An oil-based rock debris degrading bacteria and a compound bacterial agent, their cultivation method and application

Abstract

This invention provides an oil-based rock debris degrading bacterium, a compound bacterial agent, its cultivation method, and its application. The oil-based rock debris degrading bacterium includes at least one of chelating bacteria, *Chelativorans*, and xenotrophic bacteria. The chelating bacteria is classified as *Chelativorans* sp. SH6-2, with accession number CCTCC NO: M 2021290; the *Chelativorans* is classified as *Bordetella petrii* WOCB-20, with accession number CCTCC NO: M 20211089; and the xenotrophic bacteria is classified as *Xenophilus aerolatus* WOCB-11, with accession number CCTCC NO: M 20211088. The oil-based rock debris degrading bacterium of this invention can effectively degrade paraffin oil and oil-based rock debris; it can also effectively remediate pollution caused by oil-based rock debris.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to an oil-based rock fragment degrading bacterium, a screening and culture method for an oil-based rock fragment degrading bacterium, an application of an oil-based rock fragment degrading bacterium, a biological agent containing an oil-based rock fragment degrading bacterium, an application of a biological agent containing an oil-based rock fragment degrading bacterium, a compound bacterial agent, a method for preparing a compound bacterial agent, a method for applying a compound bacterial agent, a biological agent containing a compound bacterial agent, and an application of a biological agent containing a compound bacterial agent. Background Technology

[0002] In shale gas extraction, horizontal wells must use oil-based drilling fluids with stable rheological, water loss, and lubrication properties, resulting in a mixture of oil-based drilling fluid and fractured formation rock cuttings—oil-based rock cuttings. With the large-scale development of shale gas in recent years, the production of oil-based rock cuttings has increased dramatically. The highly stable system of oil-based rock cuttings, high oil content, and complex pollutant composition have increasingly attracted national attention, leading to its inclusion in the "National Hazardous Waste List." If oil-based rock cuttings mix with soil, it will alter the soil's composition and structure, causing acute or chronic poisoning of soil microorganisms and flora and fauna, and even impacting groundwater resources. Furthermore, the continuous accumulation of toxic and harmful substances will ultimately endanger human health. Therefore, research on the harmless treatment of oil-based rock cuttings is of great significance.

[0003] Currently, oil-based drilling cuttings treatment in China mainly relies on physical and chemical methods, including thermal treatment, solvent extraction, thermal distillation, in-pit sealing and burial, and stripping. However, physical and chemical treatment methods are costly and pose a risk of secondary pollution. With increasingly stringent environmental standards and stricter enforcement, there is an urgent need for low-energy, clean, safe, and low-cost oil-based drilling cuttings treatment technologies. Microbial treatment of oil-based drilling cuttings transforms complex organic compounds such as petroleum hydrocarbons into simpler organic compounds, and has become an important means of safe on-site treatment and disposal of oil-based drill cuttings, showing significant advantages compared to physical and chemical treatment technologies.

[0004] Currently, microbial treatment of oil-based rock cuttings mainly relies on the screening of highly efficient degrading strains. Existing strain screening mainly focuses on mesophilic bacteria at 25-35℃, while screening of thermophilic degrading bacteria helps to expand the library of oil-based rock cuttings degrading strains in different temperature ranges to meet the needs of different treatment environments.

[0005] For example, a patent document published on February 1, 2019, entitled "A Method for Microbial Degradation of Total Petroleum Hydrocarbons" (publication number CN 109293001 A), describes a method for microbial degradation of total petroleum hydrocarbons. This method includes the following steps: first, the bottom sediment of waste oil-based drilling fluid is dried using a blower, then the pH is adjusted to 6-8, and the water content is no more than 10%; 0.2-0.5g of SQ-1 engineered bacteria, 0.1-0.4g of SQ-2 engineered bacteria, and 1-5mL of SQ-3 engineered bacteria are added to each kilogram of solid sediment, and water is added to achieve a humidity of 20%; finally, 0.1-0.4g of NHNO and 0.1-0.3g of KHPO are added, and the mixture is then treated in a warm environment of 24℃-31℃ for 50-60 days, during which water is added to maintain the humidity of the solid sediment at 30%-40%. A patent document published on February 4, 2020, entitled "An Oil-Based Drill Cuttings Degrading Strains and Its Application," with publication number CN110747141 A, describes an oil-based drill cuttings degrading strain and its application. This strain is Bacillus sp. WJ2019, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on July 19, 2019, with accession number CCTCC NO: M2019574. This strain can grow under normal conditions using oil-based drill cuttings as the sole carbon source, without the need for artificial energy or carbon source addition. Therefore, this bacterium can be used to degrade petroleum components in oil-based drill cuttings. Within 45 days, it can achieve a degradation rate of over 48.8% for oil-based drill cuttings with a backflow oil content of 1200 mg / kg, demonstrating significant advantages such as high oil removal efficiency and short treatment cycle. Although these strains all have a certain degradation effect on oil-based drill cuttings, they can only adapt to mesophilic environments and cannot survive in high-temperature drilling fluids.

[0006] Therefore, this can further enrich the resources of high-temperature degradation strains for oil-based rock cuttings and improve the efficiency of microbial treatment of oil-based rock cuttings. 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 high-temperature degrading strain of oil-based rock cuttings capable of adapting to high-temperature processing environments, its cultivation method, and its application.

[0008] To achieve the above objectives, the present invention provides an oil-based rock debris degrading bacterium, comprising at least one of chelating bacteria, *Chelativorans*, and xenotrophic bacteria. The chelating bacteria is classified as *Chelativorans* sp. SH6-2, with accession number CCTCC NO: M 2021290; the *Chelativorans* is classified as *Bordetella petrii* WOCB-20, with accession number CCTCC NO: M 20211089; and the xenotrophic bacteria is classified as *Xenophilus aerolatus* WOCB-11, with accession number CCTCC NO: M 20211088.

[0009] In an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention, the chelating bacteria may be Gram-negative bacteria, non-spore-forming, rod-shaped, motile, and obligately aerobic.

[0010] The aforementioned Peterbauer bacteria can be Gram-negative, motile, rod-shaped, and non-spore-producing;

[0011] The heterotrophic bacteria may be Gram-negative, motile, rod-shaped, and non-spore-producing.

[0012] In an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention, the colonies formed by the chelating bacteria after culturing on tryptone soybean agar medium for 24 hours can be round, and after 48 hours the colonies are round, colorless, with a diameter of 0.5-1 mm, neat edges, flat and moist.

[0013] The colonies formed by *Petrobacter petriasis* after culturing on beef extract peptone medium for 24 hours can be round, and after 48 hours the colonies are round, white, 0.5-1 mm in diameter, with neat edges, flat and moist.

[0014] The heterotrophic bacteria formed round colonies after culturing on beef extract peptone medium for 24 hours, and after 48 hours, the colonies were round, pale yellow, 1-2 mm in diameter, with neat edges, flat and moist.

