A method for bioremediation of soil by crude oil degrading fungi

Abstract

The application discloses a method for bioremediating soil by crude oil-degrading fungi in the field of microorganisms and biotechnology, and comprises the following steps: step one, directional screening of a crude oil-degrading fungal strain; step two, function strengthening of the screened strain; step three, preparation of a repair carrier; and step four, gradient process and collaborative repair of a composite microbial population. The application realizes efficient repair of crude oil pollution through the following steps: screening and separating indigenous fungi in a crude oil pollution area, carrying out adaptive laboratory evolution on the screened strain to strengthen the degradation capacity and environmental tolerance, combining the preparation process of composite microspheres to realize adsorption-degradation efficient repair of crude oil pollution, and using gradient process and other biological methods for collaborative repair measures to further strengthen the repair effect, stabilize the natural ecological environment, avoid secondary pollution in the repair process, and reduce the risk of ecological pollution.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, specifically a method for the bioremediation of soil by crude oil-degrading fungi. Background Technology

[0002] Throughout the entire chain of the oil and petrochemical industry—from exploration and extraction to transportation, processing, storage, and sales—poor management or accidents can lead to oil spills, causing serious environmental damage. Statistics show that approximately 8 million tons of crude oil enter the environment globally each year, polluting soil, groundwater, rivers, and oceans. Sources of oil pollutants in soil mainly include crude oil spills and sludge, the accumulation of oily slag, sludge, and garbage, wastewater irrigation, air pollution and vehicle exhaust emissions, and chemical contamination.

[0003] In the field of crude oil contaminated soil remediation, various treatment methods exist, mainly including physical, chemical, and biological methods. However, these existing methods all have varying degrees of drawbacks. Physical methods, such as the use of oil spill treatment agents (like surface cleaners and chemical oil collectors), while capable of microbial degradation, temporarily slow down the degradation rate of petroleum hydrocarbons, especially for high molecular weight polycyclic aromatic hydrocarbons (PAHs), which have high residual toxicity. When dealing with high concentrations of contaminated soil, ex-situ thermal desorption is required, but this method is energy-intensive and may damage the soil structure.

[0004] Chemical methods also face many problems, such as the potential generation of secondary pollutants, like chloroform, during chemical oxidation, which can cause new harm to the environment.

[0005] While biological methods hold immense potential, they also face several limitations at present. Firstly, plant root systems are inhibited in high-concentration oil pollution (>2%), leading to a significant decrease in remediation effectiveness. Furthermore, they are only suitable for moderately polluted soils, and the remediation depth is limited by root distribution. Secondly, natural bacterial strains typically degrade only a few pollutants, making them ineffective in dealing with complex pollution scenarios, such as the coexistence of petroleum hydrocarbons and PAHs. Relying on genetically engineered strains poses ecological risks of environmental release, as illustrated in patent document CN111808770B.

[0006] Therefore, it is necessary to propose a method for the bioremediation of crude oil-contaminated soil using crude oil-degrading fungi prepared by combining indigenous strains with chemical microparticles and combining biological and chemical remediation methods, while avoiding the generation of secondary pollutants and reducing the risk of ecological pollution. Summary of the Invention

[0007] To address the aforementioned issues, the present invention aims to provide a method for the bioremediation of soil by crude oil-degrading fungi. This method involves screening and isolating indigenous fungi from crude oil-polluted areas, subjecting the screened strains to adaptive laboratory evolution to enhance their degradation and environmental tolerance, and combining this with a composite microsphere preparation process to achieve highly efficient adsorption-degradation remediation of crude oil pollution. Furthermore, it utilizes gradient processes and other biological methods as synergistic 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] To achieve the above objectives, the technical solution of the present invention is as follows: a method for bioremediation of soil by fungi that degrade crude oil, comprising the following steps:

[0009] Step 1: Targeted screening of crude oil-degrading fungal strains. Indigenous fungi are isolated from crude oil-contaminated areas. Indigenous fungi isolated are screened based on salt and low-temperature tolerance, with priority given to those exhibiting resistance to C44. 10 -C 40 Strains with broad-spectrum degradation capabilities of alkanes and 3-5 ring PAHs were screened by transcriptomics to identify strains that highly express laccase and lignin peroxidase, and enzyme activity was enhanced using inducers.

[0010] Step 2: Enhance the function of the selected strains. Through adaptive laboratory evolution technology, improve the tolerance of the selected strains to high concentrations of oil and extreme pH, ensure their activity stability in complex environments, and construct a complex microbial community containing core degradation bacteria, auxiliary bacteria and growth-promoting bacteria.

