Method for degrading grease

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for degrading grease, which comprises the following steps: culturing grease degrading bacteria and grease to be degraded at 24-30 DEG C for 10-30 days to realize the degradation of the grease; according to the present invention, the oil degrading bacterium is Domatiomyces, the Domatiomyces provided by the present invention belongs to a new family, the Domatiomyces globis or Domatiomyces germinans are two new strains, and have good degradation effects on oil, and the oil degrading bacterium is used for degrading oil, and provides a method for degrading oil.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a method for degrading oil and fat. BACKGROUND

[0002] Degradation of waste oil and fat (such as catering waste oil, industrial oil and fat waste, and animal and plant oil and fat residues) is an important issue in environmental governance and resource recycling. At present, there are many methods for treating contaminated oil and fat, but the main methods are (1) physical removal method (sedimentation method, centrifugation method, etc., suspension method, adsorption method, etc.), but the physical method is a "non-destructive" technology, that is, after the physical method is used, the pollutants are not harmless but are transferred to a new environment, and if not properly treated, secondary pollution can still occur; (2) chemical removal method (flocculation method, oxidation method, electrolysis method, etc.), but the chemical method also introduces new pollutants into the environment; (3) biodegradation method is the current mainstream technology for solving oil and fat pollution, which mainly utilizes the characteristics of microorganisms to consume oil and fat to decompose oil and fat in oil and fat wastewater. Bioremediation is a controlled or spontaneous process that utilizes the catalytic effect of microbial metabolism to degrade pollutants in the environment, thereby degrading and converting harmful substances in wastewater into harmless substances and reducing or ultimately eliminating pollution to the environment. Although the existing technology has oil and fat degrading bacteria, new biological bacteria need to be explored to enrich the types of oil and fat degrading bacteria. SUMMARY

[0003] To solve the above problems, the present application provides an oil and fat degrading bacteria Domatiomyces and its application. To achieve the above purpose, the first aspect of the present disclosure provides a fungus for degrading oil and fat, the classification name of the fungus is Domatiomyces globalis and Domatiomyces germinans , and the preservation number of the growth-promoting bacteria is

[0004] An oil and fat degrading method, wherein the oil and fat degrading bacteria and the oil and fat to be degraded are cultured at 24-30°C for 10-30 days to achieve degradation of the oil and fat. The oil and fat degrading bacteria are Domatiomyces .

[0005] Preferably, the Domatiomyces is Domatiomyces globalis or Domatiomyces germinans .

[0006] Preferably, the Domatiomyces globalis is any one of the bacteria with the preservation numbers CBS132039, CBS 132002, CBS 134915, CBS 143850, CBS 134916 and CBS 129053 of the Royal Netherlands CBS Fungus Collection.

[0007] Preferably, the oil-degrading fungus is a fungus of the genus Mortierella. Domatiomyces germinans is a fungus of the Royal CBS Fungus Collection of the Netherlands with the number CBS 134922.

[0008] Preferably, the colony with a diameter of 1 mm is added into 80 mL to 150 mL of liquid mineral medium containing the oil to be degraded.

[0009] Preferably, the volume of the liquid mineral medium is 100 mL.

[0010] Preferably, the liquid mineral medium is prepared from 10 mL of solution A and 15 mL of solution B. The formula of solution A is: 200 g of ammonium sulfate, 10 g of magnesium chloride hexahydrate, 1 g of ethylenediaminetetraacetic acid, 0.2 g of zinc sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.5 g of ferrous sulfate heptahydrate, 0.2 g of sodium molybdate dihydrate, 0.02 g of copper sulfate anhydrous, 0.04 g of chromium chloride aqueous, 0.1 g of manganese chloride dihydrate, and water to 1 L. The formula of solution B is: 155 g of dipotassium hydrogen phosphate, 85 g of disodium hydrogen phosphate dihydrate, and water to 1 L.

[0011] Preferably, the oil-degrading fungus and the oil to be degraded are cultured at 28 DEG C for 15 days.

[0012] The present application separates some fungi from some Domatia ant holes near the equator, and in the previous research, it is found that these fungi belong to a new family, which is named Domatiomycetaceae As ants can produce a large amount of secondary metabolites, including aromatic hydrocarbons and lipids and other secondary metabolites, most microorganisms cannot grow in the ant holes, but the recently discovered fungi can grow in the ant holes, so it is believed that these black yeasts have the ability to decompose oil and other metabolites. Compared with the process of degrading oil by bacteria, the bacteria produce fatty acids, which make the environment acidify, so that the bacteria quickly lose the ability to degrade, while the fungi can still maintain good activity. Because most fungi are air-borne microorganisms, they have strong tolerance to dry environment or strong acidic environment. Therefore, the research and development of fungal biodegradation of oil have gradually become a hot spot in oil pollution treatment technology. Domatiomyces Compared with the prior art, the present application has the beneficial effects that:

[0013] The present application provides fungi belonging to a new family, or Domatiomyces two new species, which are isolated from a tropical ant-plant symbiotic system and have never been reported to have oil-degrading potential. The seven strains of the two species provided by the present application all have good oil-degrading effect, and six of them are Domatiomyces globalis Domatiomyces germinans two new species, which are isolated from a tropical ant-plant symbiotic system and have never been reported to have oil-degrading potential. The seven strains of the two species provided by the present application all have good oil-degrading effect, and six of them are Domatiomyces globalis ​strains (CBS132039, CBS 132002, CBS134915, CBS 143850, CBS 134916 and CBS 129053), with degradation rates ranging from 88.45-89.68%, and one strain Domatiomyces germinans (CBS 134922), with a degradation rate of 88.52%, both belong to high-efficiency degradation strains. Therefore, the present invention is used for degrading oils and fats, and provides a method for degrading oils and fats.

[0014] Furthermore, these fungi all belong to the dematiaceous fungi family, which exhibit significant advantages in pollutant degradation, owing to their unique physicochemical properties. Their cell walls are rich in melanin, which not only provides excellent protection against UV rays and oxidative stress but also efficiently adsorbs or chelates heavy metals and organic pollutants (such as dyes), enhancing their tolerance to toxic environments. Fungi in this order are exemplary multi-extreme-tolerant microorganisms, able to maintain activity and degrade pollutants under harsh conditions such as drought, high salinity, and nutrient depletion (e.g., industrial wastewater and saline soils). This is due to their powerful molecular stress resistance mechanisms (e.g., osmoprotective proteins and antioxidant enzymes). These unique advantages make dematiaceous fungi strong candidates for bioremediation of complex and extremely polluted environments.

[0015] Domatiomyces globalis and Domatiomyces germinans Both have good ability to degrade oil. Both bacteria can grow in the range of 9℃~33℃, grow better in the range of 24℃~30℃, and 28℃ is the optimal growth temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 2 is a colony morphology diagram of the oil-degrading bacteria disclosed herein, wherein 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 CBS 134922.

[0017] Figure 2 This is the standard curve of Golden Arowana rapeseed oil drawn at a wavelength of 230nm.

[0018] Figure 3 is the oil degradation rate of the oil-degrading bacteria of the present invention. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified.

[0020] CBS132039, CBS 132002, CBS 134915, CBS 143850, CBS 134916, CBS 129053 and CBS 134922 in the present application are the strain numbers in the literature Wang MZ, Belmonte-Lopes R, Pan T, Ahmed SA, Rodrigues Lustosa BP, Quan Y, Al-Hatmi AMS, Mayer VE, Voglmayr H, Grisolia ME, de Souza Lima 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.

[0021] The information of the culture medium used in the present application is as follows: 1. The formula of potato dextrose agar medium (PDA) is: 4 g Potato extract, 20 g Dextrose, 15 g Agar (Sigma), and water is added to 1 L.

[0022] 2. The recovery medium is: PDA medium.

[0023] 3. The formula of the primary screening medium is: weigh 4 g of agar powder and 4 mL of Tween-80 into a reagent bottle, and add liquid mineral medium to 200 mL.

[0024] The liquid mineral medium was prepared as follows: each inorganic salt was weighed according to the concentration in Table 1 and Table 2, stirred until completely dissolved, and then distilled water was added to 500 mL to prepare 500 mL of A liquid and 500 mL of B liquid. 10 mL of A liquid and 15 mL of B liquid were taken into a measuring cylinder, diluted with distilled water, poured into a volumetric flask, and the measuring cylinder was rinsed 5 times with distilled water. The rinsing liquid was transferred into the volumetric flask, and distilled water was added to make up to 1 L, thereby preparing the liquid mineral medium.

[0025] Table 1 A liquid components Table 2 B liquid components Example 1 Strain screening and identification 1. Two categories of a total of 10 strains were screened from the ant nest through domestication and enrichment culture, as follows: Domatiomyces globalis Strains (CBS 135086, CBS 134924, CBS 129053, CBS 132039, CBS 143850, CBS 134916, CBS 132002, CBS 134915, CBS 129057), Domatiomyces germinans (CBS 134922).

[0026] 2. Strain recovery and subculture Prepare potato glucose agar medium (PDA), and sterilize by high-pressure steam after sealing. Prepare plates and slopes under sterile conditions in a biological safety cabinet for standby use.

[0027] Recover strains CBS 135086, CBS 134924, CBS 129053, CBS 132039, CBS 143850, CBS 134916, CBS 132002, CBS 134915, CBS 129057 and CBS 134922 in a biological safety cabinet, and incubate in a constant temperature incubator at 28°C for 15 days.

