Daily hookwe multi-bacterial source bacteria and application thereof
By screening and domesticating the LB356 bacterium source strain of Rigouwei, the difficulties in biological treatment caused by high salt and high oil content in oily wastewater from kitchen waste were solved, achieving efficient oil degradation and improving the treatment effect of oily wastewater from kitchen waste.
Patent Information
- Application Number
- CN202511648325.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-02-06
- Estimated Expiration
- 2045-11-12
AI Technical Summary
The high salt and high oil content in the oily wastewater from kitchen waste hinders the biological treatment process, affecting the stable operation and treatment effect of the system. Existing technologies make it difficult to screen out functional microorganisms that can efficiently degrade oil in a high-salt environment.
The bacterial strain LB356 of the Rhizoctonia solani was screened and domesticated. By gradually increasing the concentration of olive oil and NaCl in the culture medium, combined with dilution coating and single-strain isolation and purification, a salt-tolerant strain was obtained for the treatment of oily wastewater from kitchen waste.
Rigouwei multi-bacterial source bacterium LB356 can grow using oil as the sole carbon source in a high-salt environment, significantly improving oil removal rate and wastewater biodegradability, and is suitable for the treatment of oily wastewater from kitchen waste.
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Figure CN121109253B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Pluralibacter gergoviae bacterial source and application thereof. BACKGROUND
[0002] As a green and environmentally friendly wastewater treatment technology, aerobic biological treatment is widely used in various types of organic wastewater treatment due to its ability to effectively reduce pollutant concentration, reduce environmental load, low operating cost, automation control, and resource utilization. However, the restaurant wastewater in China is usually characterized by high salt and high oil, resulting in high salt and oil content in kitchen waste oil wastewater, which often causes biological treatment process to be hindered, affecting the stable operation of the system and the treatment effect. High concentration of Na + will increase the osmotic pressure of the system, thereby inhibiting the growth and metabolic activity of microorganisms; and high oil content will hinder oxygen transfer, weaken the metabolic function and degradation capacity of microorganisms, resulting in a decrease in organic matter removal rate and system efficiency. In addition, the discharge of insufficiently degraded oil can cause soil compaction, water eutrophication, and other environmental problems.
[0003] Therefore, an important direction of current research is to screen functional microorganisms that can efficiently degrade oil in high-salt environments for the biological treatment of kitchen waste oil wastewater, in order to improve the oil removal rate in wastewater and the overall treatment performance of the system, and achieve pollution control. SUMMARY
[0004] The present application aims to solve the above-mentioned deficiencies in the prior art, and provides a Pluralibacter gergoviae bacterial source and application thereof.
[0005] In order to achieve the above-mentioned purpose, the present application can adopt the following technical solutions:
[0006] In one aspect, the present application provides a Pluralibacter gergoviae LB356, which is deposited with the China General Microbiological Culture Collection Center, and has a deposit number of CGMCC No. 36196.
[0007] The Pluralibacter gergoviae LB356 in the present application has the following deposit information: depositing agency: China General Microbiological Culture Collection Center (CGMCC); depositing address: No. 3, Beichen West Road, Chaoyang District, Beijing; depositing date: October 16, 2025; deposit number: CGMCC No. 36196.
[0008] In another aspect, the present application further provides a screening method for the Pluralibacter gergoviae LB356, comprising the following steps:
[0009] Strain preliminary screening: the kitchen waste leachate is inoculated in olive oil inorganic salt culture medium for culture, then is transferred to fresh culture medium according to the inoculation ratio, the inoculation ratio is 1%-3%, the culture process is repeated, and the olive oil concentration is gradually increased; the degradation ability of the strain to oil is enhanced; the culture process is repeated for four rounds, and the olive oil concentration is gradually increased to 20g / L, 30g / L and 40g / L.
[0010] Salt-tolerant domestication: the bacterial liquid after preliminary screening is inoculated in the culture medium for culture; after each round of culture, the culture medium is transferred to the new culture medium according to the proportion, and multiple rounds of continuous culture are carried out, and the NaCl concentration in the culture medium is gradually increased, so as to screen the strains with strong salt-tolerant ability, and the culture is continuously carried out for four rounds, and the NaCl concentration in the culture medium is gradually increased to 20g / L, 30g / L and 40g / L.
