CN104371941A - Luteimonas capable of degrading petroleum hydrocarbon, and its application - Google Patents

Luteimonas capable of degrading petroleum hydrocarbon, and its application Download PDF

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CN104371941A
CN104371941A CN201310357439.5A CN201310357439A CN104371941A CN 104371941 A CN104371941 A CN 104371941A CN 201310357439 A CN201310357439 A CN 201310357439A CN 104371941 A CN104371941 A CN 104371941A
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luteimonas
hai
xanthomonas campestris
lianteng
oil
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薛松
信艳娟
曹旭鹏
吴佩春
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/08—Seawater, e.g. for desalination

Abstract

The invention discloses a Luteimonas capable of degrading petroleum hydrocarbon, and its application. Luteimonas dalianensis OB44-3 is preserved in China General Microbiological Culture Collection Center on January 18, 2013 with the preservation number of CGMCC No. 7165. The Luteimonas dalianensis OB44-3 is a new species of Luteimonas, is separated from petroleum polluted marine environment, can effectively degrade the petroleum hydrocarbons, and can grow with petroleum as a sole carbon source. The average degradation rate of the Luteimonas dalianensis OB44-3 on the petroleum hydrocarbons (with the initial concentration of 1000mg/L) in an artificial seawater medium within 7d is 69.0%; and the above strain grows depended on salt, belongs to marine bacteria, and has unique advantages in the control of the petroleum hydrocarbon pollution in the marine environment. The Luteimonas dalianensis OB44-3 can be used for degrading the petroleum hydrocarbons.

Description

Teng Xanthomonas campestris of one strain degradable petroleum hydrocarbon and application thereof
Technical field
The present invention relates to Teng Xanthomonas campestris and the application thereof of a strain degradable petroleum hydrocarbon.
Background technology
Oil is important energy substance, the pollution to environment all may be produced in the processes such as exploitation, transport, processing, the hydrocarbons contained in oil is referred to as petroleum hydrocarbon, mainly comprise alkane, naphthenic hydrocarbon, aromatic hydrocarbon etc., some composition can carcinogenic, mutagenesis, teratogenesis shape, and by food chain enrichment in animals and plants and human body, be listed in priority pollutant.Because petroleum hydrocarbon belongs to bio-refractory material, therefore, petroleum pollution has become a serious environmental problem, causes many countries to pay much attention to.
The Degradation of natural microbial population to petroleum hydrocarbon be present in ocean environment is one of Basic Ways of eliminating from ocean environment of petroleum hydrocarbon and other hydrocarbons pollutants.Microorganism utilizes biological self-regulatory mechanism and to the petroleum hydrocarbon class pollutant in the comprehensive purifying function treatment ocean of pollutent, makes them obtain transforming comparatively thoroughly and degrading in the metabolic processes of biology, finally generate CO 2and water, can not produce secondary pollution, be the method for the most environmental protection of the process petroleum pollution in ocean of generally acknowledging at present.
In recent years, the research of domestic and international related microorganism decomposing petroleum hydrocarbon increases gradually.Have been found that according to statistics and the microorganism of degraded oil can exceed kind more than 200, mainly comprise in the prevailing microorganism of ocean environment petroleum hydrocarbon degradation: achromobacter (Achromobacter), acinetobacter (Acinetobacter), Alkaligenes (Alcaligenes), bacillus (Bacillus), Flavobacterium (Flavobacterium), Corynebacterium (Coryneforms), Microbacterium (Microbacterium), actinomyces (Actinomycetes), Nocardia (Nocardia), Rhodopseudomonas (Pseudomonas), alkane eating bacteria belongs to (Alcanirorax), to unlink Pseudomonas (Cycloclasticus), marinobacter (Marinobacter), bacterium belongs to (Marinobacterium) in sea, whelk revolves Pseudomonas (Oleispira), Pseudomonas (Thalassospira) and micrococcus sp (Mirococcus) etc. are revolved in sea.In ocean, the fungi of energy decomposing petroleum hydrocarbon has golden Basidiomycotina (Aureobasidium), Rhodotorula sp (Rhodotorula) etc., also has a lot of mould also degradable petroleum hydrocarbon, as penicillin (Penicillium), Aspergillus (Apergillus) etc.At present become a new important directions to the research of degrading microorganism resource, increasing oil degradation microorganism is found, and has built special degrading microorganism flora resources bank in the world.Separation screening has the bacterial strain of degradation capability to petroleum hydrocarbon, is microorganisms degraded oil mechanism and the basis of petroleum hydrocarbon contaminated environment being carried out to biological restoration.
