Oil-reducing bacteria and their compound inoculants, preparation methods and applications

Abstract

This invention provides an oil-degrading bacterium, its compound bacterial agent, preparation method, and application. The oil-degrading bacterium is *Sphingopyxis terrae* subsp. *ummariensis* DDB-4, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2021243. The application of DDB-4, its bacterial suspension, its culture broth, or its fermentation products in the degradation of diesel fuel or in the degradation of pollution caused by diesel fuel is described. The preparation method of the compound bacterial agent includes mixing DDB-4, *Gordonella oleracea*, and *Alcaligenes faecalis* subsp. *phenol* to obtain the compound bacterial agent. The application method of the compound bacterial agent in the remediation of soil or water contaminated with diesel fuel and / or drilling fluid additives is also described. The strain DDB-4 of this invention can effectively and even efficiently degrade diesel fuel, and can also effectively and even efficiently degrade or remediate pollution caused by diesel fuel.

Description

Technical Field

[0001] This invention belongs to the field of environmental remediation microbial technology. Specifically, it relates to a strain of Sphingopyxis terrae subsp. ummariensis DDB-4 (oil-degrading bacterium) and its screening and culture method and application; a biological agent containing Sphingopyxis terrae subsp. ummariensis DDB-4 and its application; a compound bacterial agent and its preparation and application method; and a biological agent containing the compound bacterial agent and its application. Background Technology

[0002] Oil hydrocarbons are classified as priority environmental pollutants due to their significant potential environmental hazards. Diesel fuel, a common engine fuel and industrial feedstock, is a complex mixture of alkanes and aromatic compounds. Leaks are unavoidable during storage in tanks and pipeline transportation, leading to soil pollution, damaging soil structure, affecting soil permeability and ecosystems, and even contaminating groundwater, ultimately impacting human health. Furthermore, drilling fluid leaks or ground drains may occur during drilling, polluting site soil, rivers, and water sources. With the development of clean drilling practices, much of the land at well sites needs to be restored to farmland after drilling, necessitating the remediation of soil pollution caused by diesel fuel and drilling fluids.

[0003] Currently, contaminated soil remediation involves more than 20 professional fields, including environmental science, environmental engineering, microbiology, soil science, ecology, geography, botany, molecular biology, geology, plant nutrition, and agronomy. Various remediation technologies, such as physical, chemical, and biological methods, have been developed. Among bioremediation technologies, microbial remediation utilizes the metabolism of specific microorganisms to absorb, transform, remove, or degrade pollutants, achieving environmental purification and ecological restoration. This technology has been applied to the treatment of various organically contaminated soils, demonstrating significant application value. Domestic and international experts and scholars consider it the most valuable and vital treatment technology in the field of ecological environmental protection.

[0004] Remediation technologies for diesel and drilling fluid contaminated soils mainly include physical remediation, chemical remediation, and bioremediation, with bioremediation encompassing animal remediation, phytoremediation, and microbial remediation. Physical and chemical remediation methods typically suffer from drawbacks such as high processing costs, small digestion capacity, and secondary pollution. Bioremediation, based on microbial degradation, can oxidize and decompose pollutants, ultimately transforming them into harmless inorganic substances, making it an effective method for thoroughly degrading diesel pollutants in the environment. Typically, acclimatized microbial strains are added to the contaminated soil to accelerate the degradation process. Therefore, screening for stable and efficient microbial strains is essential for improving the efficiency of bioremediation. Currently, over 70 genera and more than 200 species of microorganisms have been reported to participate in the degradation of petroleum hydrocarbons. Bioremediation offers advantages such as economy, low consumption, large digestion capacity, ease of operation, and no secondary pollution, making it a key research area for soil system purification and remediation.

[0005] In diesel pollution control, research on bioremediation microbial resources is limited, and their degradation efficiency is uncertain. Therefore, screening for highly efficient strains for diesel pollution degradation is necessary. Furthermore, while some strains capable of treating diesel pollution remediation or drilling solid waste treatment have been reported, no reports have been found regarding the strain of this invention. Moreover, there is a lack of microbial agents capable of simultaneously remediating combined pollutants from diesel and drilling fluid at drilling sites.

[0006] Chinese patent application CN104962504A, published on October 7, 2015, discloses a strain of bacteria that degrades n-alkane components in diesel fuel and its application in the remediation of petroleum hydrocarbon pollution. The disclosed strain is *Burkholderia* sp., registered under CGMCC No. 10944, which can degrade 1843 mg / L of n-alkane in diesel fuel to 1060 mg / L within 240 hours. However, in degradation experiments using the disclosed strain, the removal efficiency of petroleum hydrocarbons was only 11% higher than the control group without the strain within 10 days, indicating a slightly insufficient degradation efficiency. Furthermore, the patent application does not cover research on the remediation of combined pollutants from diesel fuel and drilling fluid at drilling sites. Summary of the Invention

[0007] The purpose of this invention is to address at least one of the aforementioned deficiencies in the prior art. For example, one objective of this invention is to provide a novel bacterial strain capable of degrading diesel fuel, its cultivation method, and its application.

