Salt-tolerant grease degrading bacterium as well as screening method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a salt-tolerant grease degrading bacterium as well as a screening method and application thereof, the salt-tolerant grease degrading bacterium is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.35335. The salt-tolerant grease degrading bacterium obtained by the invention has good salt tolerance, and can be used for degrading oil and fat. The strain can grow by taking grease as a unique carbon source under the condition of relatively high salt concentration, shows excellent grease degradation activity, is suitable for a kitchen waste composting system with relatively high salt content, and is beneficial to efficiently decomposing organic grease and improving the composting efficiency and the quality of a final product.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to salt-tolerant oil-degrading bacteria, a screening method and an application thereof. Background Art

[0002] Aerobic composting, as an environmentally friendly treatment method, has been widely studied and applied due to its multiple advantages, including reduced environmental pollution, lower transportation costs, clean operating environments, high automation, and improved resource utilization. However, the high salt and oil content in the Chinese diet leads to high salt and oil concentrations in food waste, which often delays the composting process and affects the quality of the final compost. High concentrations of Na+ increase the osmotic pressure of the composting environment, negatively impacting microbial activity and disrupting their metabolism. High oil content inhibits the passage of oxygen required for microbial metabolism, thereby affecting microbial growth and metabolism, as well as the rate and quality of microbial decomposition of organic matter and compost. Furthermore, oil-containing organic fertilizers can also lead to a decline in soil quality, directly impacting crop growth.

[0003] Therefore, an important direction of current research is to screen out microbial species that can effectively degrade oil in a high-salt environment and use them for aerobic composting of food waste to improve the efficiency and quality of such food waste composting. Summary of the Invention

[0004] The purpose of the present invention is to solve the above-mentioned deficiencies in the prior art and to provide a salt-tolerant oil-degrading bacterium and a screening method and application thereof.

[0005] In order to achieve the above object, the present invention can adopt the following technical solutions: On the one hand, the present invention provides a salt-tolerant oil-degrading bacterium, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.35335.

[0006] The salt-tolerant oil-degrading bacteria of the present invention are deposited as follows: depository institution: General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC); deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 22, 2025; deposit number: CGMCC No. 35335; classification name: Candida tropicalis.

[0007] Another aspect of the present invention provides a method for screening salt-tolerant oil-degrading bacteria, comprising the following steps: Preliminary screening of oil-degrading bacteria: Inoculate food waste leachate into an olive oil-mineral salt medium for cultivation, then transfer the culture to fresh medium according to the inoculation ratio. Repeat the cultivation process while gradually increasing the olive oil concentration to enhance the strain's ability to degrade oil. Salt tolerance acclimation: Select the bacterial solution after the initial screening and inoculate it into the culture medium for cultivation; after each round of cultivation, transfer it to a new culture medium according to the proportion, and continue multiple rounds of cultivation while gradually increasing the NaCl concentration in the culture medium to screen strains with strong salt tolerance; Dilution and coating culture: add the pre-screened and acclimated bacterial solution into sterile water, mix well and then perform gradient dilution to prepare 10 -1 to 10 -7 Take 10 -4 to 10 -7 The diluted solution was spread on PDA plate culture medium, placed in a constant temperature incubator, and the growth morphology and size characteristics of the colonies were observed; Single bacterial isolation and purification: Select colonies with good growth status from PDA plate culture medium, inoculate them on PDA plates for streak culture, and repeat the streak operation until the colonies on the plate have consistent morphology, size and color, and there is no obvious difference in bacterial morphology under microscope. This is considered to be a pure strain; Rescreening of oil degradation ability: The purified single strain was inoculated into tributyrin solid culture medium, and a quantitative bacterial solution was added to each well for cultivation. The strain with the fastest growth rate and the largest transparent zone under the same conditions was selected as the final oil-degrading bacteria.

[0008] Furthermore, in the preliminary screening of the oil-degrading bacteria, the inoculation ratio is 1% to 3%.

[0009] Furthermore, in the preliminary screening of oil-degrading bacteria, the culture process was repeated four times to gradually increase the olive oil concentration to 20 g / L, 30 g / L, and 40 g / L.

