Strain for efficiently degrading industrial rolling oil, fungicide containing strain and application

Abstract

The invention belongs to the field of microorganisms and application thereof, and particularly provides a strain for efficiently degrading industrial rolling oil, a microbial agent containing the strain and application. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34411. The strain is separated and screened from soil, and has the capability of efficiently degrading industrial rolling oil. The discovery of the strain provides a new microbial resource for treatment of rolling waste oil and water in an industrial rolling factory, has important significance and shows a good application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of microorganisms and their applications, and in particular to a bacterial strain for efficiently degrading industrial rolling oil, a bacterial agent containing the same, and applications. Background Art

[0002] Oily wastewater generated during the industrial rolling process is a typical industrial wastewater. It primarily consists of rolling emulsion wastewater, which contains high concentrations of organic matter and oils. If discharged directly without treatment, this wastewater poses serious risks to the environment and human health. Annual discharge of emulsion wastewater from aluminum hot-rolling mills is high. The oil in this wastewater forms an oil film on the water surface, hindering the dissolution of oxygen into the water and causing a sharp drop in dissolved oxygen levels (Cao Dawei, 2016). This oil film on the water surface blocks sunlight, affecting photosynthesis in aquatic plants and disrupting the entire aquatic food chain. Furthermore, toxic components in the oil, such as polycyclic aromatic hydrocarbons, can directly poison aquatic life, leading to a decline in biodiversity. Industrial rolling oily wastewater also poses significant risks to the soil environment. Once infiltrated into the soil, the oil coats soil particles, reducing both air and water permeability and inhibiting the growth and reproduction of beneficial microorganisms. Studies have shown that petroleum hydrocarbons can reduce the effective nitrogen and phosphorus content in the soil. Polycyclic aromatic hydrocarbons have mutagenic, teratogenic and carcinogenic effects and can enter the human body through drinking water, food chains and other pathways. Long-term exposure may cause a variety of diseases (Dong Ding et al., 2017; Yang Dongmei et al., 2007).

[0003] Traditional methods for treating oily wastewater from steel rolling are mainly divided into three categories: physical, chemical and biological methods.

[0004] Physical methods primarily include gravity separation, flotation, and membrane separation. Gravity separation (e.g., using a grease trap) is suitable for removing floating oil or emulsified oil after demulsification. It is simple to operate, has low operating costs, and processes large volumes of water. However, its disadvantages are that the equipment requires a large footprint, poor treatment efficiency, and susceptibility to uneven water flow, resulting in limited effectiveness for dispersed and emulsified oil (Wang et al., 2011). Flotation introduces air into the wastewater, utilizing the adsorption of air by oil droplets to increase buoyancy and improve oil-water separation efficiency (Nikolaeva et al., 2012). Membrane separation utilizes a porous membrane to intercept tiny oil particles in oily wastewater and aggregate them into larger droplets, achieving oil-water separation through gravity (Scholz W, 2003). While membrane separation methods offer simple equipment, easy operation, and low cost, the membranes used are susceptible to corrosion and contamination, requiring timely cleaning and replacement.

[0005] Chemical methods mainly include flocculation, oxidation, and electrochemical methods. Oxidation uses chemical oxidants such as ozone and Fenton reagents to treat organic wastewater, chemically oxidizing and decomposing oils in the water (Zhang Wenlin et al., 2005). However, oxidation is costly and unsuitable for large-scale industrial processing. Flocculation involves adding flocculants that remove emulsified and dissolved oils to the wastewater, causing the oil to aggregate into flocs and achieve oil-water separation. Commonly used inorganic polymer flocculants include polyacrylamide and polyferric sulfate. Flocculation requires large equipment footprints, requires high chemical dosages, and produces sludge that is difficult to handle after wastewater treatment. Electrochemical methods have garnered widespread attention in recent years and are primarily categorized as electroflocculation, electroflotation, and electromagnetic methods. Electroflocculation processes generally achieve COD and color removal efficiencies exceeding 80%, while consuming less energy than other treatment processes. However, their disadvantage is their high treatment costs (Syam Babu D et al., 2019). Electroflotation is the most commonly used electrochemical method. It has good treatment effects, requires little space, and is simple to operate. However, it processes small volumes of water, consumes large amounts of metals and salts, and has high operating costs, making it uneconomical for treating oily cold-rolling wastewater from small steel mills. Electromagnetic methods have the advantages of not requiring the addition of other reagents and providing excellent disinfection effects. However, they have the disadvantages of high energy consumption and an immature process, resulting in relatively limited practical applications (Zhang Yi et al., 2008).

