Application of Cladophialophora psammophila in the degradation of oils and fats

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to Cladophialophora psammophila The oil-degrading bacteria and the oil to be degraded are cultured at 24 DEG C to 30 DEG C for 10d to 30d, so that the oil is degraded; the oil-degrading bacteria are Cladophialophora psammophila The strain has good oil-degrading effect, and the degradation rate is 88.06%, so the strain is a high-efficiency oil-degrading strain, and therefore, the strain is used for degrading oil, and Cladophialophora psammophila The application belongs to the technical field of microorganisms, and particularly relates to

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically involving Cladophialophora psammophila Application in the degradation of oils and fats. Background Technology

[0002] The degradation of waste oil (such as catering waste 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 the existing technologies use Bacillus subtilis, Bacillus belye, and Bacillus licheniformis in biodegradation, etc. Cladophialophora psammophila It belongs to a type of dark-colored fungus, and there are no existing technologies reporting its use in degrading oils. Summary of the Invention

[0003] To solve the above problems, the present invention provides Cladophialophora psammophila Application in the degradation of oils and fats.

[0004] Cladophialophora psammophila Application in the degradation of oils and fats.

[0005] Preferably, the Cladophialophora psammophila It is a fungus with the number CBS 110553 from the Royal Dutch CBS Fungal Collection.

[0006] Preferably, Cladophialophora psammophila The oil to be degraded was cultured with the oil at 24℃~30℃ for 10d~30d to achieve oil degradation.

[0007] 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.

[0008] Preferably, the volume of the liquid mineral culture medium is 100 mL.

[0009] Preferably, 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;

[0010] 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 1L;

[0011] Solution B is formulated as follows: 155g dipotassium hydrogen phosphate, 85g disodium hydrogen phosphate dihydrate, and water to a final volume of 1L.

[0012] Preferably, the liquid mineral culture medium is prepared from 10 mL of solution A and 15 mL of solution B.

[0013] Preferably, Cladophialophora psammophila The oil to be degraded was incubated with the culture medium at 28°C for 15 days.

[0014] This invention was obtained from petroleum-contaminated soil. Petroleum-contaminated soil naturally contains a large number of microbial communities capable of utilizing hydrocarbons as carbon sources. Bacteria (such as Pseudomonas and Bacillus) and fungi (Aspergillus fumigatus, Penicillium, and Fusarium), due to long-term exposure to hydrocarbon pollutants, have evolved enzyme systems (such as monooxygenases and dioxygenases) to metabolize petroleum hydrocarbons, enabling them to decompose complex hydrocarbon compounds. In petroleum-contaminated soil, hydrocarbons often become the sole carbon and energy source for microorganisms. This environmental selection pressure promotes the enrichment of strains with degradation capabilities. Therefore, the research and development of microbial degradation of oils has gradually become a hot topic in oil pollution remediation technology.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The oil-degrading bacteria of the present invention Cladophialophora psammophila This strain exhibits good degradation activity for oils, with a degradation rate of 88.06%, classifying it as a highly efficient degradation strain. Therefore, its application in oil degradation provides... Cladophialophora psammophila Application in the degradation of oils and fats. Attached Figure Description

[0017] Figure 1 strains Cladophialophora psammophila The growth status of CBS110553.

[0018] Figure 2 The standard curve of Golden Dragon rapeseed oil was plotted at a wavelength of 230 nm. Detailed Implementation

[0019] 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.

[0020] The strains in this invention Cladophialophora psammophila , CBS110553 See the literature Badali, H., Prenafeta-Boldu, FX, Guarro, J., Klaassen, CH, Meis, JF, & deHoog, GS (2011). Cladophialophora psammophila, a novel species of Chaetothyriales with a potential use in the bioremediation of volatilearomatic hydrocarbons. Fungal biology, 115(10), 1019–1029. https: / / doi.org / 10.1016 / j.funbio.2011.04.005

[0021] The information on the culture medium used in this invention is as follows:

[0022] 1. The formula for potato glucose agar (PDA) medium is: 4 g / L Potato extract, 20 g / L Dextrose, and 15 g / L Agar (Sigma).

[0023] 2. The formula for the resuscitation medium is: PDA medium.

[0024] 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.

[0025] 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.

