combined with IoT technology, remediation parameters such as the frequency of nutrient solution injection are controlled, enabling dynamic monitoring and intelligent regulation of the remediation process. This intelligent remediation approach can promptly adjust remediation strategies based on the actual degradation of soil pollutants, avoiding blind remediation and waste of resources, improving the accuracy and specificity of remediation, and making the entire remediation process more scientific, efficient, and energy-efficient. It provides a modern, intelligent technical approach for the remediation of crude oil-contaminated soil.

[0032] 6. Significant economic benefits. The method of the present invention makes full use of natural materials or renewable resources such as biochar, chitosan, and lignin, reducing the cost of restoration. At the same time, by optimizing the restoration process and improving the restoration efficiency, the preparation and restoration time are reduced, thereby indirectly reducing the economic losses caused by land pollution and increasing the reuse value of the land. In addition, the method minimizes the dependence on chemical agents during the restoration process, avoids the cost of additional treatment due to the generation of secondary pollutants by methods such as chemical oxidation, and improves the economic benefits of the restoration as a whole, with good market application prospects and promotion value.

[0033] Furthermore, the adsorption preparation device in step three includes a tank body, the top wall of the tank body is rotatably connected to a liquid injection pipe, the liquid injection pipe extends into the interior of the tank body, the liquid injection pipe is divided into several layers, each layer is fixedly connected to several curved pipes, the curved pipes have the same curvature direction, the outer periphery of the end of the curved pipe is fixedly connected to a limiting plate, the limiting plates are slidably fitted with a hollow cover, the outer periphery of the hollow cover is connected to several arc tubes, the arc tubes on the same hollow cover have the same curvature direction, one side of the tank body is connected to a feed pipe, one end of the feed pipe located inside the tank body is connected to a duckbill mouth, the bottom wall of the tank body is connected to an output valve, and the output valve is connected to a discharge pipe.

[0034] The beneficial effects of the basic solution are: the unique design of the adsorption preparation device makes the combination of biochar particles and chitosan bacteria balls more uniform and efficient. The ingenious layout of the multi-layer elbows and arc tubes, combined with the fine feeding of the duckbill mouth, can form an orderly and uniform mixing flow field inside the tank. When the biochar particles and chitosan bacteria balls are fully mixed in the tank, the multi-layer elbows drive the injection tube to rotate under the action of liquid pressure, and the arc tubes make the liquid evenly dispersed in the vertical direction to form a three-dimensional mixing network. This multi-dimensional mixing method ensures that each chitosan bacteria ball can be evenly surrounded by an appropriate amount of biochar particles, avoiding local overload or underload, thereby significantly improving the adsorption and loading performance of the composite microspheres, and providing a more stable carrier basis for the subsequent crude oil degradation process.

[0035] Furthermore, step three also includes adding lignin-derived slow-release fertilizer and lipopeptide biosurfactant to the composite microspheres.

[0036] The basic solution has the following beneficial effects: 1. The addition of lignin-derived slow-release fertilizers and lipopeptide biosurfactants can effectively improve the degradation efficiency of the composite microspheres. Lignin-derived slow-release fertilizers provide continuous nutritional support for fungal growth, promoting fungal reproduction and metabolic activity, enabling them to more efficiently decompose crude oil pollutants. Lipopeptide biosurfactants can reduce water surface tension, increase the solubility and bioavailability of crude oil pollutants in water, and make it easier for fungi to access and degrade highly hydrophobic petroleum hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), thereby significantly improving the degradation efficiency of the entire remediation system.

[0037] 2. The slow-release properties of lignin-derived slow-release fertilizers ensure that fungi receive the appropriate amount of nutrients under varying environmental conditions, preventing reduced activity or death due to nutrient deficiency. This allows the remediation system to maintain stable operation and efficient degradation capabilities in complex and changing soil environments (such as those with varying temperature, humidity, and pH values), enhancing the environmental adaptability and reliability of the entire method.