[0008] To achieve the above objectives, one aspect of the present invention provides a strain of Sphingopyxis terrae subsp. ummariensis DDB-4, which has been deposited at the China Center for Type Culture Collection (CCTCC) on March 17, 2021, with accession number CCTCC NO: M 2021243.

[0009] In an exemplary embodiment of the present invention, the *Sphingopyxis terrae* subsp. *ummariensis* DDB-4 is a Gram-negative bacterium, non-spore-forming, rod-shaped, motile, and obligately aerobic.

[0010] In an exemplary embodiment of the present invention, the 16S rDNA sequence of Sphingopyxis terrae subsp. ummariensis DDB-4 is shown in SEQ ID NO.1.

[0011] In an exemplary embodiment of the present invention, the *Sphingopyxis terrae* subsp. *ummariensis* DDB-4 has stress resistance and can grow in a culture medium with a salt concentration of 0.5-4%.

[0012] Another aspect of the present invention provides a screening and culturing method for Sphingopyxis terrae subsp. ummariensis DDB-4 as described above. The screening and culturing method includes: adding a sample to a sterilized first culture medium, and then enriching it at a constant temperature to obtain a culture suspension. The sample includes one or more of diesel fuel, diesel-contaminated soil, and diesel-contaminated water; spreading the culture suspension onto a second culture medium, and then culturing it at a constant temperature to obtain purified Sphingopyxis terrae subsp. ummariensis DDB-4.

[0013] Another aspect of the present invention provides an application method for using Sphingopyxis terrae subsp. ummariensis DDB-4 or its bacterial suspension or culture broth or fermentation product as described above in the degradation of diesel fuel or in the pollution caused by the degradation of diesel fuel.

[0014] Another aspect of the present invention provides a biological agent containing diesel-degrading bacteria, said biological agent comprising Sphingopyxis terrae subsp. ummariensis DDB-4 as described above, or a suspension thereof, or a culture medium thereof, or a fermentation product thereof.

[0015] Another aspect of the present invention provides a method for applying the biological agent as described above, wherein the biological agent is used to degrade diesel fuel, or to degrade the pollution caused by diesel fuel.

[0016] Another objective of this invention is to provide a composite microbial preparation capable of degrading diesel fuel and / or drilling fluid additives, or remediating soil or water contaminated by diesel fuel and / or drilling fluid additives, as well as its preparation and application methods.

[0017] To achieve the above objectives, one aspect of the present invention provides a method for preparing a compound microbial agent, the method comprising: mixing Sphingopyxis terrae subsp. ummariensis DDB-4, Sphingopyxis friendlya, and Alcaligenes faecalis phenolic subsp. to obtain a compound microbial agent.

[0018] In an exemplary embodiment of the present invention, the total viable count of the compound microbial agent is 0.2~1×10⁻⁶. 10 cfu / g, where the total viable count is the sum of the viable counts of Sphingopyxis terrae subsp. ummariensis DDB-4, Sphingopyxis friendlya, and Sphingopyxis phenolic subsp. fecalith.

[0019] In an exemplary embodiment of the present invention, the viable count of Sphingopyxis terraesubsp. ummariensis DDB-4, the viable count of Gordon's friendly bacteria, or the viable count of Alcaligenes faecalis subsp. phenolicum in the compound microbial agent is 10-90% of the total viable count.

[0020] In an exemplary embodiment of the present invention, the viable count of Sphingopyxis terraesubsp. ummariensis DDB-4 in the compound microbial agent is 10-30% of the total viable count, the viable count of Aureobasidium friendlyum is 20-40% of the total viable count, and the viable count of Alcaligenes faecalis subsp. phenol is the remainder.

[0021] In an exemplary embodiment of the present invention, the preparation method further includes, prior to the mixing step, culturing *Sphingopyxis terrae subsp. ummariensis* DDB-4, *Gordonella friendlyi*, and *Alcaligenes faecalis* subsp. *phenol* using a compound microbial culture medium, wherein the compound microbial culture medium comprises 8-12 g / L peptone, 1-3 g / L meat extract, 3-7 g / L sodium chloride, and 5-10 g / L sucrose.

[0022] Another aspect of the present invention provides a compound microbial agent, which is prepared by the method described above.

[0023] Another aspect of the present invention provides an application method in which the compound microbial agent described above is used to remediate soil or water contaminated by diesel and / or drilling fluid additives.

[0024] In an exemplary embodiment of the present invention, the application method includes: inoculating the compound microbial agent as described above at a mass concentration of 0.3 to 3% into soil or water contaminated with diesel fuel and / or drilling fluid additives.

[0025] Another aspect of the present invention provides a biological agent containing a compound microbial agent, the biological agent comprising the compound microbial agent as described above, or its microbial suspension, or its culture medium, or its fermentation product.

