Trichoderma longibrachiatum for degrading petroleum hydrocarbon in saline-alkali environment, culture and application thereof

Abstract

The application discloses a long-branched Trichoderma that can degrade petroleum hydrocarbon in a saline-alkali environment, a culture and application thereof, and relates to the technical field of microorganisms.The preservation number of the long-branched Trichoderma is CGMCC No.42182.The long-branched Trichoderma provided by the application has efficient petroleum degradation characteristics, and the application provides a practical and effective solution to the problem of petroleum pollution in saline-alkali soil, can efficiently degrade petroleum hydrocarbon in the saline-alkali soil, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a Trichoderma longifolia culture that degrades petroleum hydrocarbons in saline-alkali environments and its applications. Background Technology

[0002] Petroleum, as a strategic resource and the lifeline of industry, plays a crucial role in modern economic development. However, improper operations and leaks during oil extraction, transportation, production, and processing also cause environmental pollution. Oil spills not only increase total organic carbon in the soil and cause an imbalance in the carbon-nitrogen ratio, altering the soil's physicochemical properties and affecting soil enzyme activity, but also lead to the long-term accumulation of petroleum hydrocarbons in the soil. N-alkanes, through geochemical processes, deposit underground, posing a threat to benthic organisms. Furthermore, polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and difficult to degrade, entering the human body through the food chain and threatening human health.

[0003] Microbial remediation utilizes the natural metabolism and cometolysis of microorganisms to degrade petroleum hydrocarbons in contaminated soil and convert them into non-polluting substances, improving soil environmental conditions and making soil microorganisms more suitable for survival. It is a low-cost, low-toxicity, and environmentally friendly method for remediating petroleum pollution, and has gained widespread attention both domestically and internationally in recent years. However, the growth and remediation performance of microorganisms are often limited by conditions such as pH and salinity. Fungi are potential petroleum hydrocarbon-degrading microorganisms because they have branched hyphae, allowing them to penetrate the soil matrix more effectively than bacteria, fully contacting petroleum pollutants and degrading difficult-to-remove pollutants by secreting extracellular enzymes. Furthermore, fungi have strong environmental adaptability and can grow under various extreme environmental conditions. Therefore, fungi have profound research significance and broad application prospects in the microbial remediation of petroleum-contaminated soils. The optimal pH value for soil microorganisms is often around 7.0, and the salinity around 0.2%. High salinity and excessively acidic or alkaline environments inhibit microbial activity. Drought, water scarcity, salt accumulation, and the characteristics of high pH and high salinity in soils have become major obstacles to the microbial remediation of petroleum hydrocarbon pollution. Therefore, an efficient method is urgently needed to specifically address the problem of petroleum pollution in saline-alkali soils.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a long-branched Trichoderma, its culture, and its application in degrading petroleum hydrocarbons in saline-alkali environments to solve the problem of petroleum pollution in saline-alkali soils.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a long-branched Trichoderma ( Trichoderma longibrachiatumIt is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on September 5, 2025, with accession number CGMCC No. 42182.

[0007] Secondly, this invention provides the application of Trichoderma longifolia in the degradation of petroleum hydrocarbons.

[0008] Thirdly, the present invention provides a culture, which is the above-mentioned culture of Trichoderma longifolia. Fourthly, the present invention provides a method for degrading petroleum hydrocarbons, comprising the following steps: applying the above-mentioned Trichoderma longifolia, the above-mentioned culture, or the above-mentioned microbial agent to the material to be degraded.