[0038] 3. Lignin-derived natural slow-release nitrogen and phosphorus sources and lipopeptide biosurfactants replace traditional chemical fertilizers and surfactants, reducing the potential risk of soil and environmental contamination from chemical substances. After fulfilling their functions, these natural substances naturally degrade into environmentally friendly components, avoiding secondary pollution and aligning with the principles of green restoration and sustainable development.

[0039] 4. Lipopeptide biosurfactants have the advantages of low ecotoxicity and the ability to promote soil aggregate formation, helping to improve soil structure and physical properties. Furthermore, a favorable soil environment promotes the growth of plant roots and the reconstruction of microbial communities, thereby promoting the ecological restoration of contaminated soils and accelerating the reconstruction and stabilization of soil ecosystem functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the method for bioremediation of soil by crude oil-degrading fungi in an embodiment of the present invention.

[0041] Figure 2 Schematic diagram of the process of bioremediation of soil by using crude oil-degrading fungi in an embodiment of the present invention.

[0042] Figure 3 This is an axonometric diagram of the adsorption preparation device in an embodiment of the present invention.

[0043] Figure 4 It is a front cross-sectional view of the adsorption preparation device in an embodiment of the present invention.

[0044] The figure marks in the drawings of the specification include: 1. tank body; 2. feed pipe; 3. liquid injection pipe; 4. discharge pipe; 5. arc tube; 6. hollow cover; 7. elbow; 8. duckbill mouth; 9. output valve. DETAILED DESCRIPTION

[0045] The following is further described in detail through specific implementation methods:

[0046] Example 1

[0047] Basically as attached Figure 1 、 Figure 2 and Figure 3 A method for bioremediation of soil by using crude oil-degrading fungi, comprising the following steps:

[0048] Step 1: Targeted screening of crude oil-degrading fungal strains. Indigenous fungi are isolated from crude oil-contaminated areas and screened based on salt and low-temperature tolerance. Salt tolerance refers to a salt concentration greater than 5% and a low-temperature tolerance range of 4-15°C. Priority is given to strains with broad-spectrum degradation capabilities for C10-C40 alkanes and 3-5-ring PAHs. Transcriptomics is used to screen strains with high expression of laccase and lignin peroxidase. Resveratrol inducer is used to increase enzyme activity and enhance the oxidative degradation efficiency of PAHs.

[0049] Step 2: Enhance the function of the screened strains. Through adaptive laboratory evolution technology, the tolerance of the screened strains to high-concentration oil pollution and extreme pH is improved. High-concentration oil pollution refers to a concentration greater than 2% and an extreme pH range of 3-10, to ensure its activity and stability in complex environments. A composite bacterial community consisting of core degradation bacteria, auxiliary bacteria and growth-promoting bacteria is constructed. The core degradation bacteria are the strains with the highest crude oil degradation efficiency. The auxiliary bacteria are Trichoderma, which secretes cellulase to enhance pollutant dissolution. The growth-promoting bacteria are arbuscular mycorrhizal fungi, which coexist with plants to promote ecological restoration.

[0050] Step 3: Preparation of the repair carrier, preparing biochar particles, using chitosan to embed fungal spores of the composite bacterial community to form chitosan balls, using biochar to adsorb and load the chitosan balls in an adsorption preparation device to form composite microspheres, and modifying the surface of the composite microspheres with hydrophobic groups for targeted adsorption of crude oil pollutants. The composite microspheres have a pH-responsive release function, the hydrophobic groups are octadecyl or phenyl, and the pH-responsive release function is to slowly release spores when the soil pH is less than 6 and accelerate spore germination when the pH is 7-8. It also includes adding lignin-derived slow-release fertilizers and lipopeptide biosurfactants to the composite microspheres.