[0006] Biological treatment primarily utilizes the ability of microorganisms in oily wastewater to degrade oils, allowing them to use oils as a carbon and energy source. Furthermore, through endogenous or exogenous enzymes, microorganisms convert oils (triglycerides) into other usable biomolecules, such as fatty alkanes, fatty alkenes, and fatty acid methyl esters, through a complex metabolic process. Compared to physical or chemical treatment methods, which often suffer from high treatment costs, large footprints, complex processes, and the generation of reactants that can easily cause secondary pollution, biological treatment technology offers advantages such as low labor and material consumption, mild reaction conditions, minimal footprint, and no secondary pollution from decomposition products. Consequently, it has gradually gained favor among researchers both domestically and internationally. Biological treatment represents a hot topic, development trend, and ultimate destination for oily wastewater treatment technology. Currently, the primary biological treatment method for industrial rolling mill oily wastewater is the activated sludge process. However, the traditional activated sludge method has low direct treatment capacity and poor results for oily wastewater (You et al., 2008). Identifying highly efficient degrading bacteria from natural environments and improving activated sludge through bacterial inoculation is of great significance for the treatment of industrial rolling mill oily wastewater.

[0007] Therefore, it is necessary to continue to screen out excellent bacterial resources from nature that can efficiently degrade industrial rolling oil.

[0008] In view of this, this application is hereby filed. Summary of the Invention

[0009] The present disclosure aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the purpose of the present application is to provide a bacterial strain for efficiently degrading industrial rolling oil and a method for its use.

[0010] In order to achieve the above-mentioned purpose of this application, the following technical solutions are specially adopted:

[0011] In a first aspect, the present application provides a strain for efficiently degrading industrial rolling oil, wherein the strain is Serratia nematodiphila KY1694 strain, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.34411.

[0012] The isolated and purified strain was identified using an enrichment-high-throughput method from 500 soil samples collected from 20 different provinces and municipalities. The strain's degradation capacity was initially assessed based on the size of degradation zones observed in oil-dissolving plates. Several strains with larger degradation zones were selected from numerous samples for subsequent experiments. After evaluating the results of a series of experiments, strain KY1694 was identified as highly effective at degrading industrial rolling oil.

[0013] In one embodiment, the 16S rDNA sequence of the Serratia nematodiphila KY1694 strain is shown as SEQ ID No. 1.

[0014] The results of 16S rDNA sequencing of KY1694 in this application indicate that the base sequence of this strain is over 99% identical to that of Serratia nematodiphila, with a similarity of 99.02, confirming that KY1694 is Serratia nematodiphila. To date, there have been no reports of this strain degrading industrial rolling oil. This application is the first to demonstrate the strain's ability to efficiently degrade industrial rolling oil, suggesting that this strain has extremely high commercial value and broad market prospects.

[0015] In a second aspect, the present application provides a microbial agent, comprising the aforementioned Serratia nematodiphila KY1694 strain or a fermentation broth obtained by fermenting the aforementioned strain.

[0016] In one embodiment, the microbial agent is a solid agent or a liquid agent;

[0017] When the microbial agent is a solid agent, the effective viable count of Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / g;

[0018] When the microbial agent is a liquid agent, the effective viable count of Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / mL.

[0019] In one embodiment, the microbial agent further comprises sulfate and acetate; preferably, the sulfate is ammonium sulfate, and the acetate is sodium acetate.

[0020] In a third aspect, the present application provides use of the aforementioned Serratia nematodiphila KY1694 strain or the aforementioned microbial agent in degrading industrial rolling oil.

[0021] In one embodiment, the industrial rolling oil includes cold rolling oil and hot rolling oil.

[0022] In one embodiment, the use is for degrading industrial rolling oil contained in waste liquid or soil.

[0023] In a fourth aspect, the present application provides a method for treating industrial rolling oil, comprising treating an object to be treated containing industrial pressing oil with the aforementioned Serratia nematodiphila strain KY1694 or the aforementioned microbial agent.

[0024] In one embodiment, the method comprises using the aforementioned Serratia nematophila KY1694 together with 0.02%-0.08% ammonium sulfate by weight volume ratio and 0.02%-0.05% sodium acetate by weight volume ratio to treat an object to be treated containing industrial pressed oil.

[0025] The Serratia nematodiphila strain provided in this application, named KY1694, was deposited with the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 30, 2025, and the deposit number is CGMCC NO. 34411. The strain was tested as viable by the collection center on April 30, 2025.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The Serratia nematodiphila strain KY1694 of the present application was first obtained by the inventors through extensive screening work in soil. This strain has the ability to produce and efficiently degrade industrial rolling oil. The discovery of this strain provides a new microbial resource for solving the treatment of industrial rolling oil, enriches the bacterial resources in this field, and fills the gap in the use of microbial strains to efficiently treat industrial rolling oil. It provides a scientific and feasible solution for the treatment of related environmental problems and demonstrates great research and application value.

