A method of degrading oil

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a method for degrading oil and fat. Domatiomyces The application provides the oil and fat degrading bacteria Domatiomyces Belong to a new family, Domatiomyces globalis Or Domatiomyces germinans Two new species, and have good degrading effect on oil and fat, and the application is used for degrading oil and fat, and provides the method for degrading oil and fat.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a method for degrading oils and fats. Background Technology

[0002] The degradation of waste oil (such as waste cooking oil, industrial waste oil, animal and vegetable oil residues, etc.) is an important issue in environmental governance and resource recycling. At present, there are many methods for treating polluted oil, but the main ones are (1) physical removal methods (sedimentation, centrifugation, suspension, adsorption, etc.). However, physical methods are a "non-destructive" technology, that is, after physical treatment, the pollutants are not rendered harmless but transferred to a new environment. If not properly treated, secondary pollution can still occur; (2) chemical removal methods (flocculation, oxidation, electrolysis, etc.). However, chemical methods can also introduce new pollutants into the environment; (3) biodegradation is the current mainstream environmental protection technology for solving oil pollution. It mainly utilizes the characteristic of microorganisms to consume oil to decompose the oil in oily wastewater. Bioremediation is a controlled or spontaneous process. It uses the metabolism of microorganisms to catalyze the degradation of pollutants in the environment, thereby degrading harmful substances in wastewater and converting them into harmless substances, reducing or ultimately eliminating pollution to the environment. Although there are existing oil-degrading bacteria, it is still necessary to explore new bacteria to enrich the types of oil-degrading bacteria. Summary of the Invention

[0003] To address the above problems, the present invention provides an oil-degrading bacterium. Domatiomyces And its applications. To achieve the above objectives, the first aspect of this disclosure provides a fungus that degrades oils, the fungus being classified as... Domatiomyces globalis and Domatiomyces germinans The preservation number of the growth-promoting bacteria is [number missing].

[0004] A method for degrading oils involves culturing oil-degrading bacteria with the oil to be degraded at 24℃~30℃ for 10d~30d to achieve oil degradation.

[0005] The oil-degrading bacteria are Domatiomyces .

[0006] Preferably, the Domatiomyces for Domatiomyces globalis or Domatiomyces germinans .

[0007] Preferably, the Domatiomyces globalis It is any one of the fungi listed in the Royal Netherlands CBS Fungal Collection, with numbers CBS132039, CBS 132002, CBS134915, CBS 143850, CBS 134916 and CBS 129053.

[0008] Preferably, the Domatiomyces germinans The fungus is designated CBS134922 by the Royal Dutch CBS Fungal Collection.

[0009] Preferably, colonies with a diameter of 1 mm are added to 80 mL to 150 mL of liquid mineral culture medium containing the lipids to be degraded.

[0010] Preferably, the volume of the liquid mineral culture medium is 100 mL.

[0011] Preferably, the liquid mineral culture medium is prepared from 10 mL of solution A and 15 mL of solution B;

[0012] The formula for solution A is as follows: 200g ammonium sulfate, 10g magnesium chloride hexahydrate, 1g ethylenediaminetetraacetic acid, 0.2g zinc sulfate heptahydrate, 0.1g calcium chloride dihydrate, 0.5g ferrous sulfate heptahydrate, 0.2g sodium molybdate dihydrate, 0.02g anhydrous copper sulfate, 0.04g chromium chloride in running water, 0.1g manganese chloride dihydrate, and water to make up to 500mL;

[0013] Solution B is formulated as follows: 155g of dipotassium hydrogen phosphate, 85g of disodium hydrogen phosphate dihydrate, and water to a final volume of 500mL.

[0014] Preferably, the oil-degrading bacteria and the oil to be degraded are cultured at 28°C for 15 days.

[0015] This invention relates to fungi isolated from Domatia ant nests near the equator. Previous research revealed that these fungi belong to a new family, which we have named... Domatiomycetaceae Because ants produce large amounts of secondary metabolites, including aromatic hydrocarbons, lipids, and other secondary metabolites, most microorganisms cannot grow in anthills. However, recently discovered... Domatiomyces Fungi can grow in ant nests, leading to the belief that these black yeasts possess the ability to decompose oils and other metabolites. Compared to bacteria, which produce fatty acids during oil degradation, causing environmental acidification and quickly losing their degradation capabilities, fungi can maintain relatively good activity. This is because most fungi are aerial microorganisms, exhibiting strong tolerance to dry or highly acidic environments. Therefore, research and development on fungal biodegradation of oils has gradually become a hot topic in oil pollution control technologies.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention provides Domatiomyces It belongs to a new discipline. Domatiomyces globalis or Domatiomyces germinansThese are two novel bacterial strains, isolated from a tropical ant-plant symbiotic system. Previously, they had never been reported to have oil degradation potential. The seven strains of these two species provided in this invention all exhibit good oil degradation activity, with six of them being... Domatiomyces globalis Strains (CBS132039, CBS 132002, CBS134915, CBS 143850, CBS 134916 and CBS 129053) had a degradation rate of 88.45-89.68%, with one strain being... Domatiomyces germinans (CBS 134922), with a degradation rate of 88.52%, are all highly efficient degrading strains. Therefore, this invention is used to degrade oils and provides a method for degrading oils.