[0051] Step 4: Gradient technology and composite bacterial community synergistic remediation. For high-concentration pollution areas, that is, areas with crude oil content greater than 2%, fungi-biochar combination is used, and fungi-plant combination is used in medium and low-concentration pollution areas, and remediation parameters are adjusted through dynamic monitoring. Fungi-biochar combination includes laying a biochar adsorption layer on the surface of the contaminated soil to form a degradation buffer zone, and then spraying fungal microspheres; fungi-plant combination includes planting ryegrass in the later stage of remediation, and enhancing plant tolerance through fungal hyphae secreting indoleacetic acid, and extending the remediation depth to less than 1.5m; dynamic monitoring and adjustment of remediation parameters include implanting microbial electrochemical sensors during the remediation stage to monitor the concentrations of phenanthrene and pyrene in the soil in real time, and regulating the frequency of nutrient solution injection through Internet of Things technology.

[0052] The specific experimental process is as follows: Figure 2 As shown in Figure 1, Targeted screening experiment of crude oil degrading fungal strains

[0053] 1. Sample collection and pretreatment

[0054] Sampling location: Long-term crude oil contamination areas of Daqing Oilfield in different eras, 0-20cm surface soil around oil wells.

[0055] Pretreatment: After the oily soil is evenly mixed, 5.0 g is weighed and added to 50 mL of sterile water. After oscillation and dispersion, the mixture is allowed to stand. 100 μL of the soil suspension is then diluted to 10 -1 -10 -4 .

[0056] 2. Enrichment culture and initial screening

[0057] Enrichment medium formula: Each liter includes crude oil (C 10 -C 40 Alkanes and PAHs mixture) 10g; NaCl 50g (simulating a high-salt environment); KH2PO4 1g, MgSO4·7H2O 0.5g; agar 15g, pH 6.5

[0058] Culture conditions: The diluted solution was inoculated into the enrichment medium, cultured with shaking at 15°C and 150 rpm for 7 days, and subcultured three times.

[0059] 3. Separation and purification

[0060] Selective plate separation: The enriched solution was diluted gradiently and spread on the separation medium containing 0.1% benzopyrene, cultured at 15°C for 14 days, and single colonies were picked for purification.

[0061] Storage: The purified strain was stored on a PDA slant containing 5% NaCl.

[0062] 4. Morphological observation

[0063] Colony morphology: record color, surface features, edges, height, etc.

[0064] Mycelial morphology: Mycelia were collected using the transparent tape method and observed under an optical microscope.

[0065] 5. Determination of physiological and biochemical characteristics

[0066] Gelatin liquefaction: Inoculate gelatin culture medium and observe the liquefaction phenomenon.

[0067] Starch hydrolysis: seed starch culture medium and detect blue reaction with iodine solution.

[0068] Cellulose hydrolysis: Filter paper strips were inserted into the cellulose culture medium and the growth was observed.

[0069] 6. Molecular Biology Identification

[0070] DNA extraction: Genomic DNA was extracted using the modified CTAB method.

[0071] ITS-PCR amplification: primers ITS1 / ITS4, amplification conditions: 94°C pre-denaturation 2 min, 35 cycles (94°C 30 s, 59°C 30 s, 72°C 90 s), 72°C extension 10 min.

[0072] Sequencing and alignment: After sequencing of PCR products, the genus of close relatives was determined by NCBI BLAST alignment.

[0073] 7. Degradation performance verification

[0074] Shaking flask experiment: the strain was inoculated into liquid medium containing 1% crude oil (pH 6.5, 5% NaCl) and cultured at 15°C, 180 rpm for 21 days. The C 10 -C 40 alkane and 3-5 ring PAHs degradation rate.

[0075] Enzyme activity induction: the addition of veratryl alcohol (0.5 mM) induced laccase and lignin peroxidase activity. ABTS method and guaiacol method were used to determine enzyme activity.