[0009] The present invention has the following beneficial effects: The *Trichoderma longicornis* provided by this invention possesses highly efficient petroleum degradation characteristics. Specifically, under conditions of 30 °C, 150 r / min, pH 7.5-7.8, salinity 0.9%, and crude oil concentration 2 g / L, it can degrade 86.77% of total petroleum hydrocarbons after 14 days. Specifically, the degradation rate of n-alkanes is 91.5%; among isoprene-like alkanes, the degradation rates of pterostilbene and phytane are 85.4% and 81.9%, respectively; and among polycyclic aromatic hydrocarbons, the degradation rates of phenanthrene, methylphenanthrene, dimethylphenanthrene, and trimethylphenanthrene are 97.3%, 91.5%, 93.2%, and 93.7%, respectively. Therefore, this invention provides a practical and effective solution to the problem of petroleum pollution in saline-alkali soils, efficiently degrading petroleum in such soils. Furthermore, the *Trichoderma longicornis* provided by this invention can also be used for the degradation of kitchen grease, showing promising application prospects. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 for Trichoderma longibrachiatum 3D colony morphology and microscopic images of 39-2; Figure 2 Trichoderma longibrachiatum 39-2 Fungal 7-day oil-reducing plate; Figure 3 Trichoderma longibrachiatum 39-2 Genetic developmental tree; Figure 4 Gas chromatography-mass spectra of crude oil before and after degradation (inoculation) Trichoderma longibrachiatum39-2 crude oil chromatogram; inoculation Trichoderma longibrachiatum Chromatogram of crude oil extracted after 7 days of culture (Pr is pterostilbene, Ph is phytane)); Figure 5 Gas chromatograms and mass spectra of n-alkanes and isoprene-like alkanes before and after degradation; Figure 6 Gas chromatograms and mass spectra of phenanthrene and methylphenanthrene series before and after degradation. Detailed Implementation

[0012] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0013] In a first aspect, the present invention provides a long-branched Trichoderma ( Trichoderma longibrachiatum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on September 5, 2025, with accession number CGMCC No. 42182. The biological material is 39-2, and the classification name is *Trichoderma longifolia*. Trichoderma longibrachiatum The test results indicated that the animal was alive.

[0014] This invention obtained a highly efficient petroleum-degrading strain from the soil near the oil well platform of Changqing Oilfield in the Ordos Basin, through domestication and isolation in a culture medium with crude oil from Changqing Oilfield as the sole carbon source.

[0015] The biochemical and physicochemical characteristics of *Trichoderma longifolia* provided by this invention are as follows: Initially, the mycelium is white and cottony; upon maturity, it turns yellowish-green to dark green due to the production of conidia. It is positive for fiber hydrolysis, starch hydrolysis, gelatin liquefaction, lipid production, and hydrogen peroxide production, and negative for acid production. Initially white, it later turns yellowish-green and cottony, and spreads rapidly.

[0016] The *Trichoderma longicornis* strain described in this invention can degrade 86.77% of total petroleum hydrocarbons under high salinity and alkalinity conditions, with a degradation rate of 91.5% for n-alkanes and 85.4% and 81.9% for pterostilbene and phytane, respectively, among isoprene-like alkanes. Furthermore, *Trichoderma longicornis* strain 39-2 exhibits even higher degradation rates for aromatic hydrocarbons, with degradation rates of 97.3%, 91.5%, 93.2%, and 93.7% for phenanthrene, methylphenanthrene, dimethylphenanthrene, and trimethylphenanthrene, respectively. This invention isolates and identifies specific *Trichoderma longicornis* strains suitable for saline-alkali environments, laying the foundation for the remediation of petroleum pollution in saline-alkali environments.

[0017] Secondly, this invention provides the application of Trichoderma longifolia in the degradation of petroleum hydrocarbons: In a preferred embodiment of the present invention, the application is performed in at least one of the following (1)-(2): (1) Highly efficient degradation of total petroleum hydrocarbons; (2) Degradation of complex petroleum hydrocarbon compounds.

[0018] In a preferred embodiment of the present invention, Trichoderma longifolia is used for crude oil degradation.

[0019] In a preferred embodiment of the present invention, the petroleum hydrocarbons used are selected from crude oil from Changqing Oilfield.

[0020] In a preferred embodiment of the present invention, the application method is selected from: bacterial oil mixing.

[0021] In a preferred embodiment of the present invention, a fungal seed suspension of *Trichoderma longifolia* is used. In a preferred embodiment of the present invention, the fungal seed suspension is prepared as follows: 5 mL of 0.9% physiological saline is used to rinse the filamentous fungal spores preserved on an agar slant into 100 mL of Czapek's medium. The suspension is maintained at 28 °C and 140 r·min. -1 The seed culture of filamentous fungi was obtained by shaking culture under the specified conditions for 36 h.