Summary of the invention
The object of this invention is to provide Teng Xanthomonas campestris of a strain degradable petroleum hydrocarbon.
Teng Xanthomonas campestris provided by the present invention screens from the seawater sample of Dalian Bay tideland crude oil pollution.Confirm to belong to Teng xanthomonas (Luteimonas) novel species, called after Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3 according to its morphological specificity, cultural characters, physio-biochemical characteristics and 16S rRNA gene sequencing etc.Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 18th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCN0.7165.
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 is Gram-negative bacteria, and cell is shaft-like, and atrichia does not have mobility, and size is (1.02 ~ 1.24) μm × (0.32 ~ 0.47) μm.LB substratum is cultivated 3 days, and the bacterium colony of this bacterial strain is the feature such as small circular, smooth, protuberance, light golden yellow, translucent, neat in edge.
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 belongs to aerobic bacteria, and the pH scope that can grow is 6.0 ~ 8.0, and the most suitable growth pH value is 7.2; Can growth temperature range be 15 ~ 37 DEG C, optimum growth temperature be 30 DEG C; This bacterial strain belongs to marine microorganism, the existence of growth needs NaCl, and can grow in 2 ~ 9% (w/v) salinity range, the most suitable growth salinity is 4%.
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 catalase and oxidase positive, beta-galactosidase enzymes, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase and tryptophane desaminase are negative; The Citrate trianion of this bacterial strain utilizes positive, polychrom hydrolysis, indoles generation, V.P. reaction, gelatine liquefication and nitrate reduction negative.This bacterial strain can utilize glycogen, L-arabinose, D-Fructose, D-Psicose, Pyruvic Acid Methyl ester, formic acid, b-hydroxybutyric acid, a-ketobutyric acid, a-oxopentanoic acid, L-alanimamides, D-alanine, ALANINE, L-alanyl-glycine, altheine, Pidolidone, Serine and inosine as sole carbon source and the energy.
Lipid acid in Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 cell mainly comprises different formula 15 carbon saturated fatty acid (27.99%), ω 9c different formula 17 carbon monounsaturated fatty acids (14.86%), anteiso-formula 15 carbon saturated fatty acid (12.41%), different formula 16 carbon saturated fatty acid (10.01%), different formula 17 carbon saturated fatty acid (8.30%), different formula 11 carbon saturated fatty acid (5.99%), 16 carbon saturated fatty acids (4.36%), 3-hydroxyl different formula 13 carbon saturated fatty acid (3.05%), 3-hydroxyl different formula 11 carbon saturated fatty acid (2.77%), ω 7c-16 carbon monounsaturated fatty acids/ω 6c-16 carbon monounsaturated fatty acids (2.01%), 3-hydroxy-16 carbon saturated fatty acid (1.08%), anteiso-formula 11 carbon saturated fatty acid (1.06%) and different formula 14 carbon saturated fatty acid (1.01%).
The G+C molar content of Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 is 64.65%; The 16S rRNA gene order of this bacterial strain as shown in sequence in sequence table 1, with the pattern bacterium Luteimonas marina KCTC12327 of Teng xanthomonas of delivering t, L.lutimaris KACC14929 twith Luteimonas aestuarii KCTC22048 tthe homology of 16S rRNA gene order be 96.0%.
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 and above-mentioned type strain Luteimonas aestuarii KCTC22048 tthe tetraploid rice of DNA hybridization is low, is 14%; Simultaneously, physio-biochemical characteristics, the cell walls lipid acid composition of this bacterial strain also have notable difference with above-mentioned type strain, illustrate that Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3 CGMCC N0.7165 is a novel bacterial of Teng xanthomonas.
Another object of the present invention is to provide a kind of method of decomposing petroleum hydrocarbon.
The method of decomposing petroleum hydrocarbon provided by the present invention, is process testing sample with described Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165, petroleum hydrocarbon is degraded.
In aforesaid method, it is characterized in that described being applied as: substratum based on 2216E, Xanthomonas campestris OB44-3 bacterial classification is risen to Hai Lian and carries out enlarged culturing, to cultivate the bacterium liquid of acquisition for oil degradation microbial inoculum, join with crude oil be sole carbon source and the energy artificial seawater substratum in, the crude oil in sea water medium is degraded.
Also protection scope of the present invention is belonged to the biotechnological formulation that Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 is active fraction preparation.