[0018] Furthermore, these bacteria all belong to the dark fungi family. Dark fungi exhibit significant advantages in pollutant degradation, primarily due to their unique physicochemical properties. Their cell walls are rich in melanin, providing not only excellent protection against ultraviolet radiation and oxidative stress but also efficiently adsorbing or chelating heavy metals and organic pollutants (such as dyes), enhancing their tolerance in toxic environments. Fungi in this order are typical multi-extreme-tolerant microorganisms, maintaining activity and degrading pollutants under harsh conditions such as drought, high salinity, and nutrient deficiency (e.g., industrial wastewater, saline soil). This is attributed to their powerful molecular stress resistance mechanisms (such as osmotic pressure-protecting proteins and antioxidant enzymes). These unique advantages make dark fungi a strong candidate for bioremediation of complex and extremely polluted environments.

[0019] Domatiomyces globalis and Domatiomyces germinans Both bacteria have a good ability to degrade oils. Both bacteria can grow in the range of 9℃ to 33℃, grow better in the range of 24℃ to 30℃, and 28℃ is the optimal growth temperature. Attached Figure Description

[0020] Figure 1 The image shows the colony morphology of the lipid-degrading bacteria disclosed in this paper, where A is CBS 132002, B is CBS 132039, C is CBS 129053, D is CBS 143850, E is CBS 134916, F is CBS 134915, G is 129057, and H is CBS134922.

[0021] Figure 2 The standard curve of Golden Dragon rapeseed oil was plotted at a wavelength of 230 nm.

[0022] Figure 3 The percentage of oil degradation by the oil-degrading bacteria of this invention is given. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0024] In this invention, CBS132039, CBS 132002, CBS134915, CBS 143850, CBS 134916, CBS129053, and CBS 134922 are strain numbers from the literature Wang MZ, Belmonte-Lopes R, Pan T, Ahmed SA, RodriguesLustosa BP, Quan Y, Al-Hatmi AMS, Mayer VE, Voglmayr H, Grisolia ME, de SouzaLima BBJF, Vicente VA, Zhou SQ, Cao Y, Kang YQ, de Hoog GS (2025). A new family of ant-associated fungi in Chaetothyriales. Studies in Mycology 110:111-143. doi: 10.3114 / sim.2024.110.02.

[0025] The information on the culture medium used in this invention is as follows:

[0026] 1. The formula for Potato Glucosamine Agar (PDA) medium is: 4 g Potato extract, 20 g Dextrose, 15 g Agar (Sigma), and water to a final volume of 1 L.

[0027] 2. The resuscitation medium is PDA medium.

[0028] 3. The formula for the initial screening medium is as follows: Weigh 4g of agar powder and 4mL of Tween-80 and add them to the reagent bottle, then make up to 200mL with liquid mineral medium.

[0029] Liquid mineral culture medium is prepared as follows: Weigh each inorganic salt according to the concentrations in Table 1 and Table 2, stir until completely dissolved, and after all components are completely dissolved, add distilled water to a final volume of 500 mL to prepare 500 mL of solution A and 500 mL of solution B. Take 10 mL of solution A and 15 mL of solution B into a graduated cylinder, dilute with distilled water, pour into a volumetric flask, rinse the graduated cylinder 5 times with distilled water, transfer the rinsing solution into the volumetric flask, and add distilled water to a final volume of 1 L to prepare the liquid mineral culture medium.

[0030] Table 1. Composition of Liquid A

[0031]

[0032] Table 2 Composition of Liquid B

[0033]

[0034] Example 1

[0035] Strain screening and identification

[0036] 1. Through domestication and enrichment culture, two major categories of 10 bacterial strains were screened from ant nests, as follows:

[0037] Domatiomyces globalis strains (CBS 135086, CBS 134924, CBS 129053, CBS132039, CBS 143850, CBS 134916, CBS 132002, CBS 134915, CBS 129057), Domatiomyces germinans (CBS 134922).

[0038] 2. Strain revival and subculturing

[0039] Prepare potato glucose agar (PDA) medium, seal and autoclave. Prepare plates and slant agar using aseptic techniques within a biosafety cabinet for later use.

[0040] Bacterial strains CBS 135086, CBS134924, CBS 129053, CBS 132039, CBS143850, CBS 134916, CBS 132002, CBS 134915, CBS129057, and CBS 134922 were revived in a biosafety cabinet and incubated at 28°C for 15 days.