[0076] 8. Experimental results

[0077] Through screening of solid screening medium, 12 strains of fungi with crude oil degradation ability were isolated according to the growth rate and growth state of the colonies. About 1 cm 2 of the colony was cut with sterilized forceps at the edge of the colony and placed on a PDA solid plate. After several days of inverted culture at 28°C, the colony morphology, color, and presence or absence of exudates of each strain were observed and recorded. The colony characteristics are shown in Table 1 below:

[0078] Table 1. Colony morphology and close relative genus of 12 strains of crude oil degrading fungi

[0079]

[0080] The 12 strains of crude oil degrading fungi isolated all have the characteristics of hydrolyzing starch and degrading cellulose. In addition to strains No. 6, No. 10, No. 11, and No. 12, the remaining 8 strains of fungi all showed positive in the gelatin liquefaction experiment, indicating that strains No. 10, No. 11, and No. 12 do not produce protease, while all 12 strains of fungi have amylase and cellulase activity.

[0081] The genomic DNA of the 12 strains of crude oil degrading fungi was subjected to PCR amplification with specific primers, and specific fragments of about 500 bp were obtained. After recovery and sequencing with the kit, homology comparison was performed in NCBI BLAST to find close relatives of known fungi. The results are shown in Table 1.

[0082] During the culture process in crude oil liquid medium, the dry weight of fungal mycelia generally showed a trend of first increasing and then decreasing. This may be related to the autolysis of mycelia caused by the large consumption of nutrients in the culture medium. Therefore, when measuring the growth rate of each crude oil-degrading fungus, the data before the dry weight of mycelia began to decrease were used to calculate the average growth rate. The formula is as follows:

[0083]

[0084] The crude oil degradation rate on the 25th day of culture was used as a reference value for evaluating the crude oil degradation ability of each crude oil-degrading fungus. A 1% crude oil liquid culture medium without fungi was used as a blank control. In addition, crude oil-degrading fungi were cultured under different pH conditions, different nitrogen source conditions, and different phosphorus source conditions. The growth of the 12 fungal strains under different conditions was observed, and the strains with the best growth conditions were selected as candidate strains for the composite bacterial consortium. The results are shown in Table 2 below:

[0085] Table 2. Optimal growth conditions and degradation rates

[0086]

[0087] According to the above results, strain No. 6 has the highest crude oil degradation efficiency and was selected as the core degradation strain, while strain No. 12, belonging to the genus Trichoderma, was selected as the auxiliary strain.

[0088] Table 3. Increased enzyme activity after resveratrol induction

[0089]

[0090] As shown in Table 3, after induction by resveratrol, the laccase activity of strain 6 increased by 64.7% and the lignin peroxidase activity increased by 52.1%, verifying the enhancing effect of the inducer on the oxidative degradation of PAHs.

[0091] 2. Adaptive Laboratory Evolution (ALE) Experiment

[0092] 1. Pre-culture of strains

[0093] Strain No. 6 was inoculated into an inorganic salt medium (pH 7.0) containing 1% crude oil and cultured with shaking at 28° C. and 160 rpm until the logarithmic phase (OD600=0.6).

[0094] Strain No. 12: Inoculate into PDA medium containing 1% carboxymethyl cellulose (CMC) and culture at 28°C until the mycelium covers the plate.

[0095] 2. Pressure gradient design

[0096] Strain 6: crude oil concentration gradient 1%→3%→5% (10 passages per gradient, 30 generations in total); pH gradient, initial pH 7.0, gradually adjusted to pH 5.0 and 9.0 (pH change ±0.5 per generation).

[0097] Strain No. 12: Carbon source limitation, gradually reducing the CMC concentration from 1% to 0.5% to 0.2% to induce cellulase secretion.

[0098] 3. Subculture and screening

[0099] Strain No. 6: Each generation was cultured for 72 hours, and the top 10% of the bacterial solution with the highest growth rate (OD600) was taken for subculture.

[0100] Strain No. 12: Each generation was cultured for 7 days, and the colony with the highest cellulase activity was selected for subculture.

[0101] 4. Tolerance improvement verification

[0102] Strain 6: High-concentration crude oil tolerance, comparing growth rate (OD600) and degradation rate in 2% crude oil before and after evolution. Extreme pH adaptability, measuring survival rate (CFU count) at pH 3.0 and 10.0.