Hai Lianteng Xanthomonas campestris of the present invention (Luteimonas dalianensis) OB44-3CGMCCN0.7165 is the novel bacterial of Teng xanthomonas, be separated from the ocean environment of petroleum pollution, can effective decomposing petroleum hydrocarbon utilize petroleum hydrocarbon as the sole carbon source grown.In artificial seawater substratum, be 69.08% to the average degradation rate of petroleum hydrocarbon (starting point concentration is 1000mg/L) in 7 days; The growth of this bacterial strain simultaneously depends on the existence of salt, belongs to marine bacteria class, pollutes to controlling ocean environment Petroleum Hydrocarbon the advantage having uniqueness.Hai Lianteng Xanthomonas campestris of the present invention (Luteimonas dalianensis) OB44-3CGMCC N0.7165 can be used for the degraded of petroleum hydrocarbon.
Below in conjunction with specification drawings and specific embodiments, the invention will be further described, not limitation of the present invention.
Accompanying drawing explanation
Fig. 1 is Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165 form photo under a scanning electron microscope
Fig. 2 is the form photo of Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165 under transmission electron microscope
Fig. 3 is the 16S rRNA gene order phylogenetic tree analysis chart of Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165
Fig. 4 is that Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165 is to the degradation curve figure of petroleum hydrocarbon
Embodiment
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
The Isolation and Identification of embodiment 1 Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165
One, the separation of decomposing petroleum hydrocarbon bacterial strain
Isolation medium: (1) artificial seawater substratum (ASM): MgSO 47H 2o7.0g/L, KCl0.7g/L, KH 2pO 42.0g/L, Na 2hPO 43.0g/L, NH 4nO 31.0g/L, NaCl30g/L, Crude oil1g/L, PH7.2; Trace element solution 10mL.Trace element consists of: CaCl 220mg/L, FeCl 350mg/L, CuSO 40.5mg/L, MnCl 2.4H 2o0.5mg/L, ZnSO 4.7H 2o10mg/L.(2) solid plate isolation medium (ASM1): above-mentioned artificial seawater substratum adds 18g/L agar; (3) solid plate pure medium: 2216E(Difco).
Oil is dissolved in sherwood oil, is mixed with the petroleum oil that concentration is 100mg/mL.
Dalian Bay petroleum pollution marine site seawater sample 5mL will be picked up from, join sterilizing containing 100mL artificial seawater culture medium A SM 500mL shaking flask in, with oil (concentration 0.5 ~ 1g/L) for sole carbon source carries out enrichment culture, be placed in 28 DEG C, cultivate in 200r/min shaking table, as first time enrichment culture liquid, after 7 days, in the artificial seawater substratum that the first time enrichment culture liquid getting 5mL is transferred fresh, carry out enrichment culture again, as second time enrichment culture liquid, after 7 days, get second time enrichment culture liquid 5mL, in the artificial seawater substratum of transferring fresh, transfer 3 times.Final enrichment culture liquid is carried out 10 -1, 10 -2, 10 -3gradient dilution, on applying solid plate isolation base ASM1; Not add the artificial seawater substratum of crude oil for blank flat board, cultivate 7 ~ 10 days for 28 DEG C, picking list bacterium colony is dull and stereotyped in 2216E purifying, and line is separated single bacterium, obtains single bacterium colony of purifying.Single colony inoculation after picking purifying is in the 2216E test tube that 5mL liquid nutrient medium is housed, test tube is placed in 200r/min shaking culture case, 28 DEG C of about shaking culture 72h, get 200 μ L bacterium liquid and add in the cryopreservation tube containing 1.8mL20% aqueous glycerin solution ,-70 DEG C of placements save backup.
Result shows, without any colony growth on blank flat board, illustrate do not have additional oil as on the flat board of carbon source, the microorganism in Seawater Samples cannot grow; Accordingly, the flat board being added with oil there is bacterial strain can grow, illustrate that bacterial strain that these single bacterium colonies are corresponding can decomposing petroleum hydrocarbon be that sole carbon source grows with petroleum hydrocarbon.Picking can single bacterium colony OB44-3 of decomposing petroleum hydrocarbon for further study.
Two, the morphological specificity of bacterial strain
Above-mentioned steps one is separated the bacterial strain OB44-3 obtained to be inoculated in 2216E substratum, when Growth of Cells is to the logarithmic growth later stage, after bacterium colony size is stable, carries out the description of single bacterium colony mean state.Mainly comprise the size of bacterium colony, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), colony edge state (whether neat, irregular, radial etc.).
To be in the cell of logarithmic phase, adopt scanning electronic microscope and transmission electron microscope observation cellular form, result as depicted in figs. 1 and 2; Carry out gramstaining, in the form of the optical microphotograph Microscopic observation somatic cells of 40 × 100 times simultaneously.
