CN110669700A - Efficient petroleum hydrocarbon degrading bacterium PA16_9 and screening method and application thereof - Google Patents

Efficient petroleum hydrocarbon degrading bacterium PA16_9 and screening method and application thereof Download PDF

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CN110669700A
CN110669700A CN201911082757.9A CN201911082757A CN110669700A CN 110669700 A CN110669700 A CN 110669700A CN 201911082757 A CN201911082757 A CN 201911082757A CN 110669700 A CN110669700 A CN 110669700A
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halomonas
titaniae
anaerobic
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solution
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CN110669700B (en
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邵宗泽
王万鹏
李振宇
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China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration)
Third Institute of Oceanography MNR
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Third Institute of Oceanography MNR
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • 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
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    • 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
    • C12N1/02—Separating microorganisms from their culture media
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    • 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
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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
    • 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
    • C02F2101/36—Organic compounds containing halogen

Abstract

A high-efficiency petroleum hydrocarbon degrading bacterium PA16_9 and a screening method and application thereof relate to marine petroleum hydrocarbon degrading bacteria. A bacterial colony of efficient petroleum hydrocarbon degrading bacteria, namely, the salt monad (Halomonas titanica) HT PA16_9 is round, beige and semitransparent, the surface is smooth and wet, the edge is regular, no halo is formed, the center of the bacterial colony is slightly convex, and the diameter is 2-4 mm; the application of the tylonospora tympani can be used for degrading organic compounds under the anaerobic condition. The identified high-efficiency petroleum hydrocarbon degrading bacterium (Halomonas titanica) PA16-9 can degrade n-hexadecane with initial concentration of 3.867g/L by anaerobic and lightproof standing culture at 32 ℃, the degradation rate is 76.7-86.5 percent after 3 months, the degradation rate of pyrene under anaerobic condition reaches 58 percent, and the degradation rate under aerobic condition reaches 74.5 percent.

Description

Efficient petroleum hydrocarbon degrading bacterium PA16_9 and screening method and application thereof
Technical Field
The invention relates to marine petroleum hydrocarbon degrading bacteria, in particular to a high-efficiency petroleum hydrocarbon degrading bacterium PA16-9 and a screening method and application thereof.
Background
With the continuous development of socioeconomic of China, the demand and the dependence on petroleum are increasingly increased, so that the continuous flourishing of related industries of petroleum is guided, and the problem of increasingly serious environmental pollution is caused at present. Limited by the imperfections in the current technological processes and management concepts, petroleum products containing a large amount of hydrocarbon components flow directly or indirectly (natural leaks, accidental spills and extensive use of refined petroleum products) into the ecosphere of soil, oceans, etc. (El-sheshhtawy, Khalil, Ahmed, & Abdallah,2014), causing a series of serious and persistent pollution problems. Saturated hydrocarbon (alkane) is the main component of petroleum (Tissot & Welte,1984), leaked hydrocarbon substances are poured into gaps of soil, the permeability of the soil is influenced, the water phase, gas phase and solid phase structures of the soil of crude oil are damaged, the growth of microorganisms in the soil is influenced, the respiration of plant root systems in the soil and the absorption of water nutrients are hindered, even the plant root systems are rotten and necrotized, the growth of plants is seriously damaged, and the land cultivation value of the area is reduced. Meanwhile, the petroleum pollutants in the soil often migrate along with the flow of water, so that the polluted area is continuously enlarged. On the other hand, harmful substances in petroleum, especially polycyclic aromatic hydrocarbon substances, have carcinogenic, teratogenic, mutagenic and other effects, and can enter organisms and even human bodies through food chains to directly harm human health.
At present, the commonly used petroleum hydrocarbon pollution remediation methods mainly comprise: physical repair, chemical repair and biological repair (Guarino, Spada, & sciarrrillo, 2017). Compared with chemical and physical repair methods, biological repair has the advantages of small influence on human and environment, simple and convenient operation, low cost and the like (Cappello et al, 2015), and the biological repair cost is only 30-50% of that of traditional physical and chemical repair (Bragg, Prince, Harner, & Atlas, 1994). Bioremediation is therefore considered as the most promising environmental remediation approach with its advantages of low investment and no secondary pollution (Head & Swannell, 1999; Juhasz & Naidu, 2000).