[0011] Step 3: Preparation of the repair carrier. Biochar microparticles are prepared. Chitosan is used to encapsulate fungal spores of the complex microbial community to form chitosan microbial balls. In the adsorption preparation device, biochar is used to adsorb and load the chitosan microbial balls to form composite microspheres. Hydrophobic groups are modified on the surface of the composite microspheres to target and adsorb crude oil pollutants. The composite microspheres have pH-responsive release function.

[0012] Step 4: Gradient process and composite microbial community synergistic remediation. For high-concentration pollution areas, i.e. areas with crude oil content >2%, fungi-biochar combination is used, while for medium and low-concentration pollution areas, fungi-plant combination is used, and remediation parameters are adjusted through dynamic monitoring.

[0013] Furthermore, the salt tolerance conditions in step one refer to a salt concentration > 5% and a low temperature tolerance range of 4-15℃.

[0014] Furthermore, the inducer in step one includes resveratrol, which enhances enzyme activity to improve the oxidative degradation efficiency of PAHs by indigenous strains.

[0015] Furthermore, in step two, high-concentration oil contamination refers to a concentration >2% and an extreme pH range of 3-10.

[0016] Furthermore, the complex microbial community in step two includes core degrading bacteria, auxiliary bacteria, and growth-promoting bacteria.

[0017] Furthermore, in step two, 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, and the growth-promoting bacteria are arbuscular mycorrhizal fungi, which live in symbiosis with plants to promote ecological restoration.

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

[0019] Furthermore, the pH-responsive release function in step three involves the slow release of spores when the soil pH is <6, and the accelerated spore germination when the pH is 7-8.

[0020] Furthermore, the combined use of fungi and biochar 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 fungal-plant combination in step four includes planting ryegrass in the later stages of remediation and enhancing plant tolerance through the secretion of indoleacetic acid by fungal hyphae, extending the remediation depth to below 1.5m.

[0022] Furthermore, the dynamic monitoring and adjustment of remediation parameters in step four 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 nutrient solution injection frequency through Internet of Things technology.

[0023] The basic principle of the scheme is as follows: In the targeted screening stage of crude oil-degrading fungal strains, indigenous fungi are isolated from crude oil-contaminated areas, and salt tolerance and low-temperature tolerance are used as key screening conditions, prioritizing those with specific targets for C2C4. 10 -C 40 Fungal strains with broad-spectrum degradation capabilities for alkanes and 3-5 ring PAHs were selected. Transcriptomic screening was used to identify strains highly expressing laccase and lignin peroxidase, and resveratrol was used as an inducer to enhance enzyme activity and increase the oxidative degradation efficiency of PAHs. The principle behind this approach is to focus on screening for fungal strains that can stably survive under harsh environmental conditions (such as high salinity and low temperature) and possess the ability to efficiently degrade specific crude oil components. This lays a solid foundation for subsequent remediation work, ensuring that the selected strains can accurately and effectively degrade crude oil contaminants.

[0024] Entering the stage of strain enhancement, adaptive laboratory evolution techniques were employed to improve the tolerance of the selected strains to high concentrations of oil pollution and extreme pH levels, ensuring their activity and stability under complex environments. Simultaneously, a complex microbial community was constructed, comprising core degrading bacteria, auxiliary bacteria, and growth-promoting bacteria. This principle reflects the in-depth exploration and optimization of strain performance, strengthening the strains by simulating potentially complex pollution environments, enabling them to better adapt to varying soil conditions during actual remediation. Constructing the complex microbial community leverages the complementary strengths of different species: core degrading bacteria focus on efficiently decomposing the main components of crude oil; auxiliary bacteria enhance pollutant dissolution by secreting substances such as cellulase, breaking the tight bond between pollutants and soil particles; and growth-promoting bacteria coexist with plants to promote ecological restoration. This synergistic approach comprehensively improves remediation efficiency and quality.

[0025] In the preparation of the remediation carrier, biochar microparticles are prepared. Chitosan-encapsulated fungal spores from a composite microbial community are used to form chitosan microbial balls. These chitosan microbial balls are then loaded with biochar to form composite microspheres. Hydrophobic groups are modified on the surface of the composite microspheres to target and adsorb crude oil contaminants, and the microspheres are endowed with pH-responsive release functionality. The principle is to create a highly efficient remediation carrier integrating adsorption and degradation. Biochar has a rich porous structure and a large specific surface area, which can effectively adsorb crude oil contaminants, facilitating subsequent fungal degradation. Chitosan encapsulation of fungal spores provides protection, preventing spore inactivation under adverse external factors and controlling the spore release rate. The surface-modified hydrophobic groups can precisely target and adsorb hydrophobic contaminants in crude oil, achieving efficient contaminant enrichment. The pH-responsive release function is an adaptive design, regulating the spore release rate according to changes in soil pH, ensuring that fungal spores are activated and begin degradation at the most suitable time, avoiding the inhibition of spore activity by initial high concentrations of oil contamination, and improving the precision and effectiveness of remediation.

