Efficient petroleum degradation fungus Penicillium javanicum F5 and application thereof

Abstract

The invention relates to an efficient petroleum degradation fungus Penicillium javanicum F5 and application thereof, and belongs to the technical field of petroleum pollutant degradation. The fungus is penicillium javanicum with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M2025828. The strain F5 disclosed by the invention can grow and propagate by taking petroleum as a unique carbon source, and when the petroleum concentration is 1.0% (v / v), the survival and growth of thalli can be realized, and petroleum components can be effectively degraded. Besides, the strain F5 has relatively strong salt tolerance, can still tolerate and normally degrade petroleum under the NaCl concentration of 3.0%, and can generate an extracellular enzyme manganese peroxidase (Mnp) capable of degrading polycyclic aromatic hydrocarbon substances at the same time. The strain F5 provided by the invention has an obvious petroleum removal effect (plt; the method has the advantages of high efficiency, easiness in growth and no secondary pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of petroleum pollutant degradation, and specifically to a highly efficient petroleum-degrading fungus Penicillium javanicum F5 and its application, and more particularly to a Penicillium fungus with a significant petroleum-degrading effect that is screened from oil sludge in abandoned oil fields in Inner Mongolia Autonomous Region after multiple separation and purification. Background Art

[0002] As a raw material for chemical products, petroleum plays a vital role in numerous sectors, including energy, chemicals, and medicine, and is a vital economic lifeline for the nation. However, during extraction, transportation, loading and unloading, processing, and use, petroleum can enter the soil, water, and atmosphere through various means, including disposal and leakage. This not only wastes resources but also creates a series of environmental pollution problems. When petroleum enters water bodies such as lakes and oceans, it not only pollutes water quality but also threatens the survival of aquatic life. Petroleum contamination of soil also impacts agricultural production, human health, and even the sustainable development of ecosystems. Petroleum is a complex component, containing a wide range of polar and nonpolar alkanes, cycloalkanes, and aromatic hydrocarbons, exhibiting a wide range of physicochemical properties and biological impacts. Polycyclic aromatic hydrocarbons (PAHs), in particular, are not only difficult to degrade but are also known to be carcinogenic, teratogenic, and mutagenic. They are also a key component of the four major emerging pollutants, persistent organic pollutants (POPs), currently receiving widespread international attention. Currently, petroleum pollution has become one of the largest sources of marine pollution. If these petroleum hydrocarbons leaked into the environment cannot be transferred or removed in a timely manner, they will pose huge ecological and environmental risks and threaten human health.

[0003] Currently, methods for remediating oil pollution in the environment are generally categorized as physical, chemical, biological, and a combination of methods, with variations in approach specific to different environmental media. Among existing oil pollution remediation technologies, physical and chemical methods struggle to fully meet remediation requirements due to high costs and the potential for secondary pollution. Biological methods, with their advantages of low investment costs, minimal impact on the ecological environment, no secondary pollution, and high operability, are gradually becoming a promising research direction in the oil pollution field. Among these, the use of microorganisms such as bacteria and fungi to degrade oil holds great promise.

[0004] The unique mycelial structure and metabolic characteristics of fungi also make them an indispensable component of bioremediation. First, numerous studies have demonstrated that fungi can also degrade petroleum hydrocarbons, achieving degradation rates exceeding those of bacteria under specific conditions. Some fungi have a significant advantage in degrading polycyclic aromatic hydrocarbons. Second, fungi are larger than bacteria, allowing their mycelium to have a greater contact surface area with pollutants. Some fungi can also encapsulate pollutants through endocytosis, enabling more thorough decomposition. Furthermore, some fungi are hyperaccumulators, absorbing heavy metal ions from the environment and storing them in their fruiting bodies, making them tolerant to environments contaminated by both petroleum hydrocarbons and heavy metals. Furthermore, fungal mycelium can serve as the structural framework for a bacterial-fungal symbiotic degradation system, supporting and enriching petroleum-degrading bacteria, promoting biofilm formation and the complete degradation of petroleum components. However, the more than 30 fungal genera currently proven to be capable of degrading petroleum still suffer from limited degradation capacity and difficulty tolerating high salt concentrations. Summary of the Invention

