Salt-tolerant oil-degrading bacteria, screening method and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a salt-tolerant oil-degrading bacterium, its screening method, and its application. Background Technology

[0002] Aerobic composting, as an environmentally friendly treatment method, has been widely researched and applied due to its multiple advantages, including reducing environmental pollution, lowering transportation costs, maintaining a clean operating environment, achieving high automation, and promoting resource utilization. However, the high salt and oil content in Chinese diets leads to high concentrations of salt and oil in food waste, which often delays the composting process and affects the quality of the final compost. High concentrations of sodium... + Oil content increases the osmotic pressure of the composting environment, negatively impacting microbial activity and interfering with their metabolism. High oil content also inhibits the passage of oxygen needed for microbial metabolism, thus affecting microbial growth and metabolism, as well as the speed and quality of microbial decomposition of organic matter and compost. Furthermore, oily organic fertilizers can 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 oils in high-salt environments for aerobic composting of kitchen waste, so as to improve the efficiency and quality of such kitchen waste composting. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a salt-tolerant oil-degrading bacterium, its screening method, and its application.

[0005] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0006] In one aspect, this invention provides a salt-tolerant oil-degrading bacterium, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35335.

[0007] The preservation information of the salt-tolerant oil-degrading bacteria in this invention is as follows: Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: July 22, 2025; Preservation number: CGMCC No. 35335; Classification and name: Candida tropicalis.

[0008] Another aspect of the present invention provides a method for screening salt-tolerant oil-degrading bacteria, comprising the following steps:

[0009] Preliminary screening of grease-degrading bacteria: Leachate from kitchen waste was inoculated into olive oil inorganic salt medium for culture, and then transferred to fresh medium according to the inoculation ratio. The culture process was repeated, and the concentration of olive oil was gradually increased to enhance the strain's ability to degrade grease.

[0010] Salt tolerance acclimatization: Select the bacterial suspension after 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 ratio, and carry out multiple rounds of cultivation continuously, while gradually increasing the NaCl concentration in the culture medium to screen strains with strong salt tolerance.

[0011] Diluted plating culture: The bacterial suspension that has undergone initial screening and acclimatization is added to sterile water, mixed well, and then serially diluted to prepare 10 saturations. -1 Up to 10 -7 The dilution gradient was set to 10. -4 Up to 10 -7 The diluted solution was spread onto PDA agar plates and incubated in a constant temperature incubator. The growth morphology and size characteristics of the colonies were observed.

[0012] Single-strain isolation and purification: Select colonies with good growth status from PDA plate medium, inoculate them onto PDA plates for streaking culture, repeat the streaking operation until the colony morphology, size and color on the plate are consistent, and there is no obvious difference in bacterial morphology when observed under a microscope, then it can be regarded as obtaining a pure strain.

[0013] Re-screening of lipid degradation ability: The purified single strain was inoculated into glycerol tribose solid medium, and a quantitative bacterial solution was added to each well for cultivation. The strains that grew faster and formed larger transparent zones under the same conditions were selected as the final lipid-degrading bacteria.

[0014] Furthermore, in the preliminary screening of the oil-degrading bacteria, the inoculation ratio is 1% to 3%.

[0015] Furthermore, in the initial screening of the oil-degrading bacteria, the culture process was repeated four times, gradually increasing the concentration of olive oil to 20g / L, 30g / L, and 40g / L.

[0016] Furthermore, in the salt tolerance acclimatization process, four rounds of cultivation were carried out continuously, and the NaCl concentration in the culture medium was gradually increased to 20 g / L, 30 g / L, and 40 g / L.

[0017] Furthermore, in the dilution and coating culture, the volume of the bacterial solution is 0.5 mL to 1.5 mL and the volume of sterile water is 8.5 mL to 9.0 mL.

[0018] Furthermore, in the secondary screening of oil degradation ability, 0.8 μL to 1.2 μL of bacterial solution is added to each well.

[0019] Another aspect of the present invention provides the application of salt-tolerant oil-degrading bacteria in the degradation of olive oil.

[0020] Another aspect of the present invention provides the application of salt-tolerant oil-degrading bacteria in the treatment of oily wastewater from kitchen waste.

[0021] In another aspect, the present invention provides the application of salt-tolerant oil-degrading bacteria in aerobic composting of high-salt, high-oil kitchen waste.