[0026] Finally, in the synergistic remediation stage combining gradient processes and composite microbial communities, a combination of fungi and biochar was used for high-concentration contaminated areas (crude oil content > 2%), while a combination of fungi and plants was used for medium- and low-concentration contaminated areas. Remediation parameters were adjusted through dynamic monitoring. The core principle here is to adopt different remediation strategies according to local conditions and time. For high-concentration contaminated areas, due to the high pollutant content and risk, the combination of fungi and biochar leverages the strong adsorption capacity of biochar and the efficient degradation capacity of fungi to rapidly adsorb and decompose pollutants, curbing further diffusion. For medium- and low-concentration contaminated areas, the remediation capacity of plants was combined. Through the synergistic effect of fungi and plants, utilizing the absorption and enrichment functions of plant roots and the degradation functions of fungi, deep soil purification and ecological restoration were achieved. Simultaneously, by dynamically monitoring key parameters such as changes in pollutant concentration in the soil and using IoT technology to adjust remediation parameters such as the frequency of nutrient solution injection in real time, the remediation process can be ensured to remain in optimal condition. The remediation plan can be adjusted promptly according to the actual situation, ensuring the efficiency and adaptability of the remediation work and achieving twice the result with half the effort.

[0027] The beneficial effects of the basic approach are: 1. Significantly improved repair efficiency. Through targeted screening, salt-tolerant (adaptable to >5% salinity), low-temperature resistant (maintains activity at 4-15℃), and possessing specific C... 10 -C 40 This highly efficient fungal strain, capable of broad-spectrum degradation of alkanes and 3-5 ring PAHs, precisely targets and degrades complex components in crude oil. Compared to traditional remediation methods, it significantly accelerates the degradation rate of recalcitrant high molecular weight polycyclic aromatic hydrocarbons and other pollutants, effectively reducing soil pollutant concentrations in a shorter time and shortening the remediation cycle. This is of great significance for timely remediation of crude oil pollution and restoration of land ecological functions.

[0028] 2. Strong environmental adaptability. The selected fungal strains have been enhanced through adaptive laboratory evolution technology, significantly improving their tolerance to high concentrations of oil (>2%) and extreme pH levels (3-10). This ensures stable activity and degradation capacity in soil environments of varying regions and pollution levels, especially under harsh conditions. This strong environmental adaptability allows the method of this invention to be widely applied to various crude oil contaminated soil scenarios, whether in coastal high-salinity areas, cold northern regions, or highly acidic or alkaline soil environments. It can be effectively implemented, greatly expanding its application scope and solving the problem of significantly reduced remediation effectiveness of traditional methods in complex environments.

[0029] 3. Low ecological risk. Unlike remediation methods using genetically engineered strains, this invention prioritizes the use of natural indigenous fungal strains to construct a composite microbial community, avoiding the ecological risks that may arise from exogenous genetically engineered bacteria. These indigenous strains originally existed in crude oil contaminated areas and have adapted to the local ecological environment through long-term natural selection, without disrupting the original ecological balance of the soil. Simultaneously, by rationally combining core degradation bacteria, auxiliary bacteria, and growth-promoting bacteria, a complementary ecological remediation system is formed. While degrading crude oil pollutants, it also promotes the restoration and reconstruction of the soil ecology, which is conducive to optimizing the soil microbial community structure and improving soil fertility, achieving true green remediation and being environmentally friendly.

[0030] 4. Balancing Remediation Depth and Scope. On one hand, in terms of remediation technology, a combination of fungi and biochar 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, followed by the spraying of fungal microspheres, which 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 combination of fungi and plants is used for medium- and low-concentration pollution areas. Plants such as ryegrass are planted, and the indoleacetic acid secreted by fungal hyphae enhances the plant's tolerance, extending the remediation depth to below 1.5m. This achieves the treatment of deep soil pollutants, breaking through the limitations of traditional phytoremediation depth, which is limited by root distribution, ensuring the remediation effect across the entire soil layer, making the remediation more thorough and comprehensive.

[0031] 5. High level of intelligence. By implanting microbial electrochemical sensors during the remediation stage, the concentration changes of key pollutants such as phenanthrene and pyrene in the soil are monitored in real time. Combined with IoT technology to regulate remediation parameters such as nutrient solution injection frequency, dynamic monitoring and intelligent control of the remediation process are achieved. This intelligent remediation method can adjust the remediation strategy in a timely manner according to the actual degradation of soil pollutants, avoiding blind remediation and resource waste, improving the accuracy and targeting of remediation, and making the entire remediation process more scientific, efficient, and energy-saving. It provides a modern and intelligent technical means for the remediation of crude oil contaminated soil.

