Liuveromyces varioti source bacterium and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Pluravenilla gergoviae source bacterium and application thereof.The Pluravenilla gergoviae source bacterium is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.36196. The Pluravenilla gergoviae source bacterium has the advantages that the salt tolerance performance is achieved, and the application of the Pluravenilla gergoviae source bacterium is wide in application prospect. The strain can grow by taking grease as a unique carbon source in a high-salt-concentration environment, and shows excellent grease degradation performance. The strain is especially suitable for a kitchen waste oil-containing wastewater treatment system, can efficiently decompose organic grease in wastewater, remarkably improves the grease removal rate, improves the biodegradability of wastewater, and provides efficient microbial resources for biological treatment of kitchen waste oil-containing wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a multi-bacterial bacterium and its applications. Background Technology

[0002] Aerobic biological treatment, as a green and environmentally friendly wastewater treatment technology, is widely used in the treatment of various organic wastewaters due to its advantages such as effectively reducing pollutant concentrations, minimizing environmental impact, low operating costs, enabling automated control, and promoting resource utilization. However, catering wastewater in my country is typically characterized by high salt and high oil content, resulting in high salt and oil concentrations in oily kitchen waste. This often hinders the biological treatment process, affecting the stable operation and treatment efficiency of the system. High concentrations of sodium... + High oil content increases the osmotic pressure of the system, thereby inhibiting the growth and metabolic activity of microorganisms; while high oil content hinders oxygen transfer, weakens the metabolic function and degradation capacity of microorganisms, leading to a decrease in organic matter removal rate and system efficiency. In addition, the discharge of insufficiently degraded oil may also cause environmental problems such as soil compaction and eutrophication of water bodies.

[0003] Therefore, an important direction of current research is to screen out functional microorganisms that can efficiently degrade oils in high-salt environments for biological treatment of oily wastewater from kitchen waste, so as to improve the oil removal rate and overall treatment performance of the system and achieve pollution control. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multi-bacterial source of bacteria and its application.

[0005] To achieve the above objectives, the present invention can adopt the following technical solutions: In one aspect, this invention provides a multi-bacterial strain of Pluralibacter gergoviae LB356, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36196.

[0006] The preservation information of the *Lactobacillus ribavirinus* LB356 in this invention is as follows: Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: October 16, 2025; Preservation number: CGMCC No. 36196.

[0007] In another aspect, the present invention provides a method for screening the multi-bacterial strain LB356, comprising the following steps: Initial screening of strains: The 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 of 1% to 3%. The culture process was repeated, and the concentration of olive oil was gradually increased to enhance the strain's ability to degrade oil. Specifically, the culture process was repeated four times, and the concentration of olive oil was gradually increased to 20 g / L, 30 g / L, and 40 g / L.

[0008] Salt tolerance acclimatization: Select the bacterial culture 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, gradually increasing the NaCl concentration in the culture medium to screen strains with strong salt tolerance. Specifically, carry out four rounds of cultivation continuously, and gradually increase the NaCl concentration in the culture medium to 20 g / L, 30 g / L, and 40 g / L.

[0009] Dilution and plating culture: After initial screening and acclimatization, the bacterial suspension was added to sterile water and mixed well, then serially diluted. The volume of the bacterial suspension was 0.5 mL to 1.5 mL, and the volume of sterile water was 8.5 mL to 9.0 mL. Ten plating cultures were prepared sequentially. -1 Up to 10 -7 The dilution gradient was set to 10. -4 Up to 10 -7 The diluted solution was spread onto LB agar plates and incubated in a constant temperature incubator. The growth morphology and size characteristics of the colonies were observed. Single-strain isolation and purification: Select colonies with good growth status from LB agar plates, inoculate them onto LB agar 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. Secondary screening of strains: The purified single strains were inoculated into glycerol tribonitrile solid medium, and a quantitative bacterial solution was added to each well for cultivation. The amount of bacterial solution added to each well was 0.8 μL to 1.2 μL. The strains that grew faster and formed larger transparent zones under the same conditions were selected as the final strains.