Disclosure of Invention
The first purpose of the invention is to provide a high-efficiency petroleum hydrocarbon degrading bacterium (Halomonas titanica) HTPA16_ 9.
The second purpose of the invention is to provide a screening method of a high-efficiency petroleum hydrocarbon degrading bacterium (Halomonas titanica) HTPA16_ 9.
The third purpose of the invention is to provide a high-efficiency petroleum hydrocarbon degrading bacterium (Halomonas titanica) HTPA16_ 916S rDNA nucleotide sequence.
The fourth purpose of the invention is to provide a high-efficiency petroleum hydrocarbon degrading bacterium (Halomonas titanica) HTPA16_9 which can be used for treating hydrocarbon pollution in a relatively anoxic region in the deep layer of saline-alkali soil and in a hydrocarbon polluted sea area which is rich in salt and low in oxygen content.
The bacterial colony of the efficient petroleum hydrocarbon degrading bacterium, namely, the salt monad (Halomonas titanica) HTPA16_9 is round, beige and semitransparent, has a smooth and moist surface, regular edges, no halo, and a central micro-bulge with the diameter of 2-4 mm; the tyanic Halomonas (Halomonas titaniae) HT PA16_9 has been deposited in the China general microbiological culture Collection center at 10 and 22 months in 2019, with the following addresses: the microbial research institute of western road 1, 3, national academy of sciences, north-south, morning-yang, Beijing, zip code: 100101, accession number of collection center: CGMCC No. 18722.
The Tyrannick Halomonas (Halomonas titaniae) HT PA16_9 is screened from a pacific ocean deep sea sediment with the sample number of 45II-KW1-S37-BC29, the distance from the surface layer of the sediment is 22-26 cm, the sea area of KW1 in the 2 nd voyage section of 45 voyages of the ocean is W154 DEG 14.9909 ', N9 DEG 29.9927', the sediment is yellow brown, tasteless and weakly viscous, the surface layer is in a semi-flowing shape, the sediment is slightly powdery and sandy when rubbed by hands, the downward consistency is increased, and yellow and white lumps are rich after 10 cm.
The screening method of the tylonomonas (Halomonas titaniae) HT PA16_9 comprises the following steps:
1) taking a 156mL anaerobic bottle,after washing, adding 100mL of basic salt culture medium, introducing N2Removing oxygen in the residual space, adding cysteine (-HCl) to remove residual oxygen in the saline solution, sealing the rubber plug, adding an aluminum cover, sterilizing at 121 deg.C for 20min, cooling the system to room temperature, and adding microelement solution, vitamin solution, phosphate solution, selenite-tungstate solution, carbon source, etc. in an ultraclean bench;
in step 1), the composition of the basal salt medium may be: ammonium chloride 0.33g/L, sodium nitrate 0.88g/L, potassium chloride 0.5g/L, calcium chloride (2H)2O)0.5g/L, magnesium chloride (6H)2O)3g/L, sodium chloride 22g/L, anhydrous sodium sulfate 3g/L, PIPESBuffer5g/L, resazurin (1g/L)1mL/L, and the pH is adjusted to 6.8. The vitamin solution may have the following components: pyridoxine hydrochloride 10mg/L, thiamine hydrochloride 5mg/L, riboflavin 5mg/L, hydrochloric acid 5mg/L, calcium D- (+) -pantothenate 5mg/L, p-aminobenzoic acid 5mg/L, lipoic acid 5mg/L, biotin 2mg/L, folic acid 2mg/L, vitamin B120.1mg/L, and filtering and sterilizing. The trace element solution may have the following composition: 1.5g nitrilotriacetic acid was added to about 500mL water, the pH was adjusted to 6.5 with KOH and magnesium sulfate (7H)2O)3g, manganese sulfate (H)20.5g of O), 1g of sodium chloride and 7H of ferric sulfate2O)0.1g, cobalt chloride (6H)2O)0.1g, anhydrous calcium chloride 0.1g, zinc chloride (7H)2O)0.1g, copper sulfate (5H)2O)0.01g, aluminum potassium sulfate (12H)2O)0.01g, boric acid 0.01g, sodium molybdate (2H)2O)0.01g, adjusted to 1L with distilled water, filter sterilized. The phosphate solution may have the following composition: KH (Perkin Elmer)2PO45g of the extract was dissolved in 100mL of distilled water and autoclaved at 121 ℃ for 20 min. The selenite-tungstate solution comprises the following components: 0.5g/L of sodium hydroxide, sodium selenite (5H)2O)3mg/L, sodium tungstate (2H)2O)4mg/L, filtering and sterilizing; the carbon source may be a mixed aromatic hydrocarbon or an alkane.