[0103] Strain No. 12: Response to low nutrient stress, detecting changes in cellulase activity under 0.2% CMC conditions.

[0104] 5. Functional collaborative verification

[0105] Co-culture degradation experiment: Treatment group: Evolved strain 6 + strain 12 (ratio 5:1) inoculated into soil containing 3% crude oil. Control group: original strain 6 + strain 12, single strain 6, and single strain 12.

[0106] Detection indicators: crude oil degradation rate, soil cellulase activity and mycelium extension depth.

[0107] 6. Genomic Analysis

[0108] Targeted gene sequencing: strain 6, laccase gene (Lac) and cytochrome P450 gene (CYP450). Strain 12, cellulase gene (CMCase). Mutation analysis, comparing gene sequences before and after evolution, to identify adaptive mutation sites.

[0109] 7. Experimental Results

[0110] Through ALE evolution, the crude oil degradation rate of strain No. 6 increased by 75.3%, its survival rate in an extreme pH environment of 3.0 increased by at least 400 times, and its laccase activity increased by 64.7%. The cellulase activity of strain No. 12 increased by 93.1%, and its hyphae expansion rate at least doubled, greatly enhancing its targeted functions as a core degradation bacteria and auxiliary bacteria. The results are shown in Tables 4 and 5 below:

[0111] Table 4. Comparison of performance of strain No. 6 after ALE evolution

[0112]

[0113] Table 5. Comparison of performance of strain No. 12 after ALE evolution

[0114]

[0115] Table 6. Degradation effect of co-culture

[0116]

[0117] In the co-culture degradation experiment, it was found that the effect of the co-culture of the evolved No. 6 and No. 12 corresponded to three indicators that far exceeded the original strains, indicating that the ALE evolution experiment enhanced the corresponding functions of the strains, thereby improving their mixed degradation capabilities and making them suitable for subsequent soil remediation work.

[0118] like Figure 3 As shown, III. Preparation of composite microspheres

[0119] 1. Biochar pretreatment

[0120] Raw material processing: Corn straw was crushed to 1-2 mm, pyrolyzed at 600°C for 2 hours in oxygen-limited conditions, and ground through a 200-mesh sieve. Biochar powder was soaked in a 5% HNO3 solution for 24 hours, washed with deionized water until neutral, and dried at 60°C for later use.

[0121] 2. Preparation of chitosan solution

[0122] 2 g of chitosan was dissolved in 100 mL of 1% acetic acid solution and stirred magnetically (500 rpm, 40° C.) until completely dissolved. The solution was filtered through a 0.22 μm filter membrane to remove insoluble impurities.

[0123] 3. Preparation of Spore-Chitosan Mixture

[0124] Spore suspension: spores of strains 6 and 12 were suspended at a ratio of 5:1, and 1×10 8 The spore suspension was suspended in sterile PBS (pH 7.4) at a concentration of 100 CFU / mL. The spore suspension was mixed with chitosan solution at a volume ratio of 1:3 and ultrasonically dispersed (40 kHz, 10 min) until homogeneous.

[0125] 4. Microsphere formation and cross-linking

[0126] The biochar and chitosan solution were mixed into a biochar suspension at a mass ratio of 2:1 and supplied to the adsorption preparation device through the feed pipe 2. The spore-chitosan mixture was added layer by layer into the biochar suspension through the liquid injection pipe 3 at a speed of 30 rpm, forming a "droplet-biochar" coated structure. After completion, 5% TPP solution (volume ratio 1:10) was added to induce chitosan cross-linking and solidification to form composite microspheres.

[0127] 5. Hydrophobic group modification

[0128] 0.5% octadecyltrimethoxysilane (v / v) was added to the microsphere suspension, and the reaction was carried out at 60°C for 2 hours. After the reaction, the composite microspheres were washed with deionized water 3 times to remove unreacted reagents.