[0010] Another aspect of the present invention provides the application of LB356, a multi-bacterial progenitor, in the degradation of olive oil.

[0011] In another aspect, this invention provides the application of LB356, a multi-bacterial source of bacteria, in the treatment of oily wastewater from kitchen waste.

[0012] The technical solution provided by this invention has at least the following technical effects: The *Lactobacillus LB356* strain obtained in this invention possesses certain salt tolerance and can grow in high-salt environments using oils as the sole carbon source, exhibiting excellent oil degradation performance. This strain is particularly suitable for treating oily wastewater from kitchen waste, efficiently decomposing organic oils in the wastewater, significantly improving oil removal rates, enhancing wastewater biodegradability, and providing a highly efficient microbial resource for the biological treatment of oily wastewater from kitchen waste. Attached Figure Description

[0013] 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.

[0014] Figure 1 This is a colony morphology diagram of the multi-bacterial bacterium LB356 from an embodiment of the present invention; Figure 2 This is an embodiment of the present invention showing the clear zone and growth of the multi-bacterial strain LB356 of *Lactobacillus rubrum* on glycerol tartrate medium; Figure 3 This is a phylogenetic tree of the multi-bacterial bacterium LB356 from an embodiment of the present invention; Figure 4 This is an embodiment of the present invention showing the growth of *Lactobacillus ribavirinus* LB356 in LB medium for 48 hours. Figure 5 This is an embodiment of the invention showing the degradation effect of the multi-bacterial bacterium LB356 under different inoculation conditions; Figure 6 This is an embodiment of the degradation effect of the multi-bacterial bacterium LB356 under different pH conditions. Figure 7 This is an embodiment of the degradation effect of the multi-bacterial bacterium LB356 under different salt concentration conditions. Figure 8 This is an embodiment of the present invention showing the degradation effect of the multi-bacterial bacterium LB356 under different degradation time conditions; Figure 9 This invention demonstrates the degradation effect of the multi-bacterial bacterium LB356 on oily wastewater under different treatment conditions. Detailed Implementation

[0015] 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. All 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.

[0016] This invention discloses a Pluralibacter gergoviae LB356 and its applications. Pluralibacter gergoviae LB356 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36196.

[0017] It should be noted that the *L. LB356* bacterium in this embodiment originates from the oily leachate of food waste, while the oily leachate in this application originates from the food waste treatment system of concentrated catering areas (such as university canteens and the kitchens of large chain restaurants). The food waste in these areas is mainly composed of animal and vegetable oils (with an oil content consistently between 10% and 20%), which is highly compatible with the natural living environment of *L. LB356* (which requires oil as a carbon source). Furthermore, due to its long-term stable composition, the food waste in concentrated catering areas has formed a suitable microecological environment for the reproduction of this type of bacterial strain, thus ensuring the stability of the presence probability of *L. LB356*.

[0018] The culture medium is prepared as follows: Olive oil inorganic salt culture 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 minutes; LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1000 mL distilled water, sterilized at 121℃ for 20 minutes; LB agar medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder, 1000 mL distilled water, sterilized at 121℃ for 20 minutes; 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 minutes.

[0019] DNA was extracted from the bacterial strain using a DNA extraction kit. The primers for PCR amplification of the 16S rRNA gene were 27F and 1492R, with primer sequences of 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGCTACCTTGTTACGACT-3', respectively. The PCR system consisted of: 12.5 μL of 2×PCR Master Mix, 1.0 μL of forward primer (10 μM), 1.0 μL of reverse primer (10 μM), 1.0 μL of template DNA (10-50 ng), and 9.5 μL of sterile 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, 30 cycles followed by a final extension at 72℃ for 10 min, and finally, holding at 4℃. PCR products were detected using 1.5% agarose gel electrophoresis, 1xTAE, 150V, 100mA, for 20 minutes. After purification, the PCR products were sent to a sequencing company for sequencing.

[0020] Sequence alignment using NCBI's BLAST function in the GeneBank database revealed a similarity of over 99% with strain *Pluralibacter gergoviae*. Further characterization of the strain was performed based on 16S rRNA sequence analysis and alignment, and a phylogenetic tree was constructed using MEGA 11 software. The strain's 16S rRNA sequence was uploaded to the GeneBank database, accession number PX394027.