[0022] The technical solution provided by this invention has at least the following technical effects:

[0023] 1. The salt-tolerant oil-degrading bacteria obtained by this invention have good salt tolerance and can grow with oil as the sole carbon source under high salt concentration conditions, exhibiting excellent oil degradation activity. They are suitable for composting systems of kitchen waste with high salt content, which helps to efficiently decompose organic oils, improve composting maturity efficiency and final product quality.

[0024] 2. The method for screening salt-tolerant lipid-degrading bacteria provided by this invention is simple to operate, and the enrichment and domestication process has good repeatability. It can efficiently screen target strains with strong lipid degradation ability in a short period of time, and is suitable for large-scale microbial resource mining and screening. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a colony morphology diagram of salt-tolerant oil-degrading bacteria according to an embodiment of the present invention;

[0027] Figure 2 This is an example of the salt-tolerant oil-degrading bacteria in the tricresyl tributyl ester medium and their clear zone and growth.

[0028] Figure 3 This is a phylogenetic tree of salt-tolerant oil-degrading bacteria according to an embodiment of the present invention;

[0029] Figure 4 This is an example of the growth of salt-tolerant oil-degrading bacteria in potato glucose medium for 48 hours according to an embodiment of the present invention.

[0030] Figure 5 This describes the degradation effect of salt-tolerant oil-degrading bacteria under different inoculation conditions in the embodiments of the present invention;

[0031] Figure 6This describes the degradation effect of salt-tolerant oil-degrading bacteria under different pH conditions in the embodiments of the present invention;

[0032] Figure 7 This describes the degradation effect of salt-tolerant oil-degrading bacteria under different salt concentration conditions in the embodiments of the present invention;

[0033] Figure 8 This describes the degradation effect of salt-tolerant oil-degrading bacteria under different degradation time conditions in the embodiments of the present invention;

[0034] Figure 9 This invention demonstrates the degradation effect of salt-tolerant oil-degrading bacteria on oily wastewater under different treatment conditions.

[0035] Figure 10 This invention relates to the temperature changes of salt-tolerant oil-degrading bacteria in aerobic composting of high-salt, high-oil kitchen waste according to an embodiment of the invention.

[0036] Figure 11 This invention demonstrates the effect of salt-tolerant oil-degrading bacteria on oil degradation in aerobic composting of high-salt, high-oil kitchen waste according to an embodiment of the invention.

[0037] Figure 12 This invention demonstrates the effect of salt-tolerant oil-degrading bacteria on organic matter degradation in aerobic composting of high-salt, high-oil kitchen waste according to an embodiment of the present invention.

[0038] Figure 13 This refers to the germination index of salt-tolerant oil-degrading bacteria in aerobic composting of high-salt, high-oil kitchen waste, as described in this embodiment of the invention. Detailed Implementation

[0039] The embodiments of the present invention are described in detail below. These embodiments are provided to better illustrate the invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0040] This invention discloses a salt-tolerant oil-degrading bacterium, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35335.

[0041] It should be noted that the salt-tolerant oil-degrading bacteria in this embodiment are derived from the oily leachate of kitchen waste, while the oily leachate of kitchen waste in this application is derived from the kitchen waste treatment system of concentrated catering areas (such as university canteens and the kitchens of large chain restaurants). The kitchen waste in such areas is mainly composed of animal and vegetable oils (the oil content is stable at 10%-20%), which is highly compatible 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, due to the long-term stable composition of the kitchen waste in concentrated catering areas, a micro-ecological environment suitable for the reproduction of such strains has been formed, and the probability of the existence of salt-tolerant oil-degrading bacteria is stable.

[0042] The culture medium was: 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℃ for 20 min;

[0043] Potato glucose medium: 6 g / L potato extract powder, 20 g / L glucose, 1000 mL distilled water, sterilized at 121℃ for 20 min;

[0044] PDA medium: 6 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, 1000 mL distilled water, sterilized at 121℃ for 20 min;

[0045] Tributyric acid glyceride agar medium: 2.5 g / L meat peptone, 2.5 g / L hot casein peptone, 3 g / L yeast extract, 12 g / L agar, 10 mL / L tributyric acid glyceride, 1000 mL distilled water, sterilized at 121℃ for 20 min.

