A rapid screening method for diesel-degrading bacteria, Citrobacter GO7 and its applications

Abstract

This invention discloses a rapid screening method for diesel-degrading bacteria, Citrobacter GO7, and its applications, belonging to the field of microbial remediation technology. The rapid screening method of this invention includes the following steps: adding oily wastewater to physiological saline and shaking to obtain a bacterial suspension; inoculating the bacterial suspension into LB liquid medium for fermentation to obtain a first fermentation culture; spreading the first fermentation culture onto MSM solid medium with diesel as the sole carbon source for culture; picking single colonies from the solid medium and fermenting them in LB liquid medium to obtain a second fermentation culture; adding the second fermentation culture to MSM liquid medium containing diesel for culture; and screening for diesel-degrading strains by detecting the diesel degradation rate. This invention's method is fast, simple, and has a high success rate. The screened Citrobacter GO7 exhibits excellent degradation effects on alkanes and polycyclic aromatic hydrocarbons in oily wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial remediation technology, and in particular relates to a rapid screening method for diesel-degrading bacteria, Citrobacter GO7 and its applications. Background Technology

[0002] With industrialization, the extraction and application of oil have developed rapidly. However, the extraction, refining, and processing of oil have generated large amounts of oil-bearing soil, with diesel pollution being particularly prevalent. Diesel fuel is a complex mixture composed of alkanes, aromatics, and aliphatic substances. Because most of its compounds have low solubility in water, the biodegradability of hydrocarbons in diesel fuel is severely limited. Therefore, the decomposition and oxidation of diesel fuel in soil under natural conditions is slow. Applying physical or chemical methods to treat oil-contaminated water and soil is not only costly but can also cause secondary pollution.

[0003] Microbial treatment technology, with its low production cost, lack of secondary pollution, and ability to perform in-situ remediation, is considered a promising high-tech approach and the most ideal method. However, current traditional screening procedures are cumbersome, have low success rates, and are time-consuming. Therefore, there is an urgent need to develop a rapid screening method that can simplify the screening process, improve the success rate, and simultaneously screen for strains with good diesel degradation effects. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a rapid screening method for diesel-degrading bacteria. This screening method is simple, has a high success rate, is fast, and the selected strains have good diesel degradation effects.

[0005] Another object of the present invention is to provide a Citrobacter GO7.

[0006] Another objective of this invention is to provide a fermentation broth of Citrobacter GO7 and a method for preparing the same.

[0007] Another object of the present invention is to provide an application of the Citrobacter GO7 or the fermentation broth in the degradation of oily wastewater.

[0008] Another object of the present invention is to provide an agent for degrading oily wastewater.

[0009] Another object of the present invention is to provide a method for degrading oily wastewater.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a rapid screening method for diesel-degrading bacteria, comprising the following steps:

[0012] Oily wastewater was added to physiological saline and shaken to obtain a bacterial suspension. The bacterial suspension was inoculated into LB liquid medium for fermentation to obtain the first fermentation broth. The first fermentation broth was spread onto MSM solid medium with diesel as the sole carbon source and cultured. Single colonies from the solid medium were picked and cultured in LB liquid medium to obtain the second fermentation broth. The second fermentation broth was added to MSM liquid medium containing diesel and cultured. By detecting the diesel degradation rate of the strains, strains with diesel degradation performance were screened to obtain the strains.

[0013] The present invention also provides a Citrobacter freundii GO7 obtained by the above screening method, which was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M 20241715.

[0014] The present invention also provides a fermentation broth obtained by fermentation of the aforementioned Citrobacter GO7.

[0015] The present invention also provides a method for preparing the fermentation broth, comprising the following steps: inoculating the Citrobacter GO7 in LB liquid medium and culturing it to obtain the fermentation broth.

[0016] The present invention also provides an application of the Citrobacter GO7 or the fermentation broth in the degradation of oily wastewater.

[0017] The present invention also provides a formulation for degrading oily wastewater, the formulation comprising the above-mentioned Citrobacter GO7 or the above-mentioned fermentation broth.

[0018] The present invention also provides a method for degrading oily wastewater, comprising the following steps: treating the oily wastewater with the Citrobacter GO7 or the fermentation broth or the preparation.

[0019] Preferably, the method includes the following steps: adding the Citrobacter GO7 or the fermentation broth or the preparation to oily wastewater for cultivation.

[0020] Preferably, the total petroleum hydrocarbon concentration in the oily wastewater is 500–16000 mg / L.

[0021] Preferably, the culture conditions are: cultured at 25-37°C for 2-7 days.