[0021] The pre-activated bacterial strain was prepared into a bacterial suspension, and 2% (v / v) of the inoculum was added to LB 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 was plotted (e.g., ...). Figure 4 As shown in the figure, it is used to assess its growth patterns and metabolic activity.

[0022] Another embodiment of the present invention discloses a screening method for the multi-bacterial strain LB356, comprising the following steps: Initial screening of the strain: 1-3 ml of kitchen waste leachate was inoculated into olive oil inorganic salt medium and cultured at 25-35℃ and 130-220 rpm for 3-7 days. Afterwards, the inoculum was transferred to fresh medium at a ratio of 1%-3%, and the above culture process was repeated four times, gradually increasing the olive oil concentration to 20, 30, and 40 g / L to enhance the strain's ability to degrade oils.

[0023] 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.

[0024] 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 LB agar plates and incubated in a 30°C incubator for 48 hours. The growth morphology and size characteristics of the colonies were then observed.

[0025] Single-strain isolation and purification: Select colonies with good growth status from the above plates, inoculate them onto LB agar plates and streak them for 48 hours at 30°C; repeat the streaking operation until the colony morphology, size and color on the plates are consistent, and there is no obvious difference in bacterial morphology when observed under a microscope, then the pure strain can be considered to have been obtained.

[0026] Secondary screening of strains: The purified single strains were inoculated into glycerol tretinoin solid medium, with approximately 1 μL of bacterial culture added to each well, and incubated at 30°C for 72 hours. Figure 2 As shown in the figure. Strains that grow faster and form larger clear zones under the same conditions were selected as the final strains. The colony morphology of these strains is shown in the figure below. Figure 1 As shown, the phylogenetic tree of the strains is as follows: Figure 3 As shown.

[0027] Another embodiment of the present invention discloses the application of a multi-bacterial bacterium LB356 in the degradation of olive oil. Oil degradation effect of Rigouwei multi-bacterial protozoan LB356 under different inoculum conditions: 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 5%, reaching 74.91%.

[0028] The lipid degradation effect of Rigouwei multi-bacterial progenitor LB356 under different pH conditions: The initial pH of the culture medium was adjusted to 4-9, and the bacterial strain was inoculated at a rate of 5% (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 74.49%.

[0029] The lipid degradation effect of Rigouwei multi-bacterial progenitor LB356 under different salt concentrations: Different concentrations of salt (10–50 g / L) were added to the culture medium, and the strain was inoculated at a rate of 5% (v / v). 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 a certain degree of salt tolerance. Even at a high salt concentration of 50 g / L, it can still maintain a high degradation rate of 52.13%, indicating that it has a certain tolerance under high salt conditions.

[0030] Lipid degradation effect of Rigouwei multi-bacterial progeny LB356 at different culture times: 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 5% (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 77.87% on the sixth day.

[0031] Another embodiment of the present invention discloses the application of a multi-bacterial bacterium LB356 in the treatment of oily wastewater from kitchen waste.

[0032] The strain was inoculated at a rate of 5% (v / v) into oily wastewater from actual collected kitchen waste and cultured in a shaker at 30℃ and 160 rpm for 7 days, compared with a blank control group without inoculation. Figure 9 As shown, the experimental results indicate that the oil degradation rate in the uninoculated control group was 17.13%, while the oil degradation rate in the inoculated experimental group reached 67.95%. The *Lactobacillus LB356* strain obtained in this invention has demonstrated good application results in the treatment of oily wastewater from actual kitchen waste.

[0033] 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 multi-bacterial strain of *Pluralibacter gergoviae* LB356, characterized in that... The aforementioned Pluralibacter gergoviae LB356 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36196.

2. The application of the multi-bacterial progenitor LB356 according to claim 1 in the degradation of olive oil.

3. The application of the multi-bacterial bacterium LB356 described in claim 1 in the treatment of oily wastewater from kitchen waste.

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