[0041] 3. Initial screening

[0042] Prepare a 2% Tween-80 medium as a primary screening plate. Use a bacterial collector to inoculate 3 mm diameter mycelial blocks onto the primary screening plate and incubate at 28℃ for 30 days. Observe the growth of the mycelium and measure the size of the clear zone and mycelial zone.

[0043] Results: After screening with Tween-80 plates, 8 strains of bacteria were able to grow on the plates and produce visible clear zones. The size of the clear zones was related to the ability to degrade lipids. The results are as follows: Figure 1 .

[0044] The growth of the strain was observed on the initial screening medium, and the size of the transparent zone was measured. The diameter of the transparent zone and the diameter of the bacterial zone were randomly measured three times using a ruler, and the average value was taken. The HC (diameter of transparent zone D / diameter of bacterial zone d) was calculated, and the results are shown in Table 3.

[0045] Table 3. Strain Number and HC Value

[0046]

[0047] Example 2

[0048] Degradation experiments were conducted on the positive strains obtained from the initial screening.

[0049] 1. Drawing the standard curve

[0050] Accurately weigh 0.1000 g of Golden Dragon rapeseed oil sample (accurate to 0.1 mg), add 2 mL of chromatographically pure petroleum ether to a 25 mL beaker, and sonicate for 5 min. Quantitatively transfer the solution to a 5 mL A-grade volumetric flask using a glass rod. Rinse the beaker three times with petroleum ether (2 mL each time), combine the washings with the flask, and finally dilute to the mark to obtain a stock solution with a concentration of 20.00 mg / mL.

[0051] A series of standard solutions were prepared using the gradient dilution method: 0 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL, 0.60 mL, 0.70 mL, and 0.80 mL of stock solution were accurately transferred to five sets of 5 mL Grade A volumetric flasks, and diluted to volume with petroleum ether to obtain standard solutions with concentrations of 0 mg / mL, 0.40 mg / mL, 0.80 mg / mL, 1.20 mg / mL, 1.60 mg / mL, 2.00 mg / mL, 2.40 mg / mL, 2.80 mg / mL, and 3.20 mg / mL.

[0052] A solution with an intermediate concentration of 1.60 mg / mL was selected for full-band scanning (200 nm ~ 300 nm, scanning interval 1 nm, slit width 2 nm) to determine the maximum absorption wavelength λmax. The experiment showed that the maximum absorption value was found at a wavelength of 230 nm.

[0053] At 230 nm, using petroleum ether as a reference, the absorbance of rapeseed oil solutions of different concentrations was measured. A standard curve was plotted with oil concentration on the x-axis and OD230 on the y-axis. In subsequent experiments, the oil content in the sample can be calculated by measuring the OD230 absorbance using the standard curve. The entire experiment was conducted at a constant temperature of (25±0.5) ℃, and all glassware was treated with nitric acid-ethanol (1:3, v / v) to eliminate background interference.

[0054] 2. Determination of oil content in samples

[0055] The strains obtained from the initial screening were inoculated into conical flasks containing liquid mineral medium with a diameter of 1 mm and the oil content of 1.6 mg / mL (using rapeseed oil as the sole carbon source). The liquid mineral medium volume was 100 mL. The flasks were incubated at 28 °C on a constant temperature shaker at 100 r / min. After 15 days, the degradation rate was measured, and the strains were preserved for subsequent experiments.

[0056] Using ultraviolet spectrophotometry at room temperature, 15-day degradation culture was extracted from a conical flask by adding 15 mL of petroleum ether and shaking thoroughly. The extracted residue was then poured into a separatory funnel, shaken thoroughly, and allowed to separate into layers. The aqueous layer was transferred to a conical flask, and the petroleum ether was transferred to a 50 mL colorimetric tube. The aqueous layer was then transferred back to the separatory funnel, and the extraction was repeated once more with 15 mL of petroleum ether. After standing and separating, the petroleum ether layer was transferred to a colorimetric tube and mixed with the first extract. The two extracts were combined and diluted to 40.00 mL using a micropipette. 2.5 mL of the petroleum ether solution was added to a 50 mL colorimetric tube, and the volume was adjusted to 25 mL. The 10-fold diluted test solution was injected into a 10 mm quartz cuvette, and the absorbance was measured at 230 nm (three parallel measurements). Quantitative calculations were performed using a standard curve equation. Three replicates were set up for each sample group, and outliers were removed using the Grubbs test (α=0.05).

[0057] 3. Calculation method for oil degradation rate

[0058] A sterile liquid mineral medium containing uninoculated strains obtained from the initial screening was set up as a blank control group (BC). Parallel extraction experiments (n=5) were conducted under the exact same operating conditions as the experimental group. The absorbance of the BC group was measured by ultraviolet spectrophotometry, and the background lipid concentration C0 (mg / L) of the matrix was calculated by substituting it into the standard curve equation. The relative standard deviation should be less than 2.5%.