[0005] The primary purpose of the present invention is to address the problems of limited degradation ability and difficulty in tolerating high salt concentrations in the existing technology, and to provide a highly efficient petroleum-degrading functional fungus and its application, so that it can stably exist and efficiently degrade petroleum in a high salt concentration environment, providing a highly efficient degradation strain resource for biodegradation of petroleum pollution, which has important economic value and practical significance for the control of environmental pollution.

[0006] According to a first aspect of the present invention, a petroleum-degrading fungus, Penicillium javanicum F5, is provided. This fungus is Penicillium javanicum F5, with a deposit number of CCTCC NO: M 2025828. The Latin name is Penicillium javanicum F5, and the deposit was made with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on April 18, 2025, with a deposit number of CCTCC NO: M2025828.

[0007] According to another aspect of the present invention, there is provided the use of the petroleum-degrading fungus Penicillium javanicum F5 in degrading petroleum.

[0008] Preferably, the petroleum-degrading fungus Penicillium javanicum F5 grows and reproduces using petroleum as the sole carbon source.

[0009] Preferably, the petroleum degrading fungus Penicillium javanicum F5 is used to produce manganese peroxidase and lignin peroxidase, thereby degrading petroleum.

[0010] Preferably, the mass concentration of sodium chloride in the degradation system is less than or equal to 3.0%.

[0011] Preferably, the volume concentration of the petroleum is less than or equal to 1.0%.

[0012] Preferably, the concentration of the petroleum-degrading fungus Penicillium javanicum F5 in the degradation reaction system is 1.94 g dry weight / L-5.81 g dry weight / L.

[0013] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0014] (1) The petroleum-degrading functional fungus Penicillium javanicum F5 provided by the present invention can grow and reproduce in an environment with a petroleum concentration of 1.0% by volume using petroleum as the sole carbon source. When the petroleum concentration is 1.0% by volume, the fungus can survive and grow and effectively degrade petroleum components. The degradation rate of total petroleum hydrocarbons (TPHs) reaches more than 88% after 15 days of degradation.

[0015] (2) The petroleum-degrading functional fungus Penicillium javanicum F5 provided by the present invention has a significant effect on removing petroleum (p < 0.001), and can produce high concentrations of manganese peroxidase (with an enzyme activity of up to 943.59 U / mL) and a small amount of lignin peroxidase, which helps to degrade polycyclic aromatic hydrocarbons and has good application prospects.

[0016] (3) The oil-degrading functional fungus Penicillium javanicum F5 provided by the present invention has strong salt tolerance and can still tolerate and normally degrade oil at a NaCl concentration of 3.0%. The degradation effect is even better than that at lower NaCl concentrations, which is helpful for achieving oil pollution control under high salt concentration conditions such as in marine environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is a colony morphology diagram of the strain Penicillium javanicum F5 of the present invention on PDA solid culture medium.

[0018] Figure 2 This is a microscopic observation of hyphae and spores of the strain Penicillium javanicum F5 of the present invention.

[0019] Figure 3It is a growth curve diagram of the strain Penicillium javanicum F5 of the present invention in Sabouraud liquid culture medium.

[0020] Figure 4 The figure shows the colony diffusion of the strain Penicillium javanicum F5 of the present invention after being cultured on a petroleum solid culture medium for 9 days.

[0021] Figure 5 The present invention discloses the degradation characteristics of the strain Penicillium javanicum F5 under different NaCl concentration conditions, including (a) the remaining oil mass and (b) the oil degradation rate.