[0010] Furthermore, during the salt tolerance acclimation, four rounds of culture were performed continuously, and the NaCl concentration in the culture medium was gradually increased to 20 g / L, 30 g / L, and 40 g / L.

[0011] Furthermore, in the dilution coating culture, the volume of the bacterial liquid is 0.5 mL to 1.5 mL, and the volume of the sterile water is 8.5 mL to 9.0 mL.

[0012] Furthermore, in the oil degradation capacity re-screening, 0.8 μL to 1.2 μL of bacterial solution was added to each well.

[0013] Another aspect of the present invention provides an application of salt-tolerant oil-degrading bacteria in degrading olive oil.

[0014] Another aspect of the present invention provides an application of salt-tolerant oil-degrading bacteria in oily wastewater from restaurant kitchen waste.

[0015] Another aspect of the present invention provides an application of salt-tolerant oil-degrading bacteria in aerobic composting of high-salt and high-fat food waste.

[0016] The technical solution provided by the present invention has at least the following technical effects: 1. The salt-tolerant oil-degrading bacteria obtained in the present invention have good salt tolerance and can grow with oil as the sole carbon source under conditions of high salt concentration. They show excellent oil-degrading activity and are suitable for use in food waste composting systems with high salt content. They help to efficiently decompose organic oils, improve compost maturity efficiency and final product quality.

[0017] 2. The salt-tolerant oil-degrading bacteria screening method provided by the present invention is easy to operate, and the enrichment and acclimation process has good repeatability. It can efficiently screen out target strains with strong oil-degrading ability in a relatively short period of time, and is suitable for large-scale microbial resource mining and screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a colony morphology diagram of salt-tolerant oil-degrading bacteria according to an embodiment of the present invention; Figure 2 The transparent zone and growth condition of the salt-tolerant oil-degrading bacteria in the tributyrin culture medium according to the present invention are shown; Figure 3 is a phylogenetic tree of salt-tolerant oil-degrading bacteria according to an embodiment of the present invention; Figure 4 This is the growth of the salt-tolerant oil-degrading bacteria of the present invention in potato glucose medium for 48 hours; Figure 5 This is the degradation effect of the salt-tolerant oil-degrading bacteria of the embodiment of the present invention under different inoculation conditions; Figure 6 This is the degradation effect of the salt-tolerant oil-degrading bacteria of the embodiment of the present invention under different pH conditions; Figure 7 This is the degradation effect of the salt-tolerant oil-degrading bacteria of the embodiment of the present invention under different salt concentration conditions; Figure 8 This is the degradation effect of the salt-tolerant oil-degrading bacteria in the embodiment of the present invention under different degradation time conditions; Figure 9 The degradation effect of the salt-tolerant oil-degrading bacteria in the embodiment of the present invention on oily wastewater under different treatment conditions; Figure 10 The temperature change of the salt-tolerant oil-degrading bacteria in the aerobic composting of high-salt and high-fat food waste according to the embodiment of the present invention; Figure 11 This is the grease degradation effect of the salt-tolerant grease-degrading bacteria in the aerobic composting of high-salt and high-grease kitchen waste in the embodiment of the present invention; Figure 12 This is the organic matter degradation effect of the salt-tolerant oil-degrading bacteria in the aerobic composting of high-salt and high-fat kitchen waste in the embodiment of the present invention; Figure 13 It is the germination index of the salt-tolerant oil-degrading bacteria in the embodiment of the present invention in aerobic composting of high-salt and high-fat food waste. DETAILED DESCRIPTION

[0020] The following describes in detail embodiments of the present invention. These embodiments are provided to better illustrate the present invention, but are not intended to limit the present invention to these embodiments. Therefore, any non-essential improvements or adjustments made by those skilled in the art based on the above-described invention remain within the scope of protection of the present invention.

[0021] The embodiment of the present invention discloses a salt-tolerant oil-degrading bacterium, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 35335.