[0011] Dilution and coating culture: the bacterial liquid after preliminary screening and domestication is mixed with sterile water, gradient dilution is carried out, the volume of the bacterial liquid is 0.5mL-1.5mL, the volume of the sterile water is 8.5mL-9.0mL, 10 -1 to 10 -7 dilution gradients are prepared in turn, 10 -4 to 10 -7 dilution liquids are coated on LB agar plate culture medium, and placed in a constant temperature incubator for culture, and the growth morphology and size characteristics of the colonies are observed;
[0012] Single bacteria separation and purification: the colonies with good growth state are selected from the LB agar plate culture medium, inoculated on the LB agar plate for streak culture, and the streaking operation is repeated until the colony morphology, size and color on the plate are consistent, and the bacterial morphology observed under a microscope has no obvious difference, so that the pure strain is obtained;
[0013] Strain rescreening: the single strain purified is inoculated in tributyrin solid culture medium, and a certain amount of bacterial liquid is added dropwise to each hole for culture, the bacterial liquid added to each hole is 0.8uL-1.2uL, the strain with faster growth speed and larger transparent circle formed under the same conditions is selected as the finally obtained strain.
[0014] The application further provides an application of the daily hook Weimobacteria source bacteria LB356 in degradation of olive oil.
[0015] The application further provides an application of the daily hook Weimobacteria source bacteria LB356 in treatment of kitchen waste oil-containing wastewater.
[0016] The technical scheme provided by the application has at least the following technical effects:
[0017] The obtained daily hook multi-bacterial source bacteria LB356 has certain salt resistance, can grow in an environment with high salt concentration by taking oil as the only carbon source, and exhibits excellent oil degradation performance. The strain is particularly suitable for a kitchen garbage oil-containing wastewater treatment system, can efficiently decompose organic oil in wastewater, significantly improves the oil removal rate, improves the wastewater biodegradability, and provides efficient microbial resources for biological treatment of kitchen garbage oil-containing wastewater. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.
[0019] Figure 1 is a colony morphology diagram of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application;
[0020] Figure 2 is a transparent circle and growth condition of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application in a tributyrin medium;
[0021] Figure 3 is a phylogenetic tree of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application;
[0022] Figure 4 is a 48 h growth condition of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application in an LB medium;
[0023] Figure 5 is a degradation effect of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application under different inoculation amounts;
[0024] Figure 6 is a degradation effect of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application under different pH conditions;
[0025] Figure 7 is a degradation effect of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application under different salt concentrations;
[0026] Figure 8 is a degradation effect of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application under different degradation times;
[0027] Figure 9 is an oil-containing wastewater degradation effect of the daily hook multi-bacterial source bacteria LB356 of the embodiments of the present application under different treatment conditions. DETAILED DESCRIPTION
[0028] The embodiments of the present application are described in detail below, and the embodiments are provided to better illustrate the present application, but the present application is not limited to the embodiments. Any modification and adjustment of the embodiments made by those skilled in the art according to the above description of the present application is within the protection scope of the present application.
[0029] The present application discloses a Pluralibacter gergoviae LB356 and its application. The Pluralibacter gergoviae LB356 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 36196.
[0030] It should be noted that the Pluralibacter gergoviae LB356 in the present embodiment is from the oil-containing leachate of kitchen waste, and the oil-containing leachate of kitchen waste of the present application is derived from the kitchen waste treatment system of a concentrated dining area (such as a college canteen or a large-scale chain restaurant kitchen). The kitchen waste in such an area is mainly composed of animal and vegetable oils (the oil content is stably 10%-20%), which is extremely compatible with the natural living environment of the Pluralibacter gergoviae LB356 (the Pluralibacter gergoviae LB356 needs to rely on oil as a carbon source). In addition, due to the long-term stable composition of the kitchen waste in the concentrated dining area, a micro-ecological environment suitable for the reproduction of such strains has been formed, and the probability of the existence of the Pluralibacter gergoviae LB356 is stable.