[0026] Another aspect of the present invention provides a method for applying the biological agent containing the compound microbial agent as described above, wherein the method applies the biological agent to diesel fuel and / or drilling fluid additives, or to pollutants caused by diesel fuel and / or drilling fluid additives.

[0027] Compared with the prior art, the beneficial effects of the present invention may include at least one of the following:

[0028] (1) The *Sphingopyxis terrae* subsp. *ummariensis* DDB-4 provided by this invention can effectively and even efficiently degrade diesel fuel; it can also effectively and even efficiently degrade or remediate pollution caused by diesel fuel. For example, soil or water contaminated by diesel fuel or diesel-containing substances;

[0029] (2) Under pure culture conditions, after 14 days of inoculation, Sphingopyxis terrae subsp. ummariensis DDB-4 achieved a degradation rate of over 62.49% for petroleum hydrocarbons in diesel fuel;

[0030] (3) Sphingopyxis terrae subsp. ummariensis DDB-4 has strong stress resistance and can be used as an excellent degrading bacterium for the biological treatment of diesel and diesel-contaminated environments, with good application prospects;

[0031] (4) The compound microbial agent prepared by mixing Sphingopyxis terrae subsp. ummariensis DDB-4 with Sphingopyxis terrae and Sphingopyxis ummariensis in a predetermined ratio can efficiently and rapidly reduce the amount of diesel and / or drilling fluid additives in the environment, and degrade and remove petroleum hydrocarbons and COD (i.e. chemical oxygen demand).

[0032] (5) Under pure culture conditions, when the compound microbial agent is inoculated into soil contaminated with diesel and / or drilling fluid additives at a mass concentration of 1% for 60 days, the degradation rate of petroleum hydrocarbons can reach more than 95%, and the degradation rate of COD can reach more than 93%. Attached Figure Description

[0033] The present invention, *Sphingopyxis terrae* subsp. *ummariensis* DDB-4, has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on March 17, 2021, with accession number CCTCC NO: M 2021243.

[0034] The friendly *Gordonia sihwensis* DDB-8 of this invention has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on March 17, 2021, with accession number CCTCC NO: M 2021244.

[0035] The phenolicus subsp. phenolicus of the present invention has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on August 19, 2019, with accession number CCTCC NO: M 2019646.

[0036] The above and other objects and features of the present invention will become clearer from the following description taken in conjunction with the accompanying drawings, in which:

[0037] Figure 1A The microscopic morphology of Sphingopyxis terrae subsp. ummariensis DDB-4, an exemplary embodiment of the present invention, is shown. Figure 1B The colony morphology of Sphingopyxis terrae subsp. ummariensis DDB-4 on beef extract peptone medium is shown.

[0038] Figure 2 A phylogenetic diagram showing the 16S rDNA sequence of Sphingopyxis terrae subsp. ummariensis DDB-4, an exemplary embodiment of the present invention, is shown.

[0039] Figure 3 A schematic diagram of the process flow for a screening and cultivation method of Sphingopyxis terrae subsp. ummariensis DDB-4 according to an exemplary embodiment of the present invention is shown. Detailed Implementation

[0040] In the following description, the present invention, including Sphingopyxisterrae subsp. ummariensis DDB-4 (Diesel Degradation Bacteria-4, also known as oil-degrading bacteria), its screening and cultivation methods and applications, as well as compound bacterial agents and their preparation methods and applications, will be described in detail with reference to the accompanying drawings and exemplary embodiments.

[0041] Figure 1A The microscopic morphology of Sphingopyxis terrae subsp. ummariensis DDB-4 of the present invention is shown. Figure 1B The colony morphology of Sphingopyxis terrae subsp. ummariensis DDB-4 on beef extract peptone medium is shown. Figure 2 A phylogenetic diagram of the 16S rDNA sequence of Sphingopyxis terrae subsp. ummariensis DDB-4 of the present invention is shown.

[0042] One aspect of the present invention provides a strain of Sphingopyxis terrae subsp. ummariensis DDB-4 (hereinafter referred to as strain DDB-4), which was deposited at the China Center for Type Culture Collection on March 17, 2021, with accession number CCTCC NO: M 2021243.

[0043] In this embodiment, strain DDB-4 is a Gram-negative bacterium, non-spore-forming, rod-shaped, motile, and obligately aerobic.

[0044] In this embodiment, total DNA extracted from strain DDB-4 was used as a template to amplify the 16S rDNA fragment, and the obtained 16S sequence is shown in SEQ ID NO.1. The obtained sequence results were compared with the National Center for Biotechnology Information (NCBI) database. Strain DDB-4 showed the highest similarity (99.8%) to Sphingopyxis terrae subsp. ummariensis in the database; however, the two are different strains.

