Salt-tolerant oil-degrading bacteria, biological preparation and application thereof in degrading oil

Abstract

The application discloses a salt-tolerant oil-degrading bacterium, a biological preparation and application of the salt-tolerant oil-degrading bacterium in oil degradation, and belongs to the technical field of biological degradation. The strain of the salt-tolerant oil-degrading bacterium is named IURM G72, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC NO.29726. The salt-tolerant oil-degrading bacterium IURM G72 and the preparation thereof have good salt tolerance and oil-degrading performance, can grow with oil as the only carbon source in the case that the salt concentration is high, and have simple nutritional requirements. The degrading bacterium and the preparation thereof can effectively degrade oil in high-salt kitchen waste, and have important significance for water bodies and soil polluted by waste edible oil.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biodegradation, and particularly relates to a salt-tolerant oil-degrading bacterium, a biological agent and application thereof in degrading oil. BACKGROUND

[0002] Kitchen waste oil is prone to flow into groundwater and water bodies during the treatment process, causing water pollution. Harmful substances and chemicals in oil can harm aquatic organisms and disrupt the ecological balance. At the same time, oil can hinder the exchange of air and water in the soil, affecting the growth and development of plants. In addition, kitchen waste oil, when accumulated or not properly treated, can produce odors and harmful gases such as methane and hydrogen sulfide. These gases are harmful to human health and can also cause air pollution.

[0003] Currently, the main methods for treating kitchen waste oil include physical, chemical and biological methods. Physical and chemical methods can only remove part of the floating and dispersed oil, and it is difficult to treat emulsified and dissolved oil in water bodies. Chemical methods can effectively treat emulsified oil, but are not environmentally friendly. Biological methods are completed by microorganisms, including bacteria, fungi and other microbial populations. These microorganisms produce specific enzymes to break down oil into smaller molecules, which are then further metabolized into harmless substances such as water and carbon dioxide. The biological method is simple, low-cost and does not cause secondary pollution, so it is considered one of the important methods for deep treatment of organic matter.

[0004] Biological treatment of kitchen waste mainly includes aerobic or anaerobic fermentation treatment, which requires aerobic or anaerobic microorganisms. However, the high concentration of oil and salt in kitchen waste inhibits the activity of microorganisms, greatly affecting the fermentation process, and the fermentation products are also affected.

[0005] Therefore, it is necessary to find efficient salt-tolerant oil-degrading bacteria to degrade them to promote the biological treatment of water bodies and soil containing kitchen waste oil. SUMMARY

[0006] In view of the problems existing in the prior art, the purpose of the present application is to design a salt-tolerant oil-degrading bacterium Pseudozyma, which solves the problem of difficult degradation of kitchen waste oil and has good effect in degrading kitchen waste oil.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] In one aspect, the application provides an oil-degrading bacterium IURM G72, which is a Wickerhamiella infanticola and is deposited with the China General Microbiological Culture Collection Center, and is named as Wickerhamiella infanticola, and is deposited at 1 Qingdao West Road, Beijing, China, and has a deposit number of CGMCC NO.29726, and is deposited on January 23, 2024.

[0009] The oil-degrading bacterium has a nucleotide sequence as shown in SEQ ID NO. 1.

[0010] In a second aspect, the application provides an oil-degrading biological agent, which comprises the oil-degrading bacterium and an auxiliary material.

[0011] The oil-degrading biological agent is in a liquid or powder form.

[0012] In a third aspect, the application provides an application of the oil-degrading bacterium or the oil-degrading biological agent in degrading oil.

[0013] In a fourth aspect, the application provides an application of the oil-degrading bacterium or the oil-degrading biological agent in preparing a product for degrading oil.

[0014] In a fifth aspect, the application provides an application of the oil-degrading bacterium or the oil-degrading biological agent in treating an oil-polluted environment.

[0015] The oil-polluted environment is a water body or soil polluted by kitchen waste oil.

[0016] In a sixth aspect, the application provides an application of the oil-degrading bacterium or the oil-degrading biological agent in preparing a product for treating an oil-polluted environment.

[0017] In a seventh aspect, the application provides an application of the oil-degrading bacterium or the oil-degrading biological agent in producing organic acid by using oil.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] 1. The application provides an oil-degrading bacterium IURM G72 and a biological agent prepared therefrom, which has high salt tolerance, simple nutritional requirements, and can effectively degrade kitchen waste oil.