Result shows, the bacterial strain colony diameter that above-mentioned steps one screens is 1.5 ~ 2mm, in small circular, smooth, protuberance, yellow, glossy, translucent, neat in edge; Observe under an electron microscope, somatic cells is shaft-like, atrichia, does not have mobility, and size is (1.02 ~ 1.24) μm × (0.32 ~ 0.47) μm.
Three, the growth characteristics of bacterial strain
The bacterial strain that above-mentioned steps one screens belongs to Gram-negative, aerobic bacteria.This bacterial strain the most suitable growth pH value is 7.2, and can grow pH scope is 6.0 ~ 8.0; Optimum growth temperature is 30 DEG C, and the temperature range that can grow is 15 ~ 37 DEG C.This bacterial strain belongs to marine bacteria, and growth depends on the existence of salt, can at 2.0 ~ 9.0(w/v) growth and breeding in salinity range, the most suitable growth salinity 4%.
Four, the biochemical character of bacterial strain
1, utilization of carbon source
Utilize Biolog microorganism automatic identifying system test strain to the utilization power of 95 kinds of carbon sources.First the pure culture bacterium colony of test strains is made cell suspension, be then inoculated on GN2 identification plate and cultivate, then use Biolog Microstation software reading of data, determine utilization of carbon source situation.
2, other biochemical character
The catalase of this bacterial strain, oxydase, beta-galactosidase enzymes, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase and tryptophane deaminase active utilize API20E biochemical reagents box to detect; The Citrate trianion of bacterial strain utilizes, polychrom hydrolysis, indoles produce, V.P. reaction, gelatine liquefication and nitrate reduction reaction utilize API20NE biochemical reagents box to detect.
Above experimental result shows, the bacterial strain that above-mentioned steps one screens can utilize L-arabinose, D-Fructose, D-Psicose, glycogen, Pyruvic Acid Methyl ester, formic acid, b-hydroxybutyric acid, a-ketobutyric acid, a-oxopentanoic acid, L-alanimamides, D-alanine, ALANINE, L-alanyl-glycine, altheine, Pidolidone, Serine and inosine in 95 kinds of carbon sources as sole carbon source and the energy.The catalase of this bacterial strain and oxidase positive, beta-galactosidase enzymes, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase and tryptophane desaminase are negative; The Citrate trianion of this bacterial strain utilizes positive, polychrom hydrolysis, indoles generation, V.P. reaction, gelatine liquefication and nitrate reduction negative.
Five, compare with Teng xanthomonas type strain physiological and biochemical property
The bacterial strain physiological and biochemical property that above-mentioned steps one is screened and Teng xanthomonas type strain (L.aestuarii KCTC22048 t, L.marina KCTC12327 t, L.lutimaris KACC14929 t) physiological and biochemical property compare, the results are shown in Table shown in 1.
The bacterial strain that table 1 screens compares with the physiological and biochemical property of Teng xanthomonas type strain
Note: in table 1, numbering 1 represents the bacterial strain OB44-3 that this research screens; Numbering 2 represents type strain L.aestuarii KCTC22048 t; Numbering 3 represents type strain L.marina KCTC12327 t; Numbering 4 represents type strain L.lutimaris KACC14929 t.+, represent that result is positive ,-, represent that result is negative.
Six, strain cell fatty acid analysis
1, the preparation of solution
Solution I: 45g sodium hydroxide is dissolved in (volume ratio of first alcohol and water is 1:1) in the aqueous solution of 300mL methyl alcohol;
Solution II: be that concentrated hydrochloric acid and the 275mL methyl alcohol of 1.19g/mL is dissolved in 135mL distilled water by 190mL proportion;
Solution III: 200mL normal hexane is mixed with 200mL ether;
Solution IV: 10.8g sodium hydroxide is dissolved in 900mL distilled water;
Solution V: saturated sodium chloride solution (mass percentage is 26.5%).
2, extracting method
Get the strain culture that a transfering loop above-mentioned steps one screens and be placed in 8mL screw socket Glass tubing, add 1mL solution I, tighten blind nut, boiling water bath 5min, take out vibration 5 ~ 10s, tighten blind nut once again, continue boiling water bath 25min;
After sample cooling, add 2mL solution II, tighten blind nut vibration, precise control of temperature is 80 ± 1 DEG C subsequently, water-bath 10min, and ice bath cools; This step needs strict temperature control to be 80 ± 1 DEG C and water bath time 10min, in order to avoid alcohol acid and ring type lipid acid are damaged;
In the sample hose of cooling, add 1.25mL solution III, about quick oscillation 10min, discard lower floor's aqueous phase;
In remaining organic phase, add 3mL solution IV and several solution V, about quick oscillation 5min, it is for subsequent use that the upper organic phase of getting 2/3rds is placed in gas-chromatography sample bottle.
