CN108102979B - Degradation strain JN5 for petroleum hydrocarbons in oily sludge and application thereof - Google Patents
Degradation strain JN5 for petroleum hydrocarbons in oily sludge and application thereof Download PDFInfo
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Abstract
The invention discloses a strain JN5 for efficiently degrading petroleum hydrocarbons in oily sludge and application thereof, belonging to the technical field of bioremediation. The degrading bacteria JN5 is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.14976 and the classification number of Pseudomonas stutzeri. The strain JN5 with the petroleum hydrocarbon degradation function is used for treating oily sludge in a petroleum hydrocarbon degradation mode. The degradation rate of the bacteria on Total Petroleum Hydrocarbon (TPH) in the oily sludge within 30 days is 73.73%. The bacterium is a single strain of p-n-C10～n‑C26The straight-chain petroleum hydrocarbon has stronger degradation capability. The bacteria have optimal degradation conditions of 20-50 deg.c and pH 7-11 for petroleum pollutant. The biological method is used for the technology for restoring the oily sludge, and has the advantages of high efficiency, low cost, environmental friendliness and the like.
Description
Technical Field
The invention relates to the technical field of bioremediation, in particular to a bacterial strain for degrading and restoring oily sludge and application thereof in degrading the oily sludge.
Background
Since the 20 th century, petroleum has become one of the most important energy sources for human beings, and while being largely exploited and used, the damage of petroleum and its processed products to the environment and human beings has become more and more serious. Since the annual yield of crude oil exceeds 1 hundred million t since the innovation of China, China which is one of ten major oil producing countries in the world has more than 400 oil fields and oil and gas fields which are explored and developed and are distributed in 25 provinces (cities) and autonomous regions in the country according to the proportion of occupying about 3 percent of the area of the national soil. However, in the process of rapid development of economic technology and continuous increase of petroleum demand, the technical process for extracting oil from an oil field area is still relatively lagged behind, an environmental impact evaluation system is relatively incomplete, and meanwhile, a remediation technology for petroleum pollution is relatively lacked, so that the petroleum pollution degree of China is far higher than that of developed countries, and the petroleum pollution degree is in a state of accumulating and aggravating year by year.
The oil sludge is a main waste pollution problem commonly existing in various oil fields in China, and the oil sludge is classified as a dangerous waste (waste category HW08) by the national environmental protection Bureau of 1998, and the oil sludge and other wastes are required to be subjected to harmless treatment. The oil-containing sludge has different components and properties due to different sources. Through sampling and analyzing various kinds of oily sludge, the oily sludge generally contains 15 to 50 percent of oil. Has great harm to the environment. The oily sludge must be treated in a harmless way according to the requirements of the promotion of clean production of the people's republic of China. At present, a large amount of manpower and material resources are used for research on effective treatment of oily sludge at home and abroad, and methods such as solidification and landfill, incineration, leaching, chemical oxidation, bioremediation and the like are successively tried to treat the oily sludge. The bioremediation technology has the advantages of relatively low cost, high safety, simple operation, difficult secondary pollution, capability of implementing in-situ treatment and the like, and is considered as the most promising remediation means. But because the components of the oily sludge are complex and difficult to degrade, the research on the microbial degradation repair of the oily sludge at home and abroad is relatively less at present.
CN1594549A discloses a preparation of Pseudomonas stutzeri strain and an application technology thereof in sulfur-containing organic compound desulfurization, wherein the strain is Pseudomonas stutzeri UP-1; the strain is mainly derived from oil field sewage, oil field sludge and oil polluted soil with high sulfur content, and can be obtained by specific culture, separation and preparation means, and liquid cells, resting cells and immobilized cells of a culture medium of the strain can act on sulfur-containing organic compounds through a Kodama route to generate water-soluble sulfur-containing organic matters.
CN101074425A discloses a Pseudomonas nitroreducens and application thereof. The microorganism of the invention is the nitroreduction Pseudomonas nitroreducens S1 obtained by separating from activated sludge of a sewage treatment plant of a petrochemical plant and purifying and culturing, has high-efficiency specific degradation performance on benzothiazole, and is suitable for the biological treatment process of wastewater, waste gas, polluted water and soil containing benzothiazole.
CN104001712A discloses a method for remedying petroleum-contaminated soil by combining an alcaliella and microorganisms, which comprises the following steps: uniformly mixing the petroleum degrading microbial inoculum and soil according to the weight ratio of 1:5, scattering Alcalix arundinacea seeds, watering until plants germinate, and managing according to the normal growth requirements of the plants; the petroleum degrading microbial inoculum is prepared by mixing pseudomonas stutzeri and corynebacterium glutamicum according to the ratio of 1: 1; the soil for planting the festuca arundinacea is polluted soil with the total petroleum hydrocarbon mass fraction of 10000+5000 mg/kg. The method comprises the steps of adding a microbial inoculum into the petroleum-polluted saline-alkali soil, uniformly mixing, then sowing seeds of the Alcalix arundinacea, and watering regularly, so that the plants and microorganisms can synergistically degrade petroleum pollutants in the soil. The common tall fescue has the function of promoting the degradation of petroleum hydrocarbon and the greening function, the degradation rate and the degradation speed are improved by adding the microbial agent, and organic matters obtained after the microbial degradation are used as plant fertilizers, so that the plants and the microbes are mutually beneficial and symbiotic, the degradation time is long, and the petroleum hydrocarbon pollutants are better removed.
CN104276738A provides a rapid harmless biodegradation method aiming at waxy oil sludge, which is realized by preparing seed liquid, heating and dispersing waxy oil sludge, preparing a degradation system and biodegradation, and is specially used for the waxy oil sludge produced in the oil extraction and oil refining process, and can effectively degrade substances in the waxy oil sludge. By utilizing the biological treatment process, secondary pollution is not generated, and the environment is not influenced; the treatment process is simple, and the operation cost is low; short treatment time and high efficiency. The oil sludge conversion product has a promoting effect on the degradation of the oil sludge, which is an unexpected effect in the course of the test of the present invention.
CN105254144A discloses a harmless oil sludge treatment process, which comprises four steps of oil sludge fluidization, biological treatment, sludge dehydration and biological induction, wherein the treatment process comprises the steps of adding protected candida lipolytica Y-57 and pseudomonas putida P-101 into a biological reaction tank, carrying out aeration biological reaction, measuring absolute dry sludge, wherein the oil content of the oil sludge is less than 3%, the oil content of water is less than 130 mg/L, recycling floating oil on the upper part of a water surface through an oil collecting device, discharging bottom fine silt up to the standard, and allowing the oil sludge to enter a sludge dehydration step.
CN106565066A discloses a harmless biological treatment method for oily sludge, which comprises the following steps: 1) adding a crude oil deflocculant to deflocculate crude oil; 2) adding auxiliary materials, and performing anaerobic composting; 3) adding a microbial agent, and carrying out aerobic fermentation; 4) carrying out plant-assisted degradation; wherein the microbial agent comprises pseudomonas aeruginosa of pseudomonas, micrococcus luteus of micrococcus, bacillus subtilis of bacillus, acinetobacter juniperi of acinetobacter and acetobacter aceti of acetobacter, and the weight percentage ratio is 1:1.5:1:1.6: 2. The invention aims to provide an economical, environment-friendly and efficient oily sludge treatment method.
CN107299066A discloses a preparation method and a degradation treatment method of a microbial degradation liquid containing oily sludge, wherein the preparation method comprises the following steps: s1: preparing a first culture medium of microbial flora; s2: culturing a compound microorganism flora in the first culture medium obtained in the step S1 to obtain a first flora degradation solution, wherein the compound microorganism flora is prepared by mixing acinetobacter radiatus, bacillus subtilis, acinetobacter lofoenii, acinetobacter johnsonii, pseudomonas amygdalinus and mycobacterium according to the weight ratio of 1:1-2:0.3-0.7:1-1.4:1: 2-3; s3: preparing a second culture medium of microbial flora; s4: inoculating the first flora degradation liquid into the second culture medium for culturing to obtain a second flora degradation liquid; s5: and degrading the oily sludge by using the second flora degradation liquid to finish the microbial degradation treatment. The method obtains good degradation effect through comprehensive combination and cooperation of multiple technical characteristics such as unique microbial flora selection, culture method, degradation process operation and the like.
The biological treatment method is divided into two types, namely, nutrient substances are added into the oily sludge, aeration is carried out, and the growth and proliferation of indigenous microorganisms of the sludge are promoted, so that the pollutants are degraded. And secondly, adding a microbial agent for efficiently degrading petroleum hydrocarbon into the oily sludge. The advantages of the biological treatment technology are that: firstly, secondary pollution or pollutant transfer cannot be caused; secondly, the process is simple and the treatment cost is low; thirdly, the treatment effect is good, and the pollutant residual quantity can be greatly reduced through biochemical treatment. Research shows that the degradation rate can be improved to more than 50% by adding the petroleum hydrocarbon degrading bacteria. The oily sludge is treated by metabolic surfactant microorganisms, the solubility of petroleum hydrocarbons is improved, the degradation efficiency of the petroleum hydrocarbons can be improved, PAHs harmful substances can be removed by biological treatment, and aliphatic hydrocarbons and aromatic hydrocarbons in the oily sludge can be degraded simultaneously by biological treatment. Meanwhile, the existing microbial degradation microbial inoculum mainly comprises a compound microbial inoculum, a plurality of strains are mixed according to a certain proportion after being independently cultured and activated, and then are used as a degradation compound microbial inoculum for degrading pollutants after being cultured and activated for the second time, so that the culture process is complex in operation. In the prior art, a technical scheme for realizing excellent degradation of petroleum hydrocarbons in the oily sludge by using a single strain is not provided.
The inventor screens out a high-efficiency petroleum hydrocarbon degrading bacterium Pseudomonas stutzeri (CGMCC NO. 14976) from the oil-containing sludge in the Liaohe oil field, and further researches the degrading capability of the strain on the petroleum hydrocarbon in the oil-containing sludge so as to be applied to microbial remediation of the oil-containing sludge.
Disclosure of Invention
The invention aims to provide a petroleum hydrocarbon degrading strain and application thereof in a degradation and restoration process of oily sludge.
The invention relates to pseudomonas stutzeri and application thereof in degrading oily sludge, and the technical scheme is as follows:
the petroleum hydrocarbon degrading strain is characterized in that the strain is pseudomonas stutzeri (Pseudomonas stutzeri), is preserved in China general microbiological culture Collection center (CGMCC for short) in 2017, 12 and 1 days, and is deposited at China academy of sciences, China institute of sciences, No. 3, North Chen West Lu 1 institute of North Chen West Lu, Chaoyang, Beijing, with the preservation number of CGMCC NO. 14976.
The method for separating the efficient degrading strain from the oil-containing sludge in the Liaohe oil field to degrade the petroleum hydrocarbon pollutants in the oil-containing sludge is characterized by comprising the following steps:
10g of certain oily sludge in the Liaohe oil field is put into a conical flask filled with 100m L L B liquid culture medium, and is shaken on a constant temperature shaking table at 30 ℃ and 120rpm for enrichment culture for 24 h.
The L B culture medium comprises 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000m of distilled water L.
And inoculating the settled upper layer bacterial liquid into a screening culture medium after 24 hours, wherein the initial addition amount of the n-octadecane in the screening culture medium is 1 g/L, and continuously increasing the content of the n-octadecane in the screening culture medium until the aseptic colony grows within 5 days.
The inorganic salt culture medium comprises the following components: NaCl 30g, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g,MgSO40.5g, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution.
Solution of trace elements: CuSO40.05g，MnSO40.05g，FeSO4·7H20.05g of O and 50ml of deionized water.
Screening medium (alkane medium): adding n-octadecane as the only carbon source in the inorganic salt culture medium.
Observing the colony morphology after the colony grows out, selecting the colony with large morphological difference from the screening culture medium with the previous concentration without colony growth by using an aseptic inoculating loop, inoculating the colony on L B solid culture medium by adopting a plate streaking separation method, placing the colony in a constant temperature incubator for inverted culture for 24h at 30 ℃, selecting a single colony after the colony grows out, inoculating the single colony to L B solid culture medium by the same method, repeating the streaking separation process until a purified colony with single morphology is formed, and inoculating the purified colony on a storage culture medium inclined plane for storage in a refrigerator at 4 ℃ for later use.
The colony morphology of the strain is as follows: the colonies were yellow, flat and large, with wrinkles on the surface, as shown in FIG. 1. The thallus is rod-shaped with different lengths, and the shape of the bacterium is shown in a scanning electron microscope picture 2.
The sequence of the 16S ribosomal RNA gene of the strain is compared with a database by an analytical method, the strain is found to belong to Pseudomonas stutzeri, and a DNA sequence table of the strain is shown in a sequence table.
Before use, the strain is activated in L B liquid culture medium for 24h to prepare a strain liquid, and the strain liquid is inoculated into the oily sludge in an inoculation amount of 10%.
Preferably, the optimal conditions for degradation are as follows: the 10% inoculation amount, the temperature of 40 ℃, and the pH of 11 can lead the best degradation effect.
Compared with the prior art, the invention has the following advantages:
1. the Pseudomonas stutzeri JN5 is derived from oily sludge, so that the strain becomes a dominant flora soon after entering the oily sludge, occupies a certain ecological position, effectively promotes the degradation of petroleum hydrocarbons, optimizes a soil microbial system, improves the physical properties of soil and enhances the biological activity of the soil.
2. The Pseudomonas stutzeri JN5 is a dominant strain screened by using n-octadecane as a unique carbon source, and the strain can change the properties of petroleum hydrocarbon substances in the oily sludge in the growth and propagation process so as to change the components of the petroleum hydrocarbon substances in the oily sludge.
3. Pseudomonas stutzeri JN5 of the present invention was generated as a single strain for n-C10～n-C26The straight-chain petroleum hydrocarbon has obvious degradation capability, and GC-MS analysis shows that the strain has n-C resistance10～n-C26The degradation capability of straight-chain alkane and alkene is strong, the degradation rate reaches 100%, and the degradation efficiency of aromatic hydrocarbon is low.
4. The Pseudomonas stutzeri JN5 realizes excellent degradation of petroleum hydrocarbons in the oily sludge by using a single strain, reduces the complex steps of respectively activating and proportionally mixing strains in the preparation process of a mixed microbial inoculum, is easy to control the optimal reaction condition for degrading the petroleum hydrocarbons in the oily sludge, avoids the complex reaction products after degradation due to the degradation of the single strain, and is easy to further degrade the petroleum hydrocarbons in the oily sludge by combining other methods.
Drawings
FIG. 1 shows the colony morphology of strain JN 5.
FIG. 2 is a scanning electron micrograph of strain JN 5.
FIG. 3 shows the degradation curve of Pseudomonas stutzeri JN5 for petroleum hydrocarbons in oily sludge.
FIG. 4 shows the degradation curves of Pseudomonas stutzeri JN5 for petroleum hydrocarbons in oily sludge at different pH values.
FIG. 5 shows the degradation curves of Pseudomonas stutzeri JN5 for petroleum hydrocarbons in oily sludge at different temperatures.
Detailed Description
For better understanding of the present invention, the following examples are given as examples to illustrate the present invention, but the present invention is not limited to the following examples.
A bacterial strain for degrading petroleum hydrocarbon in oily sludge is a Pseudomonas stutzeri.
(I) Material preparation
The oily sludge is taken back from a certain place of the Liaohe oil field, stored at 4 ℃, transferred back to a laboratory and inoculated into L B culture medium for activation.
2. Culture medium:
the inorganic salt culture medium comprises the following components: 1g of n-octadecane, 30g of NaCl, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g，MgSO40.5g, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution.
Solution of trace elements: CuSO40.05g，MnSO40.05g,FeSO4·7H20.05g of O and 50ml of deionized water.
L B solid culture medium comprising tryptone 10g, yeast extract 5g, NaCl 10g, agar powder 20 and distilled water 1000m L, and adjusting pH to 7
3. Laboratory apparatus and device
Constant temperature oscillator
Electric heating constant temperature incubator
A high-pressure sterilization pot is used for sterilizing,
UV-1750 ultraviolet-visible spectrophotometer- -Shanghai Ringzhi optical technology Co., Ltd.,
the ultra-clean workbench is provided with a workbench,
OI L480 infrared spectroscopic oil tester- -Beijing Huaxia scientific Co., Ltd.,
a PCR instrument.
(II) separation, screening and domestication of strains
Taking 10g of oily sludge, putting the oily sludge into a conical flask filled with 100ml of L B liquid culture medium, shaking the oily sludge on a constant-temperature bed at 30 ℃ and 120rpm, and carrying out enrichment culture for 24 hours;
after 24 hours, taking 1ml of culture solution, coating the culture solution into a solid inorganic salt culture medium containing n-octadecane by using a coating rod, and putting the culture solution into a constant-temperature incubator for culture for 7 days;
observing the colony morphology after the colony grows out, selecting the colony with large morphological difference from the solid inorganic salt culture medium by using an aseptic inoculating loop, inoculating the colony on L B solid culture medium by adopting a plate streaking separation method, placing the colony in a constant-temperature incubator for inverted culture for 24h at 30 ℃, selecting a single colony after the colony grows out, inoculating the single colony to L B solid culture medium by the same method, repeating the streaking separation process until a purified colony with single morphology is formed, propagating the single colony in a liquid L B culture medium, storing the colony at 4 ℃, and activating the stored strain in a L B liquid culture medium before use.
The following is a detailed description of the embodiments.
Example 1: degradation repair test of Liaohe oil field oil-containing sludge
The method comprises the steps of taking oil-containing sludge in Liaohe oil field, carrying out air drying grinding, sieving by a 1mm sieve for later use, picking a single colony in a sterilized L B liquid culture medium, sealing the single colony by using a breathable sealing film, placing a conical flask in a shaking table, activating the conical flask at 120rpm and 30 ℃ for 24h, weighing 40g of the oil-containing sludge after air drying grinding and sieving, adding a Pseudomonas stutzeri JN5 activated bacterial liquid with OD600 ═ 1 according to the inoculation amount of 10%, adjusting the water-soil ratio to be 2:5 by using distilled water, sealing the conical flasks by using the breathable sealing film, placing all the conical flasks in a 30 ℃ constant temperature incubator for standing culture, sampling every 5d, measuring the content of total petroleum hydrocarbons in the oil-containing sludge by using an infrared spectrometer, wherein the degradation curve is shown in figure 3.
As can be seen from the results of GC-MS analysis of the oily sludge before and after 30 days of degradation of Pseudomonas stutzeri JN5 (Table 1), the main components in the original oily sludge were decalin, N-tetradecane, octadecene, tetramethylpentadecane, tetramethylhexadecane, indane, homoandrostane, N- (2-trifluoromethylbenzene) -3-pyridinecarboxamide oxime (C)13H10F3N3O), hexacosene and hopane. The oily sludge degraded by Pseudomonas stutzeri JN5 microorganism contains heptadecyl sulfite and 4-nitro-4' -chlorodiphenyl sulfide, which shows that the molecular structure of petroleum hydrocarbon substances in the oily sludge is changed under the action of the microorganism, and the pollutants with large molecular structures are oxidized and decomposed into small molecular structures under the action of the microorganism. Pseudomonas assattzeri JN5 for treating tetradecane, octadecene, tetramethylpentadecane and indene (C) in oily sludge19H36) Highly androstane (C)21H36O2)、C13H10F3N3The degradation capability of O and hexacosene is stronger, and the degradation rate reaches 100 percent. Illustrating the pair of the strains n-C10～n-C26The degradation capability of straight-chain alkane and alkene is stronger, and the degradation efficiency of aromatic hydrocarbon substances of decalin and hopane is lower. After 30 days of degradation of Pseudomonas stutzeri JN5, part of petroleum hydrocarbon substances in the oily sludge still remain, and the degradation time of microorganisms can be prolonged and the degradation conditions of the microorganisms can be changed for further degradation.
TABLE 1 GC-MS results of oily sludge before and after 30 days degradation of Pseudomonas stutzeri JN5
Example 2: degradation effect of Pseudomonas stutzeri JN5 on petroleum hydrocarbons in oil-containing sludge at different pH values
Taking oil-containing sludge of Liaohe oil field, air-drying, grinding and sieving by a 1mm sieve. Weighing 40g of the oily sludge in a conical flask, and adding diluted H2SO4And NaOH are used for adjusting the pH value of the soil to be 1, 3, 5, 7, 9 and 11 respectively, bacterial liquid activated by Pseudomonas stutzeri JN5 with OD600 of 1 is added according to the inoculation amount of 10%, the water-soil ratio is adjusted to be 2:5 by using distilled water, and the soil is sealed by using a breathable sealing film. Placing all the conical flasks in a constant-temperature incubator at 30 ℃ for static culture, sampling after 25 days, and determining the content of total petroleum hydrocarbons in the oily sludge by using an infrared spectrophotometer. The degradation effect of the strain Pseudomonas stutzeri JN5 on petroleum hydrocarbons in the oily sludge under different pH conditions is shown in FIG. 4. As can be seen from FIG. 4, the strain of the invention has good removal effect on petroleum hydrocarbons in the oily sludge within the pH range of 7-11, and the degradation effect is best under the condition of pH 11.
Example 3: degradation effect of Pseudomonas stutzeri JN5 on petroleum hydrocarbons in oil-containing sludge at different temperatures
Taking oil-containing sludge of Liaohe oil field, air-drying, grinding and sieving by a 1mm sieve. Weighing 40g of the oily sludge in an erlenmeyer flask, adding a Pseudomonas stutzeri JN5 activated bacterial liquid with OD600 of 1 according to the inoculation amount of 10%, adjusting the water-soil ratio to be 2:5 by using distilled water, and sealing by using a breathable sealing film. Placing the conical flasks in constant temperature incubator at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C and 50 deg.C respectively, standing for 25 days, sampling, and measuring total petroleum hydrocarbon content in the oily sludge with infrared spectrometer. The effect of the strain Pseudomonas stutzeri JN5 on the degradation of total petroleum hydrocarbons in oily sludge at different temperatures is shown in fig. 5. As can be seen from FIG. 5, the strain of the invention has certain degradation effect on petroleum hydrocarbons in the oily sludge within the range of 20-50 ℃, the degradation rate is not large within the range of 20-50 ℃, and the degradation effect is optimal under the condition of 40 ℃.
Example 4: pseudomonas stutzeri JN5 for repairing actual oily sludge
On the basis of the above examples, Pseudomonas stutzeri JN5 was used for repairing actual oily sludge, and the specific experimental steps were as follows:
(1) preparing bacterial suspension of the strain Pseudomonas stutzeri JN5, namely selecting a single bacterial colony of the strain JN5 in a L B liquid culture medium, placing the single bacterial colony in a shaking table, culturing for 24h under the conditions of 40 ℃ and 120rpm, performing fermentation culture by using a fermentation tank, inoculating the cultured seed liquid into a L B culture medium according to the inoculation amount of 5%, aerating by using an air compressor, stirring at the rotation speed of 200r/min, sampling at regular time to detect the OD600 value, and growing to the logarithmic growth phase for later use.
(2) Test oily sludge: the oily sludge collected from a certain place of the Liaohe oil field is naturally dried and sieved by a sieve with 8 meshes (a sieve with 3 mm). The oily sludge sample is put into an organic glass container, a bacterium solution activated by Pseudomonas stutzeri JN5 with OD600 ═ 1 is added according to the inoculation amount of 10 percent, the liquid-soil ratio is regulated to be 2:5 by an inorganic salt culture medium, and the container is sealed by a breathable sealing film.
(3) And (3) placing the oily sludge added with the bacterial liquid and the inorganic salt culture medium in a constant-temperature incubator for static culture, adjusting the pH value of the soil to 11 for achieving the optimal degradation effect, and maintaining the temperature at 40 ℃.
After 30 days of biodegradation treatment, the oil-containing sludge is detected, and the degradation rate of Total Petroleum Hydrocarbon (TPH) in the oil-containing sludge is 73.73%, so that the oil-containing sludge has a good degradation effect on the petroleum hydrocarbon in the oil-containing sludge.
Sequence listing
<110> Beijing university
<120> degrading strain JN5 for petroleum hydrocarbons in oily sludge and application thereof
<130>2018
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<211>1415
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<213> Pseudomonas stutzeri
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ttggatgtga aagccccggg ctcaacctgg gaactgcatc caaaactggc gagctagagt 600
atggcagagg gtggtggaat ttcctgtgta gcggtgaaat gcgtagatat aggaaggaac 660
accagtggcg aaggcgacca cctgggctaa tactgacact gaggtgcgaa agcgtgggga 720
gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtcgact agccgttggg 780
atccttgaga tcttagtggc gcagctaacg cattaagtcg accgcctggg gagtacggcc 840
gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt 900
aattcgaagc aacgcgaaga accttaccag gccttgacat gcagagaact ttccagagat 960
ggattggtgc cttcgggaac tctgacacag gtgctgcatg gctgtcgtca gctcgtgtcg 1020
tgagatgttg ggttaagtcc cgtaacgagc gcaacccttg tccttagtta ccagcacgtt 1080
aaggtgggca ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140
agtcatcatg gcccttacgg cctgggctac acacgtgcta caatggtcgg tacaaagggt 1200
tgccaagccg cgaggtggag ctaatcccat aaaaccgatc gtagtccgga tcgcagtctg 1260
caactcgact gcgtgaagtc ggaatcgcta gtaatcgtga atcagaatgt cacggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttg ctccagaagt 1380
agctagtcta accttcgggg ggacggtacc acgga 1415
Claims (7)
1. The strain JN5 for degrading and repairing oily sludge is characterized by being pseudomonas stutzeri (pseudomonas stutzeri)Pseudomonas stutzeri) The preservation unit name is China general microbiological culture Collection center with the preservation number of CGMCC NO. 14976.
2. The application of the strain JN5 for degrading and remediating the oily sludge according to claim 1, wherein the strain JN5 comprises the following components in percentage by weight: the strain JN5 is used for degrading and repairing oily sludge in a petroleum hydrocarbon degrading mode.
3. Use according to claim 2, characterized in that: the strain JN5 has the degradation condition of 20-50 ℃ and the pH value of 7-11 to petroleum hydrocarbons.
4. Use according to claim 3, characterized in that: the strain JN5 is used as a single strain for n-C10～n-C26Has obvious degradation capability on the straight-chain petroleum hydrocarbon, and the strain has n-C10～n-C26The degradation capability of straight-chain alkane and alkene is strong, the degradation rate reaches 100%, and the degradation efficiency of aromatic hydrocarbon is low.
5. Use according to claim 4, characterized in that it comprises the following steps:
(1) picking a single colony of Pseudomonas stutzeri JN5 in a sterilized conical flask of L B liquid culture medium, sealing the conical flask with a breathable sealing film, placing the conical flask in a shaking table, and activating for 24 hours at the temperature of 30 ℃ at 120rpm to prepare a seed culture solution;
(2) inoculating the cultured seed solution into a fermentation tank containing L B culture medium according to the inoculation amount of 5% for culture, aerating by adopting an air compressor, and culturing to logarithmic phase at the stirring speed of 200 r/min;
(3) air-drying and grinding the oily sludge in the Liaohe oil field, sieving the oily sludge by a 1mm sieve, adding 10 percent of inoculum size into Pseudomonas stutzeri JN5 activated bacterial liquid with OD600 being 1, adjusting the liquid-soil ratio to be 2:5 by using an inorganic salt culture medium, and sealing the opening by using a breathable sealing film;
(4) and (3) placing the oily sludge added with the bacterial liquid and the inorganic salt culture medium in a constant-temperature incubator for static culture, adjusting the pH value of the soil to 11 for achieving the optimal degradation effect, and maintaining the temperature at 40 ℃.
6. The use as claimed in claim 5, wherein the L B medium comprises tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000m L, and has a pH of 7.
7. Use according to claim 6, characterized in that: the inorganic salt culture medium comprises the following components: NaCl 30g, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g，MgSO40.5g of agar powder, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution, wherein the trace element solution comprises CuSO40.05g，MnSO40.05g，FeSO4·7H20.05g of O and 50ml of deionized water.
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| CN109576186A (en) * | 2018-12-27 | 2019-04-05 | 天津天丰泽田生物科技有限公司 | A kind of petroleum hydrocarbon degradation composite bacteria agent and preparation method thereof |
| CN111849798A (en) * | 2019-08-30 | 2020-10-30 | 上海傲江生态环境科技有限公司 | Microbial flora compound liquid and petroleum hydrocarbon polluted soil treatment method |
| CN111304114B (en) * | 2019-12-27 | 2022-03-15 | 西北大学 | Bacterial strain capable of inhibiting carbon steel corrosion and degrading crude oil, screening culture method and application thereof |
| CN114717152B (en) * | 2022-04-24 | 2024-01-09 | 北京沃太斯环保科技发展有限公司 | Composite functional microbial agent taking pseudomonas stutzeri as carrier for in-situ remediation of polluted soil and application thereof |
| CN115558624A (en) * | 2022-11-10 | 2023-01-03 | 陕西科技大学 | Low-temperature petroleum degrading bacterium, low-temperature petroleum degrading microbial inoculum and preparation method and application thereof |
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