[0046] Fungal DNA was extracted using a fungal genomic DNA extraction kit. The integrity, concentration, and purity of the extracted DNA were assessed by agarose gel electrophoresis. ITS1 (sequence 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (sequence 5'-TCCTCCGCTTATTGATATGC-3') were used as primers to amplify the ITS region sequence for fungal molecular identification. The total PCR reaction volume was 25 μL, including 12.5 μL of 2×PCR Master Mix, 1.0 μL each of forward and reverse primers, 1.0 μL of template DNA (10-50 ng), and 9.5 μL of deionized water. The PCR reaction conditions were: 85℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, for 30 cycles, with a final extension at 72℃ for 10 min, and storage at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis. After meeting the expected results, they were purified and sent to a sequencing company for sequencing.

[0047] Homology comparison of the ITS sequence of the target strain was performed in the GenBank database using NCBI's BLAST function. The results showed that its sequence similarity with *Candida tropicalis* was over 99%. Based on the ITS sequence analysis, the strain was further phylogenetically characterized, and a phylogenetic tree was constructed using MEGA 11 software (e.g., ...). Figure 3 (As shown). The ITS sequence of this strain was uploaded to the GenBank database, accession number PV973019.

[0048] The pre-activated bacterial strain was prepared into a bacterial suspension, and 2% (v / v) of the inoculum was added to potato dextrose medium. The suspension was then incubated in a shaker at 30°C and 160 rpm. Samples were taken every 6 hours from the time of inoculation to measure the optical density (OD600) of the culture medium for a total of 48 hours. The OD600 value was used to reflect the bacterial growth status, and a growth curve of the strain was plotted (e.g., ...). Figure 4 As shown in the figure, it is used to assess its growth patterns and metabolic activity.

[0049] Another embodiment of the present invention discloses a method for screening salt-tolerant lipid-degrading bacteria, comprising the following steps:

[0050] Preliminary screening of grease-degrading bacteria: 1–3 ml of kitchen waste leachate was inoculated into olive oil inorganic salt medium and cultured at 25–35℃ and 130–220 rpm in a shaker for 3–7 days. Subsequently, the inoculum was transferred to fresh medium at a ratio of 1%–3%, and the above culture process was repeated four times, with the olive oil concentration gradually increased to 20, 30, and 40 g / L to enhance the strain's ability to degrade grease.

[0051] Salt tolerance acclimatization: 1-3 ml of the bacterial suspension after initial screening was inoculated into the culture medium and continued to be cultured in a shaker at 25-35℃ and 130-220 rpm for 3-7 days. After each round of culture, the bacterial suspension was transferred to a new culture medium at a ratio of 1%-3% and cultured for four consecutive rounds. The NaCl concentration in the culture medium was gradually increased to 20, 30, and 40 g / L to screen strains with strong salt tolerance.

[0052] Dilution and plating culture: Add 1 mL of the pre-screened and acclimatized bacterial culture to 9 mL of sterile water, mix well, and then perform serial dilutions to prepare 10 plating cultures. -1 Up to 10 -7 The dilution gradient was calculated. 0.2 mL of each of the 10⁻⁶ solutions was taken. -4 Up to 10 -7 The diluted solution was spread on PDA agar plates and incubated in a 30°C incubator for 48 hours. The growth morphology and size characteristics of the colonies were then observed.

[0053] Single-strain isolation and purification: Select colonies with good growth status from the above plates, inoculate them onto PDA plates for streaking culture, and incubate at 30°C for 48 hours; repeat the streaking operation until the colony morphology, size and color on the plate are consistent, and there is no obvious difference in bacterial morphology when observed under a microscope, then it can be considered that a pure strain has been obtained.

[0054] Re-screening for lipid degradation ability: The purified single strain was inoculated into glycerol tretinoin solid medium, with approximately 1 μL of bacterial culture added to each well, and cultured at 30°C for 72 hours. Figure 2 As shown in the figure. Strains that grew faster and formed larger clear zones under the same conditions were selected as the final lipid-degrading bacteria. The colony morphology of the lipid-degrading bacteria is shown in the figure. Figure 1 As shown.

[0055] Another embodiment of the present invention discloses the application of a salt-tolerant oil-degrading bacterium in the degradation of olive oil.

