Medium-high temperature petroleum degrading bacterium, application thereof and oily sludge degrading method

Abstract

The invention belongs to the field of comprehensive treatment of oily sludge, and relates to a medium-high temperature petroleum degrading bacterium, application and an oily sludge degrading method. The medium and high temperature petroleum degrading bacterium is brevibacillus borstelensis, and the preservation number of the medium and high temperature petroleum degrading bacterium is CGMCC (China General Microbiological Culture Collection Center) No. 29416. According to the invention, petrophilic bacteria existing in oil sludge or soil polluted by petroleum are utilized to screen, enrich and enlarge high-temperature microbial bacteria in culture for degrading oily sludge. The strain screened by the method is more targeted when being used for treating the oil sludge, rapid and efficient degradation of the oil-containing sludge can be realized, and a better oil sludge treatment effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of comprehensive treatment of oily sludge, specifically relating to a medium- and high-temperature petroleum-degrading bacterium, the application of the medium- and high-temperature petroleum-degrading bacterium in the degradation of oily sludge, and a method for the degradation of oily sludge. Background Technology

[0002] Oily sludge refers to the general term for hazardous solid waste mainly composed of petroleum components generated during the petrochemical industry's oil extraction, refining, storage, and transportation processes. Its main components are crude oil and related refined oil products, mud, sand, and water, forming a black, viscous colloid after thorough emulsification and mixing. Refining plant oily sludge typically has an oil content of 10%–50% and a water content of 40%–90%, exhibiting good fluidity and containing large amounts of toxic and harmful substances such as phenols. It is severely emulsified, making three-phase separation difficult. To effectively address the pollution problem of oily sludge, it must undergo harmless treatment to remove various recalcitrant toxic and harmful substances such as phenols, anthracene, pyrene, sulfides, and benzene compounds. Oily sludge generally still contains 20%–80% oil; the extraction and recycling of this oil, as well as the utilization of its residual calorific value, will generate significant economic and social benefits. Therefore, from the perspectives of environmental protection, maintaining normal production, and energy recovery, it is essential to reduce, render harmless, and recycle oily sludge.

[0003] Conventional physicochemical methods can effectively treat most oily sludge, but in most cases, they essentially involve the transfer of pollutants from one environmental medium to another, and toxic byproducts are easily generated during the treatment process. Furthermore, high-concentration, light oily sludge cannot be completely treated using physicochemical methods. Broadly speaking, biological treatment technology refers to the use of various organisms (such as plants, oily sludge, or soil animals and microorganisms) to absorb, degrade, and transform toxic and harmful substances in oily sludge or soil. In a narrower sense, biological treatment involves microorganisms using petroleum in the oily sludge as a carbon source for degradation, ultimately mineralizing it completely and transforming it into harmless inorganic substances (CO2 and H2O). Currently, microbial treatment technologies mainly include composting and bioreactor methods. Microbial treatment technology has the lowest cost among many technologies and is environmentally friendly, with widespread applications in wastewater treatment, soil remediation, and microbial oil recovery. It is one of the main methods for the harmless treatment of oily sludge in the future. Therefore, developing high-temperature microbial treatment technology for oily sludge, with microbial treatment as the core and combined with petroleum recovery technology research, is of great significance for the low-cost reduction, harmless treatment, and resource recovery of oily sludge.

[0004] Screening thermophilic bacterial strains for high-temperature microbial treatment can accelerate the degradation of organic matter and reduce the water content of oily sludge. Most currently developed petroleum-degrading bacteria are low- to medium-temperature microorganisms, which are insufficient for treating oily sludge under high-temperature conditions. Therefore, obtaining petroleum-degrading bacteria resistant to medium and high temperatures is crucial. Due to the different compositions and characteristics of various oily sludges, the degradation of petroleum hydrocarbons is the result of the synergistic action of multiple native microorganisms; directly adding bacterial strains results in low efficiency and poor effectiveness. Under medium and high-temperature conditions, microbial growth rates are low, and the rate of petroleum degradation is even slower. Microbial treatment of oily sludge needs to be targeted to improve treatment efficiency. Through long-term natural selection, microbial communities capable of synergistically degrading this type of oily sludge already exist in oily sludge or petroleum-contaminated soil. Screening, enriching, and expanding the culture of these oily sludge microbial communities for their degradation is more targeted and can achieve rapid and efficient degradation of oily sludge. Summary of the Invention

[0005] The purpose of this invention is to provide a medium- and high-temperature petroleum-degrading bacterium screened from oily sludge, and to utilize the screened petroleum-loving bacterium to achieve efficient degradation of oily sludge.