[0028] 3. Preliminary screening Prepare 2% Tween-80 medium as a preliminary screening plate, and inoculate a 3 mm diameter mycelial block into the preliminary screening plate using a bacterium collector, and incubate in a constant temperature incubator at 28°C for 30 days. Observe the growth of the strain and measure the size of the transparent circle and the mycelial circle.

[0029] Results: After Tween-80 plate screening, 8 strains can grow on the plate and produce visible transparent circles, and the size of the transparent circle is related to the ability to degrade oil. The results are as follows:Figure 1 .

[0030] The growth of the strains was observed on the primary screening culture medium and the size of the transparent zone was measured. The diameter of the transparent zone and the diameter of the bacterial ring were randomly measured three times using a ruler and the average value was taken to calculate the HC (transparent zone diameter D / bacteria ring diameter d). The results are shown in Table 3.

[0031] Table 3 Strain numbers and HC values Example 2 The positive strains obtained from the initial screening were subjected to degradation experiments 1. Drawing of standard curve Accurately weigh 0.1000g of Golden Arowana rapeseed oil sample (accurate to 0.1mg) and add 2mL of chromatographically pure petroleum ether to a 25mL beaker. Dissolve the sample under ultrasound for 5 minutes. Transfer the solution quantitatively to a 5mL A-grade volumetric flask using a glass rod. Rinse the beaker three times with 2mL of petroleum ether, combine the washes, and transfer the volumetric flask to the mark. Finally, bring the volume to the mark to obtain a stock solution with a concentration of 20.00mg / mL.

[0032] A series of standard solutions were prepared by 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 pipetted into 5 groups of 5 mL Class A volumetric flasks, and the volume was adjusted with petroleum ether to prepare 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 standard solutions.

[0033] A 1.60 mg / mL intermediate concentration solution was selected for full-band scanning (200 nm to 300 nm, 1 nm scanning interval, 2 nm slit width) to determine the maximum absorption wavelength, λmax. The experiment determined that the maximum absorption value occurred at a wavelength of 230 nm.

[0034] The absorbance of rapeseed oil solutions of varying concentrations was measured at 230 nm, using petroleum ether as a reference. A standard curve was constructed, plotting oil concentration on the horizontal axis and OD230 on the vertical axis. In subsequent experiments, this standard curve was used to calculate the oil content of the sample by measuring OD230 absorbance. The entire experiment was conducted at a constant temperature of (25 ± 0.5)°C. All glassware was treated with nitric acid-ethanol (1:3, v / v) to eliminate background interference.

[0035] 2. Determination of oil content in samples The strains obtained by the preliminary screening were inoculated into a conical flask containing liquid mineral medium with Jinlongyu canola oil as the only carbon source, the oil content was 1.6 mg / mL, the volume of the liquid mineral medium was 100 mL, and the conical flask was cultured in a constant temperature shaker at 100 r / min and 28°C. The degradation rate was measured after 15 days of culture, and the strains were preserved and used for subsequent experiments.

[0036] Under room temperature conditions, 15 mL of petroleum ether was added to the 15-day degradation culture in the conical flask, and the residual oil was extracted by thoroughly shaking. Then, the mixture was poured into a separatory funnel, the separatory funnel was shaken thoroughly, and after the layers were separated, the water layer was moved into a conical flask, the petroleum ether was moved into a 50 mL cuvette, and the water layer was moved back into the separatory funnel. The petroleum ether was extracted again with 15 mL of petroleum ether, and after the layers were separated, the petroleum ether layer was moved into the cuvette and mixed with the first extraction liquid. After the two extraction liquids were combined, the volume was adjusted to 40.00 mL with a micro-liquid addition device. 2.5 mL of the petroleum ether solution was taken into a 50 mL cuvette, and the volume was adjusted to 25 mL. The 10-fold diluted test liquid was injected into a 10 mm quartz cuvette, and the absorbance value was measured at 230 nm (three parallel measurements). The quantitative calculation was performed by the standard curve equation, and three groups of parallel samples were set for each group of samples. Abnormal values were removed by Grubbs test method (α=0.05).

[0037] 3. Calculation method of oil degradation rate A sterile liquid mineral medium without inoculating the strains obtained by the preliminary screening was set as a blank control group (BC), and parallel extraction experiments were performed under the same operation conditions as the experimental group (n=5). The absorbance of the BC group was measured by UV spectrophotometry, and the matrix background oil concentration C0 (mg / L) was calculated by substituting the standard curve equation. The relative standard deviation should be less than 2.5%.

[0038] After the experimental group (containing bacteria system) was cultured, 50 mL of the culture solution was quantitatively extracted according to the same extraction procedure, and the absorbance value was measured after dehydration and constant volume. The residual oil concentration C1 (mg / L) was calculated.