3, analytical procedure
Adopt HP6890 gas chromatograph, be equipped with shunting/Splitless injecting samples mouth, flame ionization ditector (FID) and HP gas-chromatography chem workstation (HP CHEMSTATION ver A5.05); Chromatographic column is UItra-2 post (long 25m, internal diameter 0.2mm, thickness of liquid film 0.33 μm); Furnace temperature is second order temperature programming: starting temperature 170 DEG C, and per minute 5 DEG C rises to 260 DEG C, rises to 310 DEG C subsequently with 40 DEG C/min, maintains 1.5 minutes; Injector temperature 250 DEG C, carrier gas is hydrogen, flow velocity 0.5mL/min, split stream sampling pattern, splitting ratio 100:1, sample size 2 μ L; Detector temperature 300 DEG C, hydrogen flow rate 30mL/min, air velocity 216mL/min, supplement gas (nitrogen) flow velocity 30mL/min.
Result shows, the strain cell wall lipid acid that above-mentioned steps one screens mainly comprises isoamyl 15 carbon saturated fatty acid (27.99%), ω 9c different formula 17 carbon monounsaturated fatty acids (14.86%), anteiso-formula 15 carbon saturated fatty acid (12.41%), different formula 16 carbon saturated fatty acid (10.01%), different formula 17 carbon saturated fatty acid (8.30%), different formula 11 carbon saturated fatty acid (5.99%), 16 carbon saturated fatty acids (4.36%), 3-hydroxyl different formula 13 carbon saturated fatty acid (3.05%), 3-hydroxyl different formula 11 carbon saturated fatty acid (2.77%), ω 7c-16 carbon monounsaturated fatty acids/ω 6c-16 carbon monounsaturated fatty acids (2.01%), 3-hydroxy-16 carbon saturated fatty acid (1.08%), anteiso-formula 11 carbon saturated fatty acid (1.06%) and different formula 14 carbon saturated fatty acid (1.01%).
By the type strain (L.aestuariiKCTC22048 of lipid acid composition in this mycetocyte wall with Teng xanthomonas t, L.marina KCTC12327 t, L.lutimaris KACC14929 t) compare, result is as shown in table 2.
Table 2 screens the type strain lipid acid ratio of components of bacterial strain and Teng xanthomonas obtained comparatively
Note: in table 2, numbering 1 represents the bacterial strain OB44-3 that this research screens; Numbering 2 represents type strain L.aestuarii KCTC22048 t; Numbering 3 represents type strain L.marina KCTC12327 t; Numbering 4 represents type strain L.lutimaris KACC14929 t.
Seven, the 16S rRNA gene sequencing of bacterial strain and phylogenetic tree analysis
Extract the bacterial strain STb gene that above-mentioned steps one screens, adopt bacterial 16 S rRNA universal primer, forward primer is 5 '-AGAGTTTGATCCTGGCTCAG-3 ', and reverse primer is that 5 '-GGTTACCTTGTTACGACTT-3 ' carries out pcr amplification.PCR reaction conditions is: first 94 DEG C of 6min; 94 DEG C of 45s, 54 DEG C of 45s, 72 DEG C of 90s, totally 30 circulations; Last 72 DEG C extend 10min.
Pcr amplification product is connected on PMD18-T carrier after agarose gel electrophoresis reclaims, and send the precious biotech firm in Dalian to check order; Adopt Clustal X program (1.83), Phylogenetic Analysis is analyzed by N-J method.
Result shows, the bacterial strain 16S rRNA gene order that above-mentioned steps one screens is as shown in sequence in sequence table 1, and the Phylogenetic Analysis figure of its 16S rRNA gene order as shown in Figure 3.
According to obtain 16S rRNA gene order comparison and Phylogenetic Analysis result judge, the bacterial strain that above-mentioned steps one screens belongs to Teng xanthomonas (Luteimonas), and with the type strain Luteimonas marina KCTC12327 delivered t, L.lutimaris KACC14929 twith Luteimonas aestuarii KCTC22048 tthe homology of 16S rRNA gene order be 96.0%.
Eight, with Teng xanthomonas type strain (Luteimonas aestuarii KCTC22048 t) DNA-DNA hybridization analysis
1, the mensuration of somatic cells genomic dna purity
Extract the bacterial strain STb gene that above-mentioned steps one screens, get 10 μ L DNA sample in 1.5mL centrifuge tube, add 190 μ L0.1 × SSC solution and dissolve, cumulative volume is made to be 200 μ L, dilute 20 times by DNA sample, inhale with rifle and beat for several times, DNA sample is mixed, smashes.Ultraviolet absorption value when measuring 260nm, 280nm, 230nm and 270nm respectively with ultraviolet spectrophotometer, the DNA sample being applicable to doing DNA/DNA hybrid experiment must meet following condition:
OD(260nm)/OD(280nm) ﹥ 1.8, illustrate that protein mass content meets the requirements;
OD(260nm)/OD(230nm) ﹥ 2.0, illustrate that sugared content meets the requirements;
OD(260nm) ﹥ (270nm), illustrates that phenol content meets the requirements.