[0045] In this embodiment, the colonies formed by strain DDB-4 after culturing on beef extract peptone solid medium for 18 hours were round, yellow, less than 0.5 mm in diameter, with neat edges and raised, moist surfaces.

[0046] In this embodiment, strain DDB-4 exhibits stress resistance and can grow in a medium with a salt concentration of 0.5-4%.

[0047] Another aspect of this embodiment also provides the application of strain DDB-4 or its bacterial suspension or its culture medium or its fermentation products in the degradation of diesel fuel or in the pollution caused by the degradation of diesel fuel, which can efficiently degrade or remediate soil or water pollution caused by diesel fuel.

[0048] Figure 3 A schematic diagram of the process flow for a screening and culturing method of strain DDB-4 according to an exemplary embodiment of the present invention is shown.

[0049] Another aspect of the present invention provides a screening culture method for strain DDB-4. In a second exemplary embodiment of the present invention, as shown... Figure 3 As shown, the screening and culture method includes:

[0050] S11. Enrich the sample to obtain a bacterial suspension. Specifically, add a predetermined amount (e.g., 3-10 ml) of sample (e.g., diesel fuel) to a sterilized first culture medium and enrich it at 15-45°C to obtain a bacterial suspension. The sample may include one or more of diesel fuel, diesel-contaminated soil, and diesel-contaminated water. Here, the amount of sample and first culture medium used should be appropriate. The incubation temperature can be adjusted according to the temperature range of the target strain's application environment. For example, 5 ml of sample and 95 ml of MS medium can be used. If the target strain is used in a high-temperature environment, the incubation temperature can be 45°C; if the target strain is used in a low-temperature environment, the incubation temperature can be 15°C; if the target strain is used in a room-temperature environment, the incubation temperature can be 28°C. It should be noted that the first culture medium is a carbon-free inorganic salt medium that can be used to screen for target carbon source degrading microorganisms. For example, the first culture medium is MS medium.

[0051] Enrichment culture can be repeated several times in a separate sterilized first culture medium. That is, the enrichment culture method described above can be repeated once or multiple times. The number of times the enrichment culture is repeated can be flexibly determined according to the growth status of the target strain during enrichment. For example, the culture can be repeated 3 times to obtain a culture suspension with a higher bacterial count.

[0052] S12. The target strain is obtained by isothermal incubation of the bacterial suspension. Specifically, the bacterial suspension is formed on a second culture medium (e.g., beef extract peptone medium) and incubated at a constant temperature of 15-45°C to isolate the bacteria in the bacterial suspension.

[0053] After single colonies have grown on the second culture medium under constant temperature, strains with different colony morphologies are picked and then streaked for purification and preservation. Specifically, after culturing on the second culture medium for a certain period of time (e.g., 16-18 hours), strains with different colony morphologies are picked and streaked, and microscopic observation can be performed until the purified target strain is obtained. The purified target strain is then preserved (e.g., by inoculating the target strain into an agar slant). It should be noted that the second culture medium can be a general bacterial basal medium, which can be a liquid or solid medium, or a medium commonly used in this field, which will not be elaborated further here.

[0054] Another aspect of the present invention provides a biological agent. In a third exemplary embodiment of the present invention, the biological agent may comprise the strain DDB-4 of the present invention, a suspension thereof, a culture medium thereof, or a fermentation product thereof.

[0055] Another aspect of this embodiment provides the application of biological agents in the degradation of diesel fuel. Biological agents also have a strong degradation ability and can efficiently degrade or repair soil or water pollution caused by diesel fuel.

[0056] To better understand the above exemplary embodiments of the present invention, the strain DDB-4, its screening culture method, and its application will be further described below with reference to specific examples.

[0057] Example 1

[0058] (1) Isolation, purification and preservation of strain DDB-4

[0059] Take 5 mL of diesel sample and add it to 95 mL of sterilized MS medium. Using diesel as the sole carbon source, incubate at 28℃ and 150 rpm for 7 days with shaking. Take 5 mL of the enrichment solution and inoculate it into another identical MS medium. Repeat the enrichment culture process three times to obtain a bacterial suspension. Use beef extract peptone solid medium and employ the dilution plate method to isolate bacteria from the obtained bacterial suspension. Incubate at 28℃. After single colonies grow on the beef extract peptone medium plates, pick all strains with different colony morphologies from the plates for streak purification and preservation.

[0060] As shown in Figure 1, the isolated and purified strain DDB-4 was cultured on beef extract peptone medium. After 18 hours of culture, the colonies were round, yellow, less than 0.5 mm in diameter, with neat edges and a moist, raised appearance. Figure 1A Colony morphology was shown. Figure 1B This is a schematic diagram showing the colonies after streaking culture on a beef extract peptone plate.

[0061] (2) Amplification and phylogenetic analysis of 16S rDNA of strain DDB-4

[0062] Total DNA was extracted from strain DDB-4. Using the total DNA as a template, the 16S fragment was amplified using primers 27F and 1492R. The PCR reaction was performed using a Bio-RAD MyCycler PCR amplification instrument.

