Salt-tolerant grease degrading bacterium, biological preparation and application of biological preparation in grease degradation

Abstract

The invention discloses a salt-tolerant grease degrading bacterium, a biological agent and application of the biological agent in grease degradation, and belongs to the technical field of biodegradation. The strain name of the salt-tolerant grease degrading bacterium is IURM G72, and the salt-tolerant grease degrading bacterium is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.29726. The salt-tolerant grease degrading bacterium IURM G72 and the preparation thereof have good salt tolerance and grease degradation performance, can grow by taking grease as a unique carbon source under the condition of relatively high salt concentration, and have simple nutritional requirements. The degrading bacterium and the preparation thereof can effectively degrade grease in high-salt kitchen waste, and have important significance on water bodies, soil and the like polluted by waste edible grease.

Description

Technical Field

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

[0002] During the treatment process, waste cooking oil can easily flow into groundwater and water bodies, causing water pollution. Harmful substances and chemicals in oil can harm aquatic organisms and destroy 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, when waste cooking oil is accumulated or not properly handled, it will produce odor and harmful gases such as methane and hydrogen sulfide. These gases are harmful to human health and can also cause air pollution.

[0003] At present, the treatment methods for waste cooking oil mainly include physical, chemical and biological methods. Physical and chemical methods can only remove part of the floating oil and dispersed oil, and it is difficult to treat emulsified oil and dissolved oil in the water. Although the chemical method can treat emulsified oil well, it is not friendly to the ecological environment. The biological method is completed by microorganisms, including bacteria, fungi and other microbial communities. These microorganisms break down oil into smaller molecules by producing specific enzymes, and then further metabolize them into harmless substances such as water and carbon dioxide. The biological method has a simple treatment process, low cost, and will not cause secondary pollution. Therefore, it is considered to be one of the important methods for deep treatment of organic matter.

[0004] The biological treatment of food waste is mainly aerobic or anaerobic fermentation treatment, which requires aerobic or anaerobic microorganisms. However, the oil and salt concentrations in food waste are relatively high, which inhibits the activity of microorganisms and has a great impact on the fermentation process, and the fermentation products are also affected accordingly.

[0005] Therefore, it is necessary to find efficient salt-tolerant grease-degrading bacteria to degrade it, so as to promote the biological treatment process of water and soil containing waste cooking grease. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a salt-tolerant oil-degrading bacterium of the genus Pseudomonas, which solves the problem that kitchen waste oil is difficult to degrade and has a good effect in the application of degrading kitchen waste oil.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides an oil-degrading bacterium IURM G72, which is a yeast of the genus Wickerhamiella, deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, classified and named: Wickerhamiella infanticola, the address of the depository is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is: CGMCC NO.29726, and the deposit date is: January 23, 2024.

[0009] The salt-tolerant oil-degrading bacteria described herein have a nucleotide sequence as shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a salt-tolerant oil-degrading biological preparation, comprising the salt-tolerant oil-degrading bacteria and auxiliary materials.

[0011] The salt-resistant oil-degrading biological preparation is in liquid or powder form.

[0012] In a third aspect, the present invention provides the use of the salt-tolerant oil-degrading bacteria or the salt-tolerant oil-degrading biological agent in degrading oil.

[0013] In a fourth aspect, the present invention provides the use of the salt-tolerant oil-degrading bacteria or the salt-tolerant oil-degrading biological agent in the preparation of oil-degrading products.

[0014] In a fifth aspect, the present invention provides the use of the salt-tolerant oil-degrading bacteria or the salt-tolerant oil-degrading biological agent in treating oil-contaminated environments.

[0015] In the application, the grease-contaminated environment is water polluted by kitchen waste grease or soil polluted by kitchen waste grease.

[0016] In a sixth aspect, the present invention provides the use of the salt-tolerant oil-degrading bacteria or the salt-tolerant oil-degrading biological agent in the preparation of products for treating oil-polluted environments.