[0129] 6. Slow-release nutrient loading

[0130] Ammoniated lignin fertilizer (2% w / v) and lipopeptide surfactant (0.1% w / v) were added to the microsphere suspension and shaken for 12 hours for adsorption. The microspheres were freeze-dried at -20°C for 24 hours to obtain dry composite microspheres.

[0131] 7. pH-responsive verification

[0132] Release kinetics test: The microspheres were placed in pH 5.0, 6.0, 7.0 buffer solution and shaken at 37°C (100 rpm), and the spore release amount was determined at regular intervals (hemocytometer method).

[0133] Results required: 24-hour release rate <20% at pH 5.0, release rate >80% at pH 7.0.

[0134] 8. Experimental results

[0135] The adsorption efficiency of the prepared composite microspheres in 1g / L crude oil can reach 85%, the mechanical strength is 0.68N / ball, and the spore release amount can be dynamically adjusted according to the pH value, as shown in the following Table 7:

[0136] Table 7. Spore release kinetics of composite microspheres under different pH conditions

[0137]

[0138]

[0139] Four, soil remediation experiment

[0140] 1. Experimental group setting

[0141] Experimental group 1 (T1), composite microspheres were used alone (surface spray, dose 5g / m 2 ), covered with biochar adsorption layer (1 cm thick), experimental group 2 (T2), composite microspheres + ryegrass combined (ryegrass was planted after microsphere spraying, density 50 particles / m 2 ), control group, untreated contaminated soil.

[0142] Experimental conditions: temperature 15°C (simulating the winter environment of Daqing Oilfield), humidity maintaining soil moisture content at 20%-25%, monitoring periods 0, 7, 14, 21, and 28 days.

[0143] 2. Soil pretreatment

[0144] The contaminated soil was passed through a 2 mm sieve to remove stones and impurities, and mixed evenly. Three replicates were performed for each treatment group, and 5 kg of each soil was placed in a culture pot (20 cm in diameter and 30 cm in depth).

[0145] 3. Microsphere administration and dynamic monitoring and maintenance

[0146] The contaminated soil was treated according to the experimental group settings described above, and 50 mL of nutrient solution containing ammoniated lignin (0.1% w / v) was injected every 7 days. BES sensors were implanted in the soil to record the changes in phenanthrene and pyrene concentrations in real time. Soil samples were taken at the end of each cycle and GC-MS was used to detect C 10 -C 40 Residual amounts of alkanes and PAHs.

[0147] 4. Microbial activity detection

[0148] Laccase (Lac) activity: determined by ABTS method (unit: U / g soil).

[0149] Cellulase activity: The reducing sugar release was determined by the DNS method (unit: mg / g·h).

[0150] 5. Data Recording

[0151] Plant growth indicators: ryegrass plant height and root length (T2 group).

[0152] Soil physical and chemical properties: pH value, salinity (electrical conductivity).

[0153] 6. Experimental Results

[0154] Table 8. Comparison of crude oil degradation rates in different treatment groups (28 days)

[0155]

[0156] Table 9. Microbial activity and soil property changes (28 days)

[0157]

[0158] Table 10. Ryegrass growth indicators

[0159]

[0160] T1 group vs. C 10 -C 20 The alkane degradation rate reached 78.5%, and the T2 group (combined plants) increased to 89.2%, indicating that the plant roots and fungi synergized. Because the root secretions of ryegrass promoted the activation of microspores, the PAHs degradation rate in the T2 group (73.8%) was significantly higher than that in the T1 group (55.1%). The laccase activity of the T2 group (248.9 U / g) was 7.7 times that of the control group, confirming that the fungus-plant symbiotic system activated the degradation enzyme system. The soil salinity dropped from 6.8mS / cm to 3.5mS / cm, and the microsphere slow-release nutrient alleviated salt stress. The biomass of ryegrass in the T2 group increased 46 times in 28 days, and the root depth expanded to 18.7cm, verifying the improvement effect of the composite remediation technology on soil structure.