[0032] 6. Significant Economic Benefits. The method of this invention fully utilizes natural materials or renewable resources such as biochar, chitosan, and lignin, reducing remediation costs. Simultaneously, by optimizing the remediation process and improving efficiency, preparation and remediation time are reduced, thereby indirectly reducing economic losses caused by land pollution and increasing the land's reuse value. Furthermore, this method minimizes reliance on chemical agents during the remediation process, avoiding the additional costs associated with treating secondary pollutants generated by chemical oxidation and other methods. Overall, it enhances the economic benefits of remediation and has promising market application prospects and promotional value.

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

[0034] The beneficial effects of the basic scheme are as follows: The unique design of the adsorption preparation device enables a more uniform and efficient combination of biochar microparticles and chitosan microbial spheres. The ingenious layout of multi-layered curved tubes and arc tubes, combined with the precise feeding through the duckbill nozzle, creates an orderly and uniform mixing flow field inside the tank. When the biochar microparticles and chitosan microbial spheres are fully mixed in the tank, the multi-layered curved tubes drive the injection pipe to rotate under liquid pressure, while the arc tubes uniformly disperse the liquid vertically, forming a three-dimensional mixing network. This multi-dimensional mixing method ensures that each chitosan microbial sphere is uniformly surrounded by an appropriate amount of biochar microparticles, avoiding local overload or underloading, 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 biosurfactants to the composite microspheres.

[0036] The beneficial effects of the basic solution are: 1. The addition of lignin-derived slow-release fertilizer and lipopeptide biosurfactants can effectively improve the degradation efficiency of the composite microspheres. The lignin-derived slow-release fertilizer provides continuous nutritional support for fungal growth, promoting fungal reproduction and metabolic activities, enabling them to decompose crude oil pollutants more efficiently. Lipopeptide biosurfactants can reduce the surface tension of water, increase the solubility and bioavailability of crude oil pollutants in water, making 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 characteristics of lignin-derived slow-release fertilizers ensure that fungi can obtain adequate nutrients under different environmental conditions, avoiding reduced activity or death due to insufficient nutrition. This enables the remediation system to maintain stable operation and efficient degradation capacity in the face of complex and variable soil environments (such as different temperatures, humidity, pH values, etc.), enhancing the environmental adaptability and reliability of the entire method.

[0038] 3. By using lignin-derived natural slow-release nitrogen and phosphorus sources and lipopeptide biosurfactants to replace traditional chemical fertilizers and surfactants, the potential pollution risks of chemicals to the soil and environment are reduced. After fulfilling their functions, these natural substances can naturally degrade into environmentally harmless components, avoiding secondary pollution problems and conforming to the concepts of green remediation and sustainable development.

[0039] 4. Lipopeptide biosurfactants have advantages such as low ecotoxicity and the ability to promote soil aggregate formation, which helps improve soil structure and physical properties. At the same time, a healthy soil environment promotes plant root growth and the reconstruction of microbial communities, thereby promoting the ecological restoration of polluted soil and accelerating the reconstruction and stabilization of soil ecosystem functions. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the method for bioremediation of soil by crude oil degradation fungi in an embodiment of the present invention.

[0041] Figure 2 This is a schematic flowchart of the method for bioremediation of soil by crude oil degradation fungi in an embodiment of the present invention.

[0042] Figure 3 This is an isometric view of the adsorption preparation apparatus in an embodiment of the present invention.

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

[0044] The reference numerals in the accompanying drawings of the instruction manual include: 1. Tank body; 2. Feed pipe; 3. Injection pipe; 4. Discharge pipe; 5. Arc pipe; 6. Hollow cap; 7. Bend pipe; 8. Duckbill nozzle; 9. Output valve. Detailed Implementation

[0045] The following detailed description illustrates the specific implementation method:

[0046] Example 1

[0047] The basics are as follows: Figure 1 , Figure 2 and Figure 3 The following steps are shown: A method for bioremediation of soil by fungal degradation of crude oil.

[0048] Step 1: Targeted screening of crude oil-degrading fungal strains. Indigenous fungi were isolated from crude oil-contaminated areas. The isolated indigenous fungi were screened based on salt tolerance and low temperature tolerance. Salt tolerance was defined as a salt concentration >5%, and low temperature tolerance was defined as a temperature range of 4-15℃. Strains with broad-spectrum degradation capabilities against C10-C40 alkanes and 3-5 ring PAHs were preferentially selected. Strains expressing high levels of laccase and lignin peroxidase were screened using transcriptomics. Resveratrol was used as an inducer to enhance enzyme activity and improve the oxidative degradation efficiency of PAHs.

