A surfactant-producing petroleum-degrading bacterium KMJ-1 and its use

Abstract

The present invention belongs to the field of environmental microbiology, and specifically relates to a surfactant-producing petroleum-degrading bacterium KMJ-1 and its use. The technical points are as follows: the present invention is provided in the General Biological Center of the China Microbiological Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the deposit number is CGMCC No.21171. The surfactant-producing petroleum-degrading bacterium KMJ-1 provided by the present invention has the functions of producing surfactants and degrading petroleum. The strain has a strong ability to produce surfactants and can reduce the surface tension of pure water from 72.01 mN / m to 39.53 mN / m; the degradation rate of petroleum hydrocarbons in scum degraded by KMJ-1 strain is up to 80.06%; the degradation rate of petroleum hydrocarbons in biochemical sludge degraded by KMJ-1 strain is up to 85.88%.

Description

Technical Field

[0001] The invention belongs to the field of environmental microbiology, and in particular relates to a surfactant-producing petroleum-degrading bacterium KMJ-1 and a use thereof. Background Art

[0002] With the rapid development of social productivity, the demand for petroleum energy has been rising year by year. The amount of oily sludge generated during oil extraction, transportation and smelting has also increased day by day, and it is also facing the problem of being difficult to degrade. To address this problem, physical, chemical and biological methods can be used to degrade oily sludge, and the biological method has attracted the attention of many scholars due to its advantages of being green, environmentally friendly and free of secondary pollution. At the same time, due to the stable oil-water interface in oily sludge, the degradation ability of biological methods on oily sludge cannot be improved rapidly. Studies have shown that surfactants can effectively break the oil-water interface and accelerate the degradation of oily sludge by microorganisms. Chemical surfactants have high output, low cost and good effect, but because they are chemical reagents, they violate the "harmless" principle of treating hazardous waste. Therefore, biological surfactants have become a research hotspot for many scholars due to their green and harmless, degradable, low-hazard, mild, high-efficiency and environmentally friendly characteristics. At present, many bacterial species have been discovered that have the ability to produce biosurfactants, including Bacillus sp., Pseudomonas sp., Paenibacillus sp., etc., all of which can produce biosurfactants.

[0003] However, the current production of biosurfactants is relatively low, so it is necessary to promptly discover new high-yield and efficient surfactant-producing petroleum-degrading bacteria.

[0004] In view of the above-mentioned defects in the existing oily sludge degradation, the inventors, based on their rich experience and professional knowledge in this type of material for many years, combined with theoretical analysis, conducted research and innovation, and developed a surfactant-producing oil-degrading bacterium KMJ-1 and its uses. Summary of the invention

[0005] The purpose of the present invention is to provide a surfactant-producing and petroleum-degrading bacterium KMJ-1, which has the functions of producing surfactants and degrading petroleum.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions:

[0007] The surfactant-producing petroleum-degrading bacteria KMJ-1 provided by the present invention was screened out from the oily sludge scum of Daqing Oilfield provided by Jinling Branch of China Petrochemical Corporation, and was identified as Alcaligenes sp. It is currently preserved in the General Biological Center (CGMCC) of the China Microbiological Culture Collection Committee of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The preservation time is November 13, 2020, and the preservation number is CGMCC No. 21171.

[0008] The surfactant-producing petroleum-degrading bacteria KMJ-1 provided by the present invention has a strong ability to produce surfactants, and the surfactant can reduce the surface tension of pure water from 72.01 mN / m to 39.53 mN / m.

[0009] The degradation rate of petroleum hydrocarbons by KMJ-1 strain was up to 80.06% on the 7th day of the scum degradation test; the degradation rate of petroleum hydrocarbons by KMJ-1 strain was up to 85.88% on the 7th day of the biochemical sludge degradation test.

[0010] Another object of the present invention is to provide a use of surfactant-producing petroleum-degrading bacteria KMJ-1, which has the same technical effect.

[0011] The above technical effects of the present invention are achieved by the following technical solutions:

[0012] The surfactant-producing mobile phone degrading bacteria KMJ-1 provided by the present invention is used to prepare the surfactant-producing petroleum degrading bacteria KMJ-1 as inoculants for producing surfactants.

[0013] Furthermore, the inoculum bacteria were screened and identified by blood plate screening, oil diffusion method to determine the oil expulsion activity and characterization of the emulsification properties.

[0014] Furthermore, the raw materials for producing the surfactant include the following components in addition to the inoculum:

[0015] The raw materials for producing the surfactant include the following components in addition to the inoculum: 15-25 g sucrose, 5-15 g urea, 0.15-0.1 g MgSO4•7H2O, 0.5-1.5 g KH2PO4 and 1000 mL distilled water.

[0016] Furthermore, in addition to the inoculum, the raw materials for producing the surfactant preferably include 20 g sucrose, 4.62 g urea, 0.125 g MgSO4•7H2O, 1 g KH2PO4 and 1000 mL distilled water.

[0017] Furthermore, the temperature for producing the surfactant is 20-30°C, preferably 25°C.

[0018] Furthermore, the step of producing the surfactant also includes separation and purification of the surfactant.

[0019] Furthermore, the surfactant is separated by the following specific operations: centrifuging the fermentation liquid after 3 days of culture to remove the bacteria, adjusting the pH, standing at low temperature, taking the supernatant and adding the extractant, and drying the organic phase to obtain the crude product of the surfactant.

[0020] Furthermore, the purification of the surfactant specifically includes the following steps: dissolving the crude product of the surfactant in a 0.05 mol / L NaHCO3 solution, filtering insoluble impurities, adjusting the pH, centrifuging, collecting the precipitate, and drying to obtain the surfactant.

[0021] The present invention also provides another use of the surfactant-producing petroleum-degrading bacteria KMJ-1. The KMJ-1 strain degrades scum, and the petroleum hydrocarbon degradation rate is as high as 80.06% in 7 days; the KMJ-1 strain degrades biochemical sludge, and the petroleum hydrocarbon degradation rate is as high as 85.88% in 7 days.

[0022] The above technical objectives of the present invention are achieved by the following technical solutions:

[0023] The purpose of the surfactant-producing petroleum-degrading bacteria KMJ-1 provided by the present invention is to prepare the surfactant-producing petroleum-degrading bacteria KMJ-1 into seed liquid for degrading oil-containing sludge.

[0024] Furthermore, the seed liquid was centrifuged to remove the bacterial cells from the fermentation liquid of strain KMJ-1 cultured for 3 days, and the pH of the supernatant was adjusted to 2.0, and the supernatant was allowed to stand at 4°C for 24 hours, extracted and dried, and the obtained

[0025] Furthermore, the degradation temperature is 25°C and the degradation time is 7 days.

[0026] In summary, the present invention has the following beneficial effects:

[0027] The surfactant-producing petroleum-degrading bacteria KMJ-1 provided by the present invention has the functions of both producing surfactants and degrading petroleum. The strain has a strong ability to produce surfactants and can reduce the surface tension of pure water from 72.01 mN / m to 39.53 mN / m; the degradation rate of petroleum hydrocarbons in scum by the KMJ-1 strain is as high as 80.06%; the degradation rate of petroleum hydrocarbons in biochemical sludge by the KMJ-1 strain is as high as 85.88%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the phylogenetic tree of the 16SrDNA gene sequence of the KMJ-1 strain;

[0029] Figure 2 This is the infrared chromatogram of KMJ-1 bacterial products. DETAILED DESCRIPTION

[0030] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, a surfactant-producing petroleum-degrading bacterium KMJ-1 proposed in accordance with the present invention, its specific implementation method, characteristics and effects are described in detail as follows.

[0031] Example 1: Isolation and screening of surfactant-producing petroleum-degrading bacteria KMJ-1

[0032] The surfactant-producing petroleum-degrading bacteria KMJ-1 provided in this embodiment was identified as Alcaligenes sp. and is currently preserved in the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The preservation date is November 18, 2020, and the preservation number is CGMCC No.21171.

[0033] The separation and screening specifically includes the following steps:

[0034] (1) The strain was screened from the oily sludge scum of Jinling Petrochemical Company. 1.25 g crude oil was added to 250 mL beef extract peptone medium, sterilized at 121°C for 20 min, cooled to room temperature, and 10 g scum was added. The first generation was enriched and cultured in an oscillating incubator at 140 r / min and 25°C. After 3 days of culture, 10 mL of the first generation culture solution was taken and transferred to 250 mL of sterilized fresh beef extract peptone medium (the crude oil mass was increased to 2.5 g). The second generation was enriched and cultured in an oscillating incubator at 140 r / min and 25°C at room temperature. After 3 days, 10 mL of the second generation culture solution was taken and transferred to 250 mL of sterilized fresh beef extract peptone medium (the crude oil mass was increased to 5 g again). After 3 days, the third generation culture solution was obtained. Take 1 mL of the third generation culture solution and dilute it in a gradient of 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6, and apply 1 mL of each gradient bacterial solution on the solid culture medium. Cultivate at 25℃ for 3 days, observe the morphology of the colonies, and preliminarily identify the presence of 4 different strains, which are separated and purified by streaking.

[0035] (2) Use an inoculation loop to pick out the four strains obtained and culture them for three days. Streak the plates three times to obtain the purified strains.

[0036] (3) Use an inoculation loop to pick up a small amount of isolated and purified colonies, inoculate them into beef extract peptone medium, and culture them at 130r / min at room temperature for 3 days. Take 1µL and drop it on a blood plate, culture it at 25℃ for 48h, and observe whether there is a hemolytic zone around the colony. Only one strain produced a hemolytic zone around the colony, named KMJ-1.

[0037] (4) Determination of oil-diffusion activity by oil diffusion method: Pour 30 ml of distilled water into a 9 cm diameter culture dish, add 1 mL of liquid paraffin, wait for an oil film to form on the water surface, then drop 10 µL of fermentation liquid into the center of the oil film. The diameter of the oil-draining circle was measured to be 5.5 cm.

[0038] (5) Emulsification performance: Add 3 ml of n-hexane and 2 ml of fermentation liquid to the colorimetric tube, shake vigorously for 60 seconds, let it stand for 24 hours, and observe the height of the emulsion layer.

[0039]

[0040] The emulsification performance was measured to be 67%.

[0041] Identification of strain 16SrDNA: The genomic DNA of the strain was extracted using Shanghai Sangon E2up column bacterial genomic DNA extraction kit, and then tested by 1% agarose gel electrophoresis. Shanghai Sangon Biotechnology Technology Service Co., Ltd. was commissioned to complete the purification and sequencing of PCR products. After the sequence obtained by sequencing was entered into Gene Bank, the BLAST comparison was used to analyze the homology and draw the phylogenetic tree, see Figure 1 .

[0042] Example 2: Use of surfactant-producing petroleum-degrading bacteria KMJ-1 for producing surfactants

[0043] This embodiment provides a method for producing surfactants using surfactant-producing petroleum-degrading bacteria KMJ-1, and the specific operation is as follows:

[0044] (1) Surfactant separation: The fermentation liquid after 3 days of culture was centrifuged to remove the bacteria. The supernatant was adjusted to pH 2.0 and placed in a 4°C refrigerator for 24 hours to observe whether there was precipitation. If there was precipitation, the surfactant may contain lipopeptides or lipoproteins. Otherwise, glycolipids may be present. Take the supernatant and add an equal volume of extractant. Extract twice, combine the organic phases and dry with anhydrous NaSO4, and evaporate at 45°C until a light yellow thick substance is obtained, which is the crude surfactant product.

[0045] (2) Purification of surfactant: The crude product was dissolved in 0.05 mol / L NaHCO3 solution, insoluble impurities were filtered out, the pH was adjusted to 2.0, and the product was centrifuged at 9000 r / min at 4 °C for 15 min. The precipitate was collected and dried at 40 °C to obtain the purified surfactant.

[0046] Surfactant identification: infrared scanning analysis (FI-IR), the above-mentioned purified surfactants were mixed and pressed into transparent thin sheets using the KBr tablet method, and then subjected to infrared scanning analysis and determination, see Figure 2 As can be seen from the figure, 1022.69 and 1093.94 cm -1 The absorption band indicated the existence of an ester structure, proving that the product of KMJ-1 was a glycolipid surfactant.

[0047] Example 3: Use of surfactant-producing petroleum-degrading bacteria KMJ-1 for the degradation of oily sludge

[0048] This embodiment provides a method for using surfactant-producing petroleum-degrading bacteria KMJ-1 to degrade oily sludge, and the specific operation is as follows:

[0049] The preparation process includes the following steps:

[0050] Inoculate 1 mL of seed solution into 19 mL of degradation medium sterilized by high temperature and high pressure at a 5% inoculation rate, then add 0.1 g of oily sludge, place it in an oscillating incubator at 150 r / min and 25 °C for degradation for 7 days, and use an infrared oil meter to measure the oil content of the oily sludge every day.

[0051]

[0052] w1——determination of oil content after degradation;

[0053] w2——Determine the oil content before degradation.

[0054] The degradation rate of petroleum hydrocarbons in sludge by KMJ-1 strain was as high as 85.88% after 7 days.

[0055] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been presented as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A strain of Alcaligenes sp. KMJ-1, characterized in that: The KMJ-1 strain is preserved at the General Biological Center of China Microbiological Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC No.21171, and the preservation time is November 13, 2020.

2. The use of the KMJ-1 strain in the degradation of petroleum hydrocarbons as claimed in claim 1, characterized in that: The petroleum hydrocarbons are petroleum hydrocarbons in sludge. The KMJ-1 strain is prepared into a seed liquid for degrading the petroleum hydrocarbons in the sludge.

3. The use according to claim 2, characterized in that: The degradation temperature is 25° C. and the degradation time is 7 days.

Images