2) Taking out the pacific deep sea sediment, dissolving the pacific deep sea sediment by using an aseptic basic salt culture medium according to a proportion to form turbid liquid, injecting the turbid liquid into a sealed anaerobic culture medium through an injector, and standing and culturing for two months at room temperature in a dark place;
in step 2), the composition ratio of the anaerobic medium may be: 1mL of the microelement solution, 1mL of the vitamin solution, 1mL of the selenite-tungstate solution, 8mL of the phosphate solution, and 0.2% of the carbon source were added to 1L of the basal salt medium.
3) Sampling, coating on an anaerobic solid plate taking n-hexadecane and dodecane as unique carbon sources, placing in an anaerobic operation box, culturing at room temperature for three weeks, forming macroscopic colonies, and repeatedly separating and purifying through an HLB solid plate to obtain a strain of Telanganese Halomonas (Halomonas titaniae) HT PA16_ 9.
In step 3), the anaerobic solid plate can be added with 1.5% agar on the basis of a basic salt medium; the HLB solid plate may be composed of: 30g/L of sodium chloride, 5g/L of yeast extract, 10g/L of peptone and 15g/L of agar.
The invention extracts the genome DNA of a strain of Telanganese Halomonas (Halomonas titaniae) HT PA16_9, uses the genome DNA as a template, adopts universal 27F and 1492R primers to amplify a 16S rDNA fragment, selects a high-similarity sequence on EZBICLOd, calculates the phylogenetic distance of the sequence by MEGA-X, and constructs a phylogenetic tree.
A single colony is selected on a plate and inoculated in an anaerobic culture medium of a 30mL system, n-hexadecane is used as a unique carbon source, the initial addition amount is 0.07734g, the plate is kept at the constant temperature of 33 ℃ and kept in a dark place, and the residual n-hexadecane amount is measured after 3 months of anaerobic culture. The calculated degradation rate is about 76.7% -86.5%.
The 16S rDNA nucleotide sequence of the strain of the salt monad tympani (Halomonas titaniae) HT PA16-9 is as follows:
TGGTTACCTTGTTACGACTTCACCCCAGTGATGAAGCACACCGTGGTGATCGCCCTCTTGCGTTAGGCTAACCACTTCTGGTGCAGTCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGACCGGCTTTAAGGGATTCGCTGACTCTCGCGAGCTCGCAGCCCTTTGTACCGGCCATTGTAGCACGTGTGTAGCCCTACCCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCGACATTACTCGCTGGCAAATAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTTACAGTTCCCGAAGGCACACCAGAATCTCTTCCGGCTTCTGTAGATGTCAAGGGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAACTTCGCCACAAAGTGCTCTAGGCACCCAACGGCTGGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCACCTCAGTGTCAGTGTCAGTCCAGAAGGCCGCCTTCGCCACTGGTATTCCTCCCGATCTCTACGCATTTCACCGCTACACCGGGAATTCTACCTTCCTCTCCTGCACTCTAGCCTGACAGTTCCGGATGCCGTTCCCAGGTTGAGCCCGGGGCTTTCACAACCGGCTTATCAAGCCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTGATGTCTTTCCTACCGGGTATTAACCGATAGGCGTTCTTCCTCGCTGAAAGTGCTTTACAACCCGAGGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTCGGGCCGTGTCTCAGTCCCGATGTGGCTGATCATCCTCTCAGACCAGCTACGGATCGTTGCCTTGGTAAGCCATTACCTTACCAACTAGCTAATCCGACATAGGCTCATCCAATAGCGGGAGCCGAAGCCCCCTTTCTCCCGTAGGACGTATGCGGTATTAGCCTGGGTTTCCCCAGGTTATCCCCCACTATCGGGCAGATTCCTATGCATTACTCACCCGTCCGCCGCTCGTCAGCGGGTAGCAAGCTAGATCTGTTACCGCTCGACTTGCATGTGTTAGGCCTGCGGCGCCGCGTTCAATCTGAGCCAGGATCAAACTCTAA。
the strain of Halomonas tanicyanus (Halomonas titaniae) HT PA16_9 can be applied to degrading organic compounds under anaerobic conditions. The organic compound can be petroleum hydrocarbon compounds, and the petroleum hydrocarbon compounds can be hydrocarbon organic matters such as medium-chain and long-chain alkanes, monocyclic and polycyclic aromatic hydrocarbons, asphalt, halogenated hydrocarbons and the like.