[0049] Step 2: Enhance the function of the selected strains. Through adaptive laboratory evolution technology, improve the tolerance of the selected strains to high concentrations of oil pollution and extreme pH. High concentrations of oil pollution are defined as concentrations >2%, and extreme pH ranges are 3-10. Ensure their activity and stability in complex environments. Construct a complex microbial community containing core degrading bacteria, auxiliary bacteria, and growth-promoting bacteria. The core degrading 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 remediation carrier. Biochar microparticles are prepared. Chitosan is used to encapsulate fungal spores of the complex microbial community to form chitosan microbial balls. In the adsorption preparation device, biochar is used to adsorb and load the chitosan microbial balls to form composite microspheres. Hydrophobic groups are modified on the surface of the composite microspheres to target and adsorb crude oil pollutants. The composite microspheres have a pH-responsive release function. The hydrophobic groups are octadecyl or phenyl. The pH-responsive release function is to slowly release spores when the soil pH is <6 and accelerate spore germination at pH 7-8. It also includes adding lignin-derived slow-release fertilizer and lipopeptide biosurfactants to the composite microspheres.

[0051] Step 4: Gradient process and synergistic remediation with composite microbial communities. For high-concentration contaminated areas (i.e., areas with crude oil content >2%), a combination of fungi and biochar is used; for medium- and low-concentration contaminated areas, a combination of fungi and plants is used. Remediation parameters are adjusted through dynamic monitoring. The fungi-biochar combination includes laying a biochar adsorption layer on the surface of the contaminated soil to form a degradation buffer zone, followed by spraying fungal microspheres. The fungi-plant combination includes planting ryegrass in the later stages of remediation, and enhancing plant tolerance through the secretion of indoleacetic acid by fungal hyphae, extending the remediation depth to below 1.5m. The dynamic monitoring and adjustment of remediation parameters includes implanting microbial electrochemical sensors during the remediation stage to monitor the concentrations of phenanthrene and pyrene in the soil in real time, and controlling the nutrient solution injection frequency through Internet of Things technology.

[0052] The specific experimental procedure is as follows: Figure 2 As shown, the first experiment was a targeted screening experiment for crude oil-degrading fungal strains.

[0053] 1. Sample collection and pretreatment

[0054] Sampling locations: Long-term crude oil contamination areas in Daqing Oilfield, 0-20cm topsoil around oil wells.

[0055] Pretreatment: After thoroughly mixing the oil-contaminated soil, weigh 5.0g of the mixture, add 50mL of sterile water, shake to disperse, and let stand. Take 100μL of the soil suspension and serially dilute to 10⁻⁶. -1 -10 -4 .

[0056] 2. Enrichment culture and initial screening

[0057] Enrichment medium formulation: Each liter contains crude oil (C 10 -C 40 Mixture of alkanes and PAHs 10g; NaCl 50g (simulating a high-salt environment); KH₂PO₄ 1g; MgSO₄·7H₂O 0.5g; agar 15g; pH 6.5

[0058] Culture conditions: Inoculate the diluted solution into the enrichment medium and culture at 15°C and 150 rpm for 7 days with shaking, and subculture 3 times consecutively.

[0059] 3. Separation and purification

[0060] Selective plate isolation: The enrichment solution was serially diluted and spread onto an isolation medium containing 0.1% benzo[a]pyrene. The medium was incubated at 15°C for 14 days, and single colonies were picked for purification.

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

[0062] 4. Morphological observation

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

[0064] Hyphae morphology: Hyphae 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 with gelatin culture medium and observe the liquefaction phenomenon.

[0067] Starch hydrolysis: Starch culture medium was spotted, and the blue reaction was detected with iodine solution.

[0068] Cellulose hydrolysis: Insert filter paper strips into cellulose culture medium and observe growth.

[0069] 6. Molecular biological identification

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

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

[0072] Sequencing and alignment: After sequencing the PCR products, closely related species were determined by alignment using NCBI BLAST.

[0073] 7. Degradation performance verification

[0074] Shake-flask experiment: The strain was inoculated into liquid medium containing 1% crude oil (pH 6.5, 5% NaCl) and cultured at 15℃ and 180 rpm for 21 days. C60 was determined by GC-MS. 10 -C 40 Degradation rate of alkanes and 3-5 ring PAHs.

[0075] Enzyme activity induction: Resveratrol (0.5 mM) was added to induce laccase and lignin peroxidase activities. Enzyme activities were determined using the ABTS method and the guaiacol method.