[0022] Figure 6 The present invention relates to the degradation characteristics of the strain Penicillium javanicum F5 under different petroleum concentration conditions, including (a) the remaining petroleum mass and (b) the petroleum degradation rate.

[0023] Figure 7 The present invention shows a degradation kinetic curve of the strain Penicillium javanicum F5 in a petroleum liquid culture medium with NaCl concentrations of 0.01% and 3.0% for 15 days, including (a) changes in the remaining petroleum mass, (b) changes in fungal biomass, and (c) changes in the petroleum degradation rate.

[0024] Figure 8 The GC-MS graphs show the changes in the remaining petroleum components of the strain Penicillium javanicum F5 of the present invention after culturing in a petroleum liquid culture medium for 15 days, including (a) before degradation begins, (b) on the 3rd day of degradation, (c) on the 8th day of degradation, and (d) on the 15th day of degradation.

[0025] Figure 9 The graph shows some extracellular enzyme activities of the strain Penicillium javanicum F5 of the present invention after degradation in a petroleum liquid culture medium with a NaCl concentration of 3.0% for 15 days, including (a) lignin peroxidase (Lip) activity and (b) manganese peroxidase (Mnp) activity.

[0026] Figure 10 The present invention discloses the degradation characteristics of the strain Penicillium javanicum F5 under different bacterial liquid inoculation conditions, including (a) the remaining oil mass and (b) the oil degradation rate. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0028] The invention discloses a highly efficient petroleum-degrading fungus strain F5, which has been identified as Penicillium javanicum. The 18S rDNA sequence of the Penicillium javanicum F5 strain is SEQ ID NO: 1.

[0029] The present invention also provides a method for screening highly efficient petroleum-degrading functional fungi, the method comprising the following steps:

[0030] (1) Using waste oil sludge from oil fields in Inner Mongolia Autonomous Region as inoculum, streak the plate and inoculate it onto PDA (potato dextrose agar) plates supplemented with antibiotics suitable for fungal growth. Incubate at 30°C until colonies grow. Repeat the streak three times.

[0031] (2) Pick out colonies with different characteristics and streak them again. When a single colony with consistent characteristics grows on the newly streaked plate, pick it out and spot-graft it onto a new PDA plate. Repeat the single colony picking and spot-grafting steps several times until the colonies on the plate are purified. The isolated and purified fungi are numbered, enriched, and preserved.

[0032] (3) The screened strains were inoculated into inorganic salt solid culture medium and inorganic salt liquid culture medium containing petroleum, respectively. After a certain period of static culture and shaking culture, the growth status of the strains was observed, the residual petroleum content was measured, and their petroleum degradation ability was evaluated. Finally, the strain F5 with the best growth performance and the strongest degradation performance was screened out.

[0033] The following are specific embodiments

[0034] Example 1: Isolation and identification of petroleum-degrading fungi

[0035] (1) Sample collection and pretreatment

[0036] The oil sludge samples used to screen and isolate highly efficient petroleum-degrading fungi were collected from waste oilfields in Inner Mongolia Autonomous Region and stored in light-proof plastic barrels at room temperature. The oil used in the degradation experiments was Northwest crude oil provided by Sinopec Research Institute of Petroleum Processing Co., Ltd.

[0037] (2) Isolation and purification of strains

[0038] The main culture media and formulas involved in the implementation process are as follows:

[0039] ① Petroleum solid culture medium: MgSO4 (0.2 g), KH2PO4 (1.0 g), K2HPO4 (1.0 g), NH4NO3 (3.48 g), NaCl (30.0 g), 100× trace element concentrate (10 mL), agar 20.0 g, H2O (1000 mL), pH 7.0, autoclave at 121°C for 30 min. Pour 25 mL of culture medium onto each plate. After the medium cools and solidifies, evenly apply 100 μL of the petroleum to be degraded onto the surface.