[0017] In a seventh aspect, the present invention provides the use of the salt-tolerant oil-degrading bacteria or the salt-tolerant oil-degrading biological agent in producing organic acids from oils.

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

[0019] 1. The present invention provides a grease-degrading bacterium IURM G72 and a biological preparation made using the same, which has high salt tolerance, simple nutritional requirements, and can effectively degrade kitchen waste grease.

[0020] 2. The oil-degrading bacteria IURM G72 of the present invention and the biological preparations made therefrom have broad application prospects in treating water bodies and soils contaminated by waste edible oils and fats. When the temperature is 30°C, the pH is 7, the salt concentration is 10g / L, and the oil degradation rate is 92.25%; when the salt concentration is 30g / L, the oil degradation rate is 64.46%; when the salt concentration is 40g / L, the oil degradation rate is 54.18%. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 This is the phylogenetic tree of strain IURM G72 of the present invention;

[0023] Figure 3 The photo shows the degradation of IURM G72 using soybean oil as the sole carbon source;

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

[0025] Figure 5 It is the graph of oil degradation rate at different pH;

[0026] Figure 6 It is the graph of oil degradation rate at different temperatures;

[0027] Figure 7 This is the oil degradation rate diagram of different nitrogen sources in the culture medium;

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

[0029] Fig. 9 This is a graph showing the degradation rate of oil in kitchen waste after different treatments. DETAILED DESCRIPTION

[0030] The present invention will be further described below by means of drawings and embodiments.

[0031] Embodiment 1:

[0032] (I) Preparation of culture medium

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

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

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

[0036] (1) Add 2 mL of soybean oil and 500 μL of Tween 80 to 100 mL of inorganic salt liquid medium, mix thoroughly to form a milky white liquid, add 1.6 g of agar, mix well, and sterilize in an autoclave;

[0037] (2) Take the liquid culture medium sterilized in step (1), pour it into a plate, and obtain a soybean oil inorganic salt solid culture medium.

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

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

[0040] 1. Collect oil samples from the canteen of Changzhou University Science and Education City Campus in Changzhou City, Jiangsu Province. Weigh 5 g of the sample and add it to 100 mL of soybean oil inorganic salt liquid culture medium for enrichment. Culture it in a constant temperature shaker at 30°C and 180 r / min for 4-6 days.

[0041] 2. Take 5 mL of the culture obtained in step 1 and add it into 100 mL of new soybean oil inorganic salt liquid culture medium for subculture. Repeat this process 4-5 times.

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

[0043] 1. The culture medium of the last passage was separated by dilution and spreading on a plate, i.e., it was diluted and spread on a soybean oil inorganic salt plate and cultured in a 37°C incubator for two days.

[0044] 2. Pick out strains of different shapes and sizes from the obtained plate and place them into 5 mL soybean oil and inorganic salt liquid medium and culture them in a shaking incubator at 35°C for two days;

[0045] 3. Select the dominant culture, i.e. the bacterial solution is turbid, the oil is degraded, and it is milky white. Streak it on the LB plate and culture it in a 37°C constant temperature incubator for 1-2 days to ensure that the last separation is a single colony. Obtain the morphology of the single colony of oil-degrading bacteria IURM G72, as shown below: Figure 1 .

[0046] (IV) Identification of oil-degrading bacteria

[0047] The strain gene was amplified using the PCR method and sent to a sequencing company for sequencing.