[0076] 8. Experimental Results

[0077] Through screening on solid screening medium, based on the growth rate and state of the colonies, a total of 12 fungi with crude oil degradation capabilities were isolated. Approximately 1 cm of each strain was cut from the edge of the colony using sterile forceps. 2 The bacterial colonies were placed on PDA solid plates and incubated upside down at 28°C for several days. The colony morphology, color, and presence or absence of exudate of each strain were observed and recorded. Colony characteristics are shown in Table 1 below:

[0078] Table 1.12 Colony morphology and closely related species of crude oil degrading fungi

[0079]

[0080] All 12 isolated crude oil-degrading fungi possess the ability to hydrolyze starch and degrade cellulose. Except for strains 6, 10, 11, and 12, the other 8 fungi showed positive results in the gelatin liquefaction experiment, indicating that strains 10, 11, and 12 do not produce proteases, while all 12 fungi have amylase and cellulase activities.

[0081] PCR amplification of genomic DNA from 12 crude oil-degrading fungi was performed using specific primers, yielding a specific fragment of approximately 500 bp. This fragment was recovered and sequenced using a kit, and homology was compared with known fungal species using NCBI's BLAST algorithm. The results are shown in Table 1.

[0082] During the cultivation of crude oil in liquid culture medium, the dry weight of fungal hyphae generally showed a trend of first increasing and then decreasing. This may be related to the autolysis of hyphae caused by the large consumption of nutrients in the culture medium. Therefore, when calculating the growth rate of each crude oil-degrading fungus, the average growth rate was calculated using the data before the dry weight of hyphae began to decrease, as shown in the following formula:

[0083]

[0084] The crude oil degradation rate on day 25 of cultivation 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 fungal inoculation was used as a blank control. Furthermore, crude oil-degrading fungi were cultured under different pH conditions, different nitrogen source conditions, and different phosphorus source conditions, and the growth of 12 fungal strains under different conditions was observed. The strains with the best growth were selected as candidate strains for the complex microbial community. The results are shown in Table 2 below.

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

[0086]

[0087] Based on the above results, strain 6 has the highest crude oil degradation efficiency and was selected as the core degradation strain, while strain 12, belonging to the Trichoderma genus, was selected as the auxiliary strain.

[0088] Table 3. Enzyme activity enhancement after resveratrol induction

[0089]

[0090] In Table 3, after induction with 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] II. Adaptive Laboratory Evolution (ALE) Experiments

[0092] 1. Pre-culture of bacterial strains

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

[0094] Strain No. 12: Inoculated into PDA medium containing 1% carboxymethyl cellulose (CMC) and incubated at 28°C until mycelia covered the plate.

[0095] 2. Pressure gradient design

[0096] Strain No. 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 12: Carbon source restriction, gradually reducing CMC concentration by 1% → 0.5% → 0.2% to induce cellulase secretion.

[0098] 3. Subculture and selection

[0099] Strain No. 6: After culturing for 72 hours per generation, the top 10% of the bacterial culture with the highest growth rate (OD600) was used for subculturing.

[0100] Strain No. 12: Cultured for 7 days per generation, and selected colonies with the highest cellulase activity for subculturing.

[0101] 4. Tolerance Improvement Verification

[0102] Strain 6: Tolerance to high concentrations of crude oil; growth rate (OD600) and degradation rate in 2% crude oil were compared before and after evolution. Extreme pH adaptability was assessed, with survival rates (CFU counts) determined at pH 3.0 and 10.0.

[0103] Strain 12: Low nutrient stress response, detection of changes in cellulase activity under 0.2% CMC conditions.

[0104] 5. Functional Collaboration Verification

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

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

[0107] 6. Genome analysis

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

[0109] 7. Experimental Results

[0110] The crude oil degradation rate of strain 6, evolved through ALE, increased by 75.3%, and its survival rate under extreme pH 3.0 conditions increased by at least 400-fold, while its laccase activity increased by 64.7%. Strain 12 showed a 93.1% increase in cellulase activity and at least doubled hyphal expansion rate, significantly enhancing its targeted functions as both a core and auxiliary degradative bacterium. The results are shown in Tables 4 and 5 below.

[0111] Table 4. Performance Comparison of ALE after Evolution of Strain No. 6

[0112]

[0113] Table 5.12 Performance Comparison of ALE after Evolution

[0114]

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

[0116]

[0117] In the co-culture degradation experiment, it was found that the effects of co-culturing the evolved strains No. 6 and No. 12 far exceeded those of the original strains in all three indicators. This indicates that the ALE evolution experiment enhanced the corresponding functions of the strains, thereby improving their mixed degradation ability and making them suitable for subsequent soil remediation work.

[0118] like Figure 3 As shown in Figure 3, the preparation of composite microspheres.

[0119] 1. Biochar pretreatment

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

[0121] 2. Preparation of chitosan solution

[0122] Dissolve 2g of chitosan in 100mL of 1% acetic acid solution and stir magnetically (500rpm, 40℃) until completely dissolved. Filter the solution through a 0.22μm filter membrane to remove insoluble impurities.