[0040] ②100× concentrated solution of trace elements: FeCl3 (5.0g), CaCl2 (2.0g), ZnSO4·7H2O (1.0g), MnCl2·4H2O (0.05g), CuSO4 (0.05g), H2O (1000mL). Dissolve thoroughly and stir evenly. Store in a refrigerator at 4°C.

[0041] ③ Petroleum liquid culture medium: MgSO4 (0.2 g), KH2PO4 (1.0 g), K2HPO4 (1.0 g), NH4NO3 (3.48 g), NaCl (30.0 g), 100× concentrate of trace elements (10 mL), H2O (1000 mL), petroleum 1% (v / v), pH 7.0, high-pressure steam sterilization at 121°C for 30 min.

[0042] ④ Potato dextrose agar (PDA): Purchase a ready-made culture medium from Huankai Microorganisms. Take 40.1 g of this product and add it to 1000 mL of deionized water. Stir and boil until completely dissolved. Aliquot into Erlenmeyer flasks and sterilize by autoclaving at 121°C for 30 minutes. Ingredients: Potato (300.0 g), Glucose (20.0 g), Agar (15.0 g), Chloramphenicol (0.1 g).

[0043] ⑤ Sabouraud dextrose broth (SDB): Purchase ready-made culture medium from Huankai Microorganisms. Take 50.1 g of this product and add it to 1000 mL of deionized water. Stir and boil until completely dissolved. Aliquot into Erlenmeyer flasks and sterilize by autoclaving at 121°C for 30 minutes. Ingredients: peptone (10.0 g), glucose (40.0 g), agar (15.0 g). pH = 5.6 ± 0.2.

[0044] The retrieved sludge sample is used as an inoculum, streaked on a plate, and inoculated on a potato dextrose agar (PDA) plate supplemented with antibiotics. It is cultured at a constant temperature of 30°C until colonies grow, and the streaking is repeated three times. Carefully pick out colonies with different characteristics and streak them again. When a single colony with obvious consistent characteristics grows on the newly streaked plate, pick it out and spot-graft it on a new PDA plate. Repeat the steps of picking single colonies and spot-grafting several times until the colonies on the plate are purified, and number the purified single colonies with different characteristics.

[0045] A loop of hyphae was collected from the purified fungal plate and inoculated into pre-sterilized Sabouraud broth. The culture was then placed in a shaker at 30°C, 150 rpm for enrichment. Once the fungus has fully grown and the enrichment solution is concentrated, a certain amount of cells was collected and prepared into culture tubes using the glycerol-saline method. Three tubes of each strain were stored in a -80°C freezer.

[0046] (3) Fungal morphological observation

[0047] Colony characteristics observation: Observe the morphology of pure cultured fungal colonies and record the colony color, surface characteristics, shape and texture, spore status, colony diffusion rate and other characteristics of the strain. Figure 1 The results showed that the colonies of strain F5 were white on the front and milky white on the back. The colonies spread outward in a nearly circular shape, with a dry and rough surface and an overall powdery texture. Numerous white powdery spores could be observed with the naked eye.

[0048] Microscopic observation of hyphae and spores: Use the insert method to observe hyphae and spores microscopically. Record the characteristics of hyphae and spores, such as the presence of septa, rhizoids, spore types, etc., and compare with existing data to preliminarily determine the fungal species. Figure 2 The results showed that the hyphae of strain F5 had septa, no rhizodendrocytes, and the spores were long chain conidia, which were typical characteristics of Penicillium.

[0049] (4) 18S rDNA identification

[0050] Genomic DNA was extracted using a fungal DNA extraction kit purchased from Beijing Solebao Biotechnology Co., Ltd. Amplification was performed using the universal 18S rDNA fungal primers (NS1) SEQ ID NO: 2 and SEQ ID NO: 3 (NS8). The amplified DNA was then sent to a sequencing company for sequencing. A gene sequence of approximately 1648 bp was obtained. This sequence was then submitted to the NCBI (www.ncbi.nlm.nih.gov) database for BLAST comparison. The sample type with the highest sequence similarity was identified and its species was identified.

