CN101033453B - Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels - Google Patents

Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels Download PDF

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CN101033453B
CN101033453B CN2006100114421A CN200610011442A CN101033453B CN 101033453 B CN101033453 B CN 101033453B CN 2006100114421 A CN2006100114421 A CN 2006100114421A CN 200610011442 A CN200610011442 A CN 200610011442A CN 101033453 B CN101033453 B CN 101033453B
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klebsiella
thalline
lsse
bacterial strain
carbazole
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CN101033453A (en
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黄杰勋
邢建民
熊小超
李望良
李信
刘会洲
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Institute of Process Engineering of CAS
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Abstract

This invented Klebsiella pneumoniae LSSE-H2 was kept in China Stockpiling and Management Committee microbial strains ordinary microbial Center in Feb.24, 2006 and the preservation number is CGMCC No.1624, and its Gram staining is negative bacteria, the thalli is straight bar like with two round ends, the diameter of which is 0.3-1.0mum and the length is 0.6-6.0mum, the thalli unable to move can be alone or in pairs or arrayed in short chains having capsules formed by multi-glucide and is surrounded by fimbria and it is separated from soil polluted by dye and sewage to take off organic nitrogen from fossil fuel effectively by degradating heterocyclics containing nitrogen.

Description

Klebsiella and the application of organonitrogen in removing fossil oil thereof
Invention field
The invention belongs to biological technical field, particularly a kind of klebsiella and the application of organonitrogen in removing fossil oil thereof.
Background technology
The fluctuation in 0.02%～0.8% usually of the mass content of the elementary composition middle nitrogen of oil.The crude oil in China nitrogen content is higher, usually 0.1%～0.5%.Wherein the Daqing crude oil nitrogen content is lower, and Gudao crude is higher.Along with the exploitation of large quantities of finds, the nitrogen content of having found indivedual crude oil is up to 2% in recent years.These organic compounds containing nitrogens generally include basic cpd and non-basic cpd two big classes, it is maximum wherein to account for the non-basic cpd of major portion with carbazole and derivative thereof, accounted for about 70-75% of total nitrogen content, the existence of nitrogenous substances, can influence the performances such as color, anti-oxidative stability of oil product, so to remove non-basic cpd be vital to the quality of improving oil.Organonitrogen in the petroleum products is discharged in the atmosphere through the form of burning with oxynitride, and the discharging of these oxynitride can form acid rain and photo-chemical smog again, and environment has been caused serious pollution, and the eubiosis in the whole world in serious threat.A report of WHO's announcement in 1998 shows that global air pollution is amont the top ten large cities the most serious, appears on the list of successful candidates in the Taiyuan of China, Beijing, Urumchi, Lanzhou, Chongqing, Jinan, seven big cities, Shijiazhuang.Oxynitride is controlled relative sulfurous gas, and the research basis is relatively weaker, and country does not yet carry out overall control.But if the movable discharged nitrous oxides that produces of the energy is not added control, according to prediction, the only coal-fired oxynitride that produces just may be increased to 2,467 ten thousand tons and 2,870 ten thousand tons level with the year two thousand twenty in 2010 from 1,880 ten thousand tons level in 2000 respectively.If add the oxynitride of motor vehicle exhaust, the generation of following 20 years oxynitride also increases.Therefore, the task of following 20 years reduction of discharging oxynitride will be also severeer than reducing discharging sulfurous gas.