[0123] 3. Preparation of spore-chitosan mixture

[0124] Spore suspension: Spores of strains 6 and 12 were mixed at a ratio of 5:1, at a concentration of 1×10⁻⁶. 8 The spore suspension was suspended in sterile PBS (pH 7.4) at a concentration of 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 molding and cross-linking

[0126] Biochar and chitosan solution were mixed at a mass ratio of 2:1 to form a biochar suspension, which was supplied to the adsorption preparation device through feed pipe 2. The spore-chitosan mixture was added layer by layer to the biochar suspension through injection pipe 3 at a rotation speed of 30 rpm, forming a "droplet-biochar" coating 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. Modification with hydrophobic groups

[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 three times with deionized water to remove unreacted reagents.

[0129] 6. Loading of slow-release nutrients

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

[0131] 7. pH responsiveness verification

[0132] Release kinetics test: Microspheres were placed in buffer solutions of pH 5.0, 6.0, and 7.0, and shaken at 37°C (100 rpm). Spore release was measured at regular intervals using a hemocytometer.

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

[0134] 8. Experimental Results

[0135] The prepared composite microspheres achieved an adsorption efficiency of 85% in 1 g / L crude oil, with a mechanical strength of 0.68 N / sphere. The spore release rate could be dynamically adjusted according to pH value. The results are shown in Table 7 below.

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

[0137]

[0138]

[0139] IV. Soil Remediation Experiment

[0140] 1. Experimental group setup

[0141] Experimental group 1 (T1) used composite microspheres alone (surface spraying, dosage 5 g / m²). 2 ), covered with a biochar adsorption layer (1 cm thick), experimental group 2 (T2), composite microspheres + ryegrass combined (ryegrass planted after microsphere spraying, density 50 grains / m²). 2 (Control group, untreated contaminated soil)

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

[0143] 2. Soil pretreatment

[0144] The contaminated soil was sieved through a 2mm sieve to remove stones and impurities, and then mixed evenly. Each treatment group had 3 replicates, and each soil sample of 5kg was placed in a culture pot (20cm in diameter and 30cm in depth).

[0145] 3. Application, dynamic monitoring and maintenance of microspheres

[0146] The contaminated soil was treated according to the experimental setup described above, with 50 mL of nutrient solution containing ammoniated lignin (0.1% w / v) injected every 7 days. A BES sensor was implanted in the soil to record changes in phenanthrene and pyrene concentrations in real time. Soil samples were collected at the end of each cycle, and C was analyzed by GC-MS. 10 -C 40 Alkane and PAH residues.

[0147] 4. Microbial activity detection

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

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

[0150] 5. Data Recording

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

[0152] Soil physicochemical 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. Changes in microbial activity and soil properties (28 days)

[0157]

[0158] Table 10. Ryegrass Growth Indicators

[0159]

[0160] Group T1 vs. C 10 -C 20 The alkane degradation rate reached 78.5%, increasing to 89.2% in group T2 (with combined plants), indicating a synergistic effect between plant roots and fungi. Due to the promotion of microspore activation by ryegrass root exudates, the PAH degradation rate in group T2 (73.8%) was significantly higher than that in group T1 (55.1%). Laccase activity in group T2 (248.9 U / g) was 7.7 times that of the control group, confirming that the fungal-plant symbiotic system activated the degradation enzyme system. Soil salinity decreased from 6.8 mS / cm to 3.5 mS / cm, with the microsphere slow-release nutrient alleviating salt stress. Ryegrass biomass in group T2 increased 46-fold after 28 days, and root depth expanded to 18.7 cm, verifying the improving effect of the combined remediation technology on soil structure.

[0161] Example 2

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

[0163] The specific implementation process is as follows: Figure 4 As shown, during the mixing of the spore-chitosan mixture and the biochar suspension, it is necessary to thoroughly stir and mix to promote cross-linking and form composite microspheres with a uniform outer periphery of biochar. Simultaneously, a sufficiently long and dispersed channel is needed to inject the spore-chitosan mixture, thereby improving the efficiency of composite microsphere core formation and increasing the speed and quality of composite microsphere preparation. In the adsorption preparation device, when the injection pipe 3 introduces the spore-chitosan mixture into the tank 1, the pressurized spore-chitosan mixture first enters the bent pipe 7 in layers through the injection pipe 3. As it flows in the bent pipe 7, the pressure drives the tangential of the bend 7 to rotate, thus causing the entire injection pipe 3 to rotate horizontally. At this time, the biochar suspension enters the tank 1 through the duckbill nozzle 8 and is dispersed throughout the tank 1 by the rotating bent pipe 7, promoting uniform dispersion.