[0022] The beneficial effects of this invention are:

[0023] The screening method of this invention requires only 5-8 days to screen strains, and the diesel degradation rate of the five screened strains all reached over 80%, demonstrating excellent degradation effects on diesel. Compared with traditional screening methods, the rapid screening method of this invention has the advantages of being fast, simple, having a high success rate, and producing strains with good diesel degradation effects.

[0024] The Citrobacter GO7 strain obtained by screening in this invention is effective against C 11 ~C 15 The degradation rate of alkane reached 93.55%, C 16 ~C 19 The alkane degradation rate reached 92.45%, C 20 The degradation rate of alkanes reached 93.90%; the degradation rates of tricyclic, tetracyclic, pentacyclic, and hexacyclic aromatic hydrocarbons reached 64.30%, 65.18%, 68.52%, and 75.76%, respectively. The Citrobacter GO7 of this invention has excellent degradation effects on alkanes and polycyclic aromatic hydrocarbons in oily wastewater.

[0025] When the Citrobacter GO7 of this invention is applied to actual oily wastewater, it has an excellent degradation effect on alkanes and polycyclic aromatic hydrocarbons in the oily wastewater. The Citrobacter GO7 of this invention has a strong application prospect in the microbial remediation of oily wastewater.

[0026] Biological Preservation Information

[0027] This invention relates to Citrobacter GO7, classified and named Citrobacter freundii, which is deposited at the China Center for Type Culture Collection (CCTCC) on July 31, 2024, at Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20241715. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the steps of the rapid screening method in Example 1;

[0029] Figure 2 This is a flowchart illustrating the steps of the traditional screening method in Comparative Example 1.

[0030] Figure 3 This is a graph showing the diesel degradation effect of the strains screened by the method in Example 1;

[0031] Figure 4 The diesel degradation effect of the strains screened by the method in Comparative Example 1 is shown in the figure.

[0032] Figure 5 This is a colony morphology diagram of Citrobacter GO7;

[0033] Figure 6Phylogenetic analysis of Citrobacter GO7;

[0034] Figure 7 This is a graph showing the alkane concentrations of diesel fuel after 5 days of degradation by Citrobacter GO7 in Example 5.

[0035] Figure 8 This is a graph showing the concentration of polycyclic aromatic hydrocarbons (PAHs) in diesel fuel after 5 days of degradation by Citrobacter GO7 in Example 5.

[0036] Figure 9 The image shows the GC-MS spectrum of Citrobacter GO7 after 5 days of degradation of oily wastewater in Example 6. Detailed Implementation

[0037] This invention provides a rapid screening method for diesel-degrading bacteria, comprising the following steps:

[0038] Oily wastewater was added to physiological saline and shaken to obtain a bacterial suspension. The bacterial suspension was inoculated into LB liquid medium for fermentation to obtain the first fermentation broth. The first fermentation broth was spread onto MSM solid medium with diesel as the sole carbon source and cultured. Single colonies from the solid medium were picked and cultured in LB liquid medium to obtain the second fermentation broth. The second fermentation broth was added to MSM liquid medium containing diesel and cultured. By detecting the diesel degradation rate of the strains, strains with diesel degradation performance were screened to obtain the strains.

[0039] In this invention, oily wastewater is added to physiological saline and shaken to obtain a bacterial suspension. This invention does not specifically limit the source of the oily wastewater; as an optional embodiment, the oily wastewater comes from the high-salt oil concentration tank of Luoyang Petrochemical Plant. The volume ratio of the oily wastewater to physiological saline is preferably 0.3–0.8:40–50, more preferably 0.4–0.7:42–48, and even more preferably 0.5:45. This invention does not specifically limit the shaking method; conventional shaking methods in the art can be selected. As an optional embodiment, shaking can be performed in an air bath constant-temperature shaker. The shaking speed is preferably 100–250 rpm, more preferably 120–200 rpm, and even more preferably 180 rpm. The shaking temperature is preferably 25–37°C, more preferably 27–32°C, and even more preferably 30°C. The shaking time is preferably 3–8 min, more preferably 4–6 min, and even more preferably 5 min.

[0040] In this invention, a bacterial suspension is inoculated into LB liquid medium for fermentation to obtain a first fermentation culture broth. The volume ratio of the bacterial suspension to the LB liquid medium is preferably 1:40–200, more preferably 1:50–150, and even more preferably 1:100. The fermentation culture is preferably a shaking culture. The shaking speed of the fermentation culture is preferably 100–250 rpm, more preferably 120–200 rpm, and even more preferably 180 rpm. The fermentation temperature is preferably 25–37°C, more preferably 27–32°C, and even more preferably 30°C. The fermentation time is preferably 8–16 h, more preferably 10–14 h, and even more preferably 12 h. The LB liquid medium used in this invention is a conventional liquid medium in the art, which can be purchased commercially or prepared using conventional methods. As one possible implementation, the components of the LB liquid medium are: 10 g / L tryptone, 15 g / L NaCl, and 5 g / L yeast extract.