[0063] Reaction system (50 μl): 25 μl of 2×PCRMix solution, 1 μl each of primers 27F and 1492R (10 μM), 1 μl of DNA template, and ultrapure water to make up to 50 μl; the nucleotide sequences of primers 27F and 1492R are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0064] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 54℃ annealing for 1 min, 72℃ extension for 2 min, 30 cycles; 72℃ final extension for 8 min.

[0065] After PCR amplification products were detected by 1.0% agarose gel electrophoresis, they were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Gene sequence similarity was calculated using DNAman 6.0 software. The obtained 16S sequence is shown in SEQ ID No. 1.

[0066] The obtained sequence results were compared with those in the National Center for Biotechnology Information (NCBI) database. The 16S rDNA gene sequence of strain DDB-4 showed the highest similarity (99.8%) to *Sphingopyxis terrae* subsp. *ummariensis* in the database. Based on the NCBI comparison results, the type strain with the highest similarity was selected as the reference strain. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 6.0 software, with a bootstrap value of 1000. The resulting phylogenetic diagram is shown below. Figure 2 As shown in the image.

[0067] The strain DDB-4 was deposited at the China Center for Type Culture Collection on March 17, 2021, with accession number CCTCC NO: M 2021243.

[0068] (3) Experiment on the ability of strain DDB-4 to degrade diesel fuel

[0069] Strain DDB-4 was inoculated onto beef extract peptone liquid medium for activation culture. When the OD600 of the activated bacterial suspension reached 1.0, 1.5 mL of the bacterial suspension was taken, centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The bacterial cells were washed twice with sterile physiological saline, resuspended in 1.5 mL of sterile water, and inoculated into sterilized diesel degradation medium. The mixture was then incubated at 30℃ and 150 rpm with shaking. Three parallel experiments were repeated, and a blank control was included. Samples were collected after 14 days of incubation. The petroleum hydrocarbon content of the diesel degradation medium before and after the experiment was determined by gas chromatography (HJ 1021-2019). It should be noted that adding filtered and sterilized diesel to sterilized MS medium can prepare a diesel degradation medium with a diesel content of 1.5%.

[0070] The results showed that after 14 days of culture, the petroleum hydrocarbon degradation rate of the diesel degradation medium treated with strain DDB-4 reached 62.49%, while the petroleum hydrocarbon degradation rate of the diesel degradation medium in the blank control experiment was 0.67%.

[0071] (4) Resistance test of strain DDB-4

[0072] Acid and alkali resistance determination: Beef extract peptone medium was prepared and its pH value was determined. The pH values ​​of the beef extract peptone medium were adjusted to 4, 5, 6, 7, 8, 9, 10, and 11 using 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution, respectively. After sterilization at 121℃ for 30 min, a bacterial suspension of strain DDB-4 was added at 1% (v:v). Three replicates were set for each pH value of the beef extract peptone medium. The medium was incubated in a 28℃ constant temperature incubator with shaking at 150 r / min for 24 h. The number of bacteria was determined using a UV spectrophotometer at a wavelength of λ=600 nm.

[0073] Salt tolerance test: Beef extract peptone medium was prepared, and the pH was adjusted to 7 with 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution. NaCl solution was added to the prepared beef extract peptone medium to prepare beef extract peptone medium with salt concentrations of 0.5%, 1%, 2%, 4%, 8% and 10%, respectively. After sterilization at 121℃ for 30 min, bacterial suspension of strain DDB-4 was added at 1% (v:v). Each salt concentration of beef extract peptone medium was prepared in triplicate and incubated in a constant temperature incubator at 28℃ with shaking at 150 r / min for 24 h. The number of bacteria was determined by ultraviolet spectrophotometer at a wavelength of λ=600.

[0074] Determination of growth temperature range: Prepare beef extract peptone medium, adjust the pH to 7, sterilize at 121℃ for 30 min, and add bacterial suspension of strain DDB-4 at 1% (v:v). Place the inoculated medium in constant temperature incubators at 4℃, 15℃, 30℃, 45℃, 55℃, 60℃ and 65℃ respectively, and shake at 150 r / min for 24 h. Each temperature is set in 3 replicates. The number of bacteria is determined by ultraviolet spectrophotometer at wavelength λ=600.

[0075] The results showed that strain DDB-4 had good stress resistance, strong tolerance to acid or alkali, and could grow in the pH range of 5 to 10; it had strong salt tolerance, and could grow in a medium with a maximum salt concentration of 4%; it had a wide growth temperature range, and could grow in the temperature range of 15 to 45℃.

[0076] (5) Experiment on the treatment of diesel-containing soil by strain DDB-4

[0077] Inoculate the treated soil (contaminated with diesel fuel) with 1% (m / m) of the bacterial agent of strain DDB-4 (OD600≈1.6), and mix thoroughly. A control group without inoculation was used. The treated soil weighed 3.0 kg. The soil was placed outdoors for 60 days (from August to October 2020), maintaining a humidity of approximately 25% during this period.