[0015] In an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention, the 16S rDNA sequence of the chelate-eating bacteria may be as shown in SEQ ID NO.1;

[0016] The 16S rDNA sequence of the *Petrobacter p. rihais* is shown in SEQ ID NO. 2;

[0017] The 16S rDNA sequence of the heterotrophic bacteria is shown in SEQ ID NO.3.

[0018] In an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention, the growth temperature range of the chelate-eating bacteria can be 30-65°C, the growth temperature range of the Peterribottella bacteria can be 4-45°C, and the growth temperature range of the heterotrophic bacteria can be 4-45°C.

[0019] Another aspect of the present invention provides a method for screening and culturing oil-based rock debris degrading bacteria, the screening and culturing method comprising the following steps:

[0020] S1. Add a microbial source sample to an oil-based rock fragment sample and incubate it at a constant temperature of 25-60℃ and / or 50-60℃ for 15-30 days to obtain the first-generation enriched microbial source. The microbial source sample includes natural soil, silt and oil-contaminated soil.

[0021] S2. Add the first-generation enrichment bacterial source to another oil-based rock fragment sample and incubate at 25-60℃ and / or 50-60℃ for 7-14 days to obtain the first culture medium.

[0022] S3. The first culture medium is placed at a constant temperature of 25-60℃ and / or 50-60℃ for 7-14 days to obtain the second culture medium;

[0023] S4. Repeat step S2 at least twice to obtain enriched bacterial samples after multiple transculturing.

[0024] S5. Spread the diluted enriched bacterial sample onto tryptone soybean agar medium and incubate at 50–60°C. After repeated streak culturing, the purified chelate strain is obtained; and / or,

[0025] The diluted enriched bacterial samples were spread on beef extract peptone medium and incubated at 25–30°C. After repeated streak culture, purified Petribourt strain and heterotrophic strain were obtained.

[0026] In another aspect, the present invention provides the application of the oil-based rock cuttings degrading bacteria, or their bacterial suspension, or their culture medium, or their fermentation products as described above, in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud.

[0027] In another aspect, the present invention provides a biological agent containing oil-based rock fragment degrading bacteria, wherein the biological agent containing oil-based rock fragment degrading bacteria includes the oil-based rock fragment degrading bacteria as described above, or their bacterial suspension, or their culture medium, or their fermentation products.

[0028] In another aspect, the present invention provides the application of the above-mentioned bio-agent containing oil-based rock cuttings degrading bacteria in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud.

[0029] In another aspect, the present invention provides a method for preparing a compound microbial agent, the method comprising: mixing chelating bacteria with Peterbortella and / or heterotrophic bacteria to obtain a compound microbial agent;

[0030] The viable count of the chelating bacteria is 10% to 90% of the total viable count;

[0031] The viable count of the *Petribaudolinium* was 0% to 90% of the total viable count;

[0032] The viable count of the heterotrophic bacteria is 0% to 90% of the total viable count.

[0033] In an exemplary embodiment of the preparation method of the compound microbial agent of the present invention, the viable count of the chelating bacteria can be 40% to 60% of the total viable count;

[0034] The viable count of the *Petribauterella* can be 20% to 30% of the total viable count;

[0035] The viable count of the heterotrophic bacteria can be the remainder.

[0036] In another aspect, the present invention provides a composite microbial agent, which is prepared by the preparation method described above, and the composite microbial agent can degrade oil-based rock fragments at a temperature of 10-65°C.

[0037] In another aspect, the present invention provides a method for applying the compound microbial agent as described above in the degradation of oil-based rock debris and / or oil-based rock debris contaminated soil or water.

[0038] In an exemplary embodiment of the application method of the compound microbial agent of the present invention, the application method includes: inoculating the above-mentioned compound microbial agent at a mass concentration of 0.3% to 3% into soil or water contaminated with oil-based rock debris and / or oil-based rock debris.

[0039] In another aspect, the present invention provides a biological agent containing a compound microbial agent, wherein the biological agent containing the compound microbial agent includes the compound microbial agent as described above, or its bacterial suspension, or its culture medium, or its fermentation product.

[0040] In another aspect, the present invention provides the application of the biological agent containing the compound microbial agent as described above in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud.

[0041] Compared with the prior art, the beneficial effects of the present invention include at least one of the following:

[0042] (1) The chelating bacteria Chelativorans sp. SH6-2, Bordetella petrii WOCB-20 and Xenophilus aerolatus WOCB-11 provided by the present invention can effectively or even efficiently degrade paraffin oil and oil-based rock cuttings (mud); they can also effectively or even efficiently remediate pollution caused by oil-based rock cuttings (mud), such as soil or water contaminated by oil-based rock cuttings (mud);

[0043] (2) Under pure culture conditions, after 14 days of inoculation, the degradation rates of petroleum hydrocarbons in paraffin oil by the chelating bacteria Chelativorans sp. SH6-2, Bordetella petrii WOCB-20 and Xenophilus aerolatus WOCB-11 reached 46.55%, 40.77% and 45.33% respectively.

[0044] (3) Chelaterans sp.SH6-2 has strong temperature resistance, while Bordetella petrii WOCB-20 and Xenophilus aerolatus WOCB-11 have a wide growth temperature range and can be used as excellent degrading bacteria for the biological treatment of oil-based rock cuttings (mud) in a wide temperature range (10-65℃), with good application prospects.

[0045] (4) A compound microbial agent prepared by mixing Chelativorans sp. SH6-2, Bordetella petrii WOCB-20 and Xenophilus aerolatus WOCB-11 in a predetermined ratio can efficiently and rapidly degrade and remove petroleum hydrocarbons in the environment.

[0046] (5) Under pure culture conditions, when the compound bacterial agent is inoculated into oil-based rock cuttings at a mass concentration of 0.3-3% for 60 days, the degradation rate of petroleum hydrocarbons can reach more than 80%. Attached Figure Description

[0047] The chelating bacteria (Chelativorans sp. SH6-2) 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 29, 2021, with accession number CCTCC NO: M 2021290.

[0048] The Bordetella petrii WOCB-20 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 August 26, 2021, with accession number CCTCC NO: M 20211089.

[0049] The heterotrophic bacterium (Xenophilus aerolatus WOCB-11) 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 August 26, 2021, with accession number CCTCC NO: M 20211088.

[0050] The above and other objects and / or features of the present invention will become clearer from the following description taken in conjunction with the accompanying drawings, in which:

[0051] Figure 1A The microscopic image shows the cell morphology of Chelativorans sp. SH6-2, an oil-based rock debris degrading bacterium of the present invention.

[0052] Figure 1B The colony morphology of Chelativorans sp. SH6-2 on tryptone soybean agar medium is shown.

[0053] Figure 1C The image shows the morphology of Bordetella petrii WOCB-20 under a microscope.

[0054] Figure 1D The image shows the colony morphology of Bordetella petrii WOCB-20 on beef extract peptone agar.

[0055] Figure 1E The image shows the morphology of the heterotrophic bacterium Xenophilus aerolatus WOCB-11 under a microscope;

[0056] Figure 1F The colony morphology of Xenophilus aerolatus WOCB-11 on beef extract peptone agar medium is shown.