[0041] In this invention, the first fermentation broth is spread onto an MSM solid medium with diesel as the sole carbon source for cultivation. The preparation method of the MSM solid medium with diesel as the sole carbon source can be conventionally selected according to actual needs. As one alternative implementation, diesel is spread onto the MSM solid medium to obtain the MSM solid medium with diesel as the sole carbon source. As another alternative implementation, diesel is directly mixed with the components of the MSM solid medium, and after solidification, the MSM solid medium with diesel as the sole carbon source is obtained. The volume ratio of diesel to MSM solid medium is preferably 1:100 to 300, more preferably 1:150 to 250, and more preferably 1:200. The volume ratio of the first fermentation broth to the MSM solid medium with diesel as the sole carbon source is preferably 1:100-300, more preferably 1:150-250, and even more preferably 1:200. This invention does not specifically limit the source or type of diesel, but preferably uses #0 diesel. The MSM solid medium is a conventional solid medium in the art, which can be purchased commercially or prepared using conventional methods. As one possible implementation, the MSM solid medium consists of 0.5 g / L K₂HPO₄, 0.3 g / L KH₂PO₄, 4.0 g / L NaNO₃, 0.515 g / L MgSO₄·7H₂O, 0.01 g / L CaCl₂, 0.01 g / L FFeSO₄, and 20 g / L agar powder. The culture temperature is preferably 25-37°C, more preferably 27-32°C, and even more preferably 30°C. The culture time is preferably 1-3 days, and even more preferably 2 days.

[0042] In this invention, a single colony is picked from a solid culture medium and fermented in LB liquid culture medium to obtain a second fermentation culture broth; the volume of the LB liquid culture medium is preferably 10-1000 mL, more preferably 15-800 mL, and even more preferably 20 mL; the fermentation culture is preferably a shaking culture; the shaking speed of the fermentation culture is preferably 100-250 rpm, more preferably 120-200 rpm, and even more preferably 180 rpm; the fermentation culture temperature is preferably 25-37℃, more preferably 27-32℃, and even more preferably 30℃; the fermentation culture time is preferably 8-16 h, more preferably 10-14 h, and even more preferably 12 h.

[0043] In this invention, a second fermentation broth is added to an MSM liquid medium containing diesel oil for culturing. The MSM liquid medium containing diesel oil can be prepared using conventional methods according to actual needs. As one possible implementation, diesel oil is added to the MSM liquid medium to obtain an MSM liquid medium containing diesel oil. The volume ratio of diesel oil to MSM liquid medium is preferably 1:50 to 200, more preferably 1:75 to 150, and even more preferably 1:100. The total petroleum hydrocarbon concentration in the MSM liquid medium containing diesel oil is preferably 500 to 16000 mg / L, more preferably 8400 mg / L, 500 to 5000 mg / L, 5000 to 11800 mg / L, or 11800 to 16000 mg / L. The volume ratio of the second fermentation broth to the MSM liquid medium containing diesel is preferably 1:10 to 1000, more preferably 1:30 to 200, and even more preferably 1:50. This invention does not specifically limit the source or type of diesel, but preferably uses #0 diesel. The MSM liquid medium of this invention is a conventional liquid medium in the art, which can be purchased commercially or prepared using conventional methods. As one possible implementation, the MSM liquid medium comprises 0.5 g / L K₂HPO₄, 0.3 g / L KH₂PO₄, 4.0 g / L NaNO₃, 0.515 g / L MgSO₄·7H₂O, 0.01 g / L CaCl₂, and 0.01 g / L FeSO₄. The culture is preferably an oscillation culture; the oscillation speed is preferably 100-300 rpm, more preferably 150-250 rpm, and even more preferably 200 rpm; the culture temperature is preferably 25-37℃, more preferably 27-32℃, and even more preferably 30℃; the culture time is preferably 2-5 days, more preferably 3-4 days, and even more preferably 3 days.

[0044] In this invention, strains with diesel degradation performance are screened by detecting the diesel degradation rate of the strains. This invention does not have a specific limitation on the method for detecting the diesel degradation rate, and conventional detection methods in the field can be used. As an optional implementation method, the diesel degradation rate can be measured at 254 nm using an ultraviolet spectrophotometer. By comparing the diesel degradation rates of different strains, strains with different diesel degradation performance are screened.