[0161] Example 2

[0162] The difference from the above embodiment is that, as shown in the attached Figure 3 、 Figure 4 As shown: the adsorption preparation device in step three includes a tank body 1, the top wall of the tank body 1 is rotatably connected to a liquid injection pipe 3, the liquid injection pipe 3 extends into the interior of the tank body 1, the liquid injection pipe 3 is divided into several layers, each layer is fixedly connected to several curved pipes 7, the curved pipes 7 have the same curvature direction, the outer periphery of the end of the curved pipe 7 is fixedly connected to a limiting plate, the limiting plates are slidably fitted with a hollow cover 6, the outer periphery of the hollow cover 6 is connected to several arc tubes 5, the arc tubes 5 on the same hollow cover 6 have the same curvature direction, one side of the tank body 1 is connected to a feeding pipe 2, the end of the feeding pipe 2 located inside the tank body 1 is connected to a duckbill mouth 8, the bottom wall of the tank body 1 is connected to an output valve 9, and the output valve 9 is connected to a discharge pipe 4.

[0163] The specific implementation process is as follows: Figure 4 As shown, during the mixing process of the spore-chitosan mixture and the biochar suspension, sufficient stirring and mixing are required to promote cross-linking to form composite microspheres with uniform biochar coating on the periphery. Furthermore, sufficiently extended and dispersed pipes are required to inject the spore-chitosan mixture to improve the efficiency of forming the core of the composite microspheres, thereby improving the speed and quality of composite microsphere preparation. In the adsorption preparation device, when the spore-chitosan mixture is introduced into the tank body 1 through the injection pipe 3, the spore-chitosan mixture with a certain pressure first enters the bend pipe 7 through the injection pipe 3 in layers. When flowing in the bend pipe 7, the pressure will push the bend pipe 7 to rotate tangentially, thereby driving the horizontal rotation of the entire injection pipe 3. At this time, the biochar suspension enters the tank body 1 through the duckbill 8 and is hit by the rotating bend pipe 7 to be dispersed throughout the tank body 1, promoting uniform dispersion.

[0164] Then the spore-chitosan mixture enters the arc tube 5 through the hollow cover 6, which also drives the arc tube 5 and the hollow cover 6 to rotate in the vertical plane along the tangent direction of the arc of the arc tube 5, so that the spore-chitosan mixture finally contacts the biochar at a vertical angle, which not only stirs the biochar suspension horizontally, but also mixes and cross-links with the biochar suspension in the vertical plane to form complete and uniform composite microspheres, thereby fully utilizing the biochar material.

[0165] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0166] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for bioremediation of soil using crude oil-degrading fungi, characterized by: The following steps are included: Step 1: Directed screening of crude oil degrading fungal strains: isolate indigenous fungi from crude oil contaminated areas, screen the isolated indigenous fungi based on salt and low temperature tolerance, and give priority to those with C 10 -C 40 Strains with broad-spectrum degradation capabilities for alkanes and 3-5-ring PAHs were selected through transcriptomics to screen strains with high expression of laccase and lignin peroxidase, and inducers were used to enhance enzyme activity; Step 2: Enhance the function of the selected strains by using adaptive laboratory evolution technology to improve the tolerance of the selected strains to high-concentration oil pollution and extreme pH, ensure their activity and stability in complex environments, and construct a complex bacterial community; Step 3: Preparation of a remediation carrier, preparing biochar particles, using chitosan to embed fungal spores of a composite bacterial community to form chitosan pellets, using biochar to adsorb and load the chitosan pellets in an adsorption preparation device to form composite microspheres, and modifying the surface of the composite microspheres with hydrophobic groups to target the adsorption of crude oil pollutants, and the composite microspheres have a pH-responsive release function; Step 4: Gradient technology and composite bacterial community synergistic remediation: For high-concentration pollution areas, that is, areas with crude oil content greater than 2%, fungi-biochar combination is used, and fungi-plant combination is used for medium and low-concentration pollution areas, and remediation parameters are adjusted through dynamic monitoring.