[0022] In a preferred embodiment of the present invention, application (1) includes at least one of the following degradation methods: (1) Degradation of n-alkanes; (2) Degradation of pterostilbene and phytane in isoprene-like alkanes; (3) Degradation of aromatic components. Aromatic components include, but are not limited to, polycyclic aromatic hydrocarbons, especially phenanthrene, methylphenanthrene, dimethylphenanthrene and trimethylphenanthrene.

[0023] The *Trichoderma longifolia* strain provided by this invention can, under conditions of 30 °C, 150 r / min, pH 7.5-7.8, salinity 0.9%, and crude oil concentration 2 g / L, degrade phenanthrene, methylphenanthrene, dimethylphenanthrene, and trimethylphenanthrene in the culture medium at rates of 97.3%, 91.5%, 93.2%, and 93.7%, respectively, after a 14-day reaction, demonstrating the advantage of a strain with high degradation rate.

[0024] Thirdly, the present invention provides a culture, which is the above-mentioned culture of Trichoderma longifolia; In a preferred embodiment of the present invention, the culture refers to the fungal seed liquid, fermentation broth, fungal sludge, or extract, concentrate, or dried product of the fermentation broth of Trichoderma longifolia.

[0025] Microbial sludge can be obtained by centrifugation or static sedimentation.

[0026] The dried materials include, but are not limited to, spray-dried materials, freeze-dried materials, vacuum-dried materials, drum-dried materials, etc.

[0027] In one alternative implementation, the culture is in the form of a microbial agent, which is, for example, prepared by diluting fermentation products.

[0028] Fourthly, the present invention provides a method for degrading petroleum or oils, comprising the following steps: applying the above-mentioned Trichoderma longifolia, the above-mentioned culture, or the above-mentioned microbial agent to the substance to be degraded.

[0029] In a preferred embodiment of the present invention, the substance to be degraded is an environment contaminated by petroleum.

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] Example 1 This embodiment provides *Trichoderma longifolia*. Trichoderma longibrachiatum The isolation and identification of 39-2. The specific steps are as follows: Soil containing petroleum hydrocarbons was collected from a depth of 5-10 cm around the Changqing Oilfield. After removing stones and plant residues with tweezers, 5 g of the collected soil was weighed and added to a sterile Erlenmeyer flask containing 200 ml of sterile water. The flask was then incubated at 28 ℃ and 150 r / min on a shaker for 12 hours. After shaking, the flask was allowed to stand for 1 hour. 5 mL of the suspension was then inoculated into 45 mL of enrichment medium and incubated at 28 ℃ and 160 r / min on a shaker for 3 days. After the incubation period, 5 mL of the culture was inoculated into 45 mL of fresh enrichment medium and incubated under the same conditions for 3 days. This process was repeated three times over a 3-day cycle to obtain the fungal enrichment culture. The culture was then inoculated onto LB solid medium using the streak plate method and incubated at 30 ℃ for 48 hours.

[0033] Add 1 mL of the above fungal enrichment culture to a test tube containing 9 mL of sterile water, and repeatedly pipette and shake to mix thoroughly. The resulting dilution is 10:1. -1 Similarly, dilute the enrichment medium to 10. -7Prepare Bengal red agar medium, pour it into plates, and allow the medium to cool and solidify. Inoculate 100 μL of the corresponding dilutions onto the medium, spreading them evenly with a spreader. Repeat this process three times for each dilution. Incubate at 28 °C inverted for 5-7 days. Select fast-growing, well-developed single colonies and streak them onto fresh Bengal red agar medium. Incubate at 28 °C for 3-5 days. Repeat this process until a pure culture strain is obtained. Transfer the purified strain from the plates to Bengal red agar slant culture medium and store at -18 °C.

[0034] The purified strain was identified through biochemical and physicochemical characterization, morphological identification, and scanning identification. The results are as follows: Initially, the mycelium of this strain was white and cottony; upon maturity, it turned yellowish-green to dark green due to conidial production. It was positive for cellulose hydrolysis, starch hydrolysis, gelatin liquefaction, lipid production, and hydrogen peroxide, but negative for acid production. Initially white, it later turned yellowish-green and cottony, spreading rapidly. Figure 1 for Trichoderma longibrachiatum Morphological photographs and microscopic images of colonies of 39-2 cultured on Bengal red fixative medium after 3 days.