[0051] Based on the comprehensive characteristics of the colony, hyphae and spores, as well as the results of molecular biological identification, and compared with the information in the existing data, the isolated fungus F5 was finally determined to be Penicillium javanicum.

[0052] Example 2: Growth and spread of strains

[0053] (1) Growth curve

[0054] The fungus F5 obtained by screening was inoculated into 50 mL of sterilized Sabouraud liquid culture medium at a dosage of 5%, and cultured in a shaking table at 30°C and 150 rpm. During the period, continuous sampling was taken to monitor the changes in the dry weight of the bacteria in the 50 mL culture medium to study the growth of the strain in the Sabouraud liquid culture medium. The dry weight of the bacteria was determined by weighing, that is, the culture solution was filtered with sterile filter paper that had been weighed in advance to intercept the bacteria, and the filter paper + bacteria mass was recorded after drying at 80°C to a constant weight, and the original mass of the filter paper was subtracted to obtain the dry weight of the bacteria. The results are as follows Figure 3 In Sabouraud liquid medium, strain F5 quickly entered the logarithmic phase after 1 day, with rapid bacterial growth, and then the growth rate gradually slowed down, entering the stable phase after 5 days.

[0055] (2) Growth on petroleum solid culture medium

[0056] Prepare petroleum solid culture medium and sterilize it, pour it into plates, and pour 25mL of culture medium into each plate. After cooling and solidification, use a pipette to take 100μL of degradation petroleum and evenly spread it on the solidified plate. Pick the purified fungi with an inoculation loop and dot-inoculate them in the center of the plate. Perform three repeated experiments for each strain to control the initial dot-inoculation range. The plate without inoculation is used as a blank control and is numbered CK. Incubate at 30℃ for 9 days and record the changes in colony diameter over time. The results are as follows. Figure 4 As shown, the colonies of strain F5 exhibited a concentric structure with alternating dark and light colors, and the hyphae were dense. The colony diameter reached the plate's maximum value within 8 days, and the oil traces on the plate were completely covered by hyphae. This suggests that strain F5 can rapidly adapt to the oil environment when using oil as its sole carbon source, utilizing oil components to achieve rapid growth.

[0057] Example 3: Degradation performance test of petroleum-degrading fungi

[0058] Through experiments, the oil degradation effect and growth of oil-degrading fungi F5 under different NaCl concentrations and different oil concentrations were studied, and its degradation kinetic characteristics, changes in oil components during the degradation process and the production of extracellular enzymes were determined.

[0059] (1) Degradation characteristics under different salt concentrations

[0060] To a 100-mL Erlenmeyer flask, 50 mL of petroleum liquid medium was added. The initial pH of the medium was adjusted to 7.0, the N / P ratio to 3:1, and the NaCl concentration was adjusted to 0.01%, 0.05%, 0.5%, 1.0%, 3.0%, 5.0%, and 7.0%. The initial oil concentration was 1.0% (v / v). The medium was sterilized at 121°C for 30 min. The initial inoculum volume was 5 mL (the inoculum cell concentration was 0.0387 g dry weight / mL, and the cells were evenly distributed). The control group was inoculated with the same volume of sterile water. Three replicates were set for each treatment. The medium was shaken at 30°C and 150 rpm for 7 days. After degradation, the remaining petroleum was fully extracted with dichloromethane. The petroleum adhered to the mycelium was extracted ultrasonically. The extracted liquid was transferred to a 50-mL Erlenmeyer flask that had been previously weighed constant. After the solvent had evaporated, the remaining petroleum content was determined by weight loss. The petroleum content after sterilization of the petroleum medium was considered the initial petroleum content. The formula for calculating the oil degradation rate of fungi is as follows:

[0061]