[0078] The petroleum hydrocarbon content in the treated material was measured initially and after 60 days of treatment. The results are shown in Table 1 below.

[0079]

[0080] The results showed that after 60 days of treatment, strain DDB-4 achieved a degradation rate of 96.52% for petroleum hydrocarbons in diesel-contaminated soil. The treated soil met the Class I soil standard (TPH < 826 mg / kg) of the Soil Environmental Quality Standard for Construction Land Soil Pollution Risk Control GB36600-2018, demonstrating good degradation potential.

[0081] Another aspect of the present invention provides a method for preparing a compound microbial agent. In a fourth exemplary embodiment of the present invention, the method for preparing the compound microbial agent includes: mixing strain DDB-4, *Gordonia sihwensis*, and *Alcaligenes sfaecalis* subsp. *phenolicus* as described in the first exemplary embodiment above to obtain the compound microbial agent. *Gordonia sihwensis* is classified as *Gordonia sihwensis* DDB-8 and was deposited at the China Center for Type Culture Collection (CCTCC) on March 17, 2021, with accession number CCTCC NO: M 2021244; *Alcaligenes sfaecalis* subsp. *phenolicus* GFB-14 was classified as *Alcaligenes sfaecalis* subsp. *phenolicus* GFB-14 and was deposited at the CCTC on August 19, 2019, with accession number CCTCC NO: M 2019646.

[0082] In this embodiment, the total viable count of the compound microbial agent is 0.2~1×10⁻⁶. 10 CFU / g, the total viable count is the sum of the viable counts of strain DDB-4, *Gordonella friendlyi*, and *Alcaligenes faecalis* subsp. *phenol*, with a typical but non-restrictive total viable count of 0.2 × 10⁻⁶. 10 cfu / g, 0.6×10 10 cfu / g or 1.0×10 10 cfu / g.

[0083] In this embodiment, in the compound microbial agent, the viable count of strain DDB-4 can be 10-90% of the total viable count, the viable count of *Goldenella friendly* can be 10-90% of the total viable count, or the viable count of *Alcaligenes faecalis* subsp. *fecalobiota* can be 10-90% of the total viable count. That is, the compound microbial agent can have a viable count of strain DDB-4 accounting for 10-90% of the total viable count, with *Goldenella friendly* and *Alcaligenes faecalis* subsp. *fecalobiota* accounting for the remainder; or the compound microbial agent can have a viable count of *Goldenella friendly* accounting for 10-90% of the total viable count, with a viable count of strain DDB-4 and *Alcaligenes faecalis* subsp. *fecalobiota* accounting for 10-90% of the total viable count, with a viable count of strain DDB-4 and *Goldenella friendly* subsp. *fecalobiota* accounting for the remainder.

[0084] In this embodiment, in the compound microbial agent, the number of viable bacteria of strain DDB-4 can be 10-30% of the total number of viable bacteria, the number of viable bacteria of *Gordonella oleracea* can be 20-40% of the total number of viable bacteria, and the number of viable bacteria of *Alcaligenes faecalis* subsp. *phenol* is the remainder.

[0085] In this embodiment, the preparation method of the compound microbial agent may further include culturing strains DDB-4, Gordon's friendly bacterium, and Alcaligenes faecalis subsp. flor using a compound microbial agent culture medium before mixing them. Here, the prepared compound microbial agent culture medium may include 8-12 g / L of peptone, 1-3 g / L of meat extract, 3-7 g / L of sodium chloride, and 5-10 g / L of sucrose, with water as the solvent (e.g., distilled water or tap water).

[0086] Another aspect of the present invention provides a compound microbial agent. In a fifth exemplary embodiment of the present invention, the compound microbial agent provided by the present invention is prepared by the method for preparing a compound microbial agent as described in the fourth exemplary embodiment above.

[0087] In another aspect of this embodiment, a composite microbial agent prepared using the above-described method is also provided.

[0088] In a sixth exemplary embodiment of the present invention, the present invention provides a method for applying a compound microbial agent in the remediation of soil or water contaminated by diesel fuel and / or drilling fluid additives. The compound microbial agent can efficiently and rapidly reduce the amount of diesel fuel and / or drilling fluid additives in the environment, degrading and removing petroleum hydrocarbons and COD. In the present invention, the drilling fluid additives are complex polymers, and the content of specific components cannot be determined. However, most of them are water-soluble, so the COD value of the leachate can be used to characterize the content of drilling fluid additives in the contaminated environment. For example, drilling fluid additives may include polymers such as potassium polyacrylate, sulfonated phenolic resin, sulfonated lignite, and sulfonated tannin.