[0045] Due to the complex composition of diesel fuel, which contains a large amount of hydrophobic organic matter, microorganisms are limited in their degradation and utilization, resulting in poor biodegradability. Microbial treatment technology, with its low production cost, lack of secondary pollution, and ability to perform in-situ remediation, is considered a promising high-tech approach and the most ideal method. Our team previously screened strains with excellent diesel fuel degradation effects using traditional methods, but this was time-consuming, cumbersome, and had a low success rate. Based on this, our invention improves the screening method. Traditional methods require 25-35 days to screen strains, and the diesel fuel degradation rate of the five selected strains is below 50%. In contrast, the screening method of this invention requires only 5-8 days, and the diesel fuel degradation rate of the five selected strains exceeds 80%, demonstrating excellent degradation effects on diesel fuel. Compared to traditional screening methods, the rapid screening method of this invention is faster, simpler, has a higher success rate, and produces strains with better diesel fuel degradation effects.

[0046] The present invention also provides a Citrobacter freundii GO7 obtained by the above screening method, which was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M 20241715.

[0047] The *Citrobacter GO7* strain of this invention was identified as belonging to the genus *Citrobacter* by 16S rRNA testing, and is most closely related to *Citrobacter freundii*, and is classified as *Citrobacter freundii*. This strain is resistant to C... 11 ~C 15 The degradation rate of alkane reached 93.55%, C 16 ~C 19 The alkane degradation rate reached 92.45%, C 20 The degradation rate of alkanes reached 93.90%; the degradation rates of tricyclic, tetracyclic, pentacyclic, and hexacyclic aromatic hydrocarbons reached 64.30%, 65.18%, 68.52%, and 75.76%, respectively. The *Citrobacter GO7* strain obtained in this invention exhibits excellent degradation effects on alkanes and polycyclic aromatic hydrocarbons in oily wastewater.

[0048] The present invention also provides a fermentation broth obtained by fermentation of the aforementioned Citrobacter GO7.

[0049] In this invention, the preferred method for preparing the fermentation broth obtained by fermenting Citrobacter GO7 includes the following steps: inoculating Citrobacter GO7 into LB liquid medium and culturing to obtain the fermentation broth. The LB liquid medium of this invention is a conventional liquid medium in the art, which can be purchased commercially or prepared using conventional methods. As one possible implementation, the components of the LB liquid medium are: 10 g / L tryptone, 15 g / L NaCl, and 5 g / L yeast extract. The preferred culturing conditions are shaking culture; the preferred shaking speed is 100–250 rpm, more preferably 120–200 rpm, and more preferably 180 rpm; the preferred culturing temperature is 25–37°C, more preferably 27–32°C, and more preferably 30°C; the preferred culturing time is 8–16 h, more preferably 10–14 h, and more preferably 12 h.

[0050] The present invention also provides an application of the Citrobacter GO7 or the fermentation broth in the degradation of oily wastewater.

[0051] In this invention, the source and type of oily wastewater are not specifically limited; the sources of oily wastewater include, but are not limited to, wastewater from the refining process of petrochemical plants or wastewater from fracturing flowback fluid in oil fields; the types of oily wastewater include, but are not limited to, oily wastewater containing crude oil, gasoline, kerosene, diesel, lubricating oil, liquid paraffin, or asphalt, and other petroleum hydrocarbon pollutants; the petroleum hydrocarbon pollutants include n-alkanes, branched alkanes, cycloalkanes, or aromatics. The *Citrobacter GO7* or *Citrobacter GO7* fermentation broth described in this invention, when applied to simulated or actual oily wastewater, exhibits excellent degradation effects on both alkanes and polycyclic aromatic hydrocarbons, demonstrating strong application prospects in the microbial remediation of oily wastewater.

[0052] The present invention also provides a formulation for degrading oily wastewater, the formulation comprising the above-mentioned Citrobacter GO7 or the above-mentioned fermentation broth.

[0053] In this invention, there are no special limitations on the source and type of the oily wastewater; the sources of the oily wastewater include, but are not limited to, wastewater from the refining process of petrochemical plants or wastewater from fracturing flowback fluid in oil fields; the types of oily wastewater include, but are not limited to, oily wastewater containing crude oil, gasoline, kerosene, diesel, lubricating oil, liquid paraffin, or asphalt, and containing various petroleum hydrocarbon pollutants; the petroleum hydrocarbon pollutants include n-alkanes, branched alkanes, cycloalkanes, or aromatics. This invention does not have special limitations on the dosage form of the formulation for degrading oily wastewater, and can conventionally select according to actual needs, including but not limited to solid formulations, liquid formulations, and spray formulations. The formulation for degrading oily wastewater in this invention may also include formulation-acceptable excipients. This invention does not have special limitations on the types of excipients, including but not limited to one or more of carriers, diluents, excipients, preservatives, surfactants, and antioxidants.