[0062] Fungus F5 degrades oil under different NaCl concentrations. The remaining oil mass in the system after 7 days is as follows: Figure 5 In (a), the oil degradation rate is as follows Figure 5 (b) As can be seen, a high oil degradation rate of 46.50% can be achieved at a NaCl concentration of 0.01%, that is, when only basic nutritional needs are met without additional concentration increases. With increasing NaCl concentration, the oil degradation rate initially increases and then decreases, reaching a maximum of 47.05% at 3.0%. Further increases in NaCl concentration to 5.0% and 7.0% decreased the oil degradation rate to below 25.00%. This is likely due to the excessively high NaCl concentration, which inhibits the growth and metabolism of fungus F5. At these concentrations, there was no significant difference in oil degradation rates between the experimental and control groups. Overall, fungus F5 exhibits a certain tolerance to NaCl concentrations, even achieving the best degradation performance at 3.0%. This may be because fungus F5 was isolated from oilfield waste sludge, which contains various salts and other waste residues that have acclimated it to a certain extent, giving it a certain degree of salt tolerance. Generally speaking, the NaCl concentration in seawater is around 3.0%-3.5%, and fungus F5 still exhibits high oil degradation activity at this NaCl concentration, indicating great potential for application.

[0063] (2) Degradation characteristics under different petroleum concentrations

[0064] To a 100mL Erlenmeyer flask, add 50mL of petroleum liquid culture medium. Adjust the initial pH of the medium to 7.0, the N / P ratio to 3:1, and the NaCl concentration to 3.0%. Adjust the initial oil concentrations of the medium to 0.2%, 0.5%, 1.0%, 1.5%, and 2.0% (v / v), respectively. Autoclave sterilize at 121°C for 30 minutes. The initial inoculum size is 5mL (10% v / v). A control group is inoculated with the same volume of sterile water. Three replicates are set up for each treatment. Degrade on a shaker at 30°C and 150 rpm for 7 days. After degradation, the remaining petroleum is fully extracted with dichloromethane. Subsequent methods are the same as for the salt concentration experiments.

[0065] After 7 days of degradation under different oil concentrations, the remaining oil mass in the system was as follows: Figure 6 In (a), the oil degradation rate is as follows Figure 6 (b) in the figure. As can be seen, as the initial oil concentration gradually increases, oil degradation first increases and then decreases, reaching a peak of 44.68% at 1.0% (v / v). Further increases to 1.5% and 2.0% increase the oil degradation rate, which rapidly decreases to below 30.00%. This may be due to the high oil concentration, which, on the one hand, covers the liquid surface, hindering the fungus F5's access to oxygen and thus hindering its growth and metabolism; and, on the other hand, the higher concentration of toxic and harmful substances in the oil, exceeding the fungus' tolerance, preventing its timely degradation and causing damage. Overall, however, there was a significant difference in oil degradation between the F5 experimental and control groups, and subsequent studies used an oil concentration of 1.0% (v / v).

[0066] (3) Degradation kinetics characteristics

[0067] 50 mL of petroleum liquid culture medium was added to a 100 mL Erlenmeyer flask. The medium was initially adjusted to a pH of 7.0, an N / P ratio of 3:1, and an initial oil concentration of 1.0% (v / v). The medium was sterilized at 121°C with autoclaving for 30 min. The initial inoculum size was 5 mL (10% v / v). Experimental groups were set up every 1 day, with three replicates per group. The degradation process was carried out on a shaker at 30°C, 150 rpm, for 15 days. Degradation kinetics were performed under both low (0.01%) and high (3.0%) NaCl concentrations. Each experimental group was removed every 1 day, and the remaining oil in the flask was fully extracted with dichloromethane. The remaining oil mass at each sampling point was measured gravimetrically, and the oil degradation kinetics curve was plotted. Furthermore, the dry weight of the fungus was measured at each sampling point to analyze the growth of the fungus F5 under liquid culture conditions using petroleum as the sole carbon source.