[0057] Figure 2A phylogenetic diagram showing the 16S rDNA sequence of Chelativorans sp. SH6-2, an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention, is presented.

[0058] Figure 3 A phylogenetic diagram showing the 16S rDNA sequence of *Bordetella petrii* WOCB-20, an exemplary embodiment of the oil-based rock-degrading bacteria of the present invention.

[0059] Figure 4 A phylogenetic diagram showing the 16S rDNA sequence of the heterotrophic bacterium Xenophilus aerolatus WOCB-11, an exemplary embodiment of the oil-based rock debris degrading bacteria of the present invention. Detailed Implementation

[0060] In the following, the oil-based rock debris degrading bacteria and compound bacterial agents of the present invention, their cultivation methods and applications will be described in detail with reference to exemplary embodiments.

[0061] It should be noted that "first," "second," etc., are merely for the convenience of description and distinction, and should not be interpreted as indicating or implying relative importance.

[0062] To achieve the above objectives, the present invention provides an oil-based rock debris degrading bacterium.

[0063] In an exemplary embodiment of the present invention, an oil-based rock debris degrading bacterium includes at least one of chelating bacteria, *Petribaurus*, and xenobiotics. For example, the oil-based rock debris degrading bacterium can be: chelating bacteria, *Petribaurus*, xenobiotics, chelating bacteria + *Petribaurus*, chelating bacteria + xenobiotics, *Petribaurus* + xenobiotics, or chelating bacteria + *Petribaurus* + xenobiotics. Among them, chelating bacteria have strong temperature resistance and can survive in high-temperature environments of 30-65℃, making them suitable for treating oil-based drill cuttings in the high-temperature range; Petribourtella and xenobiotics have a wide growth temperature range and can survive in medium-temperature environments of 4-45℃, making them suitable for treating oil-based drill cuttings in the medium-temperature range; and the combined use of chelating bacteria with Petribourtella and / or xenobiotics can adapt to different treatment environment requirements and is suitable for treating oil-based drill cuttings in a wide temperature range (e.g., temperature range of 10-65℃).

[0064] The taxonomic name of the chelating bacterium *Chelativorans* sp. SH6-2 was deposited at the China Center for Type Culture Collection (CCTCC) on March 29, 2021, with accession number CCTCC NO: M 2021290. *Chelativorans* is a Gram-negative, non-spore-forming, rod-shaped, motile, and obligate aerobic bacterium. Colonies formed after 24 hours of culture on tryptone-soy agar are spherical, and after 48 hours, they are spherical, colorless, 0.5–1 mm in diameter, with regular edges, flat, and moist. The 16S rDNA sequence of *Chelativorans* is shown in SEQ ID NO. 1.

[0065] Chelateroans exhibit strong temperature tolerance, with a growth temperature range of 30–65℃. Under pure culture conditions, after 14 days of inoculation, the degradation rate of petroleum hydrocarbons in paraffin oil by Chelateroans sp. SH6-2 can reach over 46.55%.

[0066] The taxonomic name of *Bordetella petrii* is *Bordetella petrii* WOCB-20, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 26, 2021, with the accession number CCTCC NO: M 20211089. *Bordetella petrii* is a Gram-negative, motile bacterium with rod-shaped cells that do not produce spores. Colonies formed after 24 hours of culture on beef extract peptone medium are initially round, and after 48 hours, they are round, white, 0.5–1 mm in diameter, with regular edges, flat, and moist. The 16S rDNA sequence of *Bordetella petrii* is shown in SEQ ID NO. 2.

[0067] Bordetella petrii has a wide growth temperature range, from 4 to 45°C. Under pure culture conditions, after 14 days of inoculation, Bordetella petrii WOCB-20 can achieve a degradation rate of over 40.77% for petroleum hydrocarbons in paraffin oil.

[0068] The xenophilic bacterium, classified as *Xenophilus aerolatus* WOCB-11, was deposited at the China Center for Type Culture Collection (CCTCC) on August 26, 2021, with accession number CCTCC NO: M 20211088. Xenophilic bacteria can be Gram-negative, motile, rod-shaped, and do not produce spores. Colonies formed after 24 hours of culture on beef extract peptone medium are initially round, and after 48 hours, they are round, pale yellow, 1–2 mm in diameter, with regular edges, flat, and moist. The 16S rDNA sequence of the xenophilic bacterium is shown in SEQ ID NO. 3.

[0069] Xenotrophic bacteria have a wide growth temperature range, from 4 to 45°C. Under pure culture conditions, after 14 days of inoculation, the degradation rate of petroleum hydrocarbons in paraffin oil by xenophilus aerolatus WOCB-11 can reach over 45.33%.

[0070] In another aspect, the present invention provides a method for screening and culturing oil-based rock fragment degrading bacteria. In yet another exemplary embodiment of the present invention, a method for screening and culturing oil-based rock fragment degrading bacteria includes the following steps:

[0071] S1. Add a microbial source sample to an oil-based rock fragment sample and incubate at a constant temperature of 25–60℃ and / or 50–60℃ for 15–30 days to obtain the first-generation enriched microbial source. The microbial source samples include natural soil, silt and oil-contaminated soil.

[0072] S2. Add the first-generation enrichment bacterial source to another oil-based rock fragment sample at an addition ratio of 5% (w:w), and incubate at a constant temperature of 25–60℃ and / or 50–60℃ for 7–14 days to obtain the first culture medium. Here, since steps S2 to S4 are all carried out in liquid culture medium, the incubation time should not be too long; 7–14 days is more suitable.

[0073] S3. Place the first culture medium at 25-60℃ and / or 50-60℃ for 7-14 days to obtain the second culture medium.

[0074] S4. Repeat step S2 at least twice to obtain enriched bacterial samples after multiple transculturing.

[0075] S5. Spread the diluted enriched bacterial sample onto tryptone soybean agar medium and incubate at 50–60°C. After repeated streak culturing, the purified chelate strain is obtained; and / or,

[0076] The diluted enriched bacterial samples were spread on beef extract peptone medium and incubated at 25–30°C. After repeated streak culture, purified Petribourt strain and heterotrophic strain were obtained.

[0077] The tryptone-soybean agar medium includes 5–7.5 g / L tryptone, 3–7 g / L soybean peptone, 3–7 g / L sodium chloride, and 2–5 g / L brown sugar; the beef extract peptone medium includes 8–12 g / L peptone, 1–3 g / L meat extract, 3–7 g / L sodium chloride, and 5–10 g / L sucrose.

[0078] In another aspect, this invention provides the application of the oil-based rock cuttings degrading bacteria, or their bacterial suspension, or their culture medium, or their fermentation products, as described above, in the degradation of oil-based rock cuttings in petroleum contaminants or drilling mud. For example, the oil-based rock cuttings degrading bacteria can degrade oil-based rock cuttings (mud) or treat soil or water contaminated with oil-based rock cuttings. Under pure culture conditions, after 14 days of inoculation, the chelating bacteria *Chelativorans* sp. SH6-2, *Bordetella petrii* WOCB-20, and xenotrophic bacteria *Xenophilus aerolatus* WOCB-11 achieved degradation rates of over 46.55%, 40.77%, and 45.33% of petroleum hydrocarbons in paraffin oil, respectively.