[0054] The present invention also provides a method for degrading oily wastewater, preferably comprising the following steps: treating the oily wastewater with the Citrobacter GO7 or the fermentation broth or the preparation.

[0055] In this invention, there are no special limitations on the source and type of the oily wastewater; the source of the oily wastewater includes, but is not limited to, wastewater from the refining process of petrochemical plants or wastewater from fracturing flowback fluid in oil fields; the type of oily wastewater includes, but is not limited to, oily wastewater containing crude oil, gasoline, kerosene, diesel, lubricating oil, liquid paraffin, or asphalt, and other oily wastewater containing various petroleum hydrocarbon pollutants; the petroleum hydrocarbon pollutants include n-alkanes, branched alkanes, cycloalkanes, or aromatics. As an optional embodiment, the method preferably includes the following steps: adding the Citrobacter GO7 or the fermentation broth or the preparation to the oily wastewater for cultivation.

[0056] In this invention, the Citrobacter GO7 can be used directly, or it can be fermented to obtain a fermentation broth, and then the fermentation broth can be added to oily wastewater for culture; or the Citrobacter GO7 or the fermentation broth can be made into a preparation and then added to oily wastewater for culture.

[0057] In this invention, the *Citrobacter GO7*, the fermentation broth, or the preparation is added to oily wastewater for cultivation; the concentration of total petroleum hydrocarbons in the oily wastewater is preferably 500–16000 mg / L, more preferably 8400 mg / L, 500–5000 mg / L, 5000–11800 mg / L, or 11800–16000 mg / L; the amount of *Citrobacter GO7*, the fermentation broth, or the preparation can be conventionally selected according to actual needs; as an optional implementation, the viable bacterial concentration of *Citrobacter GO7* in the oily wastewater is preferably 1 × 10⁻⁶. 8 ~1×10 9 cfu / mL, more preferably 1×10⁻⁶8 ~1.12×10 8 cfu / mL or 1.12×10 8 ~1.6×10 8 cfu / mL or 1.6×10 8 ~1×10 9 cfu / mL; the culture is preferably a shaking culture; the shaking speed of the culture is preferably 100-300 rpm, more preferably 150-250 rpm, and more preferably 200 rpm; the culture temperature is preferably 25-37℃, more preferably 27-32℃, and more preferably 30℃; the culture time is preferably 2-7 days, more preferably 3-6 days, and more preferably 5 days.

[0058] The method of this invention is applied to the degradation of oily wastewater. It has excellent degradation effects on alkanes and polycyclic aromatic hydrocarbons in oily wastewater. The degradation cost is low, it does not produce secondary pollution, and it can be used for in-situ remediation. It has a strong application prospect in the remediation of oily wastewater.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Unless otherwise specified, the following embodiments are all conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] The culture medium components used in the following examples are:

[0063] The components of LB plate solid culture medium are: 10 g / L tryptone, 15 g / L NaCl, 5 g / L yeast extract, and 20 g / L agar powder.

[0064] The components of LB liquid culture medium are: 10 g / L tryptone, 15 g / L NaCl, and 5 g / L yeast extract.

[0065] The composition of MSM solid culture medium is: 0.5 g / L K2HPO4, 0.3 g / L KH2PO4, 4.0 g / L NaNO3, 0.515 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.01 g / L FeSO4, and 20 g / L agar powder.

[0066] The composition of MSM liquid culture medium is: 0.5 g / L K2HPO4, 0.3 g / L KH2PO4, 4.0 g / L NaNO3, 0.515 g / L MgSO4·7H2O, 0.01 g / L CaCl2, and 0.01 g / L FeSO4.

[0067] Example 1

[0068] A rapid screening method for diesel-degrading bacteria:

[0069] Oily wastewater samples were collected from the concentration tank in the high-salt oily wastewater treatment process of Luoyang Petrochemical Plant, at longitude: 120.7075E, latitude: 30.876716N.

[0070] S1, take 0.5 mL of oily wastewater sample into 45 mL of physiological saline, shake thoroughly in an air bath constant temperature shaker for 5 min, the conditions of the air bath constant temperature shaker are 180 rpm and 30 ℃, to prepare a suspension of bacterial solution;

[0071] S2, 0.2 mL of bacterial culture was transferred to 20 mL of LB liquid medium and fermented for 12 h at 30 °C and 180 rpm.

[0072] S3, 0.1 mL of diesel oil was spread onto 20 mL of MSM solid medium to obtain MSM solid medium with diesel oil as the sole carbon source. The fermentation broth obtained in step S2 was spread onto MSM solid medium with diesel oil as the sole carbon source in 0.1 mL and cultured at 30°C for 2 days in a constant temperature incubator.

[0073] S4. Single colonies from MSM solid medium with diesel as the sole carbon source were picked and fermented in 20 mL LB liquid medium for 12 h at 30 °C and 180 rpm.