[0059] After the experimental group (containing bacteria system) was cultured, 50 mL of culture medium was taken and quantitatively extracted according to the same extraction procedure. After dehydration and volume adjustment, the absorbance value was measured, and the concentration of remaining oil C1 (mg / L) was calculated.

[0060] The biodegradation rate (η) of the oil is calculated according to formula (1): η = [(C0 - C1) / C0] × 100% (1)

[0061] result:

[0062] According to the Lambert-Beer Law, within a certain concentration range, the absorbance of a solution is directly proportional to its concentration. Petroleum ether solutions of Golden Dragon rapeseed oil at different concentrations were prepared, and standard curves were plotted by measuring their absorbance. A Golden Dragon rapeseed oil solution with an intermediate concentration of 1.6 mg / mL was selected, and its absorbance was measured between 200 nm and 300 nm. The experiment showed that it had a maximum absorption peak at 230 nm. The absorbance of solutions of various concentrations was measured at 230 nm, and a standard curve was plotted based on the obtained absorbance values. The standard curve shows a linear relationship between absorbance and Golden Dragon rapeseed oil concentration: y = 0.4239x + 0.0095, where R0... 2 The value of 0.9979 indicates that the standard curve is reliable, and the residual oil concentration in the sample can be calculated based on the standard curve. Figure 2 .

[0063] Based on the initial screening results, strains CBS 129053, CBS 132039, CBS 134916, CBS 143850, CBS 132002, CBS 134915, CBS 134922, and CBS 129057 were able to grow on Tween-80 selection plates and produce a clear zone. However, because CBS 129057 produced a small clear zone and exhibited slow colony growth, other strains were selected for subsequent experiments. These strains were CBS 129053, CBS 132039, CBS 134916, CBS 143850, CBS 132002, CBS 134915, and CBS 129057. Seven strains of bacteria, 134922, were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid mineral medium with oil as the sole carbon source. The flasks were incubated on a shaker at 28°C and 100 rpm for 15 days. A control flask containing no inoculated microorganisms was used under the same conditions. The oil degradation rate of each strain was calculated based on the standard curve of Jinlongyu rapeseed oil. Figure 3 .

[0064] This invention uses strain resuscitation and initial screening to identify experimental strains with a transparent complete circle, thus obtaining strains capable of degrading lipids. Degradation experiments revealed the following degradation capabilities: CBS 129053 had a degradation rate of 89.68%, CBS 134916 had a degradation rate of 88.99%, CBS 134922, CBS 143850, and CBS 134915 all had degradation rates of 88.52%, and CBS 132002 and CBS 132039 had degradation rates of 87.75% and 88.45%, respectively.

[0065] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0066] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0067] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Domatiomyces In the use in degrading oil and fat, characterized in that, Will Domatiomyces Cultured at 24~30℃ for 10~30d with the oil to be degraded, realizing the degradation of oil; The Domatiomyces To Domatiomyces globalis Or Domatiomyces germinans; The Domatiomyces globalis is any one of the strains with the Dutch Royal CBS Fungus Collection accession numbers CBS 132039, CBS 132002, CBS 134915, CBS 143850, CBS 134916 and CBS 129053; The Domatiomyces germinans is the strain with the CBS number CBS 134922 of the Royal CBS Fungus Collection in the Netherlands.

2. Use according to claim 1, characterized in that, Take a colony with a diameter of 1 mm and add it to 80 mL to 150 mL of liquid mineral culture medium containing the lipids to be degraded.

3. Use according to claim 2, characterized in that, The volume of the liquid mineral culture medium is 100 mL.

4. Use according to claim 3, characterized in that, The liquid mineral culture medium is prepared by using 8 mL to 12 mL of solution A and 13 mL to 18 mL of solution B; The formula for solution A is as follows: 200g ammonium sulfate, 10g magnesium chloride hexahydrate, 1g ethylenediaminetetraacetic acid, 0.2g zinc sulfate heptahydrate, 0.1g calcium chloride dihydrate, 0.5g ferrous sulfate heptahydrate, 0.2g sodium molybdate dihydrate, 0.02g copper sulfate pentahydrate, 0.04g chromium chloride hexahydrate, 0.1g manganese chloride dihydrate, and water to make up to 500mL; Solution B is formulated as follows: 155g of dipotassium hydrogen phosphate, 85g of disodium hydrogen phosphate dihydrate, and water to a final volume of 500mL.

5. Use according to claim 4, characterized in that, The liquid mineral culture medium was prepared by using 10 mL of solution A and 15 mL of solution B.

6. Use according to claim 1, characterized in that, The Domatiomyces The oil was incubated at 28°C for 15 days.

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