[0028] (2) The present application can achieve efficient treatment of hot rolling oil and cold rolling oil by culturing the Serratia nematodiphila strain KY1694, which is suitable for large-scale production and practical engineering applications.

[0029] (3) The present application provides a method for treating industrial rolling oily wastewater using the Serratia nematodiphila strain KY1694. The strain is used for fermentation production, and the production process is simple and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] Figure 1 The results of the selective plate separation of the strains that can grow using oil as a carbon source were initially screened for this application;

[0032] Figure 2 This is the fermentation status of the strain KY1694 of this application in different rolling oils. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions of this application in conjunction with the embodiments. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0034] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0035] Detailed description of specific embodiments

[0036] In one embodiment, the present application provides a strain for efficiently degrading industrial rolling oil, which is Serratia nematodiphila KY1694 strain, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.34411.

[0037] The strain is an isolated and purified strain. This application uses the enrichment-high throughput method to screen out industrial rolling oil-efficient degrading bacteria from 500 soil samples collected from 20 different provinces and cities. This application preliminarily judges the degradation ability of the strain based on the size of the degradation circle in the oil-dissolving plate, and screens out several strains with larger degradation circles from a large number of samples for subsequent experiments. After a series of experimental results, a strain KY1694 with the ability to efficiently degrade industrial rolling oil was finally screened out. To date, there has been no report on the ability of this bacterium to degrade industrial rolling oil. This application is the first to discover the ability of this strain to efficiently degrade industrial rolling oil. This strain has extremely high commercial value and broad market prospects.

[0038] The results of 16S rDNA sequence determination of KY1694 in the present application showed that the base sequence of the strain had a high homology of more than 99% with the strain Serratia nematodiphila, with a similarity of 99.02, confirming that KY1694 was Serratia nematodiphila.

[0039] In one embodiment, the present application encompasses fermentation broths of the aforementioned strains.

[0040] In one embodiment, the present application provides a microbial agent comprising the aforementioned Serratia nematodiphila KY1694 strain or a fermentation broth obtained by fermenting the aforementioned strain.

[0041] In one embodiment, the microbial agent is a solid agent or a liquid agent;

[0042] When the microbial agent is a solid agent, the effective viable count of Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / g, for example 1×10 9CFU / g to 1×10 11 CFU / g;

[0043] When the microbial agent is a liquid agent, the effective viable count of Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / mL, for example 1×10 9 CFU / m to 3×10 9 CFU / mL.

[0044] In one embodiment, the microbial agent is in the form of dry powder, and the effective viable count of Serratia nematodiphila per gram of the microbial agent is at least 2×10 9 CFU.

[0045] In one embodiment, the microbial agent further comprises sulfate and acetate; preferably, the sulfate is ammonium sulfate, and the acetate is sodium acetate.

[0046] In specific embodiments, the strain can be prepared into a microbial agent for transportation or use as needed. For example, the composition can be prepared in liquid, solid powder, or other forms, such as an emulsion, suspension, or granules. Furthermore, the composition can also contain carriers commonly used in the preparation of microbial agents, such as solid or liquid carriers, specifically bentonite, calcium carbonate, zeolite, starch, or vegetable oil, mineral oil, and water. The microbial agent can be in the form of a liquid agent, powder, or granule.

[0047] The present application also covers fermentation cultures, fermentation culture suspensions, or fermentation culture fractions of the strains. Specifically, the culture of the strain is obtained by culturing the strain in LB medium to the logarithmic phase, i.e., obtaining a fermentation culture; the solid obtained by separating and collecting the fermentation culture and resuspending it in a solvent is obtained as a fermentation culture suspension; and the liquid obtained by separating and collecting the fermentation culture is obtained as a fermentation culture fraction.

[0048] In a specific embodiment, the microbial agent is obtained by the following steps:

[0049] The strain is fermented and cultured to obtain a fermentation product; and the fermentation product is spray-dried and pulverized to obtain the microbial agent.

[0050] In a specific embodiment, the microbial agent is in the form of dry powder, and the effective viable count of the Serratia nematodiphila KY1694 in the microbial agent is at least 10 9CFU·mL -1 , 10 10 CFU·mL -1 ; or 10 9 CFU·g -1 , 10 10 CFU·g -1 , 10 11 CFU·g -1 .