[0006] The first aspect of the present invention provides a medium- and high-temperature petroleum-degrading bacterium, wherein the medium- and high-temperature petroleum-degrading bacterium is *Brevibacillus borstelensis*, which has the accession number CGMCC No. 29416.

[0007] A second aspect of the present invention provides the application of the aforementioned medium- and high-temperature petroleum-degrading bacteria in the degradation of oily sludge.

[0008] A third aspect of the present invention provides a method for degrading oily sludge, comprising the following steps: contacting the medium- and high-temperature petroleum-degrading bacteria with the oily sludge.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1) This invention screens petroleum bacteria resistant to medium and high temperatures from oily sludge, enabling the treatment of oily sludge or petroleum-contaminated soil under medium and high temperature conditions.

[0011] 2) This invention utilizes petrophilic bacteria present in oily sludge or petroleum-contaminated soil, and screens, enriches, and expands thermophilic microorganisms for the degradation of oily sludge. The strains screened using this method are more targeted at treating this type of oily sludge, enabling rapid and efficient degradation of oily sludge and achieving superior oily sludge treatment results.

[0012] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0013] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0014] Figure 1 This is a plate image of strain BHYH-6.

[0015] Figure 2 This is a scanning electron microscope image of strain BHYH-6.

[0016] Biological Preservation Instructions

[0017] The *Brevibacillus borstelensis* BHYH-6 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on December 27, 2023, with accession number CGMCC No. 29416. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0019] The bacterial strain screened in this invention is classified as belonging to the genus Brevibacillus, specifically Brevibacillus borstelensis, named BHYH-6, and is currently deposited at the Microbial Culture Collection Center of the Chinese Academy of Sciences with accession number CGMCC No. 29416.

[0020] This bacterial strain was obtained by screening and enriching oily sludge from the petroleum refining industry. The specific method is as follows:

[0021] Step 1: Take 1-2g of oil sludge and put it into 100-200mL of sterile liquid petroleum culture medium.

[0022] Step 2: Place the liquid culture medium in a shaker and incubate for 5 to 7 days at a speed of 150 to 200 r / min and a temperature of 50 to 60℃.

[0023] Step 3: Transfer 5% to 10% of the culture to a new sterile liquid petroleum medium and continue culturing under the conditions of Step 2. Repeat this process 4 to 7 times until the liquid medium becomes noticeably turbid.

[0024] Step 4: Take 1-2 mL of the bacterial culture from multiple incubations and serially dilute it to 10⁻⁶. -6 ~10 -7 Dilute the sample and spread it onto petroleum solid culture medium. Incubate at 50-60°C for 3-5 days until colonies appear.

[0025] Step 5: Select a single colony and enrich it in beef extract peptone solid medium at 50-60°C.

[0026] The liquid petroleum culture medium described in step one uses petroleum as the sole organic component, facilitating rapid screening of petroleum-producing bacteria. The nitrogen source is at least one of ammonium sulfate, urea, and ammonium nitrate, preferably ammonium sulfate ((NH4)2SO4), and the preferred pH is 7. The specific components are: K2HPO4 (1–2 g / L), KH2PO4 (0.1–0.2 g / L), NaCl (0.04–0.05 g / L), (NH4)2SO4 (4–5 g / L), NaNO3 (4–5 g / L), MgSO4 (0.3–0.4 g / L), CaCl2 (0.1–0.2 g / L), 5 mL of concentrated trace element solution, and 10 g / L petroleum. The trace element concentrate is composed of cobalt chloride (0.1–0.2 mg / L), manganese chloride (0.1–0.2 mg / L), copper chloride (0.05–0.1 mg / L), zinc chloride (0.002–0.005 mg / L), boric acid (0.002–0.005 mg / L), EDTA (0.05–0.1 mg / L), and (NH₄)₆Mo₇O₇. 24 ·4H2O (0.005~0.01mg / L), Na2SeO3·6H2O (0.005~0.01mg / L), NiCl2 (0.002~0.005mg / L).

[0027] The solid petroleum culture medium mentioned in step three is liquid petroleum culture medium with 2% to 3% agar.

[0028] Because petroleum has a high density and viscosity, it is not easy to mix evenly with solid culture medium. Petroleum is coated on filter paper and then covered on the surface of solid culture medium coated with bacterial solution, which facilitates the growth of petroleum bacteria.

[0029] Since the culture medium is placed in a medium-high temperature (50-60℃) incubator, the agar plates are prone to water loss. The culture medium should be sealed with sealing film, and the incubation time should not be too long.

[0030] The medium- and high-temperature petroleum-degrading bacteria of the present invention can be used to degrade oily sludge.

