CN109777747B - Oil sludge petroleum degrading strain and culture method and application thereof - Google Patents
Oil sludge petroleum degrading strain and culture method and application thereof Download PDFInfo
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Abstract
The invention relates to an oil sludge petroleum degrading strain and a culture method and application thereof. A strain of oil sludge petroleum degrading bacteria ECO-17 is preserved in China general microbiological culture Collection center in 2018, 06 months and 01 days, and the strain preservation number is as follows: CGMCC No. 15837. The invention discloses a screened oil sludge petroleum degrading bacterium ECO-17 for the first time, which does not need to additionally add surfactants such as microemulsion and the like, avoids the secondary environmental pollution and the limitation on the later-stage cyclic utilization of the treated oil sludge caused by adding chemical reagents, and greatly reduces the treatment cost. The strain can treat the oil sludge waste of the storage tank with the oil content higher than 10-50%, and has good treatment effect on the oil-polluted soil and water with the oil content lower than 10%.
Description
Technical Field
The invention relates to an oil sludge petroleum degrading strain and a culture method and application thereof, belonging to the technical field of microorganisms.
Background
With the increasing demand of petroleum products, petroleum and its products enter the environment through various ways, causing serious pollution to soil and water, and threatening the health of human beings, and the research and application of petroleum-contaminated soil technology are increasingly receiving wide attention and attention. The existing treatment technology mainly comprises a physical method, a chemical method and a biological method, and because physical remediation (such as heat treatment) destroys pollutants in soil and also destroys components and structures of the soil, the existing treatment technology is expensive; the chemical remediation effect is good, but the used chemical reagent can generate secondary pollution, so that the application range of the chemical reagent is limited; the biodegradation process has the advantages of high speed, no residual toxicity, low cost and wide application.
The bioremediation technology is considered to be an oil pollution remediation method which is green, environment-friendly, free of secondary pollution, efficient and capable of thoroughly degrading pollutants and has development prospects. Researches show that the activity of petroleum degrading bacteria enzyme, environment, inorganic nutrition, fertilizer, microorganism types and the like play an important role in repairing oil-polluted sites. The method is a unique angle for carrying out microbial degradation analysis on the whole petroleum family components in petroleum geology, and needs to carry out comprehensive analysis on the whole petroleum family components, and the researches on degradation of normal paraffins, pristanes and phytanes exist in the researches, but the atlas analysis of the hopane and sterane components is difficult to identify, the content is low, and the researches are few.
Biodegradation refers to the process of decomposition of complex compounds catalyzed by organisms. In the petroleum degradation, microorganisms firstly generate catabolic enzymes through self metabolism, crack heavy hydrocarbons and crude oil, reduce the viscosity of petroleum, and in the growth and propagation processes, effective compounds such as solvents, acids, gases, surfactants, biopolymers and the like can be generated to facilitate oil displacement, and then other microorganisms are further oxidized and decomposed into small molecules to achieve the purpose of degradation.
It can be seen that screening for microorganisms capable of specifically degrading petroleum is a key to bioremediation techniques.
International patent document WO9905392A1 (application number WOGB98002217) discloses a method for removing oil from spent drilling mud comprising mixing the spent drilling mud with a microemulsion-forming surfactant and allowing the mixture to separate into phases, wherein the interfacial tension between the microemulsion phase and the conjugated polar phase is less than 10-4mNm-1. The oil may be biodegraded by inoculation with a hydrocarbon degrading microorganism (e.g. of the genera Rhodococcus, Gordona or Tsukamurella).
In the process of petroleum storage in the petrochemical industry, crude oil is precipitated by an oil storage tank to generate a large amount of hazardous waste oil sludge, the content of the crude oil in the oil sludge reaches over 10 percent, and the oil sludge is stacked in the environment to cause serious pollution and damage to ecology such as air, soil and the like. The components of the crude oil are complex, the components of Persistent Organic Pollutants (POPs) such as polycyclic aromatic hydrocarbon and the like have high toxicity and long-distance mobility, and can exist persistently in the environment, and the difficulty of restoring the high-concentration crude oil pollution by a green and environment-friendly biological method is great. The method for removing oil from waste drilling mud disclosed in international patent document WO9905392A1 needs to add microemulsion to form surfactant, the addition of chemical reagent can cause secondary pollution to the environment, the application effect and the addition cost of microemulsion are unknown for the treatment of oil sludge waste in a storage tank with oil content higher than 10% -50%, and meanwhile, the later recycling of oil-containing sand treated by using chemical surfactant microemulsion is correspondingly limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an oil sludge petroleum degrading strain and a culture method and application thereof.
The technical scheme of the invention is as follows:
a strain of fatlute petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 is preserved in China general microbiological culture Collection center in 2018 in 06 months 01, and the address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the strain preservation number: CGMCC No. 15837.
The method for culturing the oil sludge petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 comprises the following steps:
(1) taking oil sludge and petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17, streaking on a solid activation culture medium, and performing activation culture to obtain an activated strain;
(2) inoculating the activated strain prepared in the step (1) into a liquid culture medium, and performing shake culture to prepare a seed solution;
(3) transferring the seed liquid prepared in the step (2) into an amplification culture medium according to the volume percentage of 2-10%, and performing amplification culture to obtain a bacterial liquid.
Preferably, in step (1), the solid activation medium is LB solid medium, and the components are as follows:
10g of peptone, 5g of yeast extract, 10g of sodium chloride, 20g of agar, and water with a constant volume of 1L, wherein the pH value is natural.
Preferably, in step (1), the activation conditions are: and (3) carrying out inverted culture at the temperature of 28-37 ℃ for 1-2 days.
Preferably, the liquid culture medium in step (2) and the amplification culture medium in step (3) are both LB liquid culture media, and the components are as follows:
10g of peptone, 5g of yeast extract, 10g of sodium chloride and water until the volume is 1L, and the pH is natural.
According to the present invention, the shake cultivation conditions in step (2) are preferably as follows: and (3) carrying out shake culture for 1-2 days at the rotation speed of 100-200 rpm at the temperature of 28-37 ℃.
Preferably, according to the present invention, the condition of the expanded culture in the step (3) is: and (3) performing amplification culture for 1-2 days at the temperature of 28-37 ℃ and the dissolved oxygen of 20-70%.
The oil sludge petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 is applied to oil sludge petroleum degradation.
According to a preferred embodiment of the present invention, the steps of the application are as follows:
(i) mixing oil sludge and an inorganic salt culture medium according to a mass-volume ratio of 1: (5-30) mixing in unit g/ml to prepare oil sludge to be degraded;
(ii) and (3) inoculating a bacterial liquid of oil sludge degrading bacteria (Tsukamurella) ECO-17 into the oil sludge to be degraded, wherein the bacterial liquid is inoculated according to the volume mass percentage of 1-10%, and is cultured for 3-5 days in a unit ml/g under the conditions that the temperature is 28-37 ℃ and the dissolved oxygen is 20-70%.
Preferably, in step (i), the inorganic salt medium has the following composition per liter:
KNO31.5g，(NH4)SO41.5g，K2HPO41g，KH2PO41g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2O 0.01g，dH2and O is metered to 1L.
Advantageous effects
The invention discloses a screened oil sludge petroleum degrading bacterium (Tsukamurella pulmonis) ECO-17 for the first time, compared with the existing known hydrocarbon degrading microorganism (such as International patent document WO9905392A 1) of Tsukamurella for removing oil from waste drilling mud, the strain does not need to additionally add a surfactant such as microemulsion, so that the secondary environmental pollution caused by adding a chemical reagent and the limitation on the later-stage recycling of the treated oil sludge are avoided, and the treatment cost is greatly reduced. The strain can treat the oil sludge waste of the storage tank with the oil content higher than 10-50%, and has good treatment effect on the oil-polluted soil and water with the oil content lower than 10%.
Drawings
FIG. 1 is a photograph showing the results of electrophoresis after 16S rDNA amplification in the identification of oil degrading bacteria (Tsukamurella pulmonis) ECO-17;
in the figure: lane M, D2000DNA Marker; lane 17, 16SrDNA amplification fragment of Tsukamurella pulmonis ECO-17.
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
Source of biological material
A strain of oily sludge petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 is preserved in China general microbiological culture Collection center in 2018, 06 months and 01 days, and is deposited at China academy of sciences, China institute of microbiology, No. 3, Xilu No.1, North Chen, of the Chaoyang district, Beijing, with the strain preservation number of CGMCC No. 15837.
Culture medium
The inorganic salt culture medium comprises the following components per liter:
KNO31.5g，(NH4)SO41.5g，K2HPO41g，KH2PO41g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2O 0.01g，dH2o is constant volume to 1L;
the petroleum-inorganic salt solid culture medium comprises the following components per liter:
KNO31.5g，(NH4)SO41.5g，K2HPO41g，KH2PO41g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2o0.01 g, 10% petroleum, agar 20g, dH2And O is metered to 1L.
The petroleum-inorganic salt liquid culture medium comprises the following components per liter:
KNO31.5g，(NH4)SO41.5g，K2HPO41g，KH2PO41g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2o0.01 g, 10% Petroleum, dH2And O is metered to 1L.
Example 1
Collecting 2g of a sediment bottom mud sample of an oil storage tank of the southeast oilfield, placing the sediment bottom mud sample in a 150mL sterile triangular flask, adding 50mL of sterile inorganic salt culture medium and 2g of sterile glass beads, culturing the sediment bottom mud sample for 3d at 30 ℃ and 150rpm, sucking bacterial liquid, performing gradient dilution by using sterile water until the sediment bottom mud sample is respectively diluted to 10 DEG-1，10-2，10-3，10-4，10-5And (2) coating 100 mu L of sterile petroleum-inorganic salt solid culture medium respectively, standing and culturing for 3d at the temperature of 30 ℃, selecting clones with fast growth and large colonies to 50mL of petroleum-inorganic salt liquid culture medium, culturing for 3d at the temperature of 30 ℃ and 150rpm, namely absorbing 100 mu L of bacterial liquid and coating the bacterial liquid to the sterile petroleum-inorganic salt solid culture medium, and selecting monoclonals.
The single clone is sent to Shandong Shengqiang biological science and technology limited company for sequencing, the detected 16S rDNA sequence contains 1385bp, the nucleotide sequence is shown as SEQ ID NO.1, the strain is identified to belong to Tsukaamurella pulmonis and is named as oil sludge petroleum degrading bacteria (Tsukaamurella pulmonis) ECO-17, the strain is preserved in China general microbiological culture Collection center in 2018 in 06 months 01, and the microbial research institute of China academy of sciences No. 3 of West Lu 1 of the North Cheng of the Korean area, Beijing, and the strain preservation number is CGMCC No. 15837.
The strain identification process is as follows:
sample preparation: bacterial liquid;
bacterial genome DNA extraction kit: tiangen Biochemical technology (Beijing) Ltd., DP 302;
TAE buffer (50X, 1L): 242g of Tris, 57.1ml of glacial acetic acid, 37.2g of Na2EDTA.2H2O and water added to 1L;
agarose: BIOWET, AGAROSE G-10;
2 Xpfu PCR MasterMix, D2000DNA Marker, nucleic acid dye, loading buffer, etc.: tiangen Biochemical technology (Beijing) Ltd
DNA purification recovery kit: tiangen Biochemical technology (Beijing) Ltd, DP214
Centrifuge tube, rifle head consumptive material such as: the American Gene Era Biotech company (GEB)
Primer: synthesized by Suzhou Jinzhi Biotechnology Ltd, ddH2O was added to the solution to prepare a 10. mu.M solution.
1. Genomic DNA was extracted and the kit was used DP 302.
2. PCR amplification
2.1 Universal primer information
2.2 PCR amplification System Components and compositions
2.3 PCR cycle parameters
Pre-denaturation: 94 ℃ for 3 min; denaturation 94 ℃, 30s, annealing 55, 30s, extension 72 ℃, 1.5min (35 cycles total); extending at 72 ℃ for 10 min; storing at 4 ℃.
3. Agarose gel electrophoresis detection
1.0% agarose gel was prepared, and the voltage was set at 18V/cm for 20 min. And carrying out agarose electrophoresis dyeing by adopting a nucleic acid dye, and taking a picture by adopting an ultraviolet gel imaging system. The results are shown in FIG. 1.
4. Purification and recovery
And (3) carrying out agarose gel recovery on the target fragment by using a common agarose gel DNA recovery kit, and sending the recovered product to the Suzhou Jinzhi Biotech limited company for sequencing.
Sequencing splicing sequence and blast alignment:
through sequence alignment of the 16S rDNA, the strain with the closest genetic relationship is found to be the strain number NR _ 029302.1.
Example 2
The method for culturing the oil sludge petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 comprises the following steps:
(1) taking oil shale oil degrading bacteria (Tsukamurella pulmonis) ECO-17, streaking on an LB solid culture medium, inverting and activating at 35 ℃ for 2 days to prepare an activated strain;
(2) inoculating the activated strain prepared in the step (1) into an LB liquid culture medium, and performing shake culture for 2 days at the rotating speed of 150 revolutions per minute at 35 ℃ to prepare a seed solution;
(3) transferring the seed liquid prepared in the step (2) into an LB liquid culture medium according to the volume percentage of 2-10%, and carrying out expanded culture for 2 days under the conditions of 35 ℃ and 20-70% dissolved oxygen to obtain a bacterial liquid.
The detected thallus concentration of the bacteria liquid is 109cfu/ml。
Example 3
Application of oil sludge petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 in oil sludge petroleum degradation comprises the following steps:
(i) mixing oil sludge with the oil content of 10% with an inorganic salt culture medium according to the mass-volume ratio of 1: 5, mixing in unit g/ml to prepare oil sludge to be degraded;
(ii) and (3) inoculating a bacterial liquid of sludge petroleum degrading bacteria (Tsukamuramulmonis) ECO-17 into the sludge to be degraded, which is prepared in the step (i), inoculating the bacterial liquid according to the proportion of 5% by volume and mass percent, and culturing for 5 days at the temperature of 37 ℃ and the rotating speed of 200 r/min to obtain the bacterial liquid.
The weight method is adopted to detect the degradation rate of the petroleum in the oil sludge, and the degradation rate of oil sludge petroleum degrading bacteria (Tsukamurella) ECO-17 to the oil sludge with 10 percent of oil content can reach 21.9 percent within 5 days.
Comparative example
According to the method of the example 3, the same oil degradation experiment is carried out by using the known strain NR _029302.1, and the oil degradation rate of the oil sludge with the oil content of 10 percent in 5 days is only 0.35 percent by detecting the known strain NR _ 029302.1.
Analysis of results
As can be seen from the oil degradation data of the oil sludge with the oil content of 10% in the example 3 and the comparative example, the oil sludge oil degrading bacteria (Tsukamurella pulmonis) ECO-17 disclosed by the invention can directly degrade the oil in the oil sludge without using a chemical surfactant for treatment, so that the treatment components and the treatment environmental risk are greatly reduced, and the oil sludge degrading bacteria have a very significant advantage in treating oil sludge pollution compared with the existing known oil degrading bacteria.
Claims (10)
1. A strain of fatlute petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 is preserved in China general microbiological culture Collection center in 2018 in 06 months 01, and the address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the strain preservation number: CGMCC No. 15837.
2. The method for culturing the fatlute petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17 as claimed in claim 1, wherein the steps are as follows:
(1) taking oil sludge and petroleum degrading bacteria (Tsukamurella pulmonis) ECO-17, streaking on a solid activation culture medium, and performing activation culture to obtain an activated strain;
(2) inoculating the activated strain prepared in the step (1) into a liquid culture medium, and performing shake culture to prepare a seed solution;
(3) transferring the seed liquid prepared in the step (2) into an amplification culture medium according to the volume percentage of 2-10%, and performing amplification culture to obtain a bacterial liquid.
3. The culture method according to claim 2, wherein in the step (1), the solid activation medium is LB solid medium, and the components are as follows:
10g of peptone, 5g of yeast extract, 10g of sodium chloride, 20g of agar, and water with a constant volume of 1L, wherein the pH value is natural.
4. The culture method according to claim 2, wherein in the step (1), the activation conditions are: and (3) carrying out inverted culture at the temperature of 28-37 ℃ for 1-2 days.
5. The culture method according to claim 2, wherein the liquid culture medium in step (2) and the expanded culture medium in step (3) are both LB liquid culture media and have the following compositions:
10g of peptone, 5g of yeast extract, 10g of sodium chloride and water until the volume is 1L, and the pH is natural.
6. The method according to claim 2, wherein the shake culture conditions in step (2) are: and (3) carrying out shake culture for 1-2 days at the rotation speed of 100-200 rpm at the temperature of 28-37 ℃.
7. The culture method according to claim 2, wherein the condition for the expanded culture in the step (3) is: and (3) performing amplification culture for 1-2 days at the temperature of 28-37 ℃ and the dissolved oxygen of 20-70%.
8. Use of the fatlute oil-degrading bacterium (Tsukamurella pulmonis) ECO-17 as claimed in claim 1 in the degradation of fatlute oil.
9. Use according to claim 8, characterized by the steps of:
(i) mixing oil sludge and an inorganic salt culture medium according to a mass-volume ratio of 1: (5-30) mixing in unit g/ml to prepare oil sludge to be degraded;
(ii) and (3) inoculating a bacterial liquid of oil sludge degrading bacteria (Tsukamura pulmonis) ECO-17 into the oil sludge to be degraded, wherein the bacterial liquid is inoculated according to the volume mass percentage of 1-10%, and is cultured for 3-5 days in a unit ml/g under the conditions that the temperature is 28-37 ℃ and the dissolved oxygen is 20-70%.
10. The use according to claim 9, wherein in step (i), the mineral salts medium has the following composition per liter:
KNO31.5g，(NH4)SO41.5g，K2HPO41g，KH2PO41g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2O 0.01g，dH2and O is metered to 1L.
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