2. The method for bioremediation of soil by crude oil-degrading fungi according to claim 1, characterized in that: The salt tolerance condition in step 1 refers to a salt concentration greater than 5%, and a low temperature tolerance range of 4-15°C.

3. The method for bioremediation of soil by crude oil-degrading fungi according to claim 2, characterized in that: The inducer in step one includes resveratrol, which increases enzyme activity and is used to enhance the oxidative degradation efficiency of PAHs by the indigenous strain.

4. The method for bioremediation of soil by crude oil-degrading fungi according to claim 1, characterized in that: The high concentration oil pollution in step 2 refers to a concentration greater than 2% and an extreme pH range of 3-10.

5. The method for bioremediation of soil by crude oil-degrading fungi according to claim 4, characterized in that: The composite bacterial community in step 2 includes core degradation bacteria, auxiliary bacteria and growth-promoting bacteria.

6. The method for bioremediation of soil by crude oil-degrading fungi according to claim 5, characterized in that: The core degradation bacteria in step 2 are the strains with the highest crude oil degradation efficiency. The auxiliary bacteria are Trichoderma, which secretes cellulase to enhance pollutant dissolution. The growth-promoting bacteria are arbuscular mycorrhizal fungi, which coexist with plants to promote ecological restoration.

7. The method for bioremediation of soil by crude oil-degrading fungi according to claim 1, characterized in that: The hydrophobic group in step three is octadecyl or phenyl.

8. The method for bioremediation of soil using crude oil-degrading fungi according to claim 7, characterized in that: The pH-responsive release function in step three is to slowly release spores when the soil pH is less than 6 and accelerate spore germination when the pH is 7-8.

9. The method for bioremediation of soil using crude oil-degrading fungi according to claim 8, characterized in that: The adsorption preparation device in step three includes a tank body (1), the top wall of the tank body (1) is rotatably connected to a liquid injection pipe (3), the liquid injection pipe (3) extends into the interior of the tank body (1), the liquid injection pipe (3) is divided into several layers, each layer is fixedly connected to several curved pipes (7), the curved pipes (7) have the same curvature direction, the outer periphery of the end of the curved pipe (7) is fixedly connected to a limiting plate, the limiting plate is slidably matched with a hollow cover (6), the outer periphery of the hollow cover (6) is connected to several arc tubes (5), the arc tubes (5) on the same hollow cover (6) have the same curvature direction, one side of the tank body (1) is connected to a feed pipe (2), one end of the feed pipe (2) located inside the tank body (1) is connected to a duckbill (8), the bottom wall of the tank body (1) is connected to an output valve (9), and the output valve (9) is connected to a discharge pipe (4).

10. The method for bioremediation of soil by crude oil-degrading fungi according to claim 9, characterized in that: Step three also includes adding lignin-derived slow-release fertilizer and lipopeptide biosurfactant to the composite microspheres.

11. The method for bioremediation of soil using crude oil-degrading fungi according to claim 1, characterized in that: The fungus-biochar combination in step four involves laying a biochar adsorption layer on the surface of the contaminated soil to form a degradation buffer zone, and then spraying fungal microspheres.

12. The method for bioremediation of soil using crude oil-degrading fungi according to claim 11, characterized in that: The fungus-plant combination in step 4 includes planting ryegrass in the later stages of restoration and enhancing plant tolerance through the secretion of indoleacetic acid by fungal hyphae, and the restoration depth is extended to less than 1.5m.

13. The method for bioremediation of soil using crude oil-degrading fungi according to claim 12, characterized in that: The dynamic monitoring and adjustment of remediation parameters in step four includes implanting microbial electrochemical sensors during the remediation phase to monitor the concentrations of phenanthrene and pyrene in the soil in real time, and regulating the frequency of nutrient solution injection through Internet of Things technology.

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