2, the mensuration of G+C molar content
The G+C assay of the strain gene group DNA that above-mentioned steps one screens uses melting temperature (Tm) (Tm) method, with E (E.coli K12, CGMCC1.365) be control comparisons, instrument is Perkin/Elmer company Lambda35UV/VIS Spectrometer; With PTP-1 temperature digital controller temperature control.Step is as follows:
DNA sample 0.1 × SSC to be measured is diluted to OD 260nmbe worth between 0.3-0.4;
First record the OD value of 25 DEG C at wavelength 260nm, then set heating schedule, to 95 DEG C from 65 DEG C, per minute raises 1 DEG C therebetween;
OD value rises and represents that sex change starts, and record cuvette temperature and OD value, until the sex change of OD value invariant representation is complete;
According to thermal denaturation curve, draw molten chain temperature (Tm), calculate G+C mol% content.In 0.1 × SSC solution, calculation formula is: G+C mol%=G+C mol% 1.365+ 2.08 (Tm unknown-Tm 1.365).
Result shows, the G+C molar content of bacterial strain OB44-3 is 64.65%.
3, DNA-DNA cross experiment
Solution-phase refolding Speed method is adopted to measure the DNA-DNA hybrid rate of bacterial strain.Instrument is Perkin Elmer Lambda35UV/VIS Spectrophotometer.Temperature controls with PIP-1Peltier System numeral temperature controlling system.Specific experiment step is as follows:
(1) DNA sample process: the DNA sample of extraction, needs before experiment first to put in ice bath, with 40W ultrasonication (DNA sample concentration is 2.0), DNA sample is cut into 2-5 × 10 5daltonian fragment.
(2) by etc. survey DNA sample use 0.1 × SSC accurate formulation to become OD respectively 260nm1.8-2.0, and both OD 260nmvalue consistent (being accurate to 0.001);
(3) enter UV Winlab program, occur its method window, in method window, select time drives TD method, arranges page set suitable location parameter by Timed.Inst.Sample..Mensuration wavelength is 260nm, and total minute is set as 30 minutes.Optimum renaturation temperature (optimal renaturation temperature, TOR) is calculated, by the temperature-stable of cuvette at optimum renaturation temperature according to G+C mol% after measured.In 2 × SSC reaction solution, optimum renaturation temperature presses formula: TOR=0.51 × (G+C) mol%+47 calculates.
(4) each 400 μ L of bacterial classification DNA sample that Deng prepares hybridization are contained in two centrifuge tubes respectively, then to get that each 200 μ L of two strain kind DNA sample are contained in same centrifuge tube be biased sample;
(5) before unique DNA sample and hybrid dna sample test, 100 DEG C of sex change 15min are set respectively by PTP-1 temperature controlling system, are then cooled to optimum renaturation temperature.Record OD 260nmvalue, when question response proceeds to 30min, stop reading, the temperature of all processes sample all lower than TOP, finally must not obtain one and extends in time, the straight line that absorbance value reduces gradually;
(6) according to software UV Winlab, in Algorithm hurdle wherein, select Slope, draw renaturation digit rate (V), be i.e. slope (usual V is expressed as the reduced value of per minute light absorption value);
(7) according to formulae discovery homology hybrid rate:
( H ) % = 4 Vm - ( Va + Vb ) / 2 VaVb × 100 %
4, DNA-DNA results of hybridization is analyzed
Result shows, the bacterial strain DNA that above-mentioned steps one screens and the type strain Luteimonas aestuarii KCTC22048 delivered tlower, be 14%.
Based on above measurement result, identification of strains above-mentioned steps one screened is that Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on January 18th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC N0.7165.
The performance of embodiment 2, Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCCN0.7165 decomposing petroleum hydrocarbon.
Oil is dissolved in sherwood oil, filtration sterilization, add above-described embodiment 1 containing 100mL artificial seawater substratum (ASM) 500mL shaking flask in, make oil final concentration in the medium be 0.5 ~ 1g/L.