[0035] Genomic DNA was obtained from purified bacterial cells and amplified by polymerase chain reaction (PCR) using 27F / 1492R primers. The amplified products were sequenced by Guangdong Megagene Technology Co., Ltd. using an ABI 3730 sequencing platform. The sequencing results were compared on NCBI, and a phylogenetic tree was constructed using neighbor-joining based on the BLAST results.

[0036] The culture medium formulation involved in this embodiment is as follows: Enrichment medium (fungi): 1% crude oil, 6 g NH4NO3, 5 g K2HPO4, 5 g KH2PO4, 2 g MgSO4·7H2O, 2 g CaCl2, 18-20 g nutrient agar, 1000 mL distilled water, natural pH. Prepare 1% Bengal red aqueous solution and 1% streptomycin solution (prepare fresh). Add 3.3 mL of Bengal red aqueous solution to every 1000 mL of medium. After the medium melts and cools to 55-60 °C, add 3.3 mL of streptomycin to every 100 mL of medium.

[0037] Bengal Red Solid Culture Medium (Fungal Slant Preservation Medium): Weigh 36.7 g of Bengal Red Agar medium, add 1000 mL of distilled water, heat to boiling on an electric stove to fully dissolve it, and dispense into containers.

[0038] Gelatin liquefaction medium: 5 g peptone, 200 g gelatin, 20 g glucose, 1000 mL distilled water.

[0039] Starch hydrolysis medium: 2.0 g soluble starch, 10 g K2HPO4, 0.3 g K2HPO4, 0.5 g NaCl, 1 g MgCO3, 1 g KNO3, 15 g nutrient agar, pH adjusted to 7.5-7.8, 1000 mL distilled water.

[0040] Cellulose hydrolysis medium: 0.5 g MgSO4·7H2O, 0.5 g K2HPO4, 0.5 g NaCl, 1 g KNO3, a filter paper strip about 5 cm long, and 1000 mL distilled water.

[0041] Lipid-producing medium: 5 g glucose, 100 mL 10% bean sprout juice. Dispense into 50 mL Erlenmeyer flasks, 20 mL per flask, and sterilize at 115 ℃ for 20 min.

[0042] Acid-producing medium: 5 g glucose, 0.5 g sterilized CaCO3, 100 mL yeast extract, and 2 g nutrient agar.

[0043] The results are as follows: The strain 39-2 involved in this invention is *Trichoderma longifolia* (…). Trichoderma longibrachiatum The ITS sequence, as shown in SEQ ID NO: 1, has 98% similarity to the published strain (PP837572.1). This bacterium was deposited with the China General Microbiological Culture Collection Center (CGMCC) on September 5, 2025, with accession number CGMCC No. 42182. The phylogenetic tree is shown below. Figure 3 As shown.

[0044] The specific nucleotide sequence of SEQ ID NO: 1 is as follows: .

[0045] Example 2 This embodiment involves Trichoderma longifolia. Trichoderma longibrachiatum The specific steps for testing the degradation characteristics of crude oil in 39-2 are as follows: Prepare an oil-containing solid culture medium, sterilize it, and pour it into plates while still hot, allowing it to cool and solidify. Observe the diameter of fungal colonies. *Trichoderma longicornis* Trichoderma longibrachiatum 39-2 Colony morphology diagram on fungal oil-reducing medium plate after 7 days (refer to reference). Figure 2 As shown.

[0046] Take 5 mL of 0.9% physiological saline and rinse the sample stored on the slant. Trichoderma longibrachiatum39-2 filamentous fungal spores were added to 100 mL of Czapek's medium, and the pH was adjusted to 7.8 and the salinity to 0.9%. The mixture was cultured at 28 ℃ and 150 r / min with shaking for 36 h to obtain a filamentous fungal seed culture. This seed culture was then inoculated into liquid oil-soluble medium at a 1% inoculum rate, with a sterile control group included. The mixture was cultured at 28 ℃ and 150 r / min for 14 days, followed by extraction with 20 mL of petroleum ether (boiling range 60-90 °C). Each sample was extracted three times, and the extracts were combined. Crude oil extracted from uninoculated crude oil medium served as a control group.