[0048] The dominant degrading bacteria were inoculated into LB liquid culture medium and cultured for 1-2 days. Take 1.8mL of bacterial solution in a centrifuge tube, centrifuge and concentrate at 12000rpm for 1.5min; pour 1.5mL of supernatant, leaving 0.4mL in the tube; break for 1min (×3); centrifuge at 3000rpm for 30s, obtain the DNA after shaking and breaking, and perform PCR amplification. The PCR amplification system is premix: 15μL; ddw: 13.2μL; primers: ITS1 (0.75μL), ITS 4 (0.75μL); bacterial solution 0.3μL. The temperature is set as: 95℃ pre-denaturation for 10min, 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, reaction for 31 cycles, and 72℃ extension for 5min. Then perform electrophoresis and send the sample for sequencing. The nucleotide sequence of the oil-degrading bacteria is shown in SEQ ID NO.1. Sequence alignment was performed in the GeneBank database using the BLAST function of NCBI. The strain was identified to have a similarity of more than 99% with Wickerhamiella infanticola. The strain was further characterized based on ITSrRNA sequence analysis and alignment, and a phylogenetic tree was constructed using MEGA software. Figure 2 The ITS rRNA sequence of the strain was uploaded to the GeneBank database with the accession number PP702079.

[0049] (V) Training

[0050] The strain was added to 100 mL of soybean oil inorganic salt liquid medium for cultivation. It was cultured in a constant temperature shaker at 30°C and 180 r / min for 3 days. The cultured bacterial solution was turbid, the oil was degraded, and it appeared milky white. The cultured solution without bacterial solution was used as a control. Figure 3 .

[0051] Embodiment 2:

[0052] Determination method of the degradation rate of oil-degrading bacteria:

[0053] (1) Draw the soybean oil standard curve

[0054] In a 25mL volumetric flask, accurately weigh 0.1000g of soybean oil using an analytical balance, use petroleum ether as the solvent, dilute to the mark, and prepare a 0.004g / mL, i.e. 4mg / mL soybean oil petroleum ether standard solution. Take 5mL of the soybean oil standard solution to a 50mL colorimetric tube, standardize with petroleum ether, dilute 10 times, and prepare a 0.4mg / mL petroleum ether soybean oil working solution. Use a pipette to accurately draw 0.00, 6.25, 12.50, and 25.00mL of the soybean oil working solution into 4 25mL colorimetric tubes, dilute to the mark with petroleum ether, and prepare a soybean oil solution with a concentration gradient of 0.0, 0.1, 0.2, and 0.4mg / mL. Use a pipette to accurately draw 3.75, 5.00, 6.25, and 7.50 mL of soybean oil standard solution into four 25 mL colorimetric tubes, and dilute the volume to the mark with petroleum ether to prepare soybean oil solutions with a concentration gradient of 0.6, 0.8, 1.0, and 1.2 mg / mL. Finally, prepare soybean oil solutions with a concentration gradient of 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL, and use pure petroleum ether as a reference to measure their absorbance values ​​at a wavelength of 225 nm. According to the concentration and the absorbance value obtained, draw a soybean oil standard curve with the soybean oil concentration as the horizontal coordinate and the absorbance value as the vertical coordinate, as shown in the figure. Figure 4 .

[0055] (2) Determination of oil degradation rate

[0056] Take the culture medium into a centrifuge tube, weigh ammonium sulfate and add it. After removing the protein, centrifuge at 8000rpm for 15min, transfer the supernatant into a separatory funnel, add 5mL of 50% sulfuric acid to acidify it, disperse it, and make it easier to separate. Then add 15mL of petroleum ether to the separatory funnel, shake it fully for 3min, let it stand, separate it, move the water layer from the bottom into the sampling bottle, move the petroleum ether into a 25mL colorimetric tube, pour the water layer back into the separatory funnel, extract it again with 5mL of petroleum ether, let it stand, separate it, move the petroleum ether layer into the 25mL colorimetric tube used for the first extraction, and make it constant with petroleum ether. Use an ultraviolet spectrophotometer to measure its absorbance at a wavelength of 225nm, and substitute the measured data into the equation to calculate the oil concentration according to the linear regression equation of the soybean oil standard curve. Use the formula: Oil degradation rate = (initial oil concentration - oil concentration after degradation) / initial oil concentration × 100% to calculate the oil degradation rate. All experiments are set up three times.