[0051] In one embodiment, the present application provides the use of the aforementioned Serratia nematodiphila KY1694 strain or the aforementioned microbial agent in degrading industrial rolling oil in an environment. In one embodiment, the industrial rolling oil is present in environmental wastewater or waste liquid, or in soil.

[0052] In some embodiments, the industrial rolling oil is not specifically limited and may include, for example, rolling oils used in the copper hot rolling industry, the aluminum hot rolling industry, and the steel hot rolling industry. Such rolling oils may be commercially available rolling oils or may be rolling oils prepared by the enterprise as needed.

[0053] In a specific embodiment, the industrial rolling oil includes cold rolling oil and hot rolling oil, such as but not limited to: Japan Parker brand hot rolling oil, Quaker brand cold rolling oil, Quaker brand hot rolling oil, etc.

[0054] In one embodiment, the present application provides a method for treating industrial rolling oil, comprising treating the industrial rolling oil with the aforementioned Serratia nematodiphila strain KY1694 or the aforementioned microbial agent.

[0055] In one embodiment, the method comprises using the aforementioned Serratia nematophila KY1694 or the aforementioned microbial agent together with 0.02%-0.08% (w / v) ammonium sulfate and 0.02%-0.05% (w / v) sodium acetate to treat an object to be treated containing industrial pressed oil.

[0056] In one embodiment, the treatment method comprises inoculating the aforementioned nematophilic Serratia marcescens KY1694 or a microbial agent containing the same into a wastewater environment containing industrial rolling oil for fermentation, wherein the inoculation amount is 6%-10% of the culture medium volume, the fermentation time is 3-5 days, and the fermentation temperature is 28°C-35°C.

[0057] In some embodiments, the strains or microbial agents of the present application can be used for the remediation of contaminated soil, in particular, the remediation of soil containing industrial rolling oil wastewater.

[0058] In some embodiments, the strains or microbial agents of the present application can be used for the treatment of contaminated sewage, in particular, the treatment of sewage containing industrial rolling oil wastewater.

[0059] In one embodiment, the method is to use the strain or microbial agent of the present application to ferment and decompose the contaminated sample. The strain or microbial agent of the present application uses industrial rolling oil as the only carbon source.

[0060] In some embodiments, the strains or microbial agents of the present application can be used to degrade rolling oil in different rolling wastewater samples.

[0061] In some embodiments, the strain or microbial agent of the present application can be used to bioremediate soil or water contaminated by industrial rolling oil waste liquid.

[0062] In a specific embodiment, the strain of the present application is used together with ammonium sulfate and sodium acetate to form a composition. In some cases, the composition has a better degradation rate than using the strain of the present application alone or using ammonium sulfate and sodium acetate alone.

[0063] In a specific embodiment, the method of the present application comprises inoculating 0.02%-0.08% (w / v) ammonium sulfate and 0.02%-0.05% (w / v) sodium acetate together with the strain of the present application having an OD of 0.05-0.06 into an industrial press wastewater sample.

[0064] In a specific embodiment, the method of the present application comprises inoculating 0.01%-0.05% (w / v) glucose and the strain of the present application with OD=0.05-0.06 into an industrial press wastewater sample.

[0065] In one embodiment, the method of the present application comprises inoculating 0.05% (w / v) ammonium sulfate and 0.03% (w / v) sodium acetate together with the strain of the present application at OD=0.05 into an industrial press wastewater sample.

[0066] In one embodiment, the temperature condition for treating the industrial rolling oil is 28-35° C., preferably 30-35° C. In one embodiment, the time condition for treating the industrial rolling oil is 3-6 days, preferably 4-5 days.

[0067] In another embodiment, the strain of the present application is used together with glucose to form a composition. In some cases, the composition contains 0.01%-0.03% (w / v) glucose. The composition has a better degradation rate than using the strain of the present application alone.

[0068] Example

[0069] 1. Experimental Materials

[0070] 1.1 Culture medium components

[0071] R2A medium: yeast powder 0.5 g, yeast extract 0.5 g, tryptone 0.5 g, glucose 0.5 g, soluble starch 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium sulfate heptahydrate 0.05 g, agar powder 15 g, water 1 L.

[0072] Enrichment (fermentation) medium: ammonium sulfate 0.5 g, sodium chloride 0.5 g, disodium hydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.05 g, water 1 L.

[0073] Selection medium: L-glycerol tributyrate 10 g, yeast powder 0.5 g, peptone 0.5 g, tryptone 0.5 g, glucose 0.5 g, soluble starch 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium sulfate heptahydrate 0.05 g, agar powder 15 g, water 1 L.