[0020] 2、The oil-degrading bacteria IURM G72 and the biological preparation prepared by using the same have wide application prospects in treating water bodies and soil polluted by waste edible oil and the like. In water bodies, when the temperature is 30 DEG C, the pH is 7, and the salt concentration is 10 g / L, the oil-degrading rate is 92.25%; when the salt concentration is 30 g / L, the oil-degrading rate is 64.46%; and when the salt concentration is 40 g / L, the oil-degrading rate is 54.18%. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is the culture morphology of the strain;

[0022] Figure 2 It is the phylogenetic tree of the strain IURM G72 of the application;

[0023] Figure 3 It is the photo of the degradation of IURM G72 with soybean oil as the only carbon source;

[0024] Figure 4 It is the standard curve graph of oil at absorbance 225 nm;

[0025] Figure 5 It is the oil-degrading rate graph at different pH values;

[0026] Figure 6 It is the oil-degrading rate graph at different temperatures;

[0027] Figure 7 It is the oil-degrading rate graph of different nitrogen sources of the culture medium;

[0028] Figure 8 It is the oil-degrading rate graph of different initial salt concentrations of the culture medium;

[0029] Figure 9 It is the oil-degrading rate graph of different treatment of kitchen waste. DETAILED DESCRIPTION

[0030] The application will be further described below by means of the drawings and examples.

[0031] Example 1:

[0032] (I) Preparation of the culture medium

[0033] 1. Soybean oil inorganic salt liquid medium (g / L): soybean oil 10 mL, sodium nitrate (NaNO3) 1.06 g, ammonium chloride (NH4Cl) 0.66 g, potassium dihydrogen phosphate (KH2PO4) 0.5 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.1 g, dipotassium hydrogen phosphate (K2HPO4) 1.5 g, sodium chloride (NaCl) 0.35 g, pH 7.0-7.2.

[0034] 2. Soybean oil inorganic salt solid medium (g / L): soybean oil 20 mL, sodium nitrate (NaNO3) 1.06 g, ammonium chloride (NH4Cl) 0.66 g, potassium dihydrogen phosphate (KH2PO4) 0.5 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.1 g, dipotassium hydrogen phosphate (K2HPO4) 1.5 g, sodium chloride (NaCl) 0.35 g, agar powder 16 g, Tween 80 5 mL, pH 7.0-7.2.

[0035] The specific steps of the soybean oil inorganic salt solid medium used by the oil-degrading bacteria are as follows:

[0036] (1) 2 mL of soybean oil and 500 μL of Tween 80 were added to 100 mL of inorganic salt liquid medium, and after fully reacting and mixing, a milky white liquid was formed. 1.6 g of agar was mixed and then sterilized in a high-pressure sterilization pot;

[0037] (2) The liquid medium after sterilization in step (1) was poured into a plate to obtain the soybean oil inorganic salt solid medium.

[0038] 3. PDA solid medium (g / L): potato powder 5 g, glucose 20 g, agar 15 g, chloramphenicol 0.1 g, pH 5.8-6.2.

[0039] (B) Sampling, enrichment and domestication of oil-degrading bacteria

[0040] 1. Samples were collected from the oil stains in the canteen of Changzhou University in Changzhou City, Jiangsu Province, 5 g of which was weighed and added to 100 mL of soybean oil inorganic salt liquid medium for enrichment, and cultured in a constant temperature shaker at 30°C and 180 r / min for 4-6 days.

[0041] 2. 5 mL of the culture obtained in step 1 was added to a new 100 mL soybean oil inorganic salt liquid medium for subculture, and this was repeated for 4-5 times.

[0042] (Three) Isolation and purification of oil-degrading bacteria

[0043] 1. The last subcultured culture was separated by dilution and coating plate method, i.e. dilution and coating on the soybean oil inorganic salt plate, and cultured in a 37°C incubator for two days.

[0044] 2. The obtained plate was picked out to 5 mL of soybean oil inorganic salt liquid medium and cultured in a 35°C shaker for two days.

[0045] 3. The dominant culture was selected, i.e. the bacterial liquid was turbid, the oil was degraded, and it was milky white. It was streaked on a LB plate and cultured in a 37°C constant temperature incubator for 1-2 days to ensure that the last separation obtained was a single colony, and the oil-degrading bacteria IURM G72 single colony morphology diagram was obtained, as shown inFigure 1 .