The identified Thtannik Halomonas (Halomonas titaniae) HT PA16_9 can degrade n-hexadecane with initial concentration of about 3.867g/L by anaerobic and dark standing culture at 32 ℃, the degradation rate is 76.7-86.5 percent after 3 months, the degradation rate of pyrene under anaerobic condition reaches 58 percent, and the degradation rate under aerobic condition reaches 74.5 percent. Therefore, the strain of Halomonas (Halomonas titanica) HT PA16_9 can be applied to ecological restoration of hydrocarbon-polluted high-salinity soil, sea areas and other extreme environments and participate in processes of sewage treatment and the like.
Drawings
FIG. 1 shows the determination of polycyclic aromatic hydrocarbon degradation rate of a strain of Halomonas tanicutus (Halomonas titaniae) HT PA16_ 9.
FIG. 2 is a measurement of alkane degradation rate of a strain of Halomonas tanicu (Halomonas titaniae) HT PA16_ 9.
FIG. 3 is a macroscopic morphological diagram of a colony of Halomonas tanicutus (Halomonas titaniae) HT PA16_ 9.
FIG. 4 shows the morphology of a strain of Halomonas tanicutus (Halomonas titaniae) HT PA16_9 under a transmission electron microscope.
FIG. 5 is a phylogenetic analysis tree of Halomonas tanicu (Halomonas titaniae) HT PA16_9 according to the present invention.
Detailed Description
The following examples will further illustrate the present invention with reference to the accompanying drawings.
Example 1: morphological characteristics of the strains
And (3) streaking and inoculating a single colony to an HLB (hydrophile-lipophile balance) solid plate, inverting the plate in a constant-temperature incubator, and culturing for 48h at 32 ℃, wherein the colony is circular, is beige and semitransparent, has a smooth and moist surface, is regular in edge, does not have a halo, is slightly convex in the center, and has a diameter of 2-4 mm, and is shown in figure 3. And (3) taking the primary thalli to prepare a film, observing by a transmission electron microscope, wherein the film is shown in figure 4, the bacterial strain is gram-negative bacteria, facultative and aerobic, is rod-shaped, has the length of 1.6-2 mu m, is provided with pili on the periphery and mainly depends on flagella to move.
Example 2: screening and identification of strains
(1) The sample of the pacific ocean deep sea sediment is taken from a KW1 sea area (sample number 45II-KW1-S37-BC29, 22-26 cm from the surface layer of the sediment, W154 DEG 14.9909 ', N9 DEG 29.9927') of a second voyage section of 45 voyages in the ocean, the sediment is tawny, tasteless and weakly sticky, the surface layer is semi-flowing, the surface layer is slightly powdery and sandy when being rubbed by hands, the downward consistency is increased, and yellow and white lumps which are rich in below 10cm are mixed in the sample. After being collected aseptically, the sediment samples were stored at low temperature and then transported to the laboratory for the next phase of research.
(2) Taking a 156mL anaerobic bottle, cleaning, adding 100mL basic salt culture medium, introducing N2Removing oxygen in the residual space, and adding cysteine (-HCl) to remove residual oxygen in the salt solution. Sealing the rubber plug, adding an aluminum cover, and sterilizing at 121 deg.CAnd (3) performing bacteria culture for 20min, and after the system is cooled to room temperature, supplementing a trace element solution, a vitamin solution, a phosphate solution, a carbon source (mixed aromatic hydrocarbon and alkane) and the like on a super-clean workbench. Taking out the sediment sample, dissolving the sediment sample by using a sterile basic salt culture medium according to a proportion to form turbid liquid, and injecting the turbid liquid into the sealed anaerobic culture medium through an injector. Standing and culturing for two months at room temperature in a dark place, sampling, coating on a solid plate with n-hexadecane and dodecane as the only carbon source, placing in an anaerobic operation box, culturing for 3 weeks at room temperature, forming a macroscopic bacterial colony, and repeatedly separating and purifying the plate to obtain pure salt monad (Halomonas titaniae) HTPA16_9 of the Tatannik.