[0089] In this embodiment, the application method of the compound microbial agent includes inoculating the above-mentioned compound microbial agent at a mass concentration of 0.3% to 3% into soil or water contaminated by a mixture of diesel oil and / or drilling fluid additives. Here, typical but non-limiting inoculation amounts are 0.3%, 1.0%, 2.0%, or 3.0%.

[0090] To better understand the above exemplary embodiments of the present invention, the compound microbial agent and its preparation and application methods are further described below with reference to specific examples.

[0091] Example 2

[0092] A compound microbial culture medium containing 10 g / L peptone, 1.5 g / L beef extract, 5 g / L sodium chloride and 8 g / L sucrose was prepared in advance. Strains DDB-4, Friendly Gordon's and Alcaligenes faecalis subsp. phenol were cultured to form bacterial suspensions, and compound microbial agents A1 to A5 were prepared according to the proportions in Table 2 below.

[0093]

[0094] The soil mixture contaminated with diesel fuel and drilling fluid was treated using compound microbial agents A1-A5 listed in Table 2, with the specific steps as follows:

[0095] 3000g of soil mixture contaminated with diesel and drilling fluid (petroleum hydrocarbon content 15.5g / kg, COD content 560mg / L) was weighed and inoculated with compound microbial agents A1~A5 at a mass concentration of 1% (W:W). The mixture was placed in a plastic basin and mixed evenly. After treatment at 20℃~28℃ for 60 days, the petroleum hydrocarbon and COD content in the soil mixture contaminated with diesel and drilling fluid was measured, and the degradation rate of petroleum hydrocarbon and COD by the microbial agents was calculated. The degradation rate can be calculated by the following formula: V=(C1-C2) / C1; where V represents the degradation rate; C1 represents the content before treatment; and C2 represents the content after treatment. The results are shown in Table 3 below.

[0096]

[0097] The results showed that when compound microbial agents A1-A5 were inoculated into soil contaminated with diesel fuel and / or drilling fluid additives at a mass concentration of 1% for 60 days, the degradation rate of petroleum hydrocarbons was over 95% and the degradation rate of COD was over 93%.

[0098] The compound microbial agent provided by this invention has excellent degradation ability for diesel and / or drilling fluid additives, and can be widely used to degrade diesel and / or drilling fluid additives and to remediate contamination by diesel and / or drilling fluid, such as the remediation of soil contaminated by diesel and / or drilling fluid, and the remediation of water bodies contaminated by diesel and / or drilling fluid.

[0099] In summary, the beneficial effects of the present invention may include at least one of the following:

[0100] (1) The strain DDB-4 provided by the present invention can effectively or even efficiently degrade diesel fuel, or degrade or repair the pollution caused by diesel fuel;

[0101] (2) The strain DDB-4 provided by the present invention has good stress resistance, strong tolerance to acid or alkali and salt, and a wide growth temperature range.

[0102] (3) The three strains selected in the compound microbial agent prepared by the present invention are reasonable. The compound microbial agent can be obtained after the three strains are mixed evenly. The operation is simple and has wide applicability.

[0103] (4) After inoculating the compound microbial agent into the soil or water contaminated by diesel and / or drilling fluid in a certain amount, and then mixing it evenly, the content of petroleum hydrocarbons and COD can be effectively reduced.

[0104] Although the present invention has been described above in conjunction with exemplary embodiments and accompanying drawings, those skilled in the art should understand that various modifications can be made to the above embodiments without departing from the spirit and scope of the claims. sequence list <110> China National Petroleum Corporation (CNPC) Sichuan Petroleum Drilling Engineering Co., Ltd. <120> Oil-reducing bacteria and their compound inoculants, preparation methods and applications <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1389 <212> DNA <213> Sphingopyxis terrae subsp. ummariensis DBB‑4 <400> 1 ggcatgccta acacatgcaa gtcgaacgag atcttcggat ctagtggcgc acgggtgcgt 60 aacgcgtggg aatctgccct tgggtacgga ataactcaga gaaatttgtg ctaataccgt 120 ataatgtctt cggaccaaag atttatcgcc caaggatgag cccgcgtagg attagctagt 180 tggtgaggta aaagctcacc aaggcgacga tccttagctg gtctgagagg atgatcagcc 240 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattggac 300 aatgggcgaa agcctgatcc agcaatgccg cgtgagtgat gaaggcccta gggttgtaaa 360 gctcttttac ccgggatgat aatgacagta ccgggagaat aagctccggc taacttcgtg 420 ccagcagccg cggtaatacg aggggagcta gcgttgttcg gaattactgg gcgtaaagcg 480 cgcgtaggcg gttttttaag tcagaggtga aagcccgggg ctcaaccccg gaatagcctt 540 tgaaactgga aaactagaat cttggagagg tcagtggaat tccgagtgta gaggtgaaat 600 tcgtagatat tcggaagaac accagtggcg aaggcgactg actggacaag tattgacgct 660 gaggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 720 gatgataact agctgtccgg gctcatagag cttgggtggc gcagctaacg cattaagtta 780 tccgcctggg gagtacggtc gcaagattaa aactcaaagg aattgacggg ggcctgcaca 840 agcggtggag catgtggttt aattcgaagc aacgcgcaga accttaccag cgtttgacat 900 cctgatcgcg gttaccagag atggtttcct tcagttcggc tggatcagtg acaggtgctg 960 catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1020 ctcatcccta gttgccatca ttaagttggg cactctaagg aaactgccgg tgataagccg 1080 gaggaaggtg gggatgacgt caagtcctca tggcccttac gcgctgggct acacacgtgc 1140 tacaatggcg gtgacagtgg gcagcaacct cgcgagaggt agctaatctc caaaagccgt 1200 ctcagttcgg attgttctct gcaactcgag agcatgaagg cggaatcgct agtaatcgcg 1260 gatcagcatg ccgcggtgaa tacgttccca ggccttgtac acaccgcccg tcacaccatg 1320 ggagttggtt tcacccgaag gcagtgctct aacccgcaag ggaggaagct gaccacggtg 1380 atcgacagt 1389 <210> 2 <211> 19 <212> DNA <213> Artificial sequence (27F) <400> 2 agagttgatc ctggctcag 19 <210> 3 <211> 20 <212> DNA <213> Artificial sequence (1492R) <400> 3 cggttacctt gttacgactt 20