[0079] In another aspect, the present invention provides a biological agent containing oil-based rock fragment degrading bacteria. In yet another exemplary embodiment of the present invention, a biological agent containing oil-based rock fragment degrading bacteria includes the above-mentioned oil-based rock fragment degrading bacteria or its bacterial suspension or its culture medium or its fermentation product.

[0080] In another aspect, this invention provides the application of the above-mentioned bio-agent containing oil-based rock cuttings degrading bacteria in degrading petroleum contaminants or oil-based rock cuttings in drilling mud. For example, the bio-agent containing oil-based rock cuttings degrading bacteria can degrade oil-based rock cuttings (mud) or treat soil or water contaminated with oil-based rock cuttings.

[0081] In another aspect, the present invention provides a method for preparing a compound microbial agent. In yet another exemplary embodiment of the present invention, a method for preparing a compound microbial agent includes: mixing chelating bacteria with *Petribaurus* and / or xenobiotics to obtain a compound microbial agent. The viable count of the chelating bacteria is 10%–90% of the total viable count; the viable count of *Petribaurus* is 0%–90% of the total viable count; and the viable count of the xenobiotics is 0%–90% of the total viable count.

[0082] For example, the total viable count of the compound microbial agent can be 1.0–2.0 × 10⁻⁶. 10 The total viable count (cfu / g) is the sum of the viable counts of Chelativorans sp. SH6-2, Bordetella petrii WOCB-20, and Xenophilus aerolatus WOCB-11.

[0083] Preferably, the viable count of chelate-eating bacteria can be 40% to 60% of the total viable count; the viable count of Petriboulders can be 20% to 30% of the total viable count; and the viable count of heterotrophic bacteria can be the remainder.

[0084] In another aspect, the present invention provides a composite microbial agent. In yet another exemplary embodiment of the present invention, a composite microbial agent is prepared by the above-described preparation method, and the composite microbial agent is capable of degrading oil-based rock cuttings at a temperature environment of 10–65°C. For example, the composite microbial agent can be applied to degrade oil-based rock cuttings at environments such as 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C.

[0085] Inoculating oil-based rock cuttings (mud) with the compound microbial agent described above at a mass concentration of 0.3% to 3% can effectively and efficiently degrade oil-based rock cuttings. For example, under pure culture conditions, after inoculating oil-based rock cuttings with the compound microbial agent at a mass concentration of 1% for 60 days, the degradation rate of petroleum hydrocarbons can reach over 87%.

[0086] In another aspect, this invention provides the application of the composite microbial agent, or its bacterial suspension, or its culture medium, or its fermentation product as described above, in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud. For example, the composite microbial agent can degrade oil-based rock cuttings (mud) or treat soil or water contaminated with oil-based rock cuttings. A composite microbial agent prepared by mixing the chelating bacterium *Chelativorans* sp. SH6-2, *Bordetella petrii* WOCB-20, and the xenotrophic bacterium *Xenophilus aerolatus* WOCB-11 in a predetermined ratio can efficiently and rapidly degrade and remove petroleum hydrocarbons from the environment.

[0087] In another aspect, the present invention provides a biological agent containing a compound microbial agent. In yet another exemplary embodiment of the present invention, a biological agent containing a compound microbial agent includes the above-mentioned compound microbial agent or its bacterial suspension or its culture medium or its fermentation product.

[0088] In another aspect, the present invention provides the application of the biopharmaceutical containing the compound microbial agent as described above in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud. For example, the biopharmaceutical containing the compound microbial agent can degrade oil-based rock cuttings (mud) or treat soil or water contaminated with oil-based rock cuttings.

[0089] To better understand the exemplary embodiments of the present invention described above, further explanation is provided below with reference to specific examples.

[0090] Example 1

[0091] (1) Isolation, purification and preservation of chelating bacteria Chelativorans sp. SH6-2 (hereinafter referred to as "strain SH6-2"), Bordetella petrii WOCB-20 (hereinafter referred to as "strain WOCB-20") and xenotrophic bacteria WOCB-11 (hereinafter referred to as "strain WOCB-11").

[0092] Oil-based cuttings samples were collected from a shale gas drilling site in Weiyuan County, Sichuan Province, and brought back to the laboratory after cryogenic preservation. It should be noted that oil-based cuttings samples can be obtained from shale gas or shale oil drilling sites in other regions.

[0093] Take 1 kg of oil-based rock cuttings and add garden soil (or natural soil, silt, or petroleum-contaminated soil) at a 50% w:w ratio. Add ammonium bicarbonate to adjust the C / N ratio of the mixed substrate to 15:1–20:1. Adjust the moisture content to 28%–30% with distilled water. Pack the mixed substrate into a white plastic bucket, seal it with breathable sealing film, and incubate at 50°C. Stir regularly to aerate and control the moisture content at 28%–30%. After 30 days of incubation, the first generation of enriched microbial source is obtained. It should be noted that the microbial source comes from garden soil. The purpose of adding ammonium bicarbonate and adjusting the C / N ratio is to provide a better growth environment for microorganisms. Because oil-based rock cuttings have a high carbon content and a low nitrogen content, but microbial growth requires a suitable carbon and nitrogen content, i.e., a suitable C / N ratio, nitrogen needs to be artificially supplemented. Ammonium bicarbonate can also be replaced with urea or other nitrogen sources. The reason for adjusting the moisture content with distilled water is that microbial growth requires a certain level of humidity.

[0094] Take another 5.0g oil-based drilling cuttings sample, add the first-generation enrichment bacteria source to the other oil-based drilling cuttings sample at a certain ratio, and add it to 95mL of MS medium containing 1% paraffin oil. Shake and culture for 7 days at 50℃ and 140r / min to obtain the first culture medium.

[0095] Then repeat the above operation to perform a second culture for 7 days. That is, take 1 mL of the first culture medium and add 99 mL of fresh, sterilized MS medium containing 1% paraffin oil. Continue to culture with shaking at 50℃ and 140 r / min for 7 days to obtain the second culture medium.

[0096] Then repeat the above operation to perform a third culture for 7 days. That is, take 1 mL of the second culture medium and add 99 mL of fresh, sterilized MS medium containing 1% paraffin oil. Continue to culture with shaking at 50℃ and 140 r / min for 7 days to obtain the third culture medium.

[0097] Finally, 1 mL of the third culture medium was taken and serially diluted, then spread onto trypto-soy agar and beef extract peptone agar plates, and incubated at 30°C and 50°C, respectively. After colonies grew, colonies with different morphologies were selected from the plates and streaked onto trypto-soy agar and beef extract peptone agar plates. After 16 hours of incubation, a loopful was picked for repeated streaking, purification, and preservation.