[0046] (iv) Identification of lipid-degrading bacteria

[0047] The strain's genes were amplified using PCR and then sent to a sequencing company for sequencing.

[0048] The dominant degrading bacteria were inoculated into LB liquid medium and cultured for 1-2 days. 1.8 mL of the bacterial culture was transferred to a centrifuge tube and concentrated by centrifugation at 12000 rpm for 1.5 min; 1.5 mL of the supernatant was discarded, leaving 0.4 mL in a separate tube; the culture was lysed for 1 min (×3); centrifuged at 3000 rpm for 30 s to obtain the lysed DNA, which was then used for PCR amplification. The PCR amplification system was: premix: 15 μL; ddw: 13.2 μL; primers: ITS1 (0.75 μL), ITS 4 (0.75 μL); bacterial culture: 0.3 μL. The temperature settings were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for 31 cycles, followed by a 72℃ extension for 5 min. Electrophoresis was then performed, and the samples were sent for sequencing. The nucleotide sequence of the lipid-degrading bacteria is shown in SEQ ID NO.1. Sequence alignment in the GeneBank database was performed using NCBI's BLAST function. The strain was identified as having over 99% similarity to *Wickerhamiella infanticola*. Further characterization of the strain was conducted based on ITS rRNA sequence analysis and alignment. A phylogenetic tree was constructed using MEGA software. Figure 2 The ITS rRNA sequence of the strain was uploaded to the GeneBank database, accession number PP702079.

[0049] (V) Cultivation

[0050] The bacterial strain was added to 100 mL of soybean oil-inorganic salt liquid medium and cultured for 3 days in a constant temperature shaker at 30℃ and 180 rpm. The resulting bacterial solution was turbid, indicating that the oil had been degraded and turned milky white. The control group (without bacterial solution) was used. Figure 3 .

[0051] Example 2:

[0052] Methods for determining the degradation rate of lipid-degrading bacteria:

[0053] (1) Plot the standard curve of soybean oil

[0054] Accurately weigh 0.1000 g of soybean oil into a 25 mL volumetric flask using an analytical balance. Dilute to the mark with petroleum ether to prepare a 0.004 g / mL (4 mg / mL) soybean oil petroleum ether standard solution. Transfer 5 mL of the soybean oil standard solution to a 50 mL colorimetric tube, standardize with petroleum ether, and dilute 10 times to prepare a 0.4 mg / mL petroleum ether soybean oil working solution. Accurately pipette 0.00, 6.25, 12.50, and 25.00 mL of the soybean oil working solution into four 25 mL colorimetric tubes, respectively, and dilute to the mark with petroleum ether to prepare soybean oil solutions with concentration gradients of 0.0, 0.1, 0.2, and 0.4 mg / mL. Accurately pipette 3.75, 5.00, 6.25, and 7.50 mL of soybean oil standard solution into four 25 mL colorimetric tubes, respectively. Dilute to the mark with petroleum ether to prepare soybean oil solutions with concentration gradients of 0.6, 0.8, 1.0, and 1.2 mg / mL. Finally, prepare soybean oil solutions with concentration gradients of 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL. Using pure petroleum ether as a reference, measure the absorbance values ​​at a wavelength of 225 nm. Based on the concentration and absorbance values, plot a soybean oil standard curve with soybean oil concentration on the x-axis and absorbance values ​​on the y-axis, as shown below. Figure 4 .

[0055] (2) Determination of oil degradation rate

[0056] Transfer the culture medium to a centrifuge tube, add ammonium sulfate to remove proteins, centrifuge at 8000 rpm for 15 min, transfer the supernatant to a separatory funnel, add 5 mL of 50% sulfuric acid for acidification to disperse and facilitate separation. Then add 15 mL of petroleum ether to the separatory funnel and shake thoroughly for 3 min. Let stand, separate the layers, transfer the aqueous layer from the bottom to a sampling bottle, transfer the petroleum ether to a 25 mL colorimetric tube, pour the aqueous layer back into the separatory funnel, and repeat the extraction once with 5 mL of petroleum ether. Let stand, separate the layers, transfer the petroleum ether layer to the 25 mL colorimetric tube from the first extraction, and dilute to volume with petroleum ether. Measure the absorbance at 225 nm using a UV spectrophotometer. Calculate the oil concentration using the linear regression equation of the soybean oil standard curve. Calculate the oil degradation rate using the formula: Oil degradation rate = (Initial oil concentration - Degraded oil concentration) / Initial oil concentration × 100%. All experiments were performed in triplicate.