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 is accessed in above-mentioned substratum according to the inoculum size that volume ratio is 5%, 30 DEG C, rotating speed be 200r/min aerobic lucifuge condition under cultivate 7 days.Meanwhile, not add the artificial seawater substratum of oil in contrast, connect the artificial seawater substratum of molten bacillus as blank, for deducting the impact of oil volatilization not inoculate sea.
Cultivate after 7 days, the oil sample after degraded and contrast methylene dichloride and normal hexane 1:1 (V:V) 100mL are extracted oil residues wherein.Accurately measure 4mL solution from organic phase, first use anhydrous Na 2sO 4dehydration, then use 0.22 μm of organic solvent-resistant membrane filtration, dry up with nitrogen, then again dissolve with 1mL normal hexane, move in GC sample bottle.Carry out analysis with GC-FID, GC-MS to oil component residual after oil degradation to measure.
Analytical conditions for gas chromatography: gas chromatograph FL9510; Chromatographic column DB-5(30m × 0.25mm × 0.25 μm) quartz capillary column; Detector temperature 300 DEG C; Heating schedule: 100 DEG C of constant temperature 2min is that 4 DEG C/min is warmed up to 300 DEG C with temperature rise rate, and then constant temperature 20min; Carrier gas (Ar) flow 1mL/min; Injector temperature 280 DEG C; Sample size 1 μ L; Splitting ratio 1:1
Three repetitions are established in experiment, and as shown in Figure 4, result shows the degradation effect of oil, and Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3CGMCC N0.7165 was 69.08% to the average degradation rate of petroleum hydrocarbon in 7 days.
Sequence table
Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3
TGGCGGCAGGCCTACACATGCAAGTCGAACGGCAGCGCGGGGAGCTTGCTCCCTGGCGGCGAGTGGCGGACGGGTGAGGAATGCATCGGAATCTGCCCATTTGTGGGGGATAACCTCGGGAAACCGGGACTAATACCGCATACGACCTTCGGGTGAAAGCAGGGGATCTTCGGACCTTGCGCAGATGGATGAGCCGATGCCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAACTTTCCGGTTAATACCCGGAGAGAATGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTCGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATTGCATTGGATACTGGCGGGCTAGAGTGCGGTAGAGGGTAGTGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGACTGCCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAACTAGGCACTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGGGAGGACAGAGGGCTGCGAACCCGCGAGGGCAAGCCAATCCCAGAAACCTCCTCCCAGTCCGGATCGG AGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTACCACGGTGATTC 。

Claims (7)

1. Hai Lianteng Xanthomonas campestris (Luteimonas dalianensis) OB44-3, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date on January 18th, 2013, and preservation registration number is CGMCC N0.7165.
2. Hai Lianteng Xanthomonas campestris OB44-3 as claimed in claim 1, it is characterized in that described Hai Lianteng Xanthomonas campestris OB44-3 colony characteristics and physio-biochemical characteristics are: shaft-like, size is 0.3 ~ 0.4 μm × 1.0 ~ 1.2 μm, gramstaining is negative, and after LB solid medium 30 DEG C cultivates 2 ~ 3 days, bacterium colony is light golden yellow, small circular, neat in edge, smooth surface, easily provokes; Catalase and oxidase positive, V.P. reaction negative, Starch Hydrolysis is negative, and Citrate trianion utilizes positive, nitrate reduction negative, and polychrom hydrolysis is negative, and gelatine liquefication is negative.
3. Hai Lianteng Xanthomonas campestris OB44-3 as claimed in claim 1, is characterized in that the 16S rRNA sequence of described Hai Lianteng Xanthomonas campestris OB44-3 is:
TGGCGGCAGGCCTACACATGCAAGTCGAACGGCAGCGCGGGGAGCTTGCTCCCTGGCGGCGAGTGGCGGACGGGTGAGGAATGCATCGGAATCTGCCCATTTGTGGGGGATAACCTCGGGAAACCGGGACTAATACCGCATACGACCTTCGGGTGAAAGCAGGGGATCTTCGGACCTTGCGCAGATGGATGAGCCGATGCCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAACTTTCCGGTTAATACCCGGAGAGAATGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTCGTTAAGTCTGATGTGAAAGCCCTGGGCTCAACCTGGGAATTGCATTGGATACTGGCGGGCTAGAGTGCGGTAGAGGGTAGTGGAATTCCCGGTGTAGCAGTGAAATGCGTAGATATCGGGAGGAACATCCGTGGCGAAGGCGACTGCCTGGACCAGCACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAACTAGGCACTCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGGGAGGACAGAGGGCTGCGAACCCGCGAGGGCAAGCCAATCCCAGAAACCTCCTCCCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAGAT CAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTACCACGGTGATTC 。
4. the Hai Lian as described in claims 1 to 3 rises the application of Xanthomonas campestris OB44-3 in degraded ocean environment is petroleum hydrocarbon contaminated.