[0098] like Figure 1A and Figure 1B As shown, the isolated and purified strain SH6-2 was cultured on tryptone soybean agar medium. After 48 hours of culture, the colonies were round, white, 0.5–1 mm in diameter, with neat edges, flat and moist. Figure 1A The colony morphology of strain SH6-2 is shown. Figure 1B This is a schematic diagram showing the colonies of strain SH6-2 after streaking culture on tryptone soybean agar medium.

[0099] like Figure 1C and Figure 1D As shown, the isolated and purified strain WOCB-20 was cultured on beef extract peptone medium. After 24 hours of culture, the colonies were round, white, 0.5–1 mm in diameter, with neat edges, flat and moist. Figure 1C The colony morphology of strain WOCB-20 is shown. Figure 1D This is a schematic diagram showing the colonies of strain WOCB-20 after streaking culture on beef extract peptone medium.

[0100] like Figure 1E and Figure 1F As shown, the isolated and purified strain WOCB-11 was cultured on beef extract peptone medium. After 24 hours of culture, the colonies were round, pale yellow, 1–2 mm in diameter, with neat edges, flat and moist. Figure 1E The colony morphology of strain WOCB-11 is shown. Figure 1F This is a schematic diagram showing the colonies of strain WOCB-11 after streaking culture on beef extract peptone medium.

[0101] (2) Amplification and phylogenetic analysis of 16S rDNA from strains SH6-2, WOCB-20 and WOCB-11

[0102] Total DNA was extracted from the strain. Using the total DNA as a template, the 16S fragment was amplified using 27F and 1492R primers. The PCR reaction was performed using a Bio-RADMyCycler™ instrument.

[0103] Reaction system (50 μl): 25 μl of 2×PCRMix, 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.

[0104] 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.

[0105] 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 DNAman6.0 software. The sequence results are as follows: the 16S rDNA sequence of strain SH6-2 is shown in SEQ ID No. 1, the 16S rDNA sequence of strain WOCB-20 is shown in SEQ ID No. 2, and the 16S rDNA sequence of strain WOCB-11 is shown in SEQ ID No. 3.

[0106] The obtained sequence results were compared with the National Center for Biotechnology Information (NCBI) database. The results showed that the 16S rDNA gene sequence of strain SH6-2 had the highest similarity (97.0%) to *Chelativorans* sp. in the database; the 16S rDNA gene sequence of strain WOCB-20 had the highest similarity (99.6%) to *Bordetella petrii* in the database; and the 16S rDNA gene sequence of strain WOCB-11 had the highest similarity (99.9%) to *Xenophilus aerolatus* 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 for strain SH6-2 is shown below. Figure 2 As shown, strain WOCB-20 Figure 3 As shown, strain WOCB-11 Figure 4 As shown in the image.

[0107] Strain SH6-2 was deposited at the China Center for Type Culture Collection (CCTCC) on March 29, 2021, with accession number CCTCC NO: M 2021290; strain WOCB-20 was deposited at the CTCC on August 26, 2021, with accession number CCTCC NO: M 20211089; strain WOCB-11 was deposited at the CTCC on August 26, 2021, with accession number CCTCC NO: M 20211088.

[0108] (3) Experiment on the ability of strains SH6-2, WOCB-20 and WOCB-11 to degrade liquid paraffin.

[0109] Strain SH6-2 was inoculated onto tryptone soybean 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, the supernatant was discarded, the bacterial cells were washed twice with sterile physiological saline, and then resuspended in 1.5 mL of sterile water. The suspension was then inoculated into 95 mL of MS medium with a liquid paraffin concentration of 1%, and cultured at 50 °C with shaking at 140 rpm. The control was not inoculated. The experiment was repeated 3 times.

[0110] Strains WOCB-20 and WOCB-11 were 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, the supernatant was discarded, the bacterial cells were washed twice with sterile physiological saline, and then resuspended in 1.5 mL of sterile water. The bacterial suspension was then inoculated into 95 mL of MS medium with a liquid paraffin concentration of 1% and cultured at 30 °C with shaking at 140 rpm. The experiment was repeated 3 times with no bacterial inoculation as a control.

[0111] The results showed that after 14 days of culture, the degradation rates of liquid paraffin treated with strains SH6-2, WOCB-20, and WOCB-11 reached 46.55%, 40.77%, and 45.33%, respectively, while the degradation rate of petroleum hydrocarbons in the diesel degradation medium in the blank control experiment was 0.78%.

[0112] (4) Stress resistance experiments of strains SH6-2, WOCB-20 and WOCB-11

[0113] Salt tolerance determination: Tryptone-soybean liquid medium and beef extract peptone liquid medium were prepared and the pH was adjusted to 7. NaCl was added to the basal liquid medium to prepare media with salt concentrations of 0.5%, 1%, 2%, 4%, 8%, and 10%, respectively. After sterilization at 121℃ for 30 min, bacterial suspension was added to the freshly prepared medium at a ratio of 1% (v:v). Three replicates were set up. Strains WOCB-20 and WOCB-11 were added to beef extract peptone liquid medium and incubated at 30℃ with shaking at 150 rpm for 48 h. Strain SH6-2 was added to tryptone-soybean liquid medium and incubated at 50℃ with shaking at 150 rpm for 48 h. The number of bacteria was determined by ultraviolet spectrophotometer at a wavelength of λ=600 nm.

[0114] Determination of growth temperature range: Tryptone soybean liquid medium and beef extract peptone liquid medium were prepared, and the pH was adjusted to 7. After sterilization at 121℃ for 30 min, strain SH6-2 was added to tryptone soybean liquid medium at 1% (v:v), and strains WOCB-2 and WOCB-11 were added to beef extract peptone liquid medium. The inoculated media were placed in constant temperature incubators at 4℃, 15℃, 30℃, 45℃, 55℃, 65℃ and 75℃ and cultured with shaking at 150 r / min for 48 h. Each temperature was set in 3 replicates. The number of bacteria was determined by ultraviolet spectrophotometer at wavelength λ=600.

[0115] Acid and alkali resistance determination: Tryptone soybean liquid medium and beef extract peptone medium were prepared and their pH values ​​were measured. The pH values ​​of the medium were adjusted to 4, 5, 6, 7, 8, 9, 10, and 11 with 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution, respectively. After sterilization at 121℃ for 30 min, strain SH6-2 was added to tryptone soybean liquid medium at 1% (v:v), and strains WOCB-2 and WOCB-11 were added to beef extract peptone liquid medium. Three replicates were set for each pH value. The medium was incubated in a constant temperature incubator at 30℃ or 50℃ with shaking at 150 r / min for 24 h. The number of bacteria was measured using a UV spectrophotometer at a wavelength of λ = 600 nm.

[0116] The results showed that strains SH6-2, WOCB-20, and WOCB-11 exhibited good stress resistance, strong tolerance to acid and alkali, and could grow in a pH range of 5–10. Strains WOCB-20 and WOCB-11 showed strong salt tolerance, growing in media containing up to 6% salt. The growth temperature range of strains SH6-2, WOCB-20, and WOCB-11 was wide, with strain SH6-2 growing at 30℃–65℃, and strains WOCB-20 and WOCB-11 growing at 4℃–45℃.