[0068] Residual petroleum samples were collected on days 0, 3, 8, and 15 of degradation to prepare the samples for analysis. Gas chromatography-mass spectrometry (GC-MS) was used for component analysis. The GC-MS experimental conditions were as follows: an Agilent GC-MS equipped with an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm), a flame ionization detector, helium as a carrier gas at a flow rate of 1.0 mL / min, an inlet temperature of 300°C, an injection volume of 1 μl, and a split ratio of 5:1. The column temperature program was as follows: 50°C for 2 min, then increased to 280°C at a rate of 5°C / min and held for 20 min. Mass spectrometry parameters included an atmospheric pressure photoionization (EI) source, an ionization energy of 70 eV, an ion source temperature of 230°C, a quadrupole temperature of 150°C, and full scan acquisition mode over a mass range of 30–650 m / z.

[0069] The results of degradation kinetics experiments are as follows Figure 7 As shown in the figure, the degradation effect was better at a salt concentration of 3.0%. The degradation period from 0 to 3 days was a rapid degradation phase, with a rapid decrease in oil mass and a significant increase in fungal biomass. From 4 to 8 days, the degradation rate slowed slightly and the fungal biomass began to decrease, but the degradation effect remained significant, reaching a 47.97% oil degradation rate on the 8th day. From 9 to 15 days, the degradation period was a slow phase, with the remaining oil mass slowly decreasing over time and the fungal biomass continuing to decrease. Ultimately, after 15 days of degradation, a 63.25% oil degradation rate was achieved. At a salt concentration of 0.01%, the degradation rate was low from 0 to 3 days, with only a slight increase in fungal biomass. The degradation rate only increased from 4 to 8 days, reaching a 44.57% degradation rate on the 8th day. From 9 to 15 days, the degradation period continued to slow, with a degradation rate of 50.15% after 15 days.

[0070] Therefore, the oil degradation process of fungus F5 can be divided into three main stages: 0-3 days, 4-8 days, and 9-15 days. Good oil degradation was still achieved under a higher salinity of 3.0%. The NaCl concentration in seawater is generally 3.0%-3.5%. The oil degradation process and chlorine resistance of fungus F5 isolated in this study under this salinity may be helpful for research related to the removal of oil pollutants from seawater.

[0071] The GC-MS total ion current chromatograms of the remaining petroleum components at four representative sampling points are shown in Figure 2. Figure 8As shown in the figure, the remaining petroleum components decreased significantly with the increase in degradation time, indicating that the fungus F5 significantly degraded both short-chain and long-chain components of the petroleum. Calculations showed that the degradation rate of total petroleum hydrocarbons (TPHs) was 76.04% on the third day of degradation, 84.15% on the eighth day, and 88.81% on the fifteenth day. The significant degradation of petroleum components by the fungus F5 demonstrates its great potential for future applications in the microbial remediation of petroleum contamination.

[0072] (4) Degradation enzyme activity

[0073] Following the degradation kinetics experiment, the degradation experiment was conducted again at a NaCl concentration of 3.0%, with constant temperature shaking at 30°C and 150 rpm for 15 days. Every 1 day, 5 mL of fungal culture solution was aspirated and the mycelium was filtered out with sterilized filter paper. The entire process was kept on ice to prevent the enzyme protein from being inactivated. The collected filtrate was the crude enzyme solution. Using UV spectrophotometry, resveratrol and Mn were detected. 2+ The activities of lignin peroxidase (Lip) and manganese peroxidase (Mnp) were determined as oxidation substrates. The enzyme activity results of each sampling point were averaged over three measurements, and a curve of enzyme activity change over time was drawn.