[0031] The present invention also provides a method for applying medium- and high-temperature petroleum-degrading bacteria to treat oily sludge or petroleum-contaminated soil. Specifically, it is a method for degrading oily sludge, which includes the following steps: contacting the medium- and high-temperature petroleum-degrading bacteria with the oily sludge.

[0032] The contact conditions are preferably suitable for the degradation of oily sludge by medium- and high-temperature petroleum-degrading bacteria, and more preferably, the temperature is 50–60°C and the humidity is 60–70%. Under these conditions, the medium- and high-temperature petroleum-degrading bacteria can efficiently degrade oily sludge.

[0033] According to a specific embodiment of the present invention, the method for degrading oily sludge includes the following steps:

[0034] (1) Activation and enrichment of strains: The medium- and high-temperature petroleum-degrading bacteria were inoculated into a liquid culture medium and cultured at a constant temperature with shaking until the logarithmic growth phase to prepare a bacterial solution;

[0035] (2) Degradation of oily sludge: The carbon and nitrogen regulator is mixed with the oily sludge, the bacterial solution is added, the temperature is gradually increased to 50-60℃, and aerobic fermentation is carried out under stirring conditions to degrade the petroleum hydrocarbons in the oily sludge.

[0036] In step (1), the culture medium can be a conventional culture medium used for chelate cocci in the art. In one specific embodiment of the present invention, the culture medium is LB medium. The components of the LB liquid culture medium used in the present invention are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 1 L water; pH 7.0.

[0037] In step (1), the purpose of the isothermal oscillation is to activate the strain. The preferred conditions include: a temperature of 50-60°C, a rotation speed of 100-200 r / min, and a time of 30-60 h.

[0038] In step (2) of this invention, the purpose of adding a carbon-nitrogen regulator is to provide a certain carbon-nitrogen ratio and provide better degradation conditions for the microbial agent. From the perspective of economy and convenience, common high-carbon and high-nitrogen substances without special industrial and agricultural application value can be selected as carbon-nitrogen regulators. Specifically, the high-carbon substances can be straw and / or sawdust; the high-nitrogen substances can be animal manure, more preferably cow manure.

[0039] According to one specific embodiment, the weight ratio of the high carbon content material, the high nitrogen content material, and the oily sludge is (2-3):(0.5-2.5):4; preferably, the humidity is controlled at 60-70%.

[0040] According to a specific embodiment of the present invention, after the strain is activated, the OD is adjusted. 600 The value is 1, and then it is added to the oily sludge mixed with carbon and nitrogen regulator. The amount of bacterial agent added can be determined as needed to achieve a better degradation effect on the oily sludge. Preferably, the amount of bacterial solution added is 5-20% by weight of the total weight of the carbon and nitrogen regulator and the oily sludge, more preferably 10-15% by weight.

[0041] The fermentation described in step (2) can be completed in a fermentation tank. Under stirring and heating conditions, the material is mixed and heated evenly. The fermentation time is preferably 5 to 10 days, so that the moisture content and oil content decrease to the expected level.

[0042] The oily sludge described in this invention can be oily sludge from petrochemical enterprises or soil contaminated by petroleum. Both can be treated using the medium- and high-temperature petroleum-degrading bacteria of this invention to achieve superior oily sludge treatment results.

[0043] The present invention will be further described below with reference to the embodiments, but the scope of the present invention is not limited to these embodiments.

[0044] The formula for calculating the oil removal rate in this invention is as follows:

[0045] Oil removal rate (%) = ((M1×C1-M2×C2) / M1×C1)×100%

[0046] Where M1 is the mass of untreated oily sludge, C1 is the oil content of untreated oily sludge, M2 is the mass of treated oily sludge, and C2 is the oil content of treated oily sludge.

[0047] The present invention will be further described below with reference to the embodiments, but the scope of the present invention is not limited to these embodiments.

[0048] Example 1

[0049] Step 1: Take 1g of crude oil tank bottom sludge and place it in 100mL of sterile liquid petroleum culture medium. The specific components of the liquid petroleum culture medium are: K2HPO4 2g / L, KH2PO4 0.2g / L, NaCl 0.04g / L, (NH4)2SO4 4g / L, NaNO3 5g / L, MgSO4 0.4g / L, CaCl2 0.2g / L, and a concentrated trace element solution (cobalt chloride 0.1mg / L, manganese chloride 0.1mg / L, copper chloride 0.05mg / L, zinc chloride 0.003mg / L, boric acid 0.003mg / L, EDTA 0.1mg / L, (NH4)6Mo7O 24 5 mL of 4H2O 0.01 mg / L, Na2SeO3·6H2O 0.01 mg / L, NiCl2 0.002 mg / L, and petroleum (crude oil from a certain oil field, density 0.8 g / L, viscosity 1.26 mPa·S) 10 g / L.