At present, the hydrofining method that industrial denitrogenation, desulfurization are extensively adopted is a High Temperature High Pressure process that needs catalyzer, so its heat resisting and pressure resisting facility investment costliness, the process cost height.Though hydrofining can be removed non-hydrocarbon compound and alkene in the oil product effectively, improves oil quality comprehensively, and have that technology is simple, easy to operate, oil product yield advantages of higher, in general, hydrodenitrification is difficulty relatively.Lightweight oil plant denitrification percent is usually less than 25%, and heavy oils is lower.Simultaneously, in technological processs such as catalytic cracking, hydrocracking, hydrofining, the nitride of trace also can make noble metal catalyst poison even contain extremely in the raw material, causes shorten the work-ing life of catalyzer.Wherein, not only can be adsorbed in the process of catalytic pyrolysis on the activity of such catalysts site as the carbazole of main non-basic cpd, also may be the direct inhibitor of hydrodesulfurization process.The nitrogenous compound of removing carbazole and other can significantly improve the efficient of catalytic pyrolysis and the productive rate of gasoline.At last, the existence of nitrogenous compound also can cause equipment and Corrosion of Pipeline in the refining process, increases the cost of oil refining.
Biological denitrificaion utilizes the microorganism specificity to remove nitrogenous compound exactly, recent years, biocatalysis denitrogenation research obtained very big progress, microorganism can make the advanced nitrogen of oil and goods thereof become possibility with the degraded of nitrogen-containing heterocycle compound selectivity under the condition of gentleness.1993, reported first such as the Ouchiyama of Tokyo University the pathways metabolism of pseudomonas degraded carbazole.Carbazole finally enters tricarboxylic acid cycle through intermediate products such as anthranilic acid and pyrocatechols under the catalysis of dioxygenase.Other research group also separates in succession and obtains similar bacterial strain, as: pseudomonas LD2, OM1 and K23, Sphingol single-cell GTIN11, CDH-7 and KA1, the Rolls leads to bacterium RJGII.123, Xanthamonas bacterium CB2 and Janthinobacterium bacterium J3 or the like.Simultaneously, carbazole also often as the raw material of industrial production dyestuff, medicine and plastics, owing to it has tangible carinogenicity and toxicity, is discharged in the environment to cause serious ecological problem.Biological denitrificaion reaction conditions gentleness, facility investment is little, and reaction process do not have the generation of nuisance, can realize advanced nitrogen, no matter on petroleum industry still is environment remediation, all demonstrates the favorable industrial prospect.
That has at home and abroad found is more as pseudomonas and Sphingol single-cell in the denitrification microorganism of model compound with carbazole.The new bacterial strain LSSE-H2 of klebsiella of the present invention (Klebsiella pneumoniae) is the klebsiella that reported first has nitrogen removal characteristics, stronger organic solvent tolerance and higher denitrification activity are arranged, have wide prospect in industrial application and theoretical investigation and be worth.
Summary of the invention
The objective of the invention is to overcome the at present above-mentioned defective that denitride technology exists in the fossil oil that removes, separate the klebsiella bacterial strain of ability and the application of organonitrogen during removing fossil oil and provide with high reactivity degraded organic compounds containing nitrogen from occurring in nature, it is strong that this bacterium has the denitrogenation ability, it is single-minded to act on substrate, can in the process of reaction, not destroy other component in the fuel, have the stronger advantage of selectivity.
Klebsiella provided by the invention, it is characterized in that: this klebsiella is Klebsiellapneumoniae LSSE-H2, be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC NO.1624 on February 24th, 2006.
Bacterial strain LSSE-H2 thalline is shaft-like, and diameter is 0.3～1.0 micron, and length is 0.6～6.0 micron, but Individual existence or arrange with paired, short chain shape has the pod membrane that polyose is formed, and has pili around the thalline, can not move Gram-negative.
This klebsiella 16SrRNA gene order of the present invention is as follows:
TGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGAC
GGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCA
AGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCT
CACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT
TGTAAAGTACTTTCAGCGAGGAGGAAGGCNNTAAGGTTAATAACCTTNNCGATTGACGTTACTCGCAGAAGAAGCACCG
GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAG
GCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTA
GAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGG
ACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATG
TCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAC
CTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGG
CTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGNTNC
GGNCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGA
GTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGT
CGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGA
ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCG
GGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTG
The application of klebsiella provided by the invention in the degraded nitrogen-containing organic compound is especially to carbazole (CAR) and derivative thereof.It is characterized in that: described nitrogenous compound is the organic nitrogen-containing heterogeneous ring compound, and during nitrogenous compound in removing the fossil oil that accounts for main composition with carbazole (CAR), specificity ground destroys carbazole and derivative thereof, and other component in the fuel is not attacked.