[0056] The lipid degradation effect of salt-tolerant lipid-degrading bacteria under different inoculum conditions:

[0057] The strains were inoculated into olive oil-inorganic salt medium at inoculum levels of 1%, 2%, 5%, 10%, and 20% (v / v), 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, the results indicate that the lipid degradation rate of the strain was highest at an inoculum size of 10%, reaching 67.30%.

[0058] The lipid degradation effect of salt-tolerant lipid-degrading bacteria under different pH conditions:

[0059] The initial pH of the culture medium was adjusted to 4-9, and the bacterial strain was inoculated at a rate of 10% (v / v). The cultures were then incubated at 30°C and 160 rpm for 72 hours, and the lipid degradation rate of each treatment group was measured. Figure 6 As shown in the figure, the results indicate that the strain exhibits the best degradation ability at pH=7, with a degradation rate of 67.21%.

[0060] The lipid degradation effect of salt-tolerant lipid-degrading bacteria under different salt concentrations:

[0061] Different concentrations of salt (10–50 g / L) were added to the culture medium, and the strain was inoculated at a 10% (v / v) inoculum. The cultures were then incubated at 30°C and 160 rpm for 72 hours, and the lipid degradation rate was measured. Figure 7 As shown, the results indicate that this strain has good salt tolerance, maintaining a high degradation rate of 62.10% even at a high salt concentration of 50 g / L, demonstrating strong salt adaptability.

[0062] The lipid degradation effect of salt-tolerant lipid-degrading bacteria at different culture times:

[0063] Under conditions of pH=7 and salt concentration of 10 g / L, the strain was inoculated into olive oil inorganic salt medium at an inoculum rate of 10% (v / v) and cultured in a shaker at 30℃ and 160 rpm. The oil degradation rate was measured every 24 hours for 6 consecutive days. Figure 8 As shown, the experimental results indicate that the degradation rate increases over time, reaching 69.06% on the sixth day.

[0064] Another embodiment of the present invention discloses the application of salt-tolerant oil-degrading bacteria in oily wastewater from kitchen waste.

[0065] The strain was inoculated at a 10% inoculum into oily wastewater from actual collected kitchen waste and cultured for 7 days in a shaker at 30℃ and 160 rpm. A blank control group without inoculation was used for comparison. Figure 9 As shown, the experimental results indicate that the oil degradation rate in the uninoculated control group was 17.13% within 72 hours, while the oil degradation rate in the inoculated experimental group reached 64.85%. The salt-tolerant oil-degrading strains screened in this invention have demonstrated good application effects in the actual treatment of oily wastewater from kitchen waste.

[0066] Another embodiment of the present invention discloses the application of a salt-tolerant oil-degrading bacterium in aerobic composting of high-salt, high-oil kitchen waste.

[0067] Pre-treated kitchen waste (approximately 12% wet oil content and 3% salt content) was mixed with crushed corn stalks at a wet weight ratio of 4:1, and the moisture content was adjusted to approximately 60%. The mixture was then loaded into an aerobic composting reactor. The compost piles were divided into two groups: an experimental group (inoculated with 2% (v / w) of the wet weight of the compost pile containing bacterial strains) and a control group (uninoculated). Both groups underwent aerobic composting under forced ventilation for 42 days. Samples were taken every 7 days to detect changes in compost temperature, oil degradation rate, total organic matter degradation rate, and germination index. Figures 10-13 As shown, the experimental results indicate that the experimental group achieved a temperature exceeding 55℃ on the second day and maintained above 55℃ 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%. In contrast, the control group reached 55℃ on the fourth day and maintained it for only 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%. This comparison demonstrates that the salt-tolerant oil-degrading bacteria described in this invention can significantly accelerate the degradation of oil and organic matter during aerobic composting of high-salt, high-oil kitchen waste, promote rapid temperature rise and maturation of the compost, and improve overall composting efficiency, thus possessing significant practical application value.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A salt-tolerant oil-degrading bacterium, characterized in that, This strain is *Candida tropicalis*. (Tropicalis), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35335.

2. The application of the salt-tolerant oil-degrading bacteria according to claim 1 in the degradation of olive oil.

3. The application of the salt-tolerant oil-degrading bacteria according to claim 1 in the treatment of oily wastewater from kitchen waste.

4. The application of the salt-tolerant oil-degrading bacteria according to claim 1 in aerobic composting of kitchen waste.

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