[0026] Table 1. Composition of Liquid A

[0027]

[0028] Table 2 Composition of Liquid B

[0029]

[0030] Example 1

[0031] Strain screening and identification

[0032] 1. A fungal strain screened from petroleum-contaminated soil through domestication and enrichment culture. Cladophialophora psammophila , CBS110553.

[0033] 2. Strain revival and subculturing

[0034] Prepare potato glucose agar (PDA) medium, seal and autoclave. Prepare plates and slant agar using aseptic techniques within a biosafety cabinet for later use.

[0035] The bacterial strain CBS 110553 was revived in a biosafety cabinet and incubated at 28°C for 15 days.

[0036] 3. Initial screening

[0037] 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.

[0038] After screening on Tween-80 plates, this strain was able to grow on the primary screening plates and produce a visible clear zone. The size of the clear zone is related to its ability to degrade lipids, as shown in the results. Figure 1 .

[0039] 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 result was 1.50.

[0040] Example 2

[0041] 1. Drawing the standard curve

[0042] 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 to dissolve. Quantitatively transfer the solution to a 5 mL Grade A 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.

[0043] A series of standard solutions were prepared using a 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 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. The 1.60 mg / mL intermediate concentration solution 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. Experiments showed that the maximum absorbance was observed at a wavelength of 230 nm. At 230 nm, using petroleum ether as a reference, the absorbance of olive oil solutions of different concentrations was measured. The absorbance was plotted on the x-axis as oil concentration and on the OD value. 230 Plot a standard curve with y = 0 on the ordinate. In later experiments, the standard curve can be used to measure OD... 230 The oil content in the sample was calculated using the absorbance value. 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.

[0044] 2. Determination of oil content in samples

[0045] 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.

[0046] 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).

[0047] 3. Calculation method for oil degradation rate

[0048] A sterile liquid mineral culture medium without microorganisms was set up as a blank control (BC), and parallel extraction experiments (n=3) were conducted under the 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%.

[0049] 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.

[0050] The biodegradation rate (η) of oils is calculated according to formula (1):

[0051] η=[(C0-C1) / C0]×100%(1)

[0052] result

[0053] According to Beer-Lambert's 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 a standard curve was plotted by measuring their absorbance. The standard curve shows a linear relationship between absorbance and Golden Dragon rapeseed oil concentration: y = 0.4239x + 0.0095, where R² = 0.9979, indicating the reliability of the standard curve. The remaining oil concentration in the sample can be calculated based on the standard curve. Figure 2 .

[0054] Based on the fact that strain CBS 110553 obtained from the initial screening can grow on Tween-80 screening plates and has a clear zone, it was inoculated into a conical flask (250 mL) containing 100 mL of liquid mineral medium with oil as the sole carbon source. The flask was then placed in a constant temperature shaker at 28℃ and 100 r / min for 15 days. A flask of uninoculated culture medium was used as a control under the same conditions. According to the standard curve of Golden Dragon rapeseed oil, the oil degradation rate of each strain was calculated to be 88.06%, and the HC value was 1.50.

[0055] This invention uses bacterial resuscitation and initial screening to identify experimental strains with a clear zone, thus obtaining strains capable of degrading lipids. Degradation experiments show that the degradation capacity of the experimental strain is 88.06% for CBS 110553.

[0056] The oil-degrading bacteria of the present invention Cladophialophora psammophila This category belongs to dark-colored fungi. Dark-colored 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 highly efficient adsorption or chelation of heavy metals and organic pollutants (such as dyes), enhancing their tolerance to 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 strong molecular stress resistance mechanisms (such as osmotic pressure-protecting proteins and antioxidant enzymes). These unique advantages make dark-colored fungi a powerful candidate for bioremediation of complex and extremely polluted environments.

[0057] 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.

[0058] 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.

[0059] 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. Cladophialophora psammophila Its application in the degradation of oils and fats is characterized by, Will Cladophialophora psammophila The oil to be degraded was cultured with the oil at 24℃~30℃ for 10d~30d to achieve oil degradation; The Cladophialophora psammophila It is a fungus with the number CBS 110553 from the Royal Dutch CBS Fungal Collection.

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

4. The application 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 a final volume of 1L. Solution B is formulated as follows: 155g of dipotassium hydrogen phosphate, 85g of disodium hydrogen phosphate dihydrate, and water to a final volume of 1L.

5. The application 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. The application according to claim 1, characterized in that, Will Cladophialophora psammophila The oil to be degraded was cultured at 28°C for 15 days.

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