Introduce the present invention below in detail:
Klebsiella of the present invention is gathered soil sample and mud sample with China Beijing second printing and dyeing mill in the soil of dye discoloration and the mud, obtain as unique carbon source, nitrogenous source and energy selectivity cultivation separation and purification with the organic compounds containing nitrogen carbazole, its biological property is as follows:
A. morphological feature
Bacterial strain LSSE-H2 thalline is shaft-like, and thalline is straight, the two ends circle, and diameter is 0.3～1.0 micron, length is 0.6～6.0 micron, but Individual existence or arrange with paired, short chain shape has the pod membrane that polyose is formed, have pili around the thalline, can not move Gram-negative.
B. cultural characteristic
This bacterial strain LSSE-H2 soaks at glucose asparagine agar, nutrient agar medium, Sang Tasi agar, potato in five kinds of substratum such as juice agar and cultivates, and the bacterium colony of formation is canescence, bacterium colony circle, moistening, neat in edge, central protrusion, no aerial hyphae.
C. physiological and biochemical property
With reference to the method for " Bergey ' s Manual of Systematic Bacteriology " vol.IV., the physiological and biochemical property of bacterial strain LSSE-H2 is as shown in table 1.
The physiological and biochemical property of table 1 bacterial strain LSSE-H2
Feature The result The utilization of carbon source feature The result
Gelatine liquefication - Glucose +
Feature The result The utilization of carbon source feature The result
Milk solidifies - L-arabinose -
Milk peptonizes + N.F,USP MANNITOL +
The starch hydrolysis + D-fructose +
Nitrate reduction - The D-semi-lactosi +
Grow on the Mierocrystalline cellulose - The D-seminose +
H 2S produces - Trehalose +
Melanochrome produces - Maltose +
Hydrolysis of urea + The L-rhamnosyl +
Tyrosine hydrolysis - The D-sorbyl alcohol +
Indole test - Ribitol +
Decompose Vitamin C2 + Propionic acid -
Hydrolysis Tween80 + 2, the 3-butyleneglycol -
Hydrolysis Tween40 + Citric acid +
Catalase + Succsinic acid +
Arginine dihydrolase - Galactitol -
Ornithine decarboxylase - Malonate +
The glucose aerogenesis + Tartrate -
D.16SrRNA gene order
The 16SrRNA gene order of bacterial strain LSSE-H2 of the present invention is as follows:
TGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGAC
GGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCA
AGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCT
CACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT
TGTAAAGTACTTTCAGCGAGGAGGAAGGCNNTAAGGTTAATAACCTTNNCGATTGACGTTACTCGCAGAAGAAGCACCG
GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAG
GCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTA
GAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGG
ACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATG
TCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAC
CTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGG
CTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGNTNC
GGNCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGA
GTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGT
CGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGA
ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCG
GGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTG
(bacterial strain LSSE-H2 of the present invention is as shown in table 2 with the similarity of the 16SrRNA gene of relevant bacterial strain wherein by retrieval GenBank.