[0117] (5) Experiments on the treatment of oil-based cuttings by strains SH6-2, WOCB-20 and WOCB-11

[0118] Inoculate the treated material (oil-based rock cuttings) with each bacterial agent (OD600nm≈1.6) at 1% (m / m) of the oil-based rock cuttings, mix thoroughly, and repeat 3 times with an uninoculated control. The weight of oil-based rock cuttings treated with each bacterial agent is 3.0 kg. Strain SH6-2 was treated at 50℃ for 60 days (January to February 2021), and strains WOCB-20 and WOCB-11 were treated at 30℃ for 60 days (January to February 2021). During this period, maintain the humidity of the treatment system at approximately 25%.

[0119] The petroleum hydrocarbon content was determined in the initial treatment samples and inoculated and uninoculated samples after 60 days of treatment. The results are shown in Table 1 below.

[0120] Table 1. Treatment effects of the strain on oil-based rock cuttings

[0121]

[0122]

[0123] Note: Different letters indicate a significant difference (p<0.05).

[0124] The results showed that after 60 days of treatment, strain SH6-2 achieved a petroleum hydrocarbon removal rate of 83.69% from oil-based rock cuttings, strain WOCB-20 achieved 80.83%, and strain WOCB-11 achieved 81.49%. All three strains met the standards of SY / T7301-2016 "Technical Requirements for Comprehensive Utilization and Pollution Control of Oily Sludge from Onshore Oil and Gas Extraction" (TPH < 2%), demonstrating good degradation potential.

[0125] In other words, the strains SH6-2, WOCB-20 and WOCB-11 provided in this example can effectively or even efficiently degrade paraffin oil and oil-based rock cuttings (mud); or degrade or remediate pollution caused by oil-based rock cuttings (mud).

[0126] (6) Preparation method of compound microbial agent

[0127] A method for preparing a compound microbial agent includes: mixing strain SH6-2, strain WOCB-20 and strain WOCB-11 in the above example in a certain proportion to obtain a compound microbial agent.

[0128] The total viable count of the compound microbial agent is 1.0–2.0 × 10⁻⁶. 10 CFU / g, the total viable count is the sum of the viable counts of strains SH6-2, WOCB-20 and WOCB-11.

[0129] In the compound microbial agent, the viable count of strain SH6-2 can be 10-90% of the total viable count, the viable count of strain WOCB-20 can be 10-90% of the total viable count, or the viable count of strain WOCB-11 can be 10-90% of the total viable count. That is, the compound microbial agent can have strain SH6-2 accounting for 10-90% of the total viable count, with strains WOCB-20 and WOCB-11 as the remainder; or the compound microbial agent can have strain WOCB-20 accounting for 10-90% of the total viable count, with strains SH6-2 and WOCB-11 as the remainder; or the viable count of strain WOCB-11 accounting for 10-90% of the total viable count, with strains SH6-2 and WOCB-20 as the remainder.

[0130] Preferably, in the compound microbial agent, the viable count of strain SH6-2 can be 40-60% of the total viable count, the viable count of strain WOCB-20 can be 20-30% of the total viable count, and the viable count of strain WOCB-11 is the remainder.

[0131] The preparation method of the compound microbial agent may further include culturing strains SH6-2, WOCB-20, and WOCB-11 separately using a compound microbial agent culture medium before mixing them. Here, the prepared compound microbial agent culture medium may include 5–7.5 g / L tryptone, 3–7 g / L soybean peptone, 3–7 g / L sodium chloride, and 2–5 g / L brown sugar; another formulation may contain 8–12 g / L peptone, 1–3 g / L meat extract, 3–7 g / L sodium chloride, and 5–10 g / L sucrose, with water as the solvent (e.g., distilled water or tap water).

[0132] (7) Compound microbial agent

[0133] This example provides a composite microbial agent prepared using the method described above. This composite microbial agent can be used as a wide-temperature-range degrading bacterium, applied in environments ranging from 10 to 65°C to degrade oil-based rock cuttings. Alternatively, it can also be used as a high-temperature degrading bacterium, effectively degrading and treating oil-based rock cuttings when applied in high-temperature environments of 50 to 60°C.

[0134] The effective viable bacteria count in the compound microbial agent can be 1.0–2.0 × 10⁻⁶. 10 The typical but non-limiting effective viable count is 1.0 × 10⁻⁶ cfu / g. 10 cfu / g, 1.5×10 10 cfu / g or 2.0×10 10 cfu / g.

[0135] (8) Application methods of compound microbial agents

[0136] The compound microbial agent provided in this example can be applied to treat soil or water contaminated with oil-based rock cuttings and / or oil-based rock cuttings. This compound microbial agent can efficiently and rapidly reduce the amount of petroleum hydrocarbons in the environment.

[0137] In this example, the method of applying the compound microbial agent includes inoculating the above-mentioned compound microbial agent at a mass concentration of 0.3% to 3% into oil-based rock cuttings and / or oil-based rock cuttings contaminated soil or water. Here, typical but non-limiting inoculation amounts are 0.3%, 1.0%, 2.0%, or 3.0%.

[0138] (9) Experiments on the treatment of oil-based rock cuttings by different compound bacterial agents

[0139] Prepare a pre-mixed culture medium 1 containing 10 g / L peptone, 1.5 g / L beef extract, 5 g / L sodium chloride and 8 g / L sucrose, and a pre-mixed culture medium 2 containing 6 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride and 4 g / L brown sugar. Culture strains SH6-2, WOCB-20 and WOCB-11 into bacterial suspensions, and prepare compound bacterial agents A1 to A5 according to the proportions in Table 2 below.

[0140] Table 2. Compound microbial agents with different proportions

[0141]

[0142]

[0143] The soil mixture contaminated with diesel fuel and drilling fluid was treated using compound microbial agents A1 to A5 listed in Table 2, with the specific steps as follows:

[0144] 5000g of oil-based rock cuttings (petroleum hydrocarbon content 65.0g / kg) were weighed and inoculated with compound microbial agents A1 to A5 at a mass concentration of 1% (W:W). The mixtures were placed in plastic basins and thoroughly mixed. After treatment at outdoor natural temperature for 60 days, the petroleum hydrocarbon content of the oil-based rock cuttings was measured, and the degradation rate of petroleum hydrocarbons by the microbial agents was calculated. The degradation rate can be calculated using 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.

[0145] Table 3. Effects of different compound microbial agents on oil-based rock cuttings

[0146]

[0147] The results showed that when the compound microbial agents A1 to A5 were inoculated into oil-based rock cuttings at a mass concentration of 1% for 60 days, the degradation rate of petroleum hydrocarbons was over 87%.