[0047] The extract was centrifuged three times, and the supernatant was collected. Water was absorbed with Na₂SO₄ and transferred to a volumetric flask. The volume was then adjusted to 20 mL with petroleum ether. After thorough mixing, 200 μL of sample was pipetted out, and 100 μL of 0.3 mg / mL C24D50 was added as an internal standard. The sample was analyzed by GC-MS using an Agilent 6890N (GC) / 5973B (MSD) gas chromatograph-mass spectrometer. The degradation rate of each petroleum hydrocarbon component was calculated using the integrated peak area of ​​the gas chromatogram.

[0048] The results are as follows Figure 4 As shown, calculations show that Trichoderma longibrachiatum 39-2 can degrade 86.77% of petroleum hydrocarbons within 14 days under the condition of an initial crude oil concentration of 2 g / L. The degradation rates of different components were also calculated: the degradation rate of n-alkanes was 91.5%, and the degradation rates of pterostilbene and phytane among isoprene-like alkanes were 85.4% and 81.9%, respectively. Figure 5 It is worth noting that long-branched Trichoderma Trichoderma longibrachiatum 39-2 also showed higher degradation rates for aromatic components, with phenanthrene, methylphenanthrene, dimethylphenanthrene, and trimethylphenanthrene degradation rates reaching 97.3%, 91.5%, 93.2%, and 93.7%, respectively. The results are as follows... Figure 6 As shown in Table 1, the statistical results of the degradation rates of each component are presented. Table 1. Degradation rates of different components of petroleum hydrocarbons

[0049] The culture medium formulation involved in this embodiment is as follows: Oil-reducing culture medium: NH4NO3 2.00 g, MgSO4·7H2O 0.20 g, CaCl2 0.02 g, FeSO4 0.05 g, KH2PO4 0.50 g, K2HPO4 0.50 g, distilled water 1 L, pH=7.8, crude oil 1%.

[0050] Oil-containing solid culture medium: It is obtained by adding 15-20 g of nutrient agar to oil-containing culture medium.

[0051] Czapek's medium: NaNO3 3.00 g, K2HPO4 1.00 g, MgSO4·7H2O 0.50 g, KCl 0.50 g, FeSO4 0.01 g, sucrose 30.00 g, distilled water 1 L, pH = 7.8.

[0052] In summary, the fungus *Trichoderma longifolia* provided by this invention... Trichoderma longibrachiatum 39-2 can degrade petroleum hydrocarbons, and has high degradation efficiency for n-alkanes, isoprene-like alkanes, and methylphenanthrene series.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of long-branched Trichoderma ( Trichoderma longibrachiatum ), characterized in that, It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on September 5, 2025, with accession number CGMCC No. 42182.

2. The application of Trichoderma longifolia as described in claim 1 in the degradation of petroleum hydrocarbons.

3. The application according to claim 2, characterized in that, The application is an application of at least one of the following (1)-(2): (1) Highly efficient degradation of total petroleum hydrocarbons; (2) Degradation of complex petroleum hydrocarbon compounds.

4. The application according to claim 2, characterized in that, Trichoderma longifolia is used for crude oil degradation.

5. The application according to claim 2, characterized in that, The application method described is selected from: bacterial oil mixing.

6. The application according to claim 2, characterized in that, The application includes at least one of the following degradation methods: (1) Degradation of n-alkanes; (2) Degradation of pterostilbene and phytane in isoprene-like alkanes; (3) Degradation of aromatic components.

7. A culture, characterized in that, It is a culture of Trichoderma longifolia as described in claim 1.

8. The culture according to claim 7, characterized in that, The culture refers to the fungal seed liquid, fermentation broth, fungal sludge, concentrate, or dried product of Trichoderma longifolia.

9. A method for degrading petroleum hydrocarbons, characterized in that, It includes the following steps: applying the Trichoderma longifolia of claim 1 or the culture agent of any one of claims 7-8 to the material to be degraded.

10. The method according to claim 9, characterized in that, The substance to be degraded is an environment contaminated by petroleum.

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