[0050] Step 2: Place the liquid culture medium in a shaker and incubate for 5 days at a speed of 150 r / min and a temperature of 55℃.

[0051] Step 3: Transfer 5% of the culture to a new sterile liquid petroleum medium and continue culturing under the same conditions as in Step 2. Repeat this process 6 times. The liquid medium will become noticeably turbid.

[0052] Step 4: Take 1 mL of the bacterial culture from multiple cultures and dilute it serially to 10⁻⁶. -6The culture medium was coated with a mixture of [amount] times its original volume and incubated at 55°C for 3 days, during which colonies were observed to grow. Purification was performed by observing colony morphology and color, ultimately yielding the thermophilic petroleum-degrading bacterium BHYH-6. Colony morphology is shown in [reference needed]. Figure 1 .

[0053] Electron microscopy revealed that the colonies were bacilli. Figure 2 .

[0054] 16S rDNA sequencing of this strain yielded the nucleotide sequence shown in SEQ ID NO: 1. BLAST alignment of the obtained 16S rDNA sequence confirmed that the strain belongs to the genus *Brevibacillus*, and the recommended classification name is *Brevibacillus potstam*. It is currently deposited at the China General Biotechnology Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, on December 27, 2023, with accession number CGMCCNo. 29416.

[0055] Example 2

[0056] In this embodiment, the oily sludge being treated is crude oil tank bottom sludge from a certain refinery (60% water content, 24% oil content).

[0057] Step 1: Pick the BHYH-6 strain into LB liquid medium, place it on a shaker at 150 rpm and 55℃ for 2 days, and adjust the OD. 600 A value of 1 is used for the reduction treatment of sludge at the bottom of the crude oil tank.

[0058] Step 2: Mix straw, cow dung, and oil sludge in a ratio of 2:1:4, with a compost volume of 0.5L. Place the mixture in a fermentation tank, add 50mL of the bacterial solution from Step 1, start stirring, and activate the heating gradient to raise the temperature to 55℃, ensuring the compost is evenly mixed and heated. At this point, the compost has a moisture content of 55% and an oil content of 13%.

[0059] Step 3: After 7 days of fermentation, the moisture content of the compost pile decreased to 19%, and the oil content decreased to 7%. The oil removal rate was 46.2%, demonstrating a significant effect in the degradation of oily sludge.

[0060] Example 3

[0061] In this embodiment, the oily sludge being treated is sludge from the oil separator of a certain refinery (65% water content, 18% oil content).

[0062] Step 1: Pick the BHYH-6 strain into LB liquid medium, place it on a shaker at 150 rpm and 50℃ for 2 days, and adjust the OD. 600 A value of 1 is used for the reduction treatment of sludge at the bottom of the crude oil tank.

[0063] Step 2: Mix straw, cow dung, and oil sludge in a ratio of 2:1:4, forming a 1L compost. Place the compost in a fermentation tank, add 150mL of the bacterial solution from Step 1, start stirring, and activate the heating gradient to raise the temperature to 50℃, ensuring the compost is evenly mixed and heated. At this point, the compost has a moisture content of 60% and an oil content of 10%.

[0064] Step 3: After 7 days of fermentation, the moisture content of the compost pile decreased to 23%, the oil content decreased to 5.5%, and the oil removal rate was 45%, showing a significant effect in the degradation of oil sludge.

[0065] Example 4

[0066] In this embodiment, the oily sludge being treated is sludge from the oil separator of a certain refinery (65% water content, 18% oil content).

[0067] Step 1: Pick the BHYH-6 strain into LB liquid medium, place it on a shaker at 150 rpm and 50℃ for 2 days, and adjust the OD. 600 A value of 1 is used for the reduction treatment of sludge at the bottom of the crude oil tank.

[0068] Step 2: Mix straw, cow dung, and oil sludge in a ratio of 2:2:4, forming a 1L compost. Place the compost in a fermentation tank, add 150mL of the bacterial solution from Step 1, start stirring, and activate the heating gradient to raise the temperature to 50℃, ensuring the compost is evenly mixed and heated. At this point, the compost has a moisture content of 63% and an oil content of 9.4%.

[0069] Step 3: After 7 days of fermentation, the moisture content of the compost pile decreased to 24.5%, the oil content decreased to 5.1%, and the oil removal rate was 45.7%, showing a significant effect in the degradation of oil sludge.