[0022] It should be noted that the salt-tolerant oil-degrading bacteria in this embodiment are derived from the oily leachate of kitchen waste, and the oily leachate of kitchen waste in this application is derived from the kitchen waste treatment system in concentrated catering areas (such as university canteens and large chain catering kitchens). The kitchen waste in such areas is mainly composed of animal and plant oils (the oil content is stable at 10%-20%), which is highly matched with the natural living environment of salt-tolerant oil-degrading bacteria (salt-tolerant oil-degrading bacteria need to rely on oil as a carbon source). Moreover, the kitchen waste in concentrated catering areas has a long-term stable composition and has formed a microecological environment suitable for the reproduction of such strains. The probability of the existence of salt-tolerant oil-degrading bacteria is stable.

[0023] The culture medium is as follows: olive oil inorganic salt medium: ammonium sulfate 4 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium chloride 10 g / L, olive oil 10 g / L, distilled water 1000 mL, sterilized at 121°C for 20 min; Potato glucose medium: potato extract powder 6 g / L, glucose 20 g / L, distilled water 1000 mL, sterilize at 121°C for 20 min; PDA medium: potato extract powder 6 g / L, glucose 20 g / L, agar 20 g / L, distilled water 1000 mL, sterilized at 121°C for 20 min; Tributyrin agar medium: 2.5 g / L meat peptone, 2.5 g / L hot casein peptone, 3 g / L yeast extract powder, 12 g / L agar, 10 mL / L tributyrin, 1000 mL distilled water, sterilize at 121°C for 20 min.

[0024] Fungal DNA was extracted using a fungal genomic DNA extraction kit. The integrity, concentration, and purity of the extracted DNA were determined by agarose gel electrophoresis. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used to amplify the ITS region for fungal molecular identification. The PCR reaction volume was 25 μL, consisting of 12.5 μL of 2× PCR Master Mix, 1.0 μL of each forward and reverse primer, 1.0 μL of template DNA (10-50 ng), and 9.5 μL of deionized water. PCR reaction conditions were: 85°C initial denaturation for 5 min, 94°C denaturation for 30 s, 57°C annealing for 30 s, and 72°C extension for 90 s, followed by 30 cycles of extension at 72°C for 10 min, and storage at 4°C. The amplified products were detected by 1.5% agarose gel electrophoresis. If they met the expected results, they were purified and sent to a sequencing company for sequencing.

[0025] The ITS sequence of the target strain was compared with the GenBank database using the BLAST function of NCBI. The results showed that the sequence similarity with Candida tropicalis was over 99%. The strain was further characterized by phylogeny based on ITS sequence analysis, and a phylogenetic tree was constructed using MEGA 11 software (e.g. Figure 3 The ITS sequence of this strain was uploaded to the GenBank database with the accession number PV973019.

[0026] Prepare a bacterial suspension of the pre-activated strain, take a 2% (v / v) inoculum and add it to potato dextrose medium, and culture it in a shaker at 30°C and 160 rpm. Samples were taken every 6 hours from the start of inoculation to measure the optical density (OD600) of the culture solution. The measurement was continued for 48 hours. The OD600 value was used to reflect the growth of the bacteria, and a strain growth curve was drawn (e.g. Figure 4 ), used to evaluate their growth patterns and metabolic activity.

[0027] Another embodiment of the present invention discloses a method for screening salt-tolerant oil-degrading bacteria, comprising the following steps: Initial screening of oil-degrading bacteria: 1-3 ml of food waste leachate was inoculated into an olive oil-mineral salt culture medium and cultured at 25-35°C in a shaker at 130-220 rpm for 3-7 days. Subsequently, a 1%-3% inoculum was transferred to fresh culture medium. This process was repeated four times, with the olive oil concentration gradually increased to 20, 30, and 40 g / L to enhance the strain's oil-degrading ability.

[0028] Salt tolerance acclimation: Select 1-3 ml of the bacterial solution after the initial screening and inoculate it into the culture medium. Continue to incubate in a shaker at 25-35°C and 130-220 rpm for 3-7 days. After each round of culture, transfer 1%-3% of the bacterial solution to new culture medium. Perform four rounds of culture continuously and gradually increase the NaCl concentration in the culture medium to 20, 30, and 40 g / L to screen strains with strong salt tolerance.