Claims

1. A *Sphingopyxis terrae* subsp. *ummariensis* DDB-4, characterized by, The DDB-4 has been deposited at the China Center for Type Culture Collection (CCTCC) on March 17, 2021, with accession number CCTCC NO: M2021243.

2. The use of Sphingopyxis terrae subsp. ummariensis DDB-4 or a suspension thereof as described in claim 1 in the degradation of diesel fuel or in the pollution caused by the degradation of diesel fuel.

3. A biological agent containing diesel-degrading bacteria, characterized in that, The biological agent comprises Sphingopyxis terrae subsp. ummariensis DDB-4 or a suspension thereof as described in claim 1.

4. The use of a bio-agent as described in claim 3 in the degradation of diesel fuel or in the pollution caused by the degradation of diesel fuel.

5. A method for preparing a compound microbial agent, characterized in that, The preparation method includes: A compound bacterial agent was prepared by mixing Sphingopyxis terrae subsp. ummariensis DDB-4 as described in claim 1, Sphingopyxis terrae subsp. ummariensis, and Alcaligenes faecalis phenolic subsp.

6. The method for preparing the compound microbial agent according to claim 5, characterized in that, The total viable count of the compound microbial agent is 0.2~1×10⁻⁶. 10 cfu / g, where the total viable count is the sum of the viable counts of Sphingopyxis terrae subsp. ummariensis DDB-4, Sphingopyxis friendlya, and Sphingopyxis phenolic subsp. fecalith.

7. The method for preparing the compound microbial agent according to claim 6, characterized in that, In the compound microbial agent, the viable count of Sphingopyxis terrae subsp. ummariensis DDB-4 is 10-90% of the total viable count, the viable count of Aureobasidium friendlyum is 10-90% of the total viable count, or the viable count of Alcaligenes faecalis subsp. phenol is 10-90% of the total viable count.

8. The method for preparing the compound microbial agent according to claim 6, characterized in that, In the compound microbial agent, the viable count of Sphingopyxis terrae subsp. ummariensis DDB-4 is 10-30% of the total viable count, the viable count of Aureobasidium friendlyum is 20-40% of the total viable count, and the viable count of Alcaligenes faecalis subsp. phenol is the remainder.

9. The method for preparing the compound microbial agent according to claim 5, characterized in that, The preparation method further includes, prior to the mixing step, culturing Sphingopyxis terrae subsp. ummariensis DDB-4, Sphingopyxis friendly Gordonella, and Alcaligenes faecalis phenolic subsp. using a compound microbial agent culture medium, wherein the compound microbial agent culture medium comprises 8-12 g / L peptone, 1-3 g / L meat extract, 3-7 g / L sodium chloride, and 5-10 g / L sucrose.

10. A compound microbial agent, characterized in that, The compound microbial agent is prepared by the preparation method described in any one of claims 6 to 9.

11. A method for applying the compound microbial agent as described in claim 10 to the remediation of soil or water contaminated with diesel oil and / or drilling fluid.

12. The application method according to claim 11, characterized in that, The application method includes: inoculating the compound microbial agent of claim 10 at a mass concentration of 0.3-3% into soil or water contaminated with diesel and / or drilling fluid.

13. A biological agent containing a compound microbial agent, characterized in that, The biological agent comprises the compound microbial agent or its suspension as described in claim 10.

14. The use of a biopharmaceutical as described in claim 13 in the degradation of diesel oil and / or drilling fluid, or in the pollution caused by the degradation of diesel oil and / or drilling fluid.

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