[0148] In other words, the three strains selected in the compound microbial agent prepared in this example are reasonable. The compound microbial agent can be obtained by mixing the three strains evenly. The operation is simple and has wide applicability. The compound microbial agent provided in this example has excellent degradation ability for oil-based rock fragments. It can be widely used to degrade oil-based rock fragments and remediate oil-based rock fragment contamination, such as the remediation of soil contaminated by oil-based rock fragments, and the remediation of water bodies contaminated by oil-based rock fragments.

[0149] In summary, the beneficial effects of the present invention include at least one of the following:

[0150] (1) The chelating bacteria Chelativorans sp. SH6-2, Petribourtella WOCB-20 and heterotrophic bacteria WOCB-11 provided by the present invention can effectively or even efficiently degrade paraffin oil and oil-based rock cuttings (mud); they can also effectively or even efficiently remediate pollution caused by oil-based rock cuttings (mud), such as soil or water contaminated by oil-based rock cuttings (mud);

[0151] (2) Under pure culture conditions, after 14 days of inoculation, the degradation rates of petroleum hydrocarbons in paraffin oil by the chelating bacteria Chelativorans sp. SH6-2, Petribourtella WOCB-20 and xenotrophic bacteria WOCB-11 reached 46.55%, 40.77% and 45.33% respectively.

[0152] (3) Chelaterans sp.SH6-2 has strong temperature resistance, while Peterbauerella WOCB-20 and heterotrophic bacteria WOCB-11 have a wide growth temperature range and can be used as excellent degrading bacteria for the biological treatment of oil-based rock cuttings (mud) in a wide temperature range (10-65℃). Their application prospects are good.

[0153] (4) A compound microbial agent prepared by mixing Chelativorans sp. SH6-2, Petribourtella WOCB-20 and Heterophilic bacteria WOCB-11 in a predetermined ratio can efficiently and rapidly degrade and remove petroleum hydrocarbons in the environment.

[0154] (5) Under pure culture conditions, when the compound bacterial agent is inoculated into oil-based rock cuttings at a mass concentration of 0.3-3% for 60 days, the degradation rate of petroleum hydrocarbons can reach more than 80%.

[0155] 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> An oil-based rock debris degrading bacteria and a compound bacterial agent, their cultivation method and application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1386 <212> DNA <213> Chelativorans sp.SH6-2 <400> 1 ctgctgagca ccgtggcggc tgcctccttg cggttagcgc accgtcttcg ggtaaaccca 60 actcccatgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 120 tgatccgcga ttactagcga ttccaacttc atgcactcga gttgcagagt gcaatccgaa 180 ctgagatggc ttttggagat tagctcgacc tcgcggtctc gctgcccact gtcaccacca 240 ttgtagcacg tgtgtagccc agcccgtaag ggccatgagg acttgacgtc atccccacct 300 tcctctcggc ttatcaccgg cagtcccctt agagtgccca acttaatgat ggcaactaag 360 ggcgagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420 gccatgcagc acctgtcacc ggtccagccg aactgaaggc ctcgatctct caaggccgcg 480 accgggatgt caagggctgg taaggttctg cgcgttgctt cgaattaaac cacatgctcc 540 accgcttgtg cgggcccccg tcaattcctt tgagttttaa tcttgcgacc gtactcccca 600 ggcggaatgc ttaatgcgtt agctgcgcca ccgacaggca agcctgccga cggctagcat 660 tcatcgttta cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc 720 gcctcagcgt cagtttcggg ccagtgagcc gccttcgcca ctggtgttcc tccgaatatc 780 tacgaatttc acctctacac tcggaattcc actcacctct cccgaactct agattagcag 840 ttttagaggc agttccgggg ttgagccccg ggatttcacc cctaacttgc aaatccgcct 900 acgcgccctt tacgcccagt aattccgaac aacgctagcc cccttcgtat taccgcggct 960 gctggcacga agttagccgg ggcttcttct gcgggtaccg tcattatctt ccccgctgaa 1020 agagctttac aaccctaggg ccttcatcac tcacgcggca tggctggatc aggctttcgc 1080 ccattgtcca atattcccca ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc 1140 agtgtggctg atcatcctct cagaccagct acggatcgta gccttggtag gccattaccc 1200 caccaactag ctaatccgac gcgggctcat ccaactccga taaatctttc tcccggagga 1260 cgtatacggt attagctcag gtttccctga gttattccgt agagctgggt agattcccac 1320 gcgttactca cccgtctgcc gctgaccccg gagggtccgc tcgactgcat ggtaagctcc 1380 gccagt 1386 <210> 2 <211> 1433 <212> DNA <213> Bordetella petrii WOCB‑20 <400> 2 tagcgggatg ctttacacat gcaagtcgaa cggcagcgcg gacttcggtc tggcggcgag 60 tggcgaacgg gtgagtaatg tatcggaacg tgcccagtag cgggggataa ctacgcgaaa 120 gcgtggctaa taccgcatac gccctacggg ggaaagcggg ggaccttcgg gcctcgcact 180 attggagcgg ccgatatcgg attagttagt tggtggggta aaggcctacc aaggcgacga 240 tccgtagctg gtttgagagg acgaccagcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtgggg aattttggac aatgggggca accctgatcc agccatcccg 360 cgtgtgcgat gaaggccttc gggttgtaaa gcacttttgg caggaaaa acggccctgg 420 ggggcaactga cggtacctgc agaataagca ccggctaact acgtgccagc 480 agccgcggta atacgtaggg tgcaagcgtt aatcggaatt actgggcgta aagcgtgcgc 540 aggcggttcg gaagaaaga tgtgaaatcc cagggcttaa ccttggaact gcatttttaa 600 ctaccgggct agagtgtgtc agagggaggt ggaattccgc gtgtagcagt gaaatgcgta 660 720 agaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccc taaacgatgt 780 caactagctg ttggggcctt cgggccttgg tagcgcagct aacgcgtgaa gttgaccgcc 840 tggggagtac ggtcgcaaga taaaactca aaaggaattga cggggacccg cacaagcggt 900 ggatgatgtg gattaattcg atgcaacgcg aaaaacctta cctacccttg acatgtctgg 960 aatgccgaag agatttggca gtgctcgcaa ragaccgga acacaggtgc tgcatggctg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgtcat 1080 tagttgctac gaaagggcac tctaatgaga ctgccggtga caaaccggag gaaggtgggg 1140 atgacgtcaa gtcctcatgg cccttatggg tagggcttca cacgtcatac aatggtcggg 1200 acagagggct gccaacccgc gagggggagc caatcccaga aacccgatcg tagtccggat 1260 cgcagtctgc aactcgactg cgtgaagtcg gaatcgctag taatcgcgga tcagcatgtc 1320 gcggtgaata cgttcccggg tcttgtacac accgcccgtc acaccatggg agtgggtttt 1380 accagaagta gttagcctaa ccgcaagggg ggcgatacca cggtagatca date 1433 <210> 3 <211> 1430 <212> DNA <213> Xenophilus aerolatus WOCB‑11 <400> 3 aatgcgctgc ttaaatgcaa gtcgaacggt aacaggtctt cggatgctga cgagtggcga 60 acgggtgagt aatacatcgg aacgtgccca gtcgtggggg ataactacgc gaaagcgtag 120 ctaataccgc atacgatcta tggatgaaag cgggggatcg caagacctcg cgcgattgga 180 gcggccgatg gcagattagg tagttggtgg ggtaaaggct caccaagccg acgatctgta 240 gctggtctga gaggacgacc agccacactg ggactgagac acggcccaga ctcctacggg 300 aggcagcagt ggggaatttt ggacaatggg cgcaagcctg atccagccat gccgcgtgca 360 ggatgaaggc cttcgggttg taaactgctt ttgtacggaa cgaaaagtct ctttctaata 420 aagagggatc atgacggtac cgtaagaata agcaccggct aactacgtgc cagcagccgc 480 ggtaatacgt agggtgcaag cgttaatcgg aattactggg cgtaaagcgt gcgcaggcgg 540 tgatgtaaga cagttgtgaa atccccgggc tcaacctggg aactgcatct gtgactgcat 600 cgctggagtg cggcagaggg ggatggaatt ccgcgtgtag cagtgaaatg cgtagatagtg 660 cgggagaaca ccgatggcga aggcaatccc ctgggcctgc actgacgctc atgcacgaaa 720 gcgtggggag caaaggat tagataccct ggtagtccac gccctaaacg atgtcaactg 780 gttgttggga attcactttc tcagtaacga agctaacgcg tgaagttgac cgcctgggga 840 gtacggccgc aaggttgaaa ctcaaaggaa ttgacggga cccgcacaag cggtggatga 900 tgtggtttaa ttcgatgcaa cgcgaaaaac cttacccacc tttgacatgt acggaatttg 960 ccagagatgg cttagtgctc gaaagagaac cgtaacacag gtgctgcatg gctgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tcattagttg 1080 ctacattcag ttgggcactc taatgagact gccggtgaca aaccggagga aggtggggat 1140 gacgtcaagt cctcatggcc cttataggtg gggctacaca cgtcatacaa tggctggtac 1200 aaagggttgc caacccgcga gggggagcta atcccataaa gccagtcgta gtccggatcg 1260 cagtctgcaa ctcgactgcg tgaagtcgga atcgctagta atcgtggatc agaatgtcac 1320 ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac accatgggag cgggttctgc 1380 cagaagtagt tagcctaacc gcaaggaggg cgataccacg tcagctcgtg 1430