[0029] Dilution and coating culture: add 1 mL of the above-screened and acclimated bacterial solution to 9 mL of sterile water, mix well, and then perform gradient dilution to prepare 10 -1 to 10 -7 Take 0.2 mL of 10 -4 to 10 -7 The diluted solution was spread on a PDA plate culture medium, placed in a constant temperature incubator at 30°C for 48 hours, and the growth morphology and size characteristics of the colonies were observed.

[0030] Single bacterial isolation and purification: Select colonies with good growth from the above plates, inoculate them onto PDA plates for streak culture, and incubate at 30°C for 48 hours. Repeat the streak operation until the colonies on the plates are consistent in morphology, size, and color, and there are no obvious differences in bacterial morphology observed under a microscope. This is considered a pure strain.

[0031] Secondary screening of oil degradation ability: The purified single strain was inoculated into tributyrin solid culture medium, about 1 μL of bacterial solution was added to each well, and cultured at 30°C for 72 hours. Figure 2 As shown. Under the same conditions, strains with faster growth rate and larger transparent circle were screened as the final oil-degrading bacteria. The colony morphology of oil-degrading bacteria is shown in the figure below. Figure 1 shown.

[0032] Another embodiment of the present invention discloses the use of salt-tolerant oil-degrading bacteria in the degradation of olive oil. Oil degradation effect of salt-tolerant oil-degrading bacteria under different inoculation conditions: The strain was inoculated into an olive oil-mineral salt medium at 1%, 2%, 5%, 10%, and 20% (v / v) inoculum, respectively, and cultured in a shaker at 30°C and 160 rpm for 72 hours. The degradation rate was calculated by measuring the residual oil content in the medium. Figure 5 As shown in the figure, the results showed that when the inoculation amount was 10%, the oil degradation rate of the strain was the highest, reaching 67.30%.

[0033] Oil degradation effect of salt-tolerant oil-degrading bacteria under different pH conditions: The initial pH of the culture medium was adjusted to 4-9, and the strain was inoculated at a 10% (v / v) inoculation rate. The culture was carried out at 30°C and 160 rpm for 72 hours, and the oil degradation rate of each treatment group was measured. Figure 6 As shown in the figure, the results showed that the strain exhibited the best degradation ability at pH = 7, with a degradation rate of 67.21%.

[0034] Oil degradation effect of salt-tolerant oil-degrading bacteria under different salt concentration conditions: Different concentrations of salt (10-50 g / L) were added to the culture medium, and the strain was inoculated at a 10% (v / v) inoculation rate. The culture was carried out at 30°C and 160 rpm for 72 hours, and the oil degradation rate was measured. Figure 7 The results showed that the strain had good salt tolerance and could maintain a high degradation rate of 62.10% even at a high salt concentration of 50 g / L, demonstrating a strong salt adaptability.

[0035] Oil degradation effect of salt-tolerant oil-degrading bacteria at different culture times: Under the conditions of pH=7 and salt concentration of 10 g / L, the strain was inoculated into olive oil inorganic salt medium at a 10% (v / v) inoculum, cultured in a shaking incubator at 30°C and 160 rpm, and the oil degradation rate was measured every 24 hours for 6 consecutive days. Figure 8 As shown in the figure, the experimental results show that the degradation rate increases with time, and the degradation rate is 69.06% on the sixth day.

[0036] Another embodiment of the present invention discloses the use of salt-tolerant oil-degrading bacteria in oily wastewater from restaurant kitchen waste.

[0037] The strain was inoculated into the oily wastewater collected from actual kitchen waste at a 10% inoculation rate and cultured in a shaker at 30°C and 160 rpm for 7 days. The blank control group without inoculation was used for comparison. Figure 9As shown in the experimental results, the uninoculated control group had a grease degradation rate of 17.13% within 72 hours, while the inoculated experimental group had a grease degradation rate of 64.85%. The salt-tolerant grease-degrading strain screened and obtained by the present invention has shown good application results in the treatment of oily wastewater from actual restaurant waste.

[0038] Another embodiment of the present invention discloses the use of salt-tolerant oil-degrading bacteria in aerobic composting of high-salt and high-fat food waste.