[0164] Then, the spore-chitosan mixture enters the arc tube 5 through the hollow cover 6, which in turn drives the arc tube 5 and the hollow cover 6 to rotate in the vertical plane along the tangent of the arc of the arc tube 5. This allows the spore-chitosan mixture to finally contact the biochar at a vertical angle, which not only horizontally agitates the biochar suspension but also mixes and crosslinks with the biochar suspension in the vertical plane to form complete and uniform composite microspheres, making full use of the biochar material.

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

[0166] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for bioremediation of soil by fungal degradation of crude oil, characterized in that: Includes the following steps, Step 1: Targeted screening of crude oil-degrading fungal strains. Indigenous fungi are isolated from crude oil-contaminated areas. Indigenous fungi isolated are screened based on salt and low-temperature tolerance, and those with specific resistance to C2C2 are selected. 10 -C 40 Strains with broad-spectrum degradation capabilities of alkanes and 3-5 ring PAHs were screened by transcriptomics to identify strains that highly express laccase and lignin peroxidase, and enzyme activity was enhanced using inducers. Step 2: Enhance the function of the selected strains. Through adaptive laboratory evolution technology, improve the tolerance of the selected strains to high concentrations of oil and extreme pH, ensure their activity stability in complex environments, and construct a complex microbial community. The complex microbial community includes the strain with the highest crude oil degradation efficiency as the core degradation bacteria, Trichoderma as the auxiliary bacteria, and arbuscular mycorrhizal fungi as growth-promoting bacteria. The auxiliary bacteria secrete cellulase to enhance the dissolution of pollutants, and the growth-promoting bacteria promote ecological restoration through symbiosis with plants. Step 3: Preparation of the remediation carrier. Biochar microparticles are prepared. Chitosan is used to encapsulate fungal spores of the composite microbial community and cross-link them to form chitosan microbial balls. In an adsorption preparation device, biochar is used to adsorb and load the chitosan microbial balls to form composite microspheres. Lignin-derived slow-release fertilizer and lipopeptide biosurfactants are added to the composite microspheres. Hydrophobic groups are modified on the surface of the composite microspheres to target and adsorb crude oil pollutants. The composite microspheres have a pH-responsive release function. The pH-responsive release function is that when the soil pH is <6, spores are released slowly, and when the pH is 7-8, spore germination is accelerated. The hydrophobic group in step three is octadecyl or phenyl; Step 4: Gradient process and composite microbial community synergistic remediation. For high-concentration pollution areas, i.e. areas with crude oil content >2%, fungi-biochar combination is used, and for medium and low-concentration pollution areas, fungi-plant combination is used. The remediation parameters are adjusted through dynamic monitoring.

2. The method for bioremediation of soil by fungal degradation of crude oil according to claim 1, characterized in that: The salt tolerance conditions in step one refer to a salt concentration > 5% and a low temperature tolerance range of 4-15℃.

3. The method for bioremediation of soil by fungal degradation of crude oil according to claim 2, characterized in that: The inducer in step one includes resveratrol, which enhances enzyme activity to improve the oxidative degradation efficiency of PAHs by indigenous strains.

4. The method for bioremediation of soil by fungal degradation of crude oil according to claim 1, characterized in that: In step two, high-concentration oil contamination refers to a concentration >2% and an extreme pH range of 3-10.

5. The method for bioremediation of soil by fungal degradation of crude oil according to claim 1, characterized in that: The adsorption preparation device in step three includes a tank (1), the top wall of the tank (1) is rotatably connected to a liquid injection pipe (3), the liquid injection pipe (3) extends into the interior of the tank (1), the liquid injection pipe (3) is divided into several layers, each layer is fixedly connected to several bends (7), the bends (7) have the same arc direction, the outer periphery of the end of the bends (7) is fixedly connected to a limiting piece, the limiting piece is 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 arc direction, one side of the tank (1) is connected to a feeding pipe (2), one end of the feeding pipe (2) located inside the tank (1) is connected to a duckbill mouth (8), the bottom wall of the tank (1) is connected to an output valve (9), the output valve (9) is connected to a discharge pipe (4).

6. The method for bioremediation of soil by fungal degradation of crude oil according to claim 1, characterized in that: The combined use of fungi and biochar 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.

7. The method for bioremediation of soil by fungal degradation of crude oil according to claim 6, characterized in that: The fungal-plant combination in step four includes planting ryegrass in the later stage of remediation and enhancing plant tolerance through the secretion of indoleacetic acid by fungal hyphae, extending the remediation depth to below 1.5m.

8. The method for bioremediation of soil by fungal degradation of crude oil according to claim 7, characterized in that: Step four involves dynamic monitoring and adjustment of remediation parameters, including 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 nutrient solution injection frequency through Internet of Things (IoT) technology.

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