[0074] S5, take 1 mL of the fermentation broth obtained in step S4 into 50 mL of MSM liquid medium, add 0.5 mL of diesel oil, the total petroleum hydrocarbon concentration in the MSM liquid medium is 8400 mg / L, and put it in an air bath constant temperature shaker for 3 days. The conditions of the air bath constant temperature shaker are 200 rpm and 30℃.

[0075] S6, after the culture is completed, the diesel degradation rate is measured at 254 nm using a UV spectrophotometer.

[0076] Example 2

[0077] A rapid screening method for diesel-degrading bacteria:

[0078] S1. Using the same oily wastewater sample as in Example 1, 0.3 mL of the oily wastewater sample was added to 40 mL of physiological saline and thoroughly shaken in an air bath constant temperature shaker for 3 min. The conditions of the air bath constant temperature shaker were 250 rpm and 25 °C to prepare a suspended bacterial solution.

[0079] S2, 0.5 mL of bacterial culture was added to 20 mL of LB liquid medium and fermented for 8 h at 25 °C and 250 rpm.

[0080] S3, 0.2 mL of diesel oil was spread onto 20 mL of MSM solid medium to obtain MSM solid medium with diesel oil as the sole carbon source. The fermentation broth obtained in step S2 was spread onto the MSM solid medium with diesel oil as the sole carbon source in 0.2 mL and cultured at 25°C for 3 days in a constant temperature incubator.

[0081] S4. Single colonies from MSM solid medium with diesel as the sole carbon source were picked and fermented in 10 mL LB liquid medium for 8 h at 25 °C and 250 rpm.

[0082] S5, take 5 mL of fermentation culture broth into 50 mL of MSM liquid culture medium, add 1 mL of diesel oil, and place it in an air bath constant temperature shaker for 2 days. The conditions of the air bath constant temperature shaker are 300 rpm and 25℃.

[0083] S6, after the culture is completed, the diesel degradation rate is measured at 254 nm using a UV spectrophotometer.

[0084] Example 3

[0085] A rapid screening method for diesel-degrading bacteria:

[0086] S1. Using the same oily wastewater sample as in Example 1, 0.8 mL of the oily wastewater sample was added to 50 mL of physiological saline and thoroughly shaken in an air bath constant temperature shaker for 8 min. The conditions of the air bath constant temperature shaker were 100 rpm and 37 °C to prepare a suspended bacterial solution.

[0087] S2, 0.1 mL of bacterial culture was added to 20 mL of LB liquid medium and fermented for 16 h at 37 °C and 100 rpm.

[0088] S3, 0.7 mL of diesel oil was spread onto 20 mL of MSM solid medium to obtain MSM solid medium with diesel oil as the sole carbon source. The fermentation broth obtained in step S2 was spread onto the MSM solid medium with diesel oil as the sole carbon source in 0.7 mL and cultured at 37°C for 1 day in a constant temperature incubator.

[0089] S4. Single colonies on MSM solid medium with diesel as the sole carbon source were picked and fermented in 100 mL LB liquid medium for 16 h at 37 °C and 100 rpm.

[0090] S5, take 0.5 mL of fermentation culture broth into 50 mL of MSM liquid culture medium, add 0.25 mL of diesel oil, and place it in an air bath constant temperature shaker for 5 days. The conditions of the air bath constant temperature shaker are 150 rpm and 37℃.

[0091] S6, after the culture is completed, the diesel degradation rate is measured at 254 nm using a UV spectrophotometer.

[0092] Comparative Example 1

[0093] A screening method for diesel-degrading bacteria:

[0094] S1. Using the same oily wastewater sample as in Example 1, a suspension of bacteria was prepared using the same method as in Example 1. 0.1 mL of the suspension of bacteria was spread on a 20 mL blood agar plate and incubated in a 30°C incubator for 1 day.

[0095] S2, pick colonies with hemolysis zones from blood agar plates and ferment them in 20 mL LB liquid medium for 1 day at 30°C and 180 rpm.

[0096] S3: Inoculate 1 mL of the fermentation broth obtained in step S2 into 50 mL of MSM liquid medium, add 0.5 mL of diesel oil, and the total petroleum hydrocarbon concentration in the MSM liquid medium is 8400 mg / L. Incubate for 7 days in an air bath constant temperature shaker at 30°C and 200 rpm. Transfer 0.2 mL of the 7-day culture solution to 20 mL of LB liquid medium using a pipette, and ferment for 1 day in an air bath constant temperature shaker at 30°C and 180 rpm. Transfer 1 mL of the turbid culture solution after fermentation to 50 mL of MSM liquid medium, add 0.5 mL of diesel oil, and the total petroleum hydrocarbon concentration in the MSM liquid medium is 8400 mg / L. Incubate for 7 days in an air bath constant temperature shaker at 30°C and 200 rpm.