[0057] After the strain IURM G72 obtained in Example 1 was activated, the pH, temperature, nitrogen source and salt concentration were optimized. All experiments were repeated three times. Figure 5 , 6, 7, 8, and finally the optimal pH was 7, the optimal temperature was 30℃, the best nitrogen source was ammonium nitrate, and the optimal salt concentration for growth was 10g / L. The degradation rates determined by the above method were 92.21%, 93.54%, 88.73%, and 92.25%, respectively. Although the pH and culture temperature in the actual life kitchen waste oil are not the optimal conditions, the strain also has good degradation characteristics for the kitchen waste oil in life.

[0058] Embodiment 3:

[0059] After the strain IURM G72 obtained in Example 1 was activated, it was added to 100 mL of soybean oil inorganic salt liquid culture medium and cultured in a constant temperature shaker at 30°C 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 yield was compared. Figure 7 As shown. It can be seen that strain IURM G72 produces more isovaleric acid, especially when the nitrogen source is organic nitrogen. Isobutyric acid production is second; the specific production is shown in Table 1 below.

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

[0061]

[0062] Embodiment 4:

[0063] The bacterial solution of strain IURM G72 obtained in Example 1 was inoculated with 1% (v / v) inoculation amount into the food waste obtained. The food waste was taken from the supernatant of food waste in the canteen of Changzhou University, i.e., the oily waste liquid. 100 mL of the oily waste liquid was uniformly sampled in a 250 mL conical flask, and a control group CK and treatment groups T1, T2, and T3 were set. T1 was added with only 1 mL of bacterial solution, T2 treatment group was sterilized at 80 degrees for 1 hour and then cooled and then 1 mL of bacterial solution was added, and T3 treatment group was sterilized at 121 degrees for 20 minutes and then cooled and then 1 mL of bacterial solution was added. After 72 hours of culture, the degradation rate was measured according to the method of Example 2. Fig. 9 As shown, the degradation rate of CK group was 31.42%, the degradation rate of T1 group was 37.51%, the degradation rate of T2 group was 54.52%, and the degradation rate of T3 group was 82.46%. After 96 hours of culture, the degradation rate was measured, and the degradation rate of CK group was 34.53%, the degradation rate of T1 group was 54.77%, the degradation rate of T2 group was 63.59%, and the degradation rate of T3 group was 85.32%.

Claims

1. A salt-tolerant oil-degrading bacterium, the strain name is IURM G72, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.29726.

2. The salt-tolerant oil-degrading bacteria according to 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-tolerant oil-degrading biological agent, characterized in that: The invention comprises the salt-tolerant oil-degrading bacteria as claimed in claim 1 and auxiliary materials.

4. A salt-tolerant oil-degrading biological agent according to claim 3, characterized in that: The biological agent is in liquid or powder form.

5. Use of the salt-tolerant oil-degrading bacteria according to claim 1 or the salt-tolerant oil-degrading biological agent according to claim 3 in degrading oil.

6. Use of the salt-tolerant oil-degrading bacteria according to claim 1 or the salt-tolerant oil-degrading biological agent according to claim 3 in the preparation of oil-degrading products.

7. Use of the salt-tolerant oil-degrading bacteria as claimed in claim 1 or the salt-tolerant oil-degrading biological agent as claimed in claim 3 in treating an oil-contaminated environment.

8. The use according to claim 7, characterized in that The grease-contaminated environment is water polluted by kitchen waste grease or soil polluted by kitchen waste grease.

9. Use of the salt-tolerant oil-degrading bacteria according to claim 1 or the salt-tolerant oil-degrading biological agent according to claim 3 in the preparation of products for treating oil-polluted environments.

10. Use of the salt-tolerant oil-degrading bacteria according to claim 1 or the salt-tolerant oil-degrading biological agent according to claim 3 in producing organic acids from oils.

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