[0074] 1.2 Main Reagents

[0075] Industrial rolling oil: Japan Paccar hot rolling oil, Quaker cold rolling oil, Quaker hot rolling oil, and petroleum ether were purchased from Guangzhou Jinyuan Chemical Co., Ltd., and other reagents were domestically produced analytical grade.

[0076] 2. Screening of microbial strains that efficiently degrade industrial rolling oil using high-throughput methods

[0077] 2.1 Collection of soil samples

[0078] Select representative samples. The samples can come from different areas such as farmland, grassland, forest soil, etc. Collect 15-20g of samples at each point, and mark the source (province, county), collection year and month, and soil source (plant or other). Store in a -80℃ refrigerator.

[0079] 2.2 Screening of microbial strains that efficiently degrade industrial rolling oil using high-throughput methods

[0080] 1) Enrichment step: Ten soil samples were grouped together. 0.1 g of each soil sample was taken, and a total of 1 g was taken and mixed in 100 mL of pure water. 1 mL of the mixture was inoculated into a liquid enrichment medium (sterilized at 121°C for 20 min) containing 2% (w / v) Japan Parker hot-rolling oil (hereinafter referred to as "rolling oil"). The culture was shaken at 200 r / min at 30°C for 4-5 days, and changes in the color of the liquid culture medium and the amount of floating oil on the surface were observed and recorded.

[0081] 2) Second enrichment step: 1 mL of the enriched bacterial solution from the previous step was added to an enrichment liquid medium containing 2% rolling oil. The culture was shaken at 200 rpm at 30°C for 4-5 days. Changes in the liquid medium color and the amount of floating oil on the surface were observed and recorded.

[0082] 3) Enrichment Step 3: Take 1 mL of the bacterial solution enriched in Step 2 and add it to an enrichment liquid medium containing 2% rolling oil. Cultivate the culture at 30°C and 200 rpm for 4-5 days. Observe and record changes in the color of the liquid medium and the amount of floating oil on the surface.

[0083] 2.3 Select the flat hydrolysis circle for screening

[0084] (1) Take 100 μL of the bacterial solution from the last enrichment and spread it on the selective culture medium. Incubate at 30°C for 72 hours and observe whether there is a hydrolysis zone.

[0085] (2) The strain with hydrolysis zone was selected from the above culture medium and streaked on R2A medium (sterilized at 121°C for 20 min) for purification.

[0086] (3) The purified strain was inoculated onto the selective culture medium and cultured at 30°C for 72 hours. The clear zone (D) and colony diameter (d) were measured respectively. The value of the clear zone radius minus the colony radius was used for preliminary determination.

[0087] 2.4 Experimental Results

[0088] The experiment initially isolated and screened several strains that can use oil as a carbon source for growth, and the results of the selected plate are shown as follows Figure 1 The initial screening results initially indicate that specific microorganisms do grow on the carbon-limited plates. Subsequently, colonies with larger hydrolysis zones from each isolation plate will be selected for liquid shake flask oil extraction experiments.

[0089] The acclimated strains were inoculated onto selection plates for culture and the growth of the hydrolysis zone was observed. The ratio of the hydrolysis zone radius to the colony radius was comprehensively compared to determine the subsequent experimental strains, as shown in Table 1.

[0090] Table 1. Comparison of transparent zone sizes of different strains

[0091]

[0092] Titer assessment: ratio <1.0: -; 1.0≤ratio <1.5: +; 1.5≤ratio <2.0: ++; 2.0≤ratio <2.5: +++; 2.5≤ratio <3.0: ++++; ratio ≥3.0: +++++.

[0093] As can be seen from the table, KY1694 has the best potency evaluation, followed by J10. To further determine the oil-lowering effect of the strains, strains with a potency evaluation of ++ or above in the table were selected as subsequent experimental strains.

[0094] Example 3: Degradation experiment of rolling oil by initial screening of degrading bacteria

[0095] 3.1 Shake flask culture of oil-containing liquid

[0096] The strains initially screened and purified were transferred into 100 mL of fermentation medium containing 2 g of rolling oil (with the same ingredients as the enrichment medium). At the same time, a blank group without bacteria was set as a control. Liquid shake flask fermentation was carried out at 30°C and 200 r / min. The differences in degradation ability of each strain within the same degradation time were investigated, and the residual oil content and degradation rate in each bottle of fermentation liquid were determined.