[0070] Example 5

[0071] In this embodiment, the oily sludge being treated is sludge from the oil separator of a certain refinery (65% water content, 18% oil content).

[0072] Step 1: Pick the BHYH-6 strain into LB liquid medium, place it on a shaker at 150 rpm and 50℃ for 2 days, and adjust the OD. 600 A value of 1 is used for the reduction treatment of sludge at the bottom of the crude oil tank.

[0073] Step 2: Mix straw, cow dung, and oil sludge in a ratio of 1:2:4, forming a 1L compost. Place the compost in a fermentation tank, add 150mL of the bacterial solution from Step 1, start stirring, and activate the heating gradient to raise the temperature to 50℃, ensuring the compost is evenly mixed and heated. At this point, the compost has a moisture content of 70.4% and an oil content of 10.6%.

[0074] Step 3: After 7 days of fermentation, the moisture content of the compost pile decreased to 33.7%, the oil content decreased to 6.9%, and the oil removal rate was 34.9%, indicating a good degradation effect of the oil sludge.

[0075] Example 6

[0076] In this embodiment, the oily sludge being treated is crude oil tank bottom sludge from a certain refinery (60% water content, 24% oil content).

[0077] Step 1: Pick the BHYH-6 strain into LB liquid medium, place it on a shaker at 150 rpm and 55℃ for 2 days, and adjust the OD. 600 A value of 1 is used for the reduction treatment of sludge at the bottom of the crude oil tank.

[0078] Step 2: Mix straw, cow dung, and oil sludge in a ratio of 2:1:4, forming a 0.5L compost pile. Place the pile in a fermentation tank, add 50mL of the bacterial solution from Step 1, start stirring, and activate the heating gradient to raise the temperature to 40℃, ensuring the compost is evenly mixed and heated. At this point, the compost has a moisture content of 55% and an oil content of 13%.

[0079] Step 3: After 7 days of fermentation, the moisture content of the compost pile decreased to 28%, and the oil content decreased to 9.4%. The oil removal rate was 27.7%, and the degradation effect of the oil sludge was worse than that of fermentation at 55℃.

[0080] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

[0081] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

Claims

1. A medium-to-high temperature petroleum-degrading bacterium, characterized in that, The medium- and high-temperature petroleum-degrading bacteria mentioned are *Brevibacillus borstelensis*, with accession number CGMCC No. 29416.

2. The application of the medium- and high-temperature petroleum-degrading bacteria described in claim 1 in the degradation of oily sludge.

3. A method for degrading oily sludge, comprising the following steps: The medium- and high-temperature petroleum-degrading bacteria described in claim 1 are brought into contact with oily sludge.

4. The method for degrading oily sludge according to claim 3, wherein, The contact conditions include a temperature of 50–60°C and a humidity of 60–70%.

5. The method for degrading oily sludge according to claim 3, wherein, The method includes the following steps: (1) Activation and enrichment of strains: The medium- and high-temperature petroleum-degrading bacteria were inoculated into a liquid culture medium and cultured at a constant temperature with shaking until the logarithmic growth phase to prepare a bacterial solution; (2) Degradation of oily sludge: The carbon and nitrogen regulator is mixed with the oily sludge, the bacterial solution is added, the temperature is gradually increased to 50-60℃, and aerobic fermentation is carried out under stirring conditions to degrade the petroleum hydrocarbons in the oily sludge.

6. The method for degrading oily sludge according to claim 5, wherein, In step (1), the culture medium is LB medium.

7. The method for degrading oily sludge according to claim 5, wherein, In step (1), the conditions for constant temperature oscillation include: temperature of 50-60℃, rotation speed of 100-200r / min, and time of 30-60h.

8. The method for degrading oily sludge according to claim 5, wherein, In step (2), the carbon-nitrogen regulator is a high-carbon and high-nitrogen substance; The high-carbon material is preferably straw and / or wood chips; The high-nitrogen-content substance is preferably animal manure, and more preferably cow manure; The weight ratio of the high carbon content, high nitrogen content and oily sludge is (2-3):(0.5-2.5):

4.

9. The method for degrading oily sludge according to claim 5, wherein, In step (2), the amount of bacterial solution added is 5-20% by weight of the total weight of the carbon-nitrogen regulator and the oily sludge, preferably 10-15% by weight.

10. The method for degrading oily sludge according to claim 5, wherein, In step (2), the fermentation time is 5 to 10 days.

11. The method for degrading oily sludge according to any one of claims 3-10, wherein, The oily sludge refers to oily sludge from petrochemical enterprises or soil contaminated by petroleum.

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