The genomic DNA of the strain was extracted and used as a template to amplify a16 SrDNA fragment by using a universal 27F, 1492R primer, and a high similarity sequence was selected from EZBcloud (https:// www.ezbiocloud.net /), and the phylogenetic distance of the sequence was calculated by MEGA-X to construct a phylogenetic tree (NJ), see FIG. 5.
Example 3: partial hydrocarbon degradation ability measurement of Strain
(1) Preparing an anaerobic liquid culture medium, wherein the concentration of a carbon source is 0.33% (about 0.07734g) and the culture system is 30mL, scraping a proper amount of thalli from an HLB solid plate, dissolving the thalli in the liquid culture medium, uniformly mixing the thalli in a vortex manner, inoculating the thalli into the anaerobic culture medium, wherein the inoculation amount is 1%, and standing and culturing three experimental groups at the temperature of 32 ℃ in a dark place.
(2) Opening the bottle mouth after culturing for 3 months, repeatedly extracting N-hexadecane in the culture medium by using N-hexane (totally extracting for four times), combining the extract liquor, and N2After blowing, 0.5ml (about 10 drops) of n-hexadecane was secured against oxidation. Obtaining a mass spectrogram of a sample by GC-MS (gas chromatography-mass spectrometer), measuring the residual amount of the n-hexadecane, obtaining that the degradation rate is 76.7-86.5%, the anaerobic degradation rate of the pyrene reaches 58%, and the degradation rate reaches 74.5% under an aerobic condition, and referring to figures 1 and 2.
Sequence listing
<110> third oceanographic institute of natural resources department; china ocean mineral resources research and development association (China ocean affairs administration)
<120> high-efficiency petroleum hydrocarbon degrading bacterium PA16-9 and screening method and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1498
<212>DNA/RNA
<213> Halonas tatanii (Halomonas titanicae)
<400>1
tggttacctt gttacgactt caccccagtg atgaagcaca ccgtggtgat cgccctcttg 60
cgttaggcta accacttctg gtgcagtcca ctcccatggt gtgacgggcg gtgtgtacaa 120
ggcccgggaa cgtattcacc gtgacattct gattcacgat tactagcgat tccgacttca 180
cggagtcgag ttgcagactc cgatccggac tgagaccggc tttaagggat tcgctgactc 240
tcgcgagctc gcagcccttt gtaccggcca ttgtagcacg tgtgtagccc tacccgtaag 300
ggccatgatg acttgacgtc gtccccacct tcctccggtt tgtcaccggc agtctcctta 360
gagttcccga cattactcgc tggcaaataa ggacaagggt tgcgctcgtt acgggactta 420
acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctt acagttcccg 480
aaggcacacc agaatctctt ccggcttctg tagatgtcaa gggtaggtaa ggttcttcgc 540
gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcatttga 600
gttttaacct tgcggccgta ctccccaggc ggtcgactta tcgcgttaac ttcgccacaa 660
agtgctctag gcacccaacg gctggtcgac atcgtttacg gcgtggacta ccagggtatc 720
taatcctgtt tgctacccac gctttcgcac ctcagtgtca gtgtcagtcc agaaggccgc 780
cttcgccact ggtattcctc ccgatctcta cgcatttcac cgctacaccg ggaattctac 840
cttcctctcc tgcactctag cctgacagtt ccggatgccg ttcccaggtt gagcccgggg 900
ctttcacaac cggcttatca agccacctac gcgcgcttta cgcccagtaa ttccgattaa 960
cgcttgcacc ctccgtatta ccgcggctgc tggcacggag ttagccggtg cttcttctgc 1020
gagtgatgtc tttcctaccg ggtattaacc gataggcgtt cttcctcgct gaaagtgctt 1080
tacaacccga gggccttctt cacacacgcg gcatggctgg atcagggttg cccccattgt 1140
ccaatattcc ccactgctgc ctcccgtagg agttcgggcc gtgtctcagt cccgatgtgg1200
ctgatcatcc tctcagacca gctacggatc gttgccttgg taagccatta ccttaccaac 1260
tagctaatcc gacataggct catccaatag cgggagccga agcccccttt ctcccgtagg 1320