[0097] 3.2 Determination of residual oil content (gravimetric method)

[0098] The fermentation broth in each shake flask, which had been degraded by the strain, was centrifuged at 6000 rpm for 20 minutes. The supernatant was transferred to a separatory funnel and extracted twice with petroleum ether. The remaining lower layer was extracted twice more with petroleum ether. The extracted organic layers from each shake flask were collected, dried with anhydrous sodium sulfate, and transferred to weighed collection tubes. The tubes were placed in a 60°C constant temperature water bath to evaporate the organic solvent. After cooling, the tubes were weighed and the residual oil content and degradation rate of each strain were recorded and calculated. Calculation method: Degradation rate (%) = (initial oil weight - residual oil weight in sample) / initial oil weight × 100%.

[0099] 3.3 Measurement results

[0100] Table 2. Comparison of degradation rates of different strains

[0101]

[0102] It can be concluded from Table 2 that KY1694 had the highest degradation rate of 59.6%, followed by J10 at 50.2%. Therefore, KY1694 was selected as the target strain.

[0103] Example 4: 16S rDNA sequencing of target strain KY1694

[0104] 4.1 CTAB method for bacterial DNA extraction

[0105] 1. Inoculate a single colony into 5 mL of R2A and culture overnight at 30°C.

[0106] 2. Take 1 mL of seed culture solution and inoculate it into 100 mL of R2A liquid. Incubate at 37°C and 220 rpm for 16 hours.

[0107] 3. Centrifuge at 5000 rpm for 10 minutes and discard the supernatant.

[0108] 4. After adding 10 mL of TE and centrifuging to wash, dissolve the cells with 10 mL of TE, mix well, and store at -20°C for later use;

[0109] 5. Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS, mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour;

[0110] 6. Add 740 μL 5 mol / L NaCl, then add 512 μL CTAB / NaCl, mix well, and incubate at 65°C for 10 minutes;

[0111] 7. Add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 10,000 rpm for 5 minutes, and retain the supernatant;

[0112] 8. Add equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 10,000 rpm for 5 minutes, and retain the supernatant;

[0113] 9. Add 0.6 times isopropanol, mix well, centrifuge at 10000 rpm for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol;

[0114] 10. Dissolve DNA in 1 mL TE, add RNase A to a final concentration of 20 μg / mL, and store at 4°C.

[0115] 4.2 Amplification and sequencing

[0116] 16S rDNA universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 2) and 1492r (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 3) were used for PCR amplification of 16S rDNA.

[0117] PCR reaction conditions: 94°C pre-denaturation for 30 s; 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 60 s, for 30 cycles. PCR products were subjected to 1.5% agarose gel electrophoresis, recovered, purified, and sequenced.

[0118] 4.3 16S rDNA sequencing results of strain KY1694

[0119] The 16S rDNA sequence of strain KY1694 (SEQ ID No. 1) is as follows:

[0120]

[0121] Based on the obtained 16S rDNA sequence of KY1694 (SEQ ID No. 1), homologous sequences were searched in GenBank and analyzed and compared. At the same time, the sequence was aligned with the 16S RNA database recognized by the International Bacteriological Commission (Chun's lab) and combined with literature analysis to determine the taxonomic status of the target microorganism (Yoon, SH, Ha, SM, Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol. 67: 1613-1617).

[0122] The results showed that the 1423-base sequence of the strain had a similarity of 99.02 with that of Serratia nematodiphila, confirming that KY1694 was Serratia nematodiphila.

[0123] Example 5. Degradation of different rolling oils by strain KY1694

[0124] 5.1 Collection of different types of rolling oil

[0125] The rolling oils commonly used in steel mills are Japan Parker hot rolling oil, Quaker hot rolling oil, and Quaker cold rolling oil.

[0126] 5.2 Degradation experiment

[0127] 1. Group: According to the concentration of 1%, the groups were set as 1% Quaker cold rolling oil-CK, 1% Quaker cold rolling oil-KY1694, 1% Japan Parker hot rolling oil-CK, 1% Japan Parker hot rolling oil-KY1694, 1% Quaker hot rolling oil-CK, and 1% Quaker hot rolling oil-KY1694. Among them, the degradation rate of 1% Quaker hot rolling oil was low in the preliminary experiment, so 0.5% Quaker hot rolling oil-CK and 0.5% Quaker hot rolling oil-KY1694 were additionally set.

[0128] 2. Add the three different types of rolling oil collected into 100 mL of fermentation liquid culture medium respectively, and culture at 30°C and 200 r / min for 4 days. Observe the amount of floating oil in the culture medium, such as Figure 2 .

[0129] The fermentation broth in each shake flask that had been degraded by strain KY1694 was centrifuged at 6000 r / min for 20 min and then extracted twice with petroleum ether using the same method as in 3.1 for the determination of residual oil content.

[0130] 5.3 Degradation results

[0131] After 4 days of fermentation with KY1694 strain, the emulsification and surface oil volume of the three rolling oils were significantly different. Figure 2 shown. Figure 2 Figure a is a comparison of the fermentation results between the 1% Quaker cold-rolled oil CK group (blank group) and the strain KY1694 experimental group; Figure 2 Middle b is the comparison of fermentation between CK group and experimental group with 1% Japanese Parker hot rolling oil; Figure 2 Middle c is the comparison of fermentation between CK group and experimental group with 1% Quaker hot-rolled oil; Figure 2 Figure d shows the comparison of fermentation between the 0.5% Quaker hot-rolled oil CK group and the experimental group.

[0132] It can be seen that the amount of floating oil in the experimental groups of Quaker cold rolling oil and Japan Parker hot rolling oil is less than that in the blank group. Under the conditions of 1% and 0.5% Quaker hot rolling oil, the rolling oil in the blank group is in a clumping state and not dispersed, while the experimental group is dispersed, and under the 0.5% condition, the floating oil in the experimental group basically disappears.

[0133] After a series of operations including centrifugation, extraction and evaporation, the residual oil content was measured as shown in Table 3.

[0134] Table 3. Comparison of degradation rates of different rolling oils by strain KY1694

[0135]

[0136] As can be seen from the table, the KY1694 strain had the highest degradation rate for 1% Japanese Parker hot rolling oil, reaching 56.9%; followed by 1% Quaker cold rolling oil, which was 47.6%; while for Quaker hot rolling oil, the degradation rate was only 31.8% at 1% concentration, and increased to 55.4% when the concentration was reduced to 0.5%.

[0137] Example 6. Practical application of strain KY1694 in degrading rolling oil in different rolling wastewater samples

[0138] 6.1 Collection of rolling wastewater samples from different sources

[0139] Rolling wastewater was collected from three different steel mills for subsequent experiments. The water samples were galvanizing water samples from Hesteel Posco, pickling and cold rolling water samples from Tangshan Iron and Steel, and hot rolling water samples from Guotang Steel.

[0140] 6.2 Degradation of oil by strain KY1694 in HBIS Pohang galvanizing water samples

[0141] 1. Control group: CK blank group, group containing 0.03% glucose; experimental group: KY1694 group, group containing 0.01% (w / v) glucose, group containing 0.05% (w / v) ammonium sulfate and 0.03% (w / v) sodium acetate.

[0142] 2. Add 50 mL of Hesteel Posco galvanized water sample to each shake flask. Due to the varying oil content of each water sample and to ensure clear differentiation of the final results, 1 g of Quaker rolling oil (0.5 g of Quaker cold rolling oil and 0.5 g of Quaker hot rolling oil) was added to each shake flask. KY1694 was inoculated into each experimental group at an initial OD of 0.05. After incubation at 30°C and 200 rpm for 4 days, the residual oil content was determined using the same method as in 3.1. The oil degradation rate of each group was calculated and compared.

[0143] 6.3 Degradation of oil by strain KY1694 in pickling and cold rolling water samples from Tangshan Iron and Steel

[0144] 1. Control groups: CK blank group, group containing 0.03% glucose, group containing 0.05% ammonium sulfate and 0.03% sodium acetate; experimental groups: KY1694 group, KY1694 group containing 0.01% glucose, group containing 0.05% ammonium sulfate and 0.03% sodium acetate.

[0145] 2. Add 50 mL of Tangshan Iron and Steel's pickling and cold-rolling water samples to each shake flask. Then, add 1 g of Quaker rolling oil (0.5 g of Quaker cold-rolling oil and 0.5 g of Quaker hot-rolling oil) to each shake flask. Inoculate KY1694 into each experimental group at an initial OD of 0.05 and incubate at 30°C, 200 rpm for 4 days. Determine the residual oil content using the same method as in 3.1. Calculate and compare the oil degradation rate for each group.

[0146] 6.4 Degradation of oil by strain KY1694 in hot-rolled water samples from Guotang Steel

[0147] 1. Control groups: CK blank group, group containing 0.03% glucose, group containing 0.05% ammonium sulfate and 0.03% sodium acetate; experimental groups: KY1694 group, KY1694 group containing 0.01% glucose, group containing 0.05% ammonium sulfate and 0.03% sodium acetate.

[0148] 2. Add 50 mL of Guotang Steel hot-rolled water sample to each shake flask. Then, add 1 g of Quaker rolling oil (0.5 g of Quaker cold-rolled oil and 0.5 g of Quaker hot-rolled oil) to each shake flask. Inoculate KY1694 into each experimental group at an initial OD of 0.05 and incubate at 30°C, 200 rpm for 4 days. Determine residual oil content using the same method as in 3.1. Calculate and compare the oil degradation rate for each group.

[0149] 6.5 Experimental results of oil degradation by strain KY1694 in different water samples

[0150] After the degradation experiment of rolling oil by strain KY1694 in different water samples, the calculated degradation rates are shown in Tables 4, 5 and 6 below.

[0151] As shown in Table 4, the KY1694 group exhibited the highest degradation rate of 56.7% in the galvanizing water sample from Hegang Posco, demonstrating the best degradation effect. This was followed by the KY1694 group with 0.05% ammonium sulfate and 0.03% sodium acetate, which achieved a 41.9% degradation rate. Table 5 shows that the KY1694 group with 0.05% ammonium sulfate and 0.03% sodium acetate in the pickling and cold rolling water sample from Tangshan Iron and Steel Corporation exhibited the highest degradation rate of 59.9%, demonstrating the best degradation effect. Table 6 also shows that the KY1694 group with 0.05% ammonium sulfate and 0.03% sodium acetate in the hot rolling water sample from Guotang Steel Corporation also exhibited the highest degradation rate. Overall, the KY1694 group with 0.05% ammonium sulfate and 0.03% sodium acetate exhibited the best degradation effect on rolling oil in various mill wastewaters.

[0152] Table 4. Degradation rate of strain KY1694 in HBIS Pohang galvanizing water samples

[0153]

[0154] Table 5. Degradation rate of strain KY1694 in pickling and cold rolling water samples of Tangshan Iron and Steel

[0155]

[0156] Table 6. Degradation rate of strain KY1694 in hot rolling water samples of Guotang Steel

[0157]

[0158] in conclusion

[0159] This application uses a high-throughput screening method to isolate several strains that are good at degrading oils and fats from soil collected in Anhui Province. After analyzing their effects on degrading industrial rolling oils, a strain KY1694 that can efficiently degrade industrial rolling oils was identified. The results of 16S rDNA sequence determination of KY1694 showed that the base sequence of the strain had a high homology of more than 99% with the strain Serratia nematodiphila, with a similarity of 99.02, confirming that KY1694 is Serratia nematodiphila. After multiple tests in different application scenarios, the results showed that the strain can effectively degrade various types of industrial rolling oils. The application of this strain in the treatment of waste oil water from industrial rolling mills has significant social and economic value. Compared with other treatment technologies, it has obvious advantages in harmlessness, safety and economy.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A strain for efficiently degrading industrial rolling oil, characterized in that: The strain is Serratia nematodiphila KY1694 strain, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.34411.

2. The strain according to claim 1, characterized in that The 16S rDNA sequence of the strain is the sequence shown in SEQ ID NO:

1.

3. A microbial agent, characterized in that: The microbial agent contains the Serratia nematodiphila KY1694 strain according to claim 1 or claim 2, or contains a fermentation liquid obtained by fermenting the strain according to claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that The microbial agent is a solid agent or a liquid agent; When the microbial agent is a solid agent, the effective viable count of the Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / g; When the microbial agent is a liquid agent, the effective viable count of the Serratia nematodiphila KY1694 in the microbial agent is at least 1×10 9 CFU / mL.

5. The microbial agent according to claim 3 or 4, characterized in that The microbial agent further comprises sulfate and acetate; preferably, the sulfate is ammonium sulfate, and the acetate is sodium acetate.

6. Use of the Serratia nematodiphila KY1694 strain according to claim 1 or claim 2 or the microbial agent according to any one of claims 3 to 5 in degrading industrial rolling oil.

7. The use according to claim 6, characterized in that The industrial rolling oil includes cold rolling oil and hot rolling oil.

8. The use according to claim 7, characterized in that The application is to degrade industrial rolling oil contained in waste liquid or soil.

9. A method for treating industrial rolling oil, characterized in that: The method comprises treating an object to be treated containing industrial pressed oil with the Serratia nematodiphila strain KY1694 according to claim 1 or claim 2 or the microbial agent according to any one of claims 3 to 5.

10. The method according to claim 9, characterized in that The method comprises using the Serratia nematophila KY1694 according to claim 1 or claim 2, 0.02%-0.08% of ammonium sulfate by mass-weight-volume ratio, and 0.02%-0.05% of sodium acetate by weight-volume ratio to jointly treat an object to be treated containing industrial pressed oil.

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