The similarity of table 2 bacterial strain LSSE-H2 and the 16SrRNA gene of relevant bacterial strain
Login path and relevant strain name Score value (bits) The E value
gi|54887534|emb|AJ786796.1| Klebsiella sp.R-21934 partial 16SrRNA 2849 0.0
gi|6562627|emb|AJ251467.1|KOR251467 Klebsiella ornithinolytic... 2841 0.0
gi|82618463|gb|DQ277701.1| Klebsiella sp.F51-1-2 16S ribosom... 2837 0.0
gi|62529082|gb|AY972873.1| Gamma Proteobacterium BAL286 16S r... 2833 0.0
gi|3282038|emb|Y17666.1|KOY17666 Klebsiella ornithinolytica 1... 2833 0.0
gi|3282036|emb|Y17662.1|KOY17662 Klebsiella ornithinolytica 1... 2829 0.0
gi|1359900|emb|Y93216.1|KPl6SRRN2 K.planticola 16S rRNA gene (st 2825 0.0
gi|38373979|gb|AY363386.2| Klebsiella sp.rennanqilfy 18 16S r... 2821 0.0
gi|4205080|gb|U78182.1|KOU78182 Klebsiella ornithinolytica 16... 2821 0.0
gi|10334693|gb|AF181574.1|AF181574 Klebsiella planticola 16S rib 2815 0.0
gi|3282037|emb|Y17663.1|KPY17663 Klebsiella planticola 16S rRNA 2813 0.0
gi|3282033|emb|Y17659.1|KPYl7659 Klebsiella planticola 16S rRNA 2805 0.0
gi|57864391|dbj|AB004756.2| Raoultella ornithinolytica gene f... 2801 0.0
gi|57864391|dbj|AB004756.2| Raoultella ornithinolvtica gene f... 2793 0.0
gi|6562390|emb|AJ251468.1|EAE251468 Enterobacter aerogenes parti 2793 0.0
gi|84620794|gb|DQ333356.1| Raoultella planticola strain 33-4c... 2787 0.0
gi|78059508|gb|D0229101.1| Klebsiella sp.TNT2 16S ribosomal RNA 2785 0.0
gi|6693807|gb|AF129441.1|AF129441 Klebsiella ornithinolytica... 2777 0.0
gi|6693810|gb|AF129444.1|AF129444 Klebsiella trevisanii strai... 2761 0.0
gi|6693809|gb|AF129443.1|AF129443 Klebsiella planticola strai... 2761 0.0
gi|56159736|gb|AY825036.1| Enterobacter aerogenes strain NTG-... 2742 0.0
gi|1073242|gb|U39556.1|ESU39556 Enterobacter sp. 16S rRNA gene， 2736 0.0
gi|60416686|emb|AJ871856.1| Klebsiella oxytoca partial 16S rRNA 2734 0.0
gi|82792001|gb|DQ279308.1| Enterobacter sp. TM1_4 16S ribosomal 2734 0.0
gi|57472159|gb|AY873801.1| Klebsiella oxytoca isolate GR6 16S... 2734 0.0
gi|2209042|dbj|AB004750.1| Enterobacter aerogenes gene for 16... 2732 0.0
gi|78059507|gb|DQ229100.1| Klebsiella sp.TNT1 16S ribosomal RNA 2730 0.0
gi|3282055|emb|Y17670.1|KTY17670 Klebsiella terrigena 16S rRNA g 2730 0.0
gi|60416691|emb|AJ871861.1| Klebsiella oxytoca partial 16S rRNA 2726 0.0
gi|60416689|emb|AJ871859.1| Klebsiella oxytoca partial 16S rRNA 2722 0.0
gi|82792000|gb|DQ279307.1| Enterobacter sp.TM1_3 16S ribosomal 2720 0.0
gi|14549203|dbj|AB053117.1| Klebsiella oxytoca gene for 16S rRNA 2720 0.0
gi|27530901|dbj|AB098582.1| Enterobacter sp.TUT1014 gene for 16 2718 0.0
gi|61658334|gb|AY946284.1| Kluyvera cryocrescens 16S ribosomal R 2718 0.0
gi|3282039|emb|Y17667.1|KOY17667 Klebsiella oxytoca 16S rRNA gen 2716 0.0
gi|30409687|dbj|AB099403.1| Raoultella planticola gene for 16... 2714 0.0
gi|60416690|emb|AJ871860.1| Klebsiella oxytoca partial 16S rRNA 2714 0.0