acgtatgcgg tattagcctg ggtttcccca ggttatcccc cactatcggg cagattccta 1380
tgcattactc acccgtccgc cgctcgtcag cgggtagcaa gctagatctg ttaccgctcg 1440
acttgcatgt gttaggcctg cggcgccgcg ttcaatctga gccaggatca aactctaa 1498

Claims (10)

1. A bacterial colony of efficient petroleum hydrocarbon degrading bacteria, namely, the salt monad (Halomonas titanica) HT PA16_9 is round, beige and semitransparent, the surface is smooth and wet, the edge is regular, no halo is formed, the center of the bacterial colony is slightly convex, and the diameter is 2-4 mm; the tyanic Halomonas (Halomonas titanica) HT PA16_9 has been deposited in the China general microbiological culture Collection center at 10.22.2019, and the accession number of the collection center is as follows: CGMCC No. 18722.
2. The strain of Halomonas taniensis (Halomonas titaniae) HT PA16_9 as claimed in claim 1, which is selected from sediments in deep sea of the Pacific ocean, sample No. 45II-KW1-S37-BC29, 22-26 cm from the surface layer of the sediments, KW1 sea area in No. 2 voyage of Atlantic ocean 45, W154 ° 14.9909 ', N9 ° 29.9927', the sediments are yellow brown, tasteless, weak viscous, semi-flowing on the surface layer, slightly powdery and sandy by hand rubbing, downward consistency is increased, and rich yellow-white lumps are mixed at 10 cm.
3. The method for screening a strain of Halomonas taniensis (Halomonas titaniae) HT PA16_9 according to claim 1, which comprises the following steps:
1) taking a 156mL anaerobic bottle, cleaning, adding 100mL basic salt culture medium, introducing N2Removing oxygen in the residual space, adding cysteine to remove residual oxygen in the saline solution, sealing the rubber plug, adding an aluminum cover, sterilizing at the high temperature of 121 ℃ for 20min, cooling the system to room temperature, and adding a trace element solution, a vitamin solution, a phosphate solution, a selenite-tungstate solution, a carbon source and the like on an ultra-clean workbench;
2) taking out the pacific deep sea sediment, dissolving the pacific deep sea sediment by using an aseptic basic salt culture medium according to a proportion to form turbid liquid, injecting the turbid liquid into a sealed anaerobic culture medium through an injector, and standing and culturing for two months at room temperature in a dark place;
3) sampling and coating on an anaerobic solid plate taking hexadecane and dodecane as unique carbon sources, placing the plate in an anaerobic operation box for three weeks at room temperature to form macroscopic colonies, and repeatedly separating and purifying to obtain a strain of Halomonas tanaka (Halomonas titaniae) HT PA16_ 9.
4. The method for screening a strain of Halomonas taniensis (Halomonas titaniae) HT PA16_9 according to claim 3, wherein in step 1), the basic salt medium comprises the following components: 0.33g/L of ammonium chloride, 0.88g/L of sodium nitrate, 0.5g/L of potassium chloride, 0.5g/L of calcium chloride, 3g/L of magnesium chloride, 22g/L of sodium chloride, 3g/L of anhydrous sodium sulfate, 5g/L of PIPESBuffer, 1mL/L of Resazurin, and the pH value is adjusted to 6.8; the vitamin solution comprises the following components: pyridoxine hydrochloride 10mg/L, thiamine hydrochloride 5mg/L, riboflavin 5mg/L, hydrochloric acid 5mg/L, calcium D- (+) -pantothenate 5mg/L, p-aminobenzoic acid 5mg/L, lipoic acid 5mg/L, biotin 2mg/L, folic acid 2mg/L, vitamin B120.1mg/L, and filtering and sterilizing.