[0057] After activating the strain IURM G72 obtained in Example 1, the pH, temperature, nitrogen source, and salt concentration were optimized. All experiments were performed in triplicate. Figure 5 , 6The optimal pH was determined to be 7, the optimal temperature to be 30℃, the optimal nitrogen source to be ammonium nitrate, and the optimal salt concentration for growth to be 10 g / L. The degradation rates, measured using the above methods, were 92.21%, 93.54%, 88.73%, and 92.25%, respectively. Although the pH and culture temperature measured in actual household kitchen waste oil were not optimal, this strain still exhibited good degradation characteristics for household kitchen waste oil.

[0058] Example 3:

[0059] After activating the strain IURM G72 obtained in Example 1, it was added to 100 mL of soybean oil inorganic salt liquid medium and cultured in a constant temperature shaker at 30℃ and 180 r / min for 4-6 days. The culture medium changed from transparent to milky white. The content of some organic acids in the culture medium with different nitrogen sources was determined by HPLC. The nitrogen sources were ammonium sulfate, ammonium nitrate, and peptone, respectively. The yields were compared as follows: Figure 7 As shown in the table, strain IURM G72 produces a relatively high amount of isovaleric acid, especially when the nitrogen source is organic nitrogen. Isobutyric acid production is the second highest; specific yields are shown in Table 1 below.

[0060] Table 1. Yields (g / L) of short-chain fatty acids from different nitrogen sources

[0061]

[0062] Example 4:

[0063] The bacterial suspension of strain IURM G72 obtained in Example 1 was inoculated into kitchen waste at an inoculation rate of 1% (v / v). The kitchen waste was taken from the supernatant of kitchen waste from the canteen of Changzhou University, which is oily waste liquid. 100 mL of the oily waste liquid was evenly sampled into 250 mL Erlenmeyer flasks, and control group CK and treatment groups T1, T2, and T3 were set up. T1 was treated with only 1 mL of bacterial suspension; T2 was treated with 1 mL of bacterial suspension added after sterilization at 80°C for 1 hour and cooling; and T3 was treated with 1 mL of bacterial suspension added after sterilization at 121°C for 20 minutes and cooling. After culturing for 72 hours, the degradation rate was measured according to the method in Example 2. Figure 9 As shown, the degradation rate was 31.42% in group CK, 37.51% in group T1, 54.52% in group T2, and 82.46% in group T3. After 96 hours of cultivation, the degradation rates were measured as follows: 34.53% in group CK, 54.77% in group T1, 63.59% in group T2, and 85.32% in group T3.

Claims

1. A salt-tolerant oil-degrading bacterium, named Wickerhamiella infanticola, strain IURM G72, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.29726.

2. The salt-tolerant oil-degrading bacteria as described in claim 1, characterized in that, The nucleotide sequence of the salt-tolerant oil-degrading bacteria is shown in SEQ ID NO.

1.

3. A salt-resistant lipid degradation biological agent, characterized in that, It contains the salt-tolerant oil-degrading bacteria and excipients as described in claim 1.

4. The salt-tolerant lipid degradation biological agent as described in claim 3, characterized in that, The biological agent is in liquid or powder form.

5. The application of the salt-tolerant oil-degrading bacteria as described in claim 1 or the salt-tolerant oil-degrading biological agent as described in claim 3 in the degradation of oils.

6. The application of the salt-tolerant oil-degrading bacteria as described in claim 1 or the salt-tolerant oil-degrading biological agent as described in claim 3 in the preparation of oil-degrading products.

7. The application of the salt-tolerant oil-degrading bacteria as described in claim 1 or the salt-tolerant oil-degrading biological agent as described in claim 3 in the treatment of oil-polluted environments.

8. The application as described in claim 7, characterized in that, The grease-contaminated environment refers to water bodies or soil contaminated by grease from kitchen waste.

9. The application of the salt-tolerant oil-degrading bacteria as described in claim 1 or the salt-tolerant oil-degrading biological agent as described in claim 3 in the preparation of products for treating oil-polluted environments.

10. The application of a salt-tolerant lipid-degrading bacterium as described in claim 1 or a salt-tolerant lipid-degrading biological agent as described in claim 3 in the production of organic acids from lipids.

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