[0039] The oil biodegradation rate (η) was calculated according to formula (1): η = [(C0 - C1) / C0] × 100% (1) Results: According to the Lambert-Beer law, within a certain concentration range, the absorbance of a solution is proportional to its concentration. Petroleum ether solutions of different concentrations of Golden Arowana rapeseed oil were prepared, and a standard curve was drawn by measuring their absorbance. A Golden Arowana rapeseed oil solution with an intermediate concentration of 1.6 mg / mL was selected to measure its absorbance between 200 nm and 300 nm. The experiment showed that it had a maximum absorption peak at a wavelength of 230 nm. The absorbance of solutions of various concentrations was measured at a wavelength of 230 nm, and then a standard curve was drawn based on the obtained absorbance values. According to the standard curve, its absorbance is linearly related to the concentration of Golden Arowana rapeseed oil: y=0.4239x+0.0095, where R 2 =0.9979 shows that the standard curve is reliable. The residual oil concentration in the sample can be calculated based on the standard curve. Figure 2 .

[0040] According to the initial screening, the strains CBS 129053, CBS 132039, CBS 134916, CBS 143850, CBS 132002, CBS 134915, CBS 134922, and CBS129057 were able to grow on Tween-80 screening plates and had clear zones. However, since the clear zones produced by CBS 129057 were small and the colony grew slowly, other strains were selected for subsequent experiments. Seven strains were inoculated into a 250 mL conical flask containing 100 mL of liquid mineral culture medium with oil as the sole carbon source, and cultured in a constant temperature shaker at 28°C and 100 r / min for 15 days. A bottle of culture medium without microorganisms was used as a control under the same conditions. The oil degradation rate of each strain was calculated based on the standard curve of Jinlongyu rapeseed oil. Figure 3 .

[0041] The present invention screens out experimental strains with transparent full circles through strain recovery and initial screening to obtain strains capable of degrading oil and fat. Degradation experiments show that the degradation capabilities of the experimental strains are as follows: the degradation rate of CBS 129053 is 89.68%, the degradation rate of CBS 134916 is 88.99%, the degradation rates of CBS 134922, CBS 143850, and CBS 134915 are all 88.52%, and the degradation rates of CBS 132002 and CBS 132039 are 87.75% and 88.45%, respectively.

[0042] It is to be understood that the numerical ranges recited in the claims are intended to include every integer value within the range and any fraction of the values within the range. In other words, the numerical ranges are intended to include every value from the lower limit to the upper limit, inclusive of the lower and upper limits, as well as any intervening incremental values. Preferred embodiments of the application are described herein.

[0043] Although preferred embodiments of the application have been described herein, it will be apparent to those skilled in the art that various modifications and changes can be made without departing from the spirit and scope of the application. Accordingly, it is intended that all claims be interpreted to include all equivalent processes, procedures, compositions, and compounds that fall within the scope of the claims. The following examples are provided to further illustrate the application.

[0044] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described herein.

Claims

1. A method for degrading oils and fats, characterized in that: The oil-degrading bacteria and the oil to be degraded are cultured at 24°C to 30°C for 10 days to 30 days to achieve oil degradation; The oil-degrading bacteria are Domatiomyces .

2. The method according to claim 1, characterized in that described Domatiomyces for Domatiomyces globalis or Domatiomyces germinans .

3. The method according to claim 2, characterized in that described Domatiomyces globalis The fungus may be any one of the fungi numbers CBS 132039, CBS 132002, CBS 134915, CBS 143850, CBS 134916 and CBS 129053 of the CBS Fungus Collection of the Netherlands.

4. The method according to claim 2, characterized in that described Domatiomyces germinans It is a fungus with the number CBS 134922 of the Royal Netherlands CBS Fungus Collection.

5. The method according to claim 1, wherein 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 oil to be degraded.

6. The method according to claim 5, characterized in that The volume of the liquid mineral culture medium was 100 mL.

7. The method according to claim 6, characterized in that The liquid mineral culture medium is prepared from 8 mL to 12 mL of solution A and 13 mL to 18 mL of solution B; The formula of liquid A is: 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 running chromium chloride, 0.1g manganese chloride dihydrate, and make up to 1L with water; The formula of liquid B is: 155g of dipotassium hydrogen phosphate, 85g of disodium hydrogen phosphate dihydrate, and water to make up to 1L.

8. The method according to claim 5, characterized in that The liquid mineral culture medium is prepared from 10 mL of liquid A and 15 mL of liquid B.

9. The method according to claim 1, characterized in that The oil-degrading bacteria and the oil to be degraded were cultured at 28°C for 15 days.

10. Use of the bacteria according to claim 3 or claim 4 in degrading oils and fats.

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