[0031] The culture medium is configured as follows:
[0032] 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, 121℃ sterilization for 20 minutes;
[0033] LB culture medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, distilled water 1000 mL, 121℃ sterilization for 20 minutes;
[0034] LB agar culture medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 18 g / L, distilled water 1000 mL, 121℃ sterilization for 20 minutes;
[0035] Tributyrin agar culture medium: meat peptone 2.5 g / L, hot casein peptone 2.5 g / L, yeast extract powder 3 g / L, agar 12 g / L, tributyrin 10 mL / L, distilled water 1000 mL, 121℃ sterilization for 20 minutes.
[0036] The strain DNA was extracted using a DNA extraction kit. The 16S rRNA gene PCR amplification primers were 27F and 1492R, and the primer sequences were 27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-TACGGCTACCTTGTTACGACT-3'. The PCR system included: 2x PCR Master Mix 12.5 μL, forward primer (10 μM) 1.0 μL, reverse primer (10 μM) 1.0 μL, template DNA (10-50 ng) 1.0 μL, sterile deionized water 9.5 μL. The PCR reaction conditions were: 85°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 57°C annealing for 30 seconds, 72°C extension for 90 seconds, 30 cycles of 72°C extension for 10 minutes, and finally at 4°C. The PCR product was detected by 1.5% agarose gel electrophoresis, 1x TAE, 150V, 100mA, 20 minutes electrophoresis observation. The PCR product was purified and sent to a sequencing company for sequencing.
[0037] The sequence alignment was performed in the GeneBank database by using the BLAST function of NCBI, and it was identified that the similarity with the strain Pluralibacter gergoviae was more than 99%. Based on the 16S rRNA sequence analysis and comparison of the strains, a phylogenetic tree was constructed by using MEGA 11 software. The 16S rRNA sequence of the strain was uploaded to the GeneBank database, and the accession number was PX394027.
[0038] The pre-activated strain was prepared into a bacterial liquid, 2% (v / v) inoculation amount was added into the LB culture medium, and the culture was placed in a shaking bed at 30°C and 160 rpm. Sampling was performed every 6 hours from inoculation, the optical density (OD600) of the culture solution was determined, a total of 48 hours of continuous determination was performed, the OD600 value was used to reflect the growth condition of the bacterial body, and a strain growth curve (as shown in Figure 4 ) was drawn, which was used to evaluate the growth rule and metabolic activity.
[0039] Another embodiment of the present application discloses a screening method of a daily hook bacterial source strain LB356, comprising the following steps:
[0040] Strain preliminary screening: 1-3 ml of kitchen waste leachate was inoculated in an olive oil inorganic salt culture medium, and the culture was performed in a 130-220 rpm shaking bed at 25-35°C for 3-7 days. Then, the inoculation ratio was 1%-3% to the fresh culture medium, the above culture process was repeated for four rounds, and the olive oil concentration was gradually increased to 20, 30 and 40 g / L, so as to enhance the oil degradation capacity of the strain.
[0041] Salt-tolerant acclimation: select the bacteria liquid 1-3 ml after preliminary screening, inoculate in the culture medium, continue to be cultured in 25-35 DEG C, 130-220 rpm shaking bed for 3-7 days; after each round of culture, transfer to the new culture medium at a ratio of 1%-3%, and continuously culture for four rounds, and gradually increase the NaCl concentration in the culture medium to 20, 30, 40 g / L, so as to screen the strains with strong salt tolerance.
[0042] Dilution coating culture: 1 mL of the above screened and acclimated bacteria liquid is mixed with 9 mL of sterile water, and gradient dilution is carried out to prepare 10 -1 to 10 -7 dilution gradient. 0.2 mL of 10 -4 to 10 -7 dilution liquid is coated on LB agar plate culture medium, and placed in a 30 DEG C constant temperature incubator for 48 hours, and the growth morphology and size characteristics of the colonies are observed.