5. As claimed inSolving 3 the screening method of a strain of Halomonas taniensis HT PA16_9, which is characterized in that in the step 1), the selenite-tungstate solution comprises the following components: 0.5g/L of sodium hydroxide, 3mg/L of sodium selenite and 4mg/L of sodium tungstate, and filtering and sterilizing; the trace element solution comprises the following components: firstly, 1.5g of nitrilotriacetic acid is added into about 500mL of water, the pH value is adjusted to 6.5 by KOH for dissolution, 3g of magnesium sulfate, 0.5g of manganese sulfate, 1g of sodium chloride, 0.1g of ferric sulfate, 0.1g of cobalt chloride, 0.1g of anhydrous calcium chloride, 0.1g of zinc chloride, 0.01g of copper sulfate, 0.01g of aluminum potassium sulfate, 0.01g of boric acid and 0.01g of sodium molybdate, the volume is adjusted to 1L by distilled water, and filtration sterilization is carried out; the phosphate solution comprises the following components: KH (Perkin Elmer)2PO4Dissolving 5g of the extract in 100mL of distilled water, and autoclaving at 121 ℃ for 20 min; the carbon source may be a mixed aromatic hydrocarbon or an alkane.
6. The method for screening a strain of Halomonas taniensis (Halomonas titaniae) HT PA16_9 according to claim 3, wherein in step 2), the anaerobic culture medium comprises the following components in proportion: 1mL of the microelement solution, 1mL of the vitamin solution, 1mL of the selenite-tungstate solution, 8mL of the phosphate solution, and 0.2% of the carbon source were added to 1L of the basal salt medium.
7. The method for screening a strain of Halomonas taniensis (Halomonas titaniae) HT PA16_9 according to claim 3, wherein in step 3), said anaerobic solid plate is supplemented with 1.5% agar based on a basal salt medium.
8. The 16S rDNA nucleotide sequence of the salt monad tympani (Halomonas titaniae) HT PA16_9 of claim 1 is as follows:
TGGTTACCTTGTTACGACTTCACCCCAGTGATGAAGCACACCGTGGTGATCGCCCTCTTGCGTTAGGCTAACCACTTCTGGTGCAGTCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGACCGGCTTTAAGGGATTCGCTGACTCTCGCGAGCTCGCAGCCCTTTGTACCGGCCATTGTAGCACGTGTGTAGCCCTACCCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCGACATTACTCGCTGGCAAATAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTTACAGTTCCCGAAGGCACACCAGAATCTCTTCCGGCTTCTGTAGATGTCAAGGGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAACTTCGCCACAAAGTGCTCTAGGCACCCAACGGCTGGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCACCTCAGTGTCAGTGTCAGTCCAGAAGGCCGCCTTCGCCACTGGTATTCCTCCCGATCTCTACGCATTTCACCGCTACACCGGGAATTCTACCTTCCTCTCCTGCACTCTAGCCTGACAGTTCCGGATGCCGTTCCCAGGTTGAGCCCGGGGCTTTCACAACCGGCTTATCAAGCCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTGATGTCTTTCCTACCGGGTATTAACCGATAGGCGTTCTTCCTCGCTGAAAGTGCTTTACAACCCGAGGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTCGGGCCGTGTCTCAGTCCCGATGTGGCTGATCATCCTCTCAGACCAGCTACGGATCGTTGCCTTGGTAAGCCATTACCTTACCAACTAGCTAATCCGACATAGGCTCATCCAATAGCGGGAGCCGAAGCCCCCTTTCTCCCGTAGGACGTATGCGGTATTAGCCTGGGTTTCCCCAGGTTATCCCCCACTATCGGGCAGATTCCTATGCATTACTCACCCGTCCGCCGCTCGTCAGCGGGTAGCAAGCTAGATCTGTTACCGCTCGACTTGCATGTGTTAGGCCTGCGGCGCCGCGTTCAATCTGAGCCAGGATCAAACTCTAA。
9. the use of a strain of Halomonas tanicutus (Halomonas titaniae) HT PA16_9 according to claim 1 for the anaerobic degradation of organic compounds.
10. The use of claim 9, wherein the organic compound is a petroleum hydrocarbon compound selected from the group consisting of medium and long chain alkanes, monocyclic and polycyclic aromatic hydrocarbons, pitch, and halogenated hydrocarbon organics.
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