gi|3282034|emb|Y17660.1|KOY17660 Klebsiella oxytoca 16S rRNA gen 2714 0.0
gi|83033247|gb|DQ294284.1| Klebsiella oxytoca strain 5 16S ri... 2712 0.0
gi|47132404|gb|AY601679.1| Klebsiella oxytoca strain HP1 16S... 2710 0.0
gi|60416693|emb|AJ871863.1| Klebsiella oxytoca partial 16S rRNA 2710 0.0
gi|60416692|emb|AJ871862.1| Klebsiella oxytoca partial 16S rRNA 2710 0.0
gi|3282035|emb|Y17661.1|KOY17661 Klebsiella oxytoca 16S rRNA gen 2710 0.0
gi|1359899|emb|X93215.1|KP16SRRN1 K.planticola 16S rRNA gene(st 2708 0.0
gi|30409691|dbj|AB099407.1| Raoultella planticola gene for 16... 2708 0.0
gi|78059509|gb|DQ229102.1| Klebsiella sp.TNT3 16S ribosomal RNA 2706 0.0
gi|12044275|gb|AF310218.1| Kluyvera cryocrescens 16S ribosomal R 2706 0.0
gi|23955492|gb|AF543283.1| Klebsiella oxytoca strain ChDC OS3... 2698 0.0
gi|2584741|emb|Z96078.1|ECZ96078 Enterobacter cancerogenus LMG 2 2698 0.0
gi|30409686|dbj|AB099402.1| Enterobacter aerogenes gene for 16S 2698 0.0
gi|2209046|dbj|AB004754.1| Klebsiella oxytoca gene for 16S ribos 2696 0.0
gi|71842215|gb|DQ133596.1| Pantoea agglomerans strain B1 16S... 2694 0.0
gi|70888002|gb|DQ100465.1| Klebsiella sp.GR9 16S ribosomal RNA 2694 0.0
gi|6693808|gb|AF129442.1|AF129442 Klebsiellaterrigena strain... 2690 0.0
gi|2209041|dbj|AB004749.1| Enterobacter amnigenus gene for 16... 2690 0.0
gi|23955505|gb|AF543296.1| Klebsiella oxytoca strain ChDC OS4... 2688 0.0
Table 2 shows that the 16SrRNA gene of the new bacterial strain of LSSE-H2 of the present invention is no identical bacterial strain in GenBank, illustrates that this bacterial strain is separated first.This Pseudomonas is in the Klebsiella branch of evolving.Morphological specificity: thalline is shaft-like, and thalline is straight, the two ends circle, and diameter is 0.3～1.0 micron, length is 0.6～6.0 micron, but Individual existence or arrange with paired, short chain shape; No aerial mycelium.
LSSE-H2 bacterial strain of the present invention both can be cultivated in nutritional medium, also can cultivate in the selective medium of carbazole as sole carbon source, nitrogenous source and the energy.The thalline of growing in the selective medium has highly active denitrogenation ability.Bacterial strain all can be grown well at pH4-9, and is growth temperature 25-40 ℃, aerobic.
The application method of bacterial strain LSSE-H2 of the present invention has: (1) can be directly used in the denitrogenation of oil and products thereof with carbazole as carbon source, nitrogenous source and the energy; (2) make resting cell with cryodesiccated thalline and carry out denitrogenation; (3) cell fragment or the cell extract with this bacterium carries out denitrogenation; (4) carry out denitrogenation with the biological activity of purifying in this bacterium; (5) this bacterium can make the denitrogenation biological catalyst with absorption or the immobilized cell that is embedded on the carrier.