[0043] Single bacteria separation and purification: the well-grown colonies are selected from the above plate, inoculated on LB agar plate and streaked, and cultured at 30 DEG C for 48 hours; repeat the streaking operation until the colony morphology, size and color on the plate are consistent, and the bacterial morphology observed under the microscope has no obvious difference, that is, the pure strain is obtained.
[0044] Strain rescreening: the purified single strain is inoculated in tributyrin solid culture medium, about 1 μL of bacteria liquid is added to each hole, and cultured at 30 DEG C for 72 hours, as shown in Figure 2 . The strains with faster growth rate and larger transparent circle under the same conditions are selected as the final obtained strains, the colony morphology of the strain is shown in Figure 1 , and the phylogenetic tree of the strain is shown in Figure 3 .
[0045] Another embodiment of the application discloses application of the bacterium LB356 from Rhodococcus globerulus in degrading olive oil,
[0046] Oil degradation effect of the bacterium LB356 from Rhodococcus globerulus under different inoculation amounts:
[0047] The strains are inoculated into olive oil inorganic salt culture medium at inoculation amounts of 1%, 2%, 5%, 10% and 20% (v / v), and cultured in a shaking bed at 30 DEG C and 160 rpm for 72 hours. The degradation rate is calculated by measuring the residual oil content in the culture medium. As shown in Figure 5 , the results show that when the inoculation amount is 5%, the oil degradation rate of the strain is the highest, reaching 74.91%.
[0048] Oil degradation effect of the bacterium LB356 from Rhodococcus globerulus under different pH conditions:
[0049] The initial pH of the culture medium was adjusted to 4-9, the strain was inoculated at a 5% (v / v) inoculation amount, and was cultured at 30 DEG C and 160 rpm for 72 hours, and the oil degradation rate of each treatment group was determined. Figure 6 As shown in the table, the results show that the strain exhibits the best degradation capacity at pH = 7, and the degradation rate is 74.49%.
[0050] Oil degradation effect of bacterial source strain LB356 of Rikiovia multivorans under different salt concentrations:
[0051] Different concentrations of salt (10-50 g / L) were added to the culture medium, and the strain was inoculated at a 5% (v / v) inoculation amount, and was cultured at 30 DEG C and 160 rpm for 72 hours, and the oil degradation rate was determined. Figure 7 As shown in the table, the results show that the strain has a certain salt tolerance, and 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.
[0052] Oil degradation effect of bacterial source strain LB356 of Rikiovia multivorans under different culture times:
[0053] Under the conditions of pH = 7 and salt concentration of 10 g / L, the strain was inoculated into an olive oil inorganic salt culture medium at a 5% (v / v) inoculation amount, and was cultured in a 30 DEG C, 160 rpm shaker, and the oil degradation rate was determined every 24 hours for 6 days. As shown in the table, Figure 8 The experimental results show that the degradation rate increases with time, and the degradation rate is 77.87% on the sixth day.
[0054] Another embodiment of the present application discloses the application of the bacterial source strain LB356 of Rikiovia multivorans in the treatment of kitchen waste oil-containing wastewater.
[0055] The strain was inoculated into the actual collected kitchen waste oil-containing wastewater at a 5% (v / v) inoculation amount, and was cultured in a 30 DEG C, 160 rpm shaker for 7 days, and a blank control group without inoculation was used for comparison. As shown in the table, Figure 9 The experimental results show that the oil degradation rate of the control group without inoculation is 17.13%, and the oil degradation rate of the experimental group inoculated with the strain is 67.95%. The bacterial source strain LB356 of Rikiovia multivorans screened in the present application exhibits good application effect in the treatment of actual kitchen waste oil-containing wastewater.
[0056] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Various changes and improvements can be made to the present application without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the present application. The scope of the present application is defined by the appended claims and their equivalents.
Claims
1. A Pluralibacter gergoviae LB356, characterized by, The Pluralibacter gergoviae LB356, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 36196.
2. Application of the Pluralibacter gergoviae LB356 in degrading olive oil according to claim 1.
3. Application of the Pluralibacter gergoviae LB356 in treating kitchen waste containing oil wastewater according to claim 1.
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