5. apply as claimed in claim 4, it is characterized in that described being applied as: take 2216E as seed culture medium, rise Xanthomonas campestris OB44-3 bacterial classification to Hai Lian to cultivate, obtain bacteria suspension by cultivating in strain inoculation to seed culture medium 2216E, bacteria suspension is with 0.5 ~ 2 × 10 6the inoculum size of individual cell/mL substratum is inoculated in seed culture medium, 30 DEG C, rotating speed be 200r/min cultivate 48h, obtain bacterium liquid, to cultivate the bacterium liquid of acquisition for oil degradation microbial inoculum, join containing the seawater sample of crude oil 0.5 ~ 1g/L or with crude oil be sole carbon source artificial seawater substratum (ASM) in, the crude oil in sea water medium is degraded.
6. apply as claimed in claim 5, it is characterized in that each component final concentration of described artificial seawater substratum (ASM) is: MgSO 47H 2o7.0g/L, KCl0.7g/L, KH 2pO 42.0g/L, Na 2hPO 43.0g/L, NH 4nO 31.0g/L, NaCl30g/L, Crude oil1g/L, PH7.2; Trace element solution 10mL.Trace element consists of: CaCl 220mg/L, FeCl 350mg/L, CuSO 40.5mg/L, MnCl 2.4H 2o0.5mg/L, ZnSO 4.7H 2o10mg/L.
7. apply as claimed in claim 4, it is characterized in that described application is carried out as follows:
(1) 5 ~ 10g crude oil is dissolved in 5 ~ 10mL sherwood oil, filtration sterilization, joins containing in 100mL artificial seawater substratum (ASM) 500mL shaking flask, make crude oil final concentration in the medium be 0.5 ~ 1g/L.
(2) Hai Lian is risen Xanthomonas campestris OB44-3 seed and be inoculated into 2216E substratum, 30 DEG C, rotating speed be 200r/min cultivate 24h, obtain bacterium liquid, be 5% volume ratio by the bacterium liquid of acquisition by inoculum size, join in above-mentioned artificial seawater substratum (ASM), 30 DEG C, rotating speed be 200r/min aerobic lucifuge condition under cultivate 7 days.Meanwhile, not add the artificial seawater substratum of oil in contrast, rise the artificial seawater of Xanthomonas campestris OB44-3 as blank not inoculate Hai Lian, for deducting the impact of oil volatilization.
CN201310357439.5A 2013-08-15 2013-08-15 Luteimonas capable of degrading petroleum hydrocarbon, and its application Pending CN104371941A (en)

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CN110079479A (en) * 2019-04-29 2019-08-02 北京润世能源技术有限公司 One plant of rattan Flavimonas W-Y14 and its application
CN110846248A (en) * 2019-11-19 2020-02-28 天津大学 Artificial mixed bacteria system for degrading polycyclic aromatic hydrocarbon and application method thereof
CN111763631A (en) * 2020-03-10 2020-10-13 云南天腾化工有限公司 Pheretima aspergillum and its application
CN112662601A (en) * 2021-01-28 2021-04-16 山东九一生物科技股份有限公司 Screening method of microorganisms for repairing petroleum-polluted soil
CN112679057A (en) * 2020-11-18 2021-04-20 山东高科联合环保科学研究院有限公司 Marine microorganism reduction harmless treatment process for oil-containing sludge of refinery plant

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079479A (en) * 2019-04-29 2019-08-02 北京润世能源技术有限公司 One plant of rattan Flavimonas W-Y14 and its application
CN110079479B (en) * 2019-04-29 2022-03-22 北京润世能源技术有限公司 Tenebriomonas lutescens W-Y14 and application thereof
CN110846248A (en) * 2019-11-19 2020-02-28 天津大学 Artificial mixed bacteria system for degrading polycyclic aromatic hydrocarbon and application method thereof
CN111763631A (en) * 2020-03-10 2020-10-13 云南天腾化工有限公司 Pheretima aspergillum and its application
CN112679057A (en) * 2020-11-18 2021-04-20 山东高科联合环保科学研究院有限公司 Marine microorganism reduction harmless treatment process for oil-containing sludge of refinery plant
CN112679057B (en) * 2020-11-18 2022-07-19 山东高科联合环保科学研究院有限公司 Marine microorganism reduction harmless treatment process for oil-containing sludge of refinery
CN112662601A (en) * 2021-01-28 2021-04-16 山东九一生物科技股份有限公司 Screening method of microorganisms for repairing petroleum-polluted soil

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