[0039] The pre-treated kitchen waste (wet weight fat content of about 12%, salt content of about 3%) was mixed evenly with crushed corn stalks at a wet weight ratio of 4:1, the moisture content was adjusted to about 60%, and the mixture was placed in an aerobic composting reactor. The piles were divided into two groups: an experimental group (inoculated with 2% (v / w) of the wet weight of the pile of strains) and a control group (not inoculated). Both groups of piles were aerobically composted under forced ventilation conditions for 42 days. Samples were taken every 7 days to detect changes in pile temperature, fat degradation rate, total organic matter degradation rate, and germination index. Figure 10-13 As shown, the experimental results show that the temperature of the experimental group exceeded 55°C on the second day and remained above 55°C for 14 days. After 42 days of composting, the oil degradation rate was 86.40%, the organic matter decomposition rate was 40.01%, and the germination index was 83.13%. The control group only reached 55°C on the fourth day and only maintained it for 10 days. After 42 days of composting, the oil degradation rate was 76.13%, the organic matter decomposition rate was 33.02%, and the germination index was 74.25%. From this comparison, it can be seen that the salt-tolerant oil-degrading bacteria of the present invention can significantly accelerate the degradation of oil and organic matter in the aerobic composting process of high-salt and high-fat food waste, promote rapid temperature rise and maturity of compost, improve overall composting efficiency, and have good practical application value.

[0040] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A salt-tolerant oil-degrading bacterium, characterized in that It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.35335.

2. A method for screening salt-tolerant oil-degrading bacteria according to claim 1, characterized in that: The steps include: Initial screening of oil-degrading bacteria: food waste leachate was inoculated into an olive oil-mineral salt medium for cultivation, then transferred to fresh medium according to the inoculation ratio, and the cultivation process was repeated while gradually increasing the olive oil concentration. To enhance the strain's ability to degrade oil; Salt tolerance acclimation: Select the bacterial solution after the initial screening and inoculate it into the culture medium for cultivation; after each round of cultivation, transfer it to a new culture medium according to the proportion, and continue multiple rounds of cultivation while gradually increasing the NaCl concentration in the culture medium to screen strains with strong salt tolerance; Dilution and coating culture: add the pre-screened and acclimated bacterial solution into sterile water, mix well and then perform gradient dilution to prepare 10 -1 to 10 -7 The dilution gradient was 10 -4 to 10 -7 The diluted solution was spread on PDA plate culture medium, placed in a constant temperature incubator, and the growth morphology and size characteristics of the colonies were observed; Single bacterial isolation and purification: Select colonies with good growth status from PDA plate culture medium, inoculate them on PDA plates for streak culture, and repeat the streak operation until the colonies on the plate have consistent morphology, size and color, and there is no obvious difference in bacterial morphology under microscope. This is considered to be a pure strain; Rescreening of oil degradation ability: The purified single strain was inoculated into tributyrin solid culture medium, and a quantitative bacterial solution was added to each well for cultivation. The strain with the fastest growth rate and the largest transparent zone under the same conditions was selected as the final oil-degrading bacteria.

3. The screening method according to claim 2, characterized in that In the preliminary screening of the oil-degrading bacteria, the inoculation ratio is 1% to 3%.

4. The screening method according to claim 2, wherein In the preliminary screening of oil-degrading bacteria, the culture process was repeated four times, and the olive oil concentration was gradually increased to 20 g / L, 30 g / L, and 40 g / L.

5. The screening method according to claim 2, characterized in that During the salt tolerance acclimation, four rounds of culture were performed continuously, and the NaCl concentration in the culture medium was gradually increased to 20 g / L, 30 g / L, and 40 g / L.

6. The screening method according to claim 2, wherein In the dilution coating culture, the volume of the bacterial liquid is 0.5 mL to 1.5 mL, and the volume of the sterile water is 8.5 mL to 9.0 mL.

7. The screening method according to claim 2, wherein In the oil degradation capacity rescreening, 0.8 μL to 1.2 μL of bacterial solution was added to each well.

8. Use of the salt-tolerant oil-degrading bacteria according to claim 1 in degrading olive oil.

9. Use of the salt-tolerant oil-degrading bacteria according to claim 1 in oily wastewater from restaurant kitchen waste.

10. Use of the salt-tolerant oil-degrading bacteria according to claim 1 in aerobic composting of high-salt and high-fat food waste.

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