[0074] The results are as follows Figure 9 As shown, as the degradation process progressed, Lipase activity generally showed an increasing and then decreasing trend, reaching a peak of 9.08 U / mL on day 3 and maintaining a 7 U / mL activity between days 5 and 10. However, the activity remained low, indicating that fungus F5 can produce Lipase, but its contribution was very limited. Mnpase activity also showed an increasing and then decreasing trend, showing high activity between days 10 and 14, reaching a peak of 943.59 U / mL on day 13. The high Mnpase activity of fungus F5 in a petroleum environment suggests that crude oil significantly induces Mnpase production in fungus F5. Both Lipase and Mnpase can act on aromatic rings, cleaving the benzene ring structure in aromatic hydrocarbons. This is often the first and most critical reaction in the degradation of aromatic hydrocarbons. In summary, fungus F5 not only possesses the ability to degrade aromatic hydrocarbons, but also Mnpase plays a major role in the degradation process.

[0075] (5) Degradation characteristics at different bacterial concentrations

[0076] To a 100-mL Erlenmeyer flask, 50 mL of petroleum liquid medium was added. The medium was adjusted to an initial pH of 7.0, an N / P ratio of 3:1, a NaCl concentration of 3.0%, and an initial oil concentration of 1.0% (v / v). The medium was then autoclaved at 121°C for 30 min. Initial inoculum sizes were set at 5% (1.94 g dry weight / L), 10% (3.87 g dry weight / L), 15% (5.81 g dry weight / L), 25% (9.68 g dry weight / L), and 40% v / v (15.48 g dry weight / L). A control group was inoculated with the same volume of sterile water. Three replicates were performed for each treatment. The medium was shaken at 30°C and 150 rpm for 7 days. After degradation, the remaining oil was fully extracted with dichloromethane. Subsequent methods were used for the salt concentration experiments.

[0077] Fungus F5 degrades oil under different inoculation conditions. The remaining oil mass and oil degradation rate in the system after 7 days are as follows: Figure 10 As can be seen, with increasing bacterial inoculum size, the remaining oil mass first decreases and then increases. When the bacterial inoculum size was 10% (v / v), the oil degradation rate was the highest, at 39.47%. This was followed by the 5% and 15% experimental groups, at 34.79% and 34.59%, respectively. The results showed that the oil degradation rates of each experimental group decreased to a certain extent. This may be due to the fact that the bacteria in the inoculum used in the experiment were in the form of mycelial balls and were not fully dispersed, which reduced the contact efficiency between the bacteria and the oil during the shaker degradation process. During the experiments with different bacterial inoculum sizes, the same bacterial solution was inoculated in different experimental groups. When the bacterial dry weight was the same, the influence of bacterial morphology on the overall degradation effect was the same in different experimental groups, so the results of the optimal bacterial inoculum size were reliable. From the overall trend, the oil degradation effect was better when the bacterial inoculum size was 10%, that is, the initial bacterial inoculum concentration was 3.87g dry weight / L.

[0078] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A petroleum-degrading fungus, Penicillium javanicum F5, characterized in that: The petroleum-degrading fungus Penicillium javanicum F5 is a Penicillium javanicum with a preservation number of CCTCC NO: M 2025828.

2. Use of the petroleum-degrading fungus Penicillium javanicum F5 according to claim 1 in degrading petroleum.

3. The use according to claim 2, characterized in that The petroleum-degrading fungus Penicillium javanicumF5 grows and reproduces using petroleum as the sole carbon source.

4. The use according to claim 2, characterized in that The petroleum-degrading fungus Penicillium javanicum F5 is used to produce manganese peroxidase and lignin peroxidase, thereby degrading petroleum.

5. The use according to claim 2, characterized in that The mass concentration of sodium chloride in the degradation system is less than or equal to 3.0%.

6. The use according to claim 2, characterized in that The volume concentration of the petroleum is less than or equal to 1.0%.

7. The use according to claim 2, characterized in that The concentration of the petroleum-degrading fungus Penicillium javanicum F5 in the degradation reaction system is 1.94 g dry weight / L-5.81 g dry weight / L.

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