CN111705007A - New azospirillum species for degrading heavy oil and microbial preparation thereof - Google Patents
New azospirillum species for degrading heavy oil and microbial preparation thereof Download PDFInfo
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Abstract
The invention discloses a new azospirillum species for degrading heavy oil and a microbial preparation thereof. The new species of Azospirillum solenii provided by the invention is Azospirillum oleraceum (Azospirillum oleraceum), and the representative strain is RWY-5-1TThe 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 inventionTCGMCC No.17324 belongs to a new strain of Azospirillum. Azospirillum oleraceum RWY-5-1TCGMCC No.17324 not only has nitrogen fixation function, but also has degradation function of heavy crude oil, and has wide comprehensive application prospect in the aspects of microbial oil extraction, soil bioremediation, soil texture improvement, crop growth promotion and the like.
Description
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 which are difficult to biodegrade, colloid, asphaltene and other heavy components, 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 recombinant bacteria 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-5-1T。
The invention claims oil-decomposing Azospirillum olyeichium RWY-5-1TBelongs 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 aspectT. The remaining ingredients may be adjuvants or carriers commonly used in the art.
In a third aspect, the invention claims the Azospirillum oleraceum RWY-5-1TOr 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 solenii (or a microbial preparation containing said Azospirillum solenii) 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 bacterial species according to the protocol from the reference "Stackelbrandt E, Ebers J.2006, Tarsonomic parameters revisited: Tarnised gold standards. Microbiol Today,33: 152-.
Experiments prove that the Azospirillum oleraceum RWY-5-1 provided by the inventionTCGMCC No.17324 belongs to a new strain of Azospirillum. Azospirillum oleraceum RWY-5-1TCGMCC 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 oleecicum
According to the biological materials (strains): RWY-5-1T
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 inventionTTransmission electron micrograph (D).
FIG. 2 shows a strain RWY-5-1 of the present inventionTThe degradation effect of the heavy oil is shown.
FIG. 3 shows a strain RWY-5-1 of the present inventionTPhylogenetic 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 19922T: the detailed information of the strain is recorded in the following website: https:// www.dsmz.de/transactions/details/culture/DSM-19922. html? tx _ dsmzresources _ pi 5% 5breturn pid% 5D 304. Meanwhile, the strain is described in "International Journal of Systematic and evolution Microbiology (2009),59: 761-765".
A.thiophilum DSM 21654T: the detailed information of the strain is recorded in the following website: https:// www.dsmz.de/transactions/culture/DSM-21654. html? tx _ dsmzresources _ pi 5% 5breturn pid% 5D 304. Meanwhile, the strain is described in "International Journal of Systematic and evolution Microbiology (2010), 60: 2832-2837".
A.lipoferum NBRC 102290T: the detailed information of the strain is recorded in the following website: https:// www.jcm.riken.jp/cgi-bin/jcm/jcm _ cc? CC ═ NBRC&NUM 102290. Meanwhile, the strain is described in "Canadian Journal of Microbiology (1978),24: 967-.
A.humicireducens KACC 16605T: the detailed information of the strain can be inquired in the following websites: http:// www.cctcc.org/news _ show1. php? newsid 88. Meanwhile, the strain is described in "International journal of Systematic and evolution Microbiology (2013),63: 2618-.
Example 1 Strain RWY-5-1TSeparation screening and identification of
1. Strain RWY-5-1TSeparation and screening of
The strain RWY-5-1 of the inventionTThe 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-1Magnesium sulfate heptahydrate 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-5-1T。
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-5-1-1TPhysiological and biochemical characteristic detection of strain
Strain RWY-5-1TAfter 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) of 7 (0, 1, 2, 3, 4, 5, 6) concentration gradients; 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 a pig. int J Syst Evol. Microbiol,68: 1969-. Of bacterial strainsThe physiological and biochemical functions were performed using the detection kit API 20E, API ZYM, manufactured by Meriella, France, and the detection system GEN III, manufactured by BiOLOG, USA. 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 brasilense sp. nov. Can J. Microbiol 24:967 + 980). 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-5-1TThe cells are slightly bent and short rod-shaped, have the length of 2.95-4.0 micrometers and the width of 1.5-1.9 micrometers, are facultative anaerobic, and have single-ended flagellum movement (see figure 1), contain polyhydroxybutyrate, and after the bacterial strains are cultured on an R2A culture medium at 30 ℃ for 48 hours, the bacterial colonies are milky, round, smooth and slightly convex, and have the diameter of 0.8-1.0 millimeters, the growth tolerance range of the bacterial strains is 15-37 ℃, 0-3% NaCl, and the pH is 6-8.5, the optimal growth conditions are 25 ℃, 0% NaCl, and the pH is 7.5, oxidase, catalase, β -galactosidase, α -glucosidase, casein, urease production and nitrate reduction, and esculin hydrolysis are positive, lipase (C14), cystine arylaminase, arginine dihydrolase production and citric acid utilization, gelatin liquefaction, indole production and Mackanka plate growth are negative bacterial strains RWY-5-1TThe physiological and biochemical characteristics of the strain are different from those of the azospirillum closely-sourced species, and are shown in the 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 inventionTThe strain is obviously different from reported near-source strains and is a new species of azospirillum.
3. Strain RWY-5-1TDetection of the cytochemical characteristics of
Detection of Strain RWY-5-1 by gas chromatography (6890; Hewlett Packard) analysisTThe fatty acid component of cell walls of (Sasser M.1990, Identification of bacterial by gas chromatography of cellular fartty acids. USFCC News Lett,20: 1-6.). In the strain RWY-5-1 of the inventionTCoenzyme Q (Q-10) having 10 isoprene unit side chains is the predominant respiratory quinone type in the respiratory chain. Comparative invention Strain RWY-5-1TStrain a. picis DSM 19922 as representative of closely related speciesT、A.thiophilum DSM 21654T、A.lipoferumNBRC 102290T、A.humicireducens KACC 16605TSee table 2 for fatty acid components of (a). The results show that the strain RWY-5-1 of the inventionTThe main fatty acid of (B) is C18:1ω7c/C18:1Omega.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 inventionTAnd has a unique feature different from other closely related species, e.g. C16:1Omega 9c is the unique component, and simultaneously, as shown in Table 2, the strain RWY-5-1TThe content of many fatty acid components is also different from other closely-sourced species, so that the strain RWY-5-1 of the inventionTIs 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 "-" tableNot detected; 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 C12:0aldehyde (unknown component), C16:1iso I/C14:03-OH; general feature 3 includes C16:1ω7c/C16:1Omega 6 c; general characteristics 8 include C18:1ω7c/C18:1ω6c。
4. Detection of nitrogen fixation ability of strain
Strain RWY-5-1TThe nitrogen-fixing ability of (1) is determined using acetylene reduction (Baldani, J.I., Reis, V.M., Videira, S.S., et al 2014, The art of isolating nitrogen-fixing bacteria from non-limiting plants using N-free semi-soluble 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 and is injected into a reaction gas storage bottle, 100 mu l of gas sample is taken from the reaction gas storage bottle and is 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 in the same manner. 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 formulaTNitrogenase activity:
the results show that the strain RWY-5-1TThe azotase activity is 22nmol C2H4/h˙108cells. To further determine the strain RWY-5-1 of the present inventionTThe nitrogen fixing ability of the strain RWY-5-1TThe nifH gene encoding nitrogen-fixing reductase was amplified in the genomic DNA of (Poly F., Monroz)ier L.J., Bally R.2001, Improvement in the RFLP plasmid for interpreting the diversity of nifH genes in microorganisms 152: 95-103) fragment (332bp) and sequencing, comparing it with the NIFH gene sequence disclosed on GenBank, the result shows that it is similar to A.brasilense Gr29TThe 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 inventionTHas nitrogen fixation capacity and is different from other known species.
5. Detection of heavy crude oil degradation capability of strain
The strain RWY-5-1 of the inventionTThe 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 phase by inoculationTThe bacterial liquid is cultured in 4ml to 100ml of culture medium for 14 days at 30 ℃, a blank control group is arranged, and the degradation effect of crude oil 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 temperatureTThe 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-5-1TThe 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-5-1TSignificant degradation capability on 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 inventionTThe 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-5-1THas 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 extractedTThe 16S rRNA gene sequence (GeneBank accession No. MK332580) was amplified, cloned and sequenced, and the resulting sequence (SEQ ID No.1) was aligned online in an International authoritative bacteria taxonomic analysis database (http:// www.ezbiocloud.net /) (Kim OS, Cho YJ, Lee K, et al.2012, Integrated EzTaxon-e: a prokarstic 16S rRNA gene sequence database databasewith phylotypes being present in unknown species. int J Syst ol Microbiol,62:716 Evo 721.). The results show that the strain RWY-5-1 of the inventionTThe 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 19922T(similarity 96.0%), A. thiophilum DSM 21654T(95.9%) and A.humipireducens KACC 16605T(95.9%). As can be seen, the strain RWY-5-1 of the inventionTThe 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 as representatives to construct phylogenetic trees (Thompson J.D, highgins D.G, Gibson T.J.1994, CLUSTAL W: optimizing The sensitivity of multiple sequence alignment, position-specific gap primers and weight matrix reagents Res. 22:4673-4680.Saitou N, New M.1987, position-specific gap primers: a new method for expressing genomic probes 406. Evool, 4: S-coating, K.425. yield K.52. M.1980. M.52. molecular analysis of molecular genes, M.52. molecular analysis of molecular analysis, M.1987. molecular analysis of molecular analysis, K.D.52. molecular analysis of molecular analysis, M.52. molecular analysis of molecular analysis, M.52. M.31. M.D.M.M.J Mol Evol,16:111-120, Felsenstein J1985, conjugation limