CN111705007B

CN111705007B - New azospirillum species for degrading heavy oil and microbial preparation thereof

Info

Publication number: CN111705007B
Application number: CN201910203022.0A
Authority: CN
Inventors: 蔡曼; 孟俞辰; 张笑鹃; 周宇光
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-03-18
Filing date: 2019-03-18
Publication date: 2022-06-21
Anticipated expiration: 2039-03-18
Also published as: CN111705007A

Abstract

The invention discloses a new azospirillum species for degrading heavy oil and a microbial preparation thereof. The new species of Azospirillum firmum provided by the invention is Azospirillum oleamicum, and the representative strain is RWY-5-1^TThe preservation number of the strain in China general microbiological culture Collection center is CGMCC No. 17324. The Azospirillum oleraceum RWY-5-1 provided by the invention^TCGMCC No.17324 belongs to a new strain of Azospirillum. Azospirillum oleraceum RWY-5-1^TCGMCC No.17324 not only has the nitrogen fixation function, but also has the degradation function of heavy crude oil, and has wide comprehensive application prospect in the aspects of microbial oil recovery, soil bioremediation, soil texture improvement, crop growth promotion and the like.

Description

New azospirillum species for degrading heavy oil and microbial preparation thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a new azospirillum species for degrading heavy oil and a microbial preparation thereof.

Background

Azospirillum (Azospirillum) is a group of alpha-Proteobacteria (Alphaproteobacteria) in the bacterial domain. In 1978, Tarrand et al discovered that Azospirillum brasilense (Azospirillum brasilense) was described and reclassified as Azospirillum lipolyticum (Spirillum lipoferum), so far this genus was first proposed and comprised two species, which became typical species of this genus due to the time preference for the discovery of Azospirillum lipolyticum. At present, the genus contains 18 species, most of which are sourced from cultivated soil, and also have environments separated from spring water, a biological battery and the like, and most of representative strains of the genus have the nitrogen fixation capacity, so that the genus has the potential of promoting plant growth.

The heavy oil is heavy crude oil, namely crude oil with the relative density of 0.90-1.00 g/cubic centimeter, has high density and viscous property, contains a large amount of macromolecular polycyclic aromatic hydrocarbons, colloid, asphaltene and other heavy components which are difficult to biodegrade, is difficult to extract, and is difficult to degrade once accumulated in the environment. However, with the continuous increase of energy demand and the increasing consumption of conventional oil and gas resources, the proportion and the status of heavy oil in the petroleum industry are greatly improved, and the problems of low recovery rate, overflow, dripping, leakage and the like in the transportation and processing processes are inevitable at present. For example, 70-80% of the crude oil yield of the Bohai Bay oil field which has a major oil spill accident in 2011 belongs to heavy oil. In addition, as the second largest petroleum import country in the world, heavy crude oil is imported from great quantities in Venezuela and other countries in China, and the risk of oil spill accidents in transportation is greatly increased. Once leakage occurs, the influence on the environment is more serious and lasting than that of the conventional crude oil and fuel oil, and the treatment of heavy oil pollution is more difficult. In recent years, microbial oil recovery and pollutant microbial remediation technologies, which take microbial degradation as a core and have the obvious advantages of high efficiency, environmental protection, economy and the like, have been widely concerned by researchers. Although a plurality of crude oil degrading bacteria are separated in various habitats at present, the reports on the heavy oil degrading bacteria are less; meanwhile, there is only one report on the degradation of light crude oil by Azospirillum (Muratova, A.Y, Turkovskaya, O.V., Antonyuk, L.P., et al, 2005.oil-oxidizing potential of assisted rhizobacterium of the genus Azospirillum. microbiology,74,2: 210-.

Disclosure of Invention

The invention aims to provide a new azospirillum species for degrading heavy oil and a microbial preparation thereof.

In a first aspect, the invention claims Azospirillum oleraceum RWY-5-1^T。

The invention claims oil-decomposing Azospirillum olyeichium RWY-5-1^TBelongs to a new strain of Azospirillum, and the preservation number of the new strain in China general microbiological culture collection center is CGMCC No. 17324.

In a second aspect, the invention claims a microbial preparation.

The microbial preparation claimed by the invention contains Azospirillum oleraceum RWY-5-1 as described in the first aspect^T. The remaining ingredients may be adjuvants or carriers commonly used in the art.

In a third aspect, the invention claims the Azospirillum oleraceum RWY-5-1^TOr the use of the microbial preparation in any one of:

(a1) degrading petroleum or crude oil;

further, the petroleum is heavy petroleum; the crude oil is heavy crude oil;

(a2) microbial oil recovery;

(a3) biological nitrogen fixation;

(a4) soil remediation and/or soil reclamation;

(a5) promoting the growth of plants.

Untreated petroleum is known as crude oil.

The microbial repairing technology of soil pollution is that under the stress of pollution, some organic matter capable of complexing or decomposing and converting pollutant is secreted from body to reduce the mobility and change polarity of pollutant and make it difficult to enter body; or the pollutants are decomposed and converted into non-toxic and harmless substances in vitro under the action of extracellular enzymes secreted by the microorganisms. The method increases the number of resistant microorganisms in the soil, not only can reduce the pollution degree, but also can improve the fertility of the soil, and is an economic and effective technology.

The microbial soil improving technology is to apply organic fertilizer and microbial mixture for fast releasing soil nutrients to soil. The microorganisms can rapidly and efficiently decompose organic matters in the soil to accelerate the growth and the propagation of the microorganisms, fix molecular nitrogen in the air and convert the molecular nitrogen into ammonia nitrogen which can be absorbed by plants, and simultaneously decompose insoluble P, K in the soil into soluble elements, so that the soluble elements are easily absorbed and utilized by the plants to improve the soil; in addition, after the crops are harvested, the effective microbial population is directly sprayed on the stubble, so that the stubble above and below the ground surface can be rapidly decomposed, and the aims of increasing the soil fertility, improving the soil structure and fully maintaining and utilizing the soil moisture are fulfilled.

In a fourth aspect, the invention claims the use, in particular the use of Azospirillum oleraceum (or a microbial preparation containing said Azospirillum oleraceum), of an Azospirillum oleospirillum oleraceum in any of:

(a1) degrading petroleum or crude oil;

further, the petroleum is heavy petroleum; the crude oil is heavy crude oil;

(a2) microbial oil recovery;

(a3) biological nitrogen fixation;

(a4) soil remediation and/or soil reclamation;

(a5) promoting the growth of plants.

Wherein the Azospirillum olysospira (Azospirillum oleraceum) is a gram-negative rod-shaped bacterium, and the 16S rRNA gene sequence of the strain has 98.7% or more homology with SEQ ID No. 1.

98.7% was used as a threshold for the division of the bacterial species according to the general references "Stackelbrandt E, Ebers J.2006, Tarxonomic parameters revisited: tarnished gold standards. Microbiol Today,33: 152-" from the references ".

Experiments prove that the Azospirillum oleraceum RWY-5-1 provided by the invention^TCGMCC No.17324 belongs to a new strain of Azospirillum. Azospirillum oleuisticium RWY-5-1-1^TCGMCC No.17324 not only has nitrogen fixation function, but also has the degradation function of heavy crude oil and petroleum, and has wide comprehensive application prospect in the aspects of microbial oil recovery, soil bioremediation, soil texture improvement, crop growth promotion and the like.

Deposit description

The strain name: oil-decomposing azospirillum

Latin name: azospirillum oleraceum

According to the biological materials (strains): RWY-5-1^T

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 3, 8 months in 2019

Registration number of the preservation center: CGMCC No.17324

Drawings

FIG. 1 shows a strain RWY-5-1 of the present invention^TTransmission electron micrograph (D).

FIG. 2 shows a strain RWY-5-1 of the present invention^TThe degradation effect of the heavy oil is shown.

FIG. 3 shows a strain RWY-5-1 of the present invention^TPhylogenetic tree based on 16S rRNA gene sequences.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

A.picis DSM 19922^T: the detailed information of the strain is recorded in the following website: https:// www.dsmz.de/transactions/details/culture/DSM-19922. htmltx _ dsmzresources _ pi 5% 5 Breturpid% 5D ═ 304. Meanwhile, the strain is described in "International Journal of Systematic and evolution Microbiology (2009),59: 761-765".

A.thiophilum DSM 21654^T: detailed information of the strainRecorded in the following website: https:// www.dsmz.de/transactions/details/culture/DSM-21654. htmltx _ dsmzresources _ pi 5% 5 Breturpid% 5D ═ 304. Meanwhile, the strain is described in "International Journal of Systematic and evolution Microbiology (2010), 60: 2832-.

A.lipoferum NBRC 102290^T: the detailed information of the strain is recorded in the following website: https:// www.jcm.riken.jp/cgi-bin/jcm/jcm _ ccCC ═ NBRC&NUM 102290. Meanwhile, the strain is described in "Canadian Journal of Microbiology (1978),24: 967-.

A.humicireducens KACC 16605^T: the detailed information of the strain can be inquired in the following websites: http:// www.cctcc.org/news _ show1.phpnewsid 88. Meanwhile, the strain is described in "International Journal of Systematic and evolution Microbiology (2013),63: 2618-.

Example 1 Strain RWY-5-1^TSeparation screening and identification of

1. Strain RWY-5-1^TSeparation and screening of

The strain RWY-5-1 of the invention^TThe crude oil produced liquid separated from the single north block of the Yumen oil field comprises the following specific separation operations:

pre-treating a crude oil sample: the crude oil sample was washed repeatedly with sterile isooctane and the washed supernatant was transferred to a sterile container. And collecting cells in all the supernatant by using an organic filter membrane with the aperture of 0.22 micron, putting the filter membrane into 5ml of sterile physiological saline, and washing the membrane by fully shaking to form a bacterial suspension for bacteria separation and later use.

Strain isolation medium: R2A medium was used, the solvent was water, the solutes and concentrations were as follows: glucose 0.5 g.L^-10.5 g.L of soluble starch^-1、

Peptone 0.5 g.L^-10.5 g.L yeast extract^-1Acid hydrolyzed casein 0.5 g.L^-10.3 g.L of sodium pyruvate^-1Dipotassium hydrogen phosphate 0.3 g.L^-1Seven, sevenMagnesium sulfate hydrate 0.05 g.L^-115 g.L of agar^-1(ii) a The pH value is 7.2.

The separation and screening method comprises the following steps: the bacterial suspension obtained by the pretreatment was spread on a plate of R2A medium and cultured at 30 ℃ for 7 days. Selecting single bacterial colonies which grow well, have complete bacterial lawn and different morphological characteristics, and streaking and purifying the single bacterial colonies on the same isolation culture medium plate to obtain different pure bacterial strains for subsequent research. Meanwhile, the obtained pure strains are preserved by using two modes of 10% (v/v) skim milk vacuum freeze drying and 15% (v/v) glycerol ultralow temperature liquid nitrogen. The obtained strain is numbered as RWY-5-1^T。

When the strain is grown on R2A culture medium at 30 ℃ and is studied in morphological, physiological, biochemical, cytochemical and gene level, other special cases will be explained.

2、RWY-5-1-1^TPhysiological and biochemical characteristic detection of strain

Strain RWY-5-1^TAfter 3 days of growth under culture conditions, cell morphology was observed using a transmission electron microscope. The growth temperature detection range is 4, 10, 15, 20, 25, 30, 37, 42 and 45 ℃; the detection range of the growth salt concentration (NaCl) is 0-6% (0-6g/100ml) and 7 (0, 1, 2, 3, 4, 5, 6) concentration gradients are adopted; growth pH was measured in 10 (4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10) gradients (Meng, Y.C., Liu, H.C., Zhou Y.G., et al 2018, Vibrio ganlei sp.nov., a novel member of viral acid isolated from saw down in a pig pen. int J Syst Evol. Microbiol,68:1969-1974.) ranging between 4 and 10. The physiological and biochemical functions of the strain are completed by using detection kits API 20E and API ZYM produced by French Meriella, and a detection system GEN III produced by American BiOLOG. The growth detection of the MacConkey culture medium is to inoculate a strain on a MacConkey plate, culture for 7 days at 30 ℃, and observe the color of a bacterial colony and the change of a turbid circle around the bacterial colony. Biotin requirements were as per Tarrand et al (Tarrand, j.j., Krieg, N.R.&Dobereiner, J.1978, A taxonomic student of the Spirillum lipoferum group, with descriptions of a new gene, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) com. nov. and Azospirillum brasilens sp. nov. Can J. Microbiol 24:967-The method is used for detection. Other strain physiological characteristics, including gram stain profile, cell contents, motility, oxygen demand, catalase activity, casein hydrolysis activity, were performed according to the handbook of general bacterial systems identification (east elegans bead, zeisis. 2001. manual of general bacterial systems identification. Beijing: scientific Press).

The identification result shows that the strain RWY-5-1^TThe cells are gram-negative bacteria, slightly bent, short rods, 2.95-4.0 microns long and 1.5-1.9 microns wide, facultative anaerobic, with single-ended flagellar movement (see FIG. 1), and contain polyhydroxybutyrate. After the bacterial strain is cultured on an R2A culture medium for 48 hours at the temperature of 30 ℃, the bacterial colony is milk white, round, smooth and slightly convex, and the diameter is 0.8-1.0 mm. The growth tolerance range of the strain is 15-37 ℃, 0-3% NaCl and pH 6-8.5, and the optimal growth condition is 25 ℃, 0% NaCl and pH 7.5. The production of oxidase, catalase, beta-galactosidase, alpha-glucosidase, casein, urease, nitrate reduction and esculin hydrolysis are all positive; lipoidase (C14), cystine arylamine, arginine double hydrolase production and citric acid utilization, gelatin liquefaction, indole production and MacConkey plate growth were negative. Strain RWY-5-1^TThe differences of physiological and biochemical characteristics of the strain and the strain represented by the azospirillum close-source species are shown in a table 1.

TABLE 1 difference table of physiological and biochemical characteristics of strains and azospirillum near-source strains

Note: in the table, + indicates positive, and-indicates negative.

As can be seen from the results shown in Table 1, the strain RWY-5-1 of the present invention^TThe strain is obviously different from reported near-source strains and is a new species of azospirillum.

3. Strain RWY-5-1^TDetection of the cytochemical characteristics of

Detection of Strain RWY-5-1 by gas chromatography (6890; Hewlett Packard) analysis^TThe fatty acid component of cell walls of (Sasser M.1990, Identification of bacterial by gas chromatography of cellular fasts acids. USFCC News Lett,20: 1-6.). In the strain RWY-5-1 of the invention^TCoenzyme Q (Q-10) having 10 isoprene unit side chains is the predominant respiratory quinone type in the respiratory chain. Comparative invention Strain RWY-5-1^TStrain a. picis DSM 19922 as representative of closely related species^T、A.thiophilum DSM 21654^T、A.lipoferum NBRC 102290^T、A.humicireducens KACC 16605^TSee table 2 for fatty acid components of (a). The results show that the strain RWY-5-1 of the invention^TThe main fatty acid of (B) is C_18:1ω7c/C_18:1Omega.6c, accounting for 72.72% of the total content, this major component also being consistent with other species of azospirillum. However, the strain RWY-5-1 of the present invention^TAnd has a unique feature different from other closely related species, e.g. C_16:1Omega 9c is the unique component, and simultaneously, as shown in Table 2, the strain RWY-5-1^TThe content of many fatty acid components is also different from other closely-sourced species, so that the strain RWY-5-1 of the invention^TIs a new species of azospirillum.

TABLE 2 fatty acid characteristic difference table between the strain and the azospirillum near-source strain

Note: in the table, "-" indicates no detection; the generalized characteristic components refer to 2 or 3 fatty acid components that cannot be separated by the gas chromatography MIDI system. General characteristics 2 include C_12:0aldehyde (unknown component), C_16:1iso I/C_14:03-OH; general feature 3 includes C_16:1ω7c/C_16:1Omega 6 c; general characteristics 8 include C_18:1ω7c/C_18:1ω6c。

4. Detection of nitrogen fixation ability of strain

Strain RWY-5-1^TThe nitrogen fixation ability of (b) was determined using acetylene reduction (Baldani, j.i., Reis)V.M., video, S.S., et al.2014, The art of attenuating nitrile-attenuating bacteria from non-leguminous plants using N-free semi-solid media: a reactive guide for microbiology. plant and Soil,384: 413-431). The method comprises the following specific steps: NFb semisolid culture medium is prepared and put into an anaerobic tube with a ventilating rubber plug, and strain culture is inoculated and cultured for 36h at 30 ℃ in the dark. The butyl rubber plug was replaced, 10% (v/v) of the gas in the anaerobic tube was replaced with acetylene, and the culture was continued for 36 h. The ethylene content in the anaerobic tube was then measured every 4 hours for a total of 24 hours. During detection, 2ml of gas is extracted from the anaerobic tube, pumped into a reaction gas storage bottle, 100 μ l of gas sample is taken from the reaction gas storage bottle and injected into a gas chromatograph 7820A (Agilent), and N in the instrument is controlled₂，H₂And the flow rates of the drying air were 40, 40 and 400ml min, respectively^-1And recording the peak area of ethylene in the sample according to the display value of the instrument screen. The peak area of 100. mu.l of ethylene standard gas was measured by the same method. Meanwhile, the CFU value of the strain is calculated by a dilution plate coating method. Finally, the strain RWY-5-1 is calculated according to the following formula^TNitrogenase activity:

the results show that the strain RWY-5-1^TThe azotase activity is 22nmol C₂H₄/h˙10⁸cells. To further determine the strain RWY-5-1 of the present invention^TNitrogen fixation ability of (2), from strain RWY-5-1^TAmplifying and sequencing a nifH gene (Poly F., Monrozier L.J., Bally R.2001, Improvement in the RFLP process for interpreting the diversity of nifH genes in soil. Res. Microbiol 152: 95-103) fragment (332bp) encoding nitrogen fixing reductase in genomic DNA of (GenBank), and comparing the sequence with the NIFH gene sequence of Azospirillum disclosed in GenBank, the result shows that the sequence is consistent with A.brasilense Gr29^TThe similarity is 96.6% at most, and the similarity with the nifH gene sequence of other species in the genus is 85.5% -96.3%. Further illustrates the strain RWY-5-1 of the invention^THas nitrogen fixing capacity and is combined with other known speciesDifferent.

5. Detection of heavy crude oil degradation capability of strain

The strain RWY-5-1 of the invention^TThe degradation rate of the heavy crude oil is calculated by a gravimetric method (Shedunculus sieboldii, Yi Hua, Penghui, and the like. 2004. the degradation of the mixed bacteria to the petroleum. the journal of applied and environmental biology, 10,2: 210-214.). The method comprises the following specific steps: NFb liquid culture medium is prepared, 1 percent (1g/100ml) of heavy crude oil sample (relative density 0.94) from Yumen oil field in China is added, and the mixture is sterilized for later use. RWY-5-1 growing to logarithmic growth phase by inoculation^TThe bacterial liquid is cultured in a culture medium of 4ml to 100ml at 30 ℃ for 14 days, a blank control group is arranged, and the crude oil degradation effect is shown in figure 2. As can be seen from the figure: after being cultured, the non-inoculated strain RWY-5-1 is cultured at normal temperature^TThe residual oil in the blank control group culture medium is still a clear and visible solid heavy crude oil, and the phenomena of emulsification and dispersion are avoided; and inoculated with the strain RWY-5-1^TThe residual oil is obviously liquid oil drops with extremely small volume and is dispersed on the surface layer of the culture medium in the experimental group culture medium, which indicates that the strain RWY-5-1^TSignificant degradation ability to heavy crude oil. Further, in order to calculate the degradation rate of the crude oil, the residual crude oil in the culture bottles of the experimental group and the control group is fully extracted by using petroleum ether and a separating funnel respectively, the obtained crude oil component is dried at 68 ℃, cooled to constant weight in a drier, weighed, and the degradation rate of the crude oil is calculated according to the following formula. M0 is the weight of the blank control oil residue, and M1 is the weight of the experimental oil residue.

Calculated that the strain RWY-5-1 of the invention^TThe crude oil degradation rate of (2) was 36.2%. The actual degradation effect graph (figure 2) and the crude oil degradation rate of 36.2 percent both prove that the strain RWY-5-1^THas the functions of degrading and emulsifying heavy crude oil and petroleum.

6. Determination of phylogenetic position of strain

The strain RWY-5-1 of the invention is extracted^TAmplifying, cloning and sequencing the genome DNA of the strain to obtain the 16S rRNA gene sequenceIn this column (GeneBank accession MK332580), the resulting sequence (SEQ ID No.1) was aligned online in the International authoritative bacteria taxonomic database (http:// www.ezbiocloud.net /) (Kim OS, Cho YJ, Lee K, et al 2012, reducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes which is present in unknown specific species. int J Syst Evol. Microbiol 62:716 721.). The results show that the strain RWY-5-1 of the invention^TThe species with the highest similarity to the species of Azospirillum, wherein the species representative strain with the highest pairwise similarity in the sequences is Azospirillum picis DSM 19922^T(similarity 96.0%), A. thiophilum DSM 21654^T(95.9%) and A.humipireducens KACC 16605^T(95.9%). As can be seen, the strain RWY-5-1 of the invention^TThe highest similarity of the 16S rRNA gene sequence to known species is 96.0%, which is significantly below the defined threshold of 98.7% for different species of bacteria. To further clarify The phylogenetic status of The strains, 16S rRNA gene sequences of all strains of Azospirillum were selected to construct phylogenetic trees (Thompson J.D, highgins D.G, Gibson T.J.1994, CLUSTAL W: optimizing The sensitivity of developmental multiple sequence alignment, position-specific gap dependencies and weight matrix choice Res.22: 4673-4680.Saitou N, New M.1987, position-specific gap-correlation method: a new method for expressing The genomic sequences, molecular 406 Evool, 4: S-425, K.425. Pat. No. 3. Evogenetic analysis, gradient analysis, No. 3. Evogenetic analysis, No. 4: M.1987, molecular analysis, gradient analysis, K.120. Pat. 5. Evogenetic analysis, gradient analysis, M.7, molecular analysis, No. 4: M.425. 425. K.120. expression, sample analysis, molecular, as shown in FIG. 3, the strain RWY-5-1 of the present invention^TForm a branch alone and gather in the azospirillum, which fully indicates that the azospirillum is a new species of the azospirillum.

In conclusion, the strain RWY-5-1 of the invention^THas a plurality of obvious and significant different characteristics with the prior azospirillum, includingPhenotype, physiology, biochemistry and cytochemistry. Meanwhile, phylogenetic analysis based on gene level further proves that the strain is different from various species of the existing azospirillum, and fully proves that the strain RWY-5-1 of the invention^TRepresents a new species of Azospirillum, named Azospirillum oleraceum. Meanwhile, the detection of the nitrogen fixation capacity and the crude oil (petroleum) degradation capacity also proves that the strain has the nitrogen fixation capacity and the heavy crude oil (petroleum) degradation capacity, is a multifunctional new azospirillum species, and has wide application prospects in the aspects of soil remediation, soil improvement, crop growth promotion and the like. Azospirillum oleraceum RWY-5-1^THas been preserved in China general microbiological culture collection management center in 2019, 3, 8 and the preservation number is CGMCC No. 17324.

<110> institute of microbiology of Chinese academy of sciences

<120> a new species of Azospirillum azotobacter for degrading heavy oil and its microbial preparation

<130> GNCLN190415

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1443

<212> DNA

<213> Azospirillum oleiclasticum

<400> 1

gagtttgatc ctggctcaga acgaacgctg gcggcatgcc taacacatgc aagtcgaacg 60

aaggcttcgg ccttagtggc gcacgggtga gtaacacgtg ggaacctgcc tttcggttcg 120

ggataacgtt tggaaacgaa cgctaacacc ggatacgccc ttcggggaaa gttcacgccg 180

agagatgggc ccgcgtcgga ttaggtagtt ggtgaggtaa tggctcacca agcctgcgat 240

ccgtagctgg tctgagagga tgatcagcca cactgggact gagacacggc ccagactcct 300

acgggaggca gcagtgggga atattggaca atgggcgcaa gcctgatcca gcaatgccgc 360

gtgagtgatg aaggccttag ggttgtaaag ctctttcgca cgcgacgatg atgacggtag 420

cgtgagaaga agccccggct aacttcgtgc cagcagccgc ggtaatacga agggggctag 480

cgttgttcag aattactggg cgtaaagggc gcgtaggcgg cctgtttagt cagaagtgaa 540

agccccgggc tcgacctggg aacagctttt gatactggca ggcttgagtt ccggagagga 600

tggtggaatt cccagtgtag aggtgaaatt cgtagatatt gggaagaaca ccggtggcga 660

aggcggccat ctggacggac actgacgctg aggcgcgaaa gcgtggggag caaacaggat 720

tagataccct ggtagtccac gccgtaaacg atgagtgcta gacgctgggg ggcttgcctt 780

tcggtgtcgc agctaacgca ttaagcactc cgcctgggga gtacggccgc aaggttaaaa 840

ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 900

cgcgcagaac cttaccagct cttgacatgt ccgtcgcggt tgggagagat cccatccttc 960

ggttcggccg gacggaacac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt 1020

tgggttaagt cccgcaacga gcgcaacccc caccgccagt tgccaccatt tggttgggca 1080

ctctggtgga accgccggtg acaagccgga ggaaggcggg gatgacgtca agtcctcatg 1140

gcccttatga gctgggctac acacgtgcta caatggcggt gacagtgggt cgcgaagccg 1200

cgaggtggag ccaatcccca aaagccgtct cagttcggat cgcactctgc aactcgggtg 1260

cgtgaagttg gaatcgctag taatcgcgga tcagcacgcc gcggtgaata cgttcccggg 1320

ccttgtacac accgcccgtc acaccatggg agttggcttt acccgaagac ggtgcgctaa 1380

ccgcaaggag gcagccggcc acggtcaggt cagcgactgg ggtgaagtcg taacaaggta 1440

acc 1443

Claims

1. Oil-decomposing azospirillum (A. sp.), (B. sp.), (Azospirillum oleiclasticum）RWY-5-1-1^TThe preservation and organization of the strain in China general microbiological culture preservation management centerThe number is CGMCC No. 17324.

2. A microbial preparation comprising the oil-solubilizing azospirillum (A) of claim 1 (A)Azospirillum oleiclasticum）RWY-5-1-1^T。

3. The oil-solubilizing azospirillum(s) (of claim 1)Azospirillum oleiclasticum）RWY-5-1-1^TOr the use of a microbial preparation according to claim 2 for the degradation of petroleum.

4. Use according to claim 3, characterized in that: the petroleum is heavy petroleum.

5. The oil-solubilizing azospirillum(s) (of claim 1)Azospirillum oleiclasticum）RWY-5-1-1^TOr the use of a microbial preparation according to claim 2 for the degradation of crude oil.

6. Use according to claim 5, characterized in that: the crude oil is heavy crude oil.

7. The oil-solubilizing azospirillum(s) (of claim 1)Azospirillum oleiclasticum）RWY-5-1-1^TOr the use of a microbial preparation according to claim 2 in microbial oil recovery.

8. The oil-solubilizing azospirillum(s) (of claim 1)Azospirillum oleiclasticum）RWY-5-1-1^TOr the use of a microbial preparation according to claim 2 for biological nitrogen fixation.

9. The oil-solubilizing azospirillum of claim 1 (a), (b), (c), (d) and (d)Azospirillum oleiclasticum）RWY-5-1-1^TOr the use of a microbial preparation according to claim 2 for soil remediation and/or soil improvement.