Claims

1. An oil-based rock debris degrading bacterium, characterized in that, The oil-based rock debris degrading bacteria include at least one of chelate-eating bacteria, *Petribochalcogenide*, and xenobiotics, wherein... The classification name of the chelating bacteria is chelating bacteria. Chelativorans sp. SH6-2, its accession number is CCTCC NO: M 2021290; The classification name of the bacteria mentioned is *Petribobotilia*. Bordetella petrii WOCB-20, with accession number CCTCC NO: M 20211089; The classification of the xenobiotics is xenobiotics. Xenophilus aerolatus WOCB-11, with accession number CCTCC NO: M 20211088.

2. The oil-based rock debris degrading bacteria according to claim 1, characterized in that, The chelating bacteria are Gram-negative bacteria, non-spore-forming, rod-shaped, motile, and obligately aerobic. The aforementioned Peterbauer bacteria are Gram-negative, motile, rod-shaped, and do not produce spores; The heterotrophic bacteria are Gram-negative, motile, rod-shaped, and do not produce spores.

3. The oil-based rock debris degrading bacteria according to claim 1, characterized in that, The colonies formed by the chelating bacteria after culturing on tryptone soybean agar medium for 24 hours were round, and after 48 hours the colonies were round, colorless, with a diameter of 0.5~1 mm, neat edges, flat and moist. The colonies of *Petricobacterium* cultured on beef extract peptone medium for 24 hours were round, and after 48 hours, the colonies were round, white, 0.5-1 mm in diameter, with neat edges, flat and moist. The heterotrophic bacteria formed round colonies after culturing on beef extract peptone medium for 24 hours, and after 48 hours, the colonies were round, pale yellow, 1-2 mm in diameter, with neat edges, flat and moist.

4. The oil-based rock debris degrading bacteria according to claim 1, characterized in that, The 16S rDNA sequence of the chelating bacteria is shown in SEQ ID NO.1; The 16S rDNA sequence of the *Petricobacterium* is shown in SEQ ID NO.2; The 16S rDNA sequence of the heterotrophic bacteria is shown in SEQ ID NO.

3.

5. The oil-based rock debris degrading bacteria according to claim 1, characterized in that, The growth temperature range of the chelate-eating bacteria is 30~65℃, the growth temperature range of the Peterbauerella is 4~45℃, and the growth temperature range of the heterotrophic bacteria is 4~45℃.

6. The application of an oil-based rock cuttings degrading bacterium or its suspension as described in any one of claims 1 to 5 in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud.

7. A biological agent containing oil-based rock debris degrading bacteria, characterized in that, The biological agent containing oil-based rock fragment degrading bacteria includes the oil-based rock fragment degrading bacteria or its suspension as described in any one of claims 1 to 5.

8. The application of a biological agent of oil-based rock cuttings degrading bacteria as described in claim 7 in the degradation of petroleum contaminants or oil-based rock cuttings in drilling mud.

9. A method for preparing a compound microbial agent, characterized in that, The preparation method includes: mixing chelating bacteria with Petribourtella and / or xenotrophic bacteria to obtain a compound bacterial agent; The viable count of the chelating bacteria is 10% to 90% of the total viable count; The viable count of the *Petrobacter p.* was 0% to 90% of the total viable count. The viable count of the heterotrophic bacteria is 0% to 90% of the total viable count; The classification name of the chelating bacteria is chelating bacteria. Chelativorans sp. SH6-2, its accession number is CCTCC NO: M 2021290; The classification name of the bacteria mentioned is *Petribobotilia*. Bordetella petrii WOCB-20, with accession number CCTCC NO: M 20211089; The classification of the xenobiotics is xenobiotics. Xenophilus aerolatus WOCB-11, with accession number CCTCC NO: M 20211088.

10. The method for preparing the compound microbial agent according to claim 9, characterized in that, The viable count of the chelating bacteria is 40% to 60% of the total viable count; The viable count of the *Petrobacter* strain is 20% to 30% of the total viable count; The viable count of the heterotrophic bacteria is the remainder.

11. A compound microbial agent, characterized in that, The composite microbial agent is prepared by the preparation method described in claim 9 or 10, and the composite microbial agent can degrade oil-based rock fragments at a temperature of 10~65℃.

12. A method for applying the composite microbial agent as described in claim 11 to degrade oil-based rock debris and / or oil-based rock debris contaminated soil or water.

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

14. A biological agent containing a compound microbial agent, characterized in that, The biological agent containing the compound microbial agent includes the compound microbial agent or its bacterial suspension as described in claim 11.

15. The application of a biological agent containing a compound microbial agent as described in claim 14 in the degradation of petroleum contaminants or oil-based cuttings in drilling mud.

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