[0097] S4. Spread 0.1 mL of diesel oil onto 20 mL of MSM solid medium to obtain MSM solid medium with diesel oil as the sole carbon source. Spread 0.1 mL of the bacterial solution obtained in step S3 onto MSM solid medium with diesel oil as the sole carbon source and incubate in a constant temperature incubator at 30°C for 7 days.

[0098] S5, pick a single colony from MSM solid medium with diesel as the sole carbon source and streak it onto 20 mL LB solid medium. Incubate at 30°C for 2 days.

[0099] S6. Pick a single colony from LB solid medium and transfer it to 20 mL of LB liquid medium for fermentation for 1 day. The fermentation conditions are 30℃ and 180 rpm.

[0100] S7. Take 1 mL of the fermentation broth obtained in step S6 and put it into 50 mL of MSM liquid medium for culture. Then add 0.5 mL of diesel oil. The total petroleum hydrocarbon concentration in the MSM liquid medium is 8400 mg / L. Culture for 7 days in an air bath constant temperature shaker at 30°C and 200 rpm.

[0101] S8, after the culture is completed, the diesel degradation rate is determined by ultraviolet spectrophotometry.

[0102] Experimental Example 1

[0103] Strains from the same source of wastewater (collected from the thickening tank of the high-salt, oily wastewater treatment process at Luoyang Petrochemical Plant, longitude: 120.7075E, latitude: 30.876716N) were screened using the methods of Example 1 and Comparative Example 1, respectively, and the diesel degradation rate of the screened strains was determined. The experimental flow chart using the method of Example 1 is shown below. Figure 1 As shown, diesel-degrading strains were rapidly screened through a series of steps including preparation of bacterial suspension, fermentation culture, oil plate culture, and shake-flask degradation experiments. The experimental flowchart using the method in Comparative Example 1 is shown below. Figure 2 As shown, diesel-degrading strains were screened through a series of processes including preparation of bacterial suspension, blood agar plates, fermentation culture, first shake-flask degradation experiment, fermentation culture, second shake-flask degradation experiment, oil agar plates, LB solid medium, fermentation culture, and a third shake-flask degradation experiment. The diesel degradation rates of the strains obtained by the two methods are shown in Table 1. Figure 3-4 As shown.

[0104] Table 1. Diesel degradation rates of strains obtained by screening methods in Example 1 and Comparative Example 1.

[0105]

[0106] From Table 1 and Figure 3 As can be seen, the five strains screened using the screening method of Example 1 exhibited high degradation rates for diesel fuel, all exceeding 80%. Strain GO7 showed the highest degradation rate at 92.73%, while strain GO1 showed the lowest at 80.51%. This demonstrates that the strains screened using the method of this invention have excellent degradation effects on diesel fuel.

[0107] From Table 1 and Figure 4 As can be seen, among the five strains screened using the screening method of Comparative Example 1 that exhibit diesel degradation activity, strain YS-5 showed the highest diesel degradation rate at 40.74%, while strain YS-3 showed the lowest degradation rate at 7.6%. In contrast, the five strains screened using the traditional screening method in Comparative Example 1 all showed diesel degradation rates below 50%, indicating poor performance in diesel degradation.

[0108] The above results show that the screening method of the present invention requires only 5-8 days to screen strains, and the diesel degradation rate of the five screened strains all reached over 80%, demonstrating excellent degradation effects on diesel. In contrast, the traditional screening method requires 25-35 days, and the diesel degradation rate of the five screened strains is all below 50%. Compared with the traditional screening method, the rapid screening method of the present invention is faster, simpler, and has a higher success rate.

[0109] Example 4: Identification of the strain

[0110] The strain GO7, screened using the method in Example 1, was isolated and purified on LB solid medium. The colony morphology is as follows: Figure 5 As shown. Total DNA was extracted from GO7 strain using a bacterial genomic DNA extraction kit. Using this total DNA as a template, the full-length 16S rDNA was amplified by PCR using universal bacterial primers 27F and 1492R. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced gene sequences were submitted to the NCBI database for sequence alignment. Ten strains with high gene sequence similarity were selected, and a phylogenetic tree was constructed using MEGA 7.0 software with the neighbor-joining method. The results are shown below. Figure 6 As shown.

[0111] Depend on Figure 5 As can be seen, strain GO7 forms regular, round, milky-white colonies on LB solid medium. These colonies are opaque, slightly raised in the center, and have a smooth, moist surface.

[0112] Depend on Figure 6 As can be seen, strain GO7 was identified as belonging to the genus *Citrobacter* by 16S rRNA, and is most closely related to *Citrobacter freundii*, and is classified as *Citrobacter freundii*. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 31, 2024, with accession number CCTCC NO: M 20241715.

[0113] Example 5: Simulated Degradation of Oily Wastewater

[0114] 0.5 mL of diesel fuel was added to 50 mL of MSM liquid medium to prepare simulated high-concentration oily wastewater, with a total petroleum hydrocarbon concentration of 8400 mg / L. Citrobacter GO7 was fermented in LB liquid medium for 12 h at 180 rpm and 30 °C, resulting in a viable cell concentration of 7.16 × 10⁻⁶ cells / mL. 9CFU / mL. 1 mL of fermentation broth was added to 50 mL of simulated high-concentration oily wastewater and cultured with shaking for 5 days at 200 rpm and 30°C. The degradation characteristics of alkanes and polycyclic aromatic hydrocarbons were determined by GC-MS. Each sample was performed in triplicate. A blank group and a control group were set up. The blank group consisted of simulated high-concentration oily wastewater without the addition of Citrobacter GO7 fermentation broth, while the control group consisted of simulated high-concentration oily wastewater with 1 mL of sterilized Citrobacter GO7 fermentation broth added. All other culture conditions and procedures were the same as described above. Results are as follows: Figure 7-8 As shown.

[0115] Depend on Figure 7 It can be seen that strain GO7 is resistant to C 11 -C 15 The degradation rate of alkane reached 93.55%, C 16 -C 19 The alkane degradation rate reached 92.45%, C 20 The degradation rate of the above alkanes reached 93.90%. The results show that the alkane degradation rate of Citrobacter GO7 is above 90%, and it has excellent degradation effect on alkanes with C11 and above.

[0116] Depend on Figure 8 It can be seen that the degradation rates of tricyclic, tetracyclic, pentacyclic, and hexacyclic aromatic hydrocarbons by strain GO7 were 64.30%, 65.18%, 68.52%, and 75.76%, respectively. The results indicate that the addition of Citrobacter GO7 significantly promoted the degradation of polycyclic aromatic hydrocarbons in oily wastewater.

[0117] Example 6: Degradation of actual oily wastewater

[0118] The oily wastewater was collected from the Luoyang Petrochemical Wastewater Plant. The total concentration of petroleum hydrocarbons in the oily wastewater was measured to be 509.6 mg / L.

[0119] Citrobacter GO7 was fermented in LB liquid medium for 10 h at 180 rpm and 30 °C, with a viable cell concentration of 5.6 × 10⁻⁶. 9 CFU / mL. 1 mL of fermentation broth was added to 50 mL of oily wastewater and incubated for 5 days in an air bath shaker at 200 rpm and 30°C. The oily substances were significantly consumed. The degradation of total petroleum hydrocarbons in the wastewater was determined by GC-MS. A blank control group was set up, consisting of oily wastewater without the addition of Citrobacter GO7 fermentation broth; all other culture conditions and procedures were the same as described above. Results are as follows: Figure 9 As shown.

[0120] Depend on Figure 9It can be seen that, compared with the control group, most of the substances (alkanes and polycyclic aromatic hydrocarbons) in the oily wastewater were removed, and Citrobacter GO7 has an excellent degradation effect on total petroleum hydrocarbons in oily wastewater.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Citrobacter freundii ( Citrobacter freundii GO7, characterized in that, It was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M 20241715.

2. The fermentation broth obtained by fermentation of Citrobacter freundii GO7 as described in claim 1.

3. The method for preparing the fermentation broth according to claim 2, characterized in that, The process includes the following steps: inoculating the *Citrobacter freundii* GO7 into LB liquid medium and culturing it to obtain a fermentation broth.

4. The application of the Citrobacter freundii GO7 of claim 1 or the fermentation broth of claim 2 in the degradation of oily wastewater.

5. A formulation for degrading oily wastewater, characterized in that, The formulation comprises Citrobacter freundii GO7 as described in claim 1 or the fermentation broth as described in claim 2.

6. A method for degrading oily wastewater, characterized in that, The process includes the following steps: treating oily wastewater with the Citrobacter freundii GO7 as described in claim 1, the fermentation broth as described in claim 2, or the preparation as described in claim 5.

7. The method according to claim 6, characterized in that, The steps include: adding the Citrobacter freundii GO7 or the fermentation broth or the preparation to oily wastewater for cultivation.

8. The method according to claim 7, characterized in that, The total petroleum hydrocarbon concentration in the oily wastewater is 500~16000 mg / L.

9. The method according to claim 7, characterized in that, The cultivation conditions are: incubation at 25~37℃ for 2~7 days.

Images