Klebsiella provided by the invention obtains from the nature screening and separating; No matter this klebsiella is growth conditions or resting cell, no matter be free cell or immobilized cell, no matter be complete thalline or broken thalline fragment, no matter be that the thick liquid of cell or the enzyme component of separation and purification all can show highly active denitrogenation ability, particularly the nitrogen in oil and products thereof has solved existing oil hydrogenation technique processing cost height, has been not easy to remove shortcoming such as nitrogen in the heterogeneous ring compound; This bacterial strain is by the nitrogen-containing heterocycle compound in the organic compound in the fuel of optionally degrading, and do not destroy other component, greatly kept the combustion heat value of fuel.This bacterium has many good characteristics as the industrial application bacterial strain, and the ability of organic solvent-resistant is strong, and the denitrification activity height has wide industrial application potentiality.
Description of drawings
Accompanying drawing 1 is the chemical structural drawing of a few class nitrogenous compounds in the fuel
Accompanying drawing 2 is a carbazole biological denitrificaion pathways metabolism synoptic diagram;
Accompanying drawing 3 is that LSSE-H2 bacterial strain of the present invention is at the denitrogenation situation synoptic diagram of aqueous phase to carbazole;
Accompanying drawing 4 is the 16SrRNA gene order of LSSE-H2 bacterial strain of the present invention.
Embodiment
The screening of embodiment 1:LSSE-H2 bacterial strain
Second printing and dyeing mill gathers and is made sample by the soil of dye discoloration and mud from the BeiJing, China.Get 5 gram pedotheques and be suspended in 25 milliliters the physiological saline, put in the shaking table and mix after 30-60 minute, leave standstill, inhale with supernatant liquor (water sample can directly be got supernatant liquor and be added in the selective medium) in 100 milliliters selective medium.Consisting of of selective medium: 1000 milliliters of deionized waters, 2.2g Na2HPO412H2O, 0.8g KH2PO4,0.015g MgSO47H2O, 0.015g FeCl37H2O, 0.01g CaCl2 and 0.05g yeast powder, the carbazole (CAR) that wherein adds 3mmol/L is as carbon source, nitrogenous source and the energy.Put shaking table with 180 rev/mins, after 30 ℃ of one weeks of cultivation, switching 1ml nutrient solution is in fresh selective medium, and same switching serves as to transfer three times at interval with a week.Well-grown nutrient solution is applied to on the solid plate of carbazole as sole carbon source and nitrogenous source, the single bacterium colony that grows inoculates in the liquid nutrient medium, choose the high bacterium colony of degrading activity and on solid plate, carry out the mono-clonal separation, separating the single bacterium colony that obtains is inoculated in respectively in the selective medium that CAR is 6mmol/L, put shaking table with 180 rev/mins, cultivated three to five days for 30 ℃.Get nutrient solution 0.7ml, regulating its PH with 1M hydrochloric acid is 1.0, with the ethyl acetate oscillation extraction of 0.7ml.Extraction liquid is analyzed with high performance liquid phase.The result has chosen the bacterial strain that a strain has high reactivity denitrogenation ability to carbazole (CAR), name for klebsiella be Klebsiella pneumoniae LSSE-H2, be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC NO.1624 on February 24th, 2006.
The acquisition of embodiment 2:LSSE-H2 grown cell with induce
The dull and stereotyped LSSE-H2 bacterial strain bacterium colony of cultivating of picking selectivity is inoculated in 20 milliliters the selection substratum.The composition of substratum: 1000 milliliters of deionized waters, 2.2g Na2HPO412H2O, 0.8g KH2PO4,0.015gMgSO47H2O, 0.015g FeCl37H2O, 0.01g CaCl2 and 0.05g yeast powder, the carbazole (CAR) that wherein adds 6mmol/L is as carbon source, nitrogenous source and the energy.30 ℃, cultivate after 24-48 hour for 180 rev/mins, again it is added in the selection substratum of 500 milliliters of carbazoles that are added with 6mmol/L, after 48-72 hour, centrifugal acquisition thalline.Thalline is placed 50 milliliters physiological saline, shaking table mixing 2-4 hour, the centrifugal thalline of obtaining once more.
The acquisition of the active stem cell of embodiment 3:LSSE-H2
The dull and stereotyped LSSE-H2 bacterium colony of cultivating of picking selectivity is inoculated in 20 milliliters the selective medium.The composition of substratum: 1000 milliliters of deionized waters, 2.2g Na2HPO412H2O, 0.8g KH2PO4,0.015gMgSO47H2O, 0.015g FeCl37H2O, 0.01g CaCl2 and 0.05g yeast powder, the carbazole (CAR) that wherein adds 6mmol/L is as carbon source, nitrogenous source and the energy.30 ℃, cultivate after 24-48 hour for 150 rev/mins, again it is added in the basic medium of 500 milliliters of carbazoles that are added with 12mmol/L, after 48-72 hour, centrifugal acquisition thalline.Thalline is placed 50 milliliters physiological saline, shaking table mixing 2-4 hour, the centrifugal thalline of obtaining once more.Vacuum freezedrying obtains to have active stem cell.
Embodiment 4: bacterial strain LSSE-H2 is in the denitrogenation of aqueous phase
Add carbazole in the selected liq substratum as unique carbon source and nitrogenous source, make concentration reach 6mmol/L.Picking is selected the dull and stereotyped LSSE-H2 bacterial strain of cultivating, and adds in 20 milliliters the selection substratum.The composition of substratum: 1000 milliliters of deionized waters, 2.2g Na2HPO412H2O, 0.8g KH2PO4,0.015g MgSO47H2O, 0.015g FeCl37H2O, 0.01g CaCl2 and 0.05g yeast powder, the carbazole (CAR) that wherein adds 6mmol/L is as carbon source, nitrogenous source and the energy.30 ℃, cultivate after 24-48 hour for 180 rev/mins, be inoculated in the selection substratum of the carbazole that contains 6mmol/L of 100mL with 4% inoculum size again, sampling at interval in per 8 hours, with using high pressure liquid chromatographic analysis behind isopyknic ethyl acetate oscillation extraction, degraded in 32 hours 90% carbazole, carbazole is by degraded (as shown in Figure 3) fully after 56 hours.
Example 5: bacterial strain LSSE-H2 removes denitrogenation in simulated system
Add CAR in the dodecane, make concentration reach 1mmol/L.Picking is selected the dull and stereotyped LSSE-H2 bacterial strain of cultivating, and adds in 20 milliliters the selection substratum.The composition of substratum: 1000 milliliters of deionized waters, 2.2gNa2HPO412H2O, 0.8g KH2PO4,0.015g MgSO47H2O, 0.015g FeCl37H2O, 0.01gCaCl2 and 0.05g yeast powder, the carbazole (CAR) that wherein adds 6mmol/L is as carbon source, nitrogenous source and the energy.30 ℃, cultivate after 24-48 hour for 180 rev/mins, with water oil ratio 1: 3 mimic oil phase and yeast culture thing are mixed.30 ℃, to cultivate 24-48 hour for 180 rev/mins, nitrogen is all removed.
The 16S rDNA sequence number of klebsiella LSSE-H2
1 TGAACGCTGG CGGCAGGCCT AACACATGCA AGTCGAGCGG TAGCACAGAG
51 AGCTTGCTCT CGGGTGACGA GCGGCGGACG GGTGAGTAAT GTCTGGGAAA
101 CTGCCTGATG GAGGGGGATA ACTACTGGAA ACGGTAGCTA ATACCGCATA
151 ACGTCGCAAG ACCAAAGTGG GGGACCTTCG GGCCTCATGC CATCAGATGT
201 GCCCAGATGG GATTAGCTAG TAGGTGGGGT AATGGCTCAC CTAGGCGACG
251 ATCCCTAGCT GGTCTGAGAG GATGACCAGC CACACTGGAA CTGAGACACG
301 GTCCAGACTC CTACGGGAGG CAGCAGTGGG GAATATTGCA CAATGGGCGC
351 AAGCCTGATG CAGCCATGCC GCGTGTATGA AGAAGGCCTT CGGGTTGTAA
401 AGTACTTTCA GCGAGGAGGA AGGCNNTAAG GTTAATAACC TTNNCGATTG
451 ACGTTACTCG CAGAAGAAGC ACCGGCTAAC TCCGTGCCAG CAGCCGCGGT
501 AATACGGAGG GTGCAAGCGT TAATCGGAAT TACTGGGCGT AAAGCGCACG
551 CAGGCGGTCT GTTAAGTCAG ATGTGAAATC CCCGGGCTCA ACCTGGGAAC
601 TGCATTTGAA ACTGGCAGGC TTGAGTCTTG TAGAGGGGGG TAGAATTCCA
651 GGTGTAGCGG TGAAATGCGT AGAGATCTGG AGGAATACCG GTGGCGAAGG
701 CGGCCCCCTG GACAAAGACT GACGCTCAGG TGCGAAAGCG TGGGGAGCAA
751 ACAGGATTAG ATACCCTGGT AGTCCACGCT GTAAACGATG TCGACTTGGA
801 GGTTGTTCCC TTGAGGAGTG GCTTCCGGAG CTAACGCGTT AAGTCGACCG
851 CCTGGGGAGT ACGGCCGCAA GGTTAAAACT CAAATGAATT GACGGGGGCC
901 CGCACAAGCG GTGGAGCATG TGGTTTAATT CGATGCAACG CGAAGAACCT
951 TACCTACTCT TGACATCCAG AGAACTTAGC AGAGATGCTT TGGTGCCTTC
1001 GGGAACTCTG AGACAGGTGC TGCATGGCTG TCGTCAGCTC GTGTTGTGAA
1051 ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTTATCCT TTGTTGCCAG
1101 CGNTNCGGNC GGGAACTCAA AGGAGACTGC CAGTGATAAA CTGGAGGAAG
1151 GTGGGGATGA CGTCAAGTCA TCATGGCCCT TACGAGTAGG GCTACACACG
1201 TGCTACAATG GCATATACAA AGAGAAGCGA CCTCGCGAGA GCAAGCGGAC
1251 CTCATAAAGT ATGTCGTAGT CCGGATTGGA GTCTGCAACT CGACTCCATG
1301 AAGTCGGAAT CGCTAGTAAT CGTAGATCAG AATGCTACGG TGAATACGTT
1351 CCCGGGCCTT GTACACACCG CCCGTCACAC CATGGGAGTG GGTTGCAAAA
1401 GAAGTAGGTA GCTTAACCTT CGGGAGGGCG CTTACCACTT TGTGATTCAT
1451 GACTGGGGTG

Claims (2)

1. a strain klebsiella, it is characterized in that: this klebsiella is Klebsiella pneumoniaeLSSE-H2, be preserved in that " diameter is 0.3～1.0 micron; length is 0.6～6.0 micron for China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; its preserving number is CGMCC NO.1624; the Gram-negative of this klebsiella; thalline is shaft-like; thalline is straight, two ends circle on February 24th, 2006, but Individual existence or with in pairs, the short chain shape is arranged, and has the pod membrane that polyose is formed, and has pili around the thalline, can not move, the 16SrRNA gene order of this klebsiella is as follows:
TGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGAC
GGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCA
AGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCT
CACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT
TGTAAAGTACTTTCAGCGAGGAGGAAGGCNNTAAGGTTAATAACCTTNNCGATTGACGTTACTCGCAGAAGAAGCACCG
GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAG
GCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTA
GAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGG
ACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATG
TCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAC
CTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGG
CTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGNTNC
GGNCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGA
GTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGT
CGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGA
ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCG
GGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTG。
2. the application of described this bacterium of Cray Bai Shi of claim 1 organonitrogen in removing fossil oil, it is characterized in that: described organonitrogen is the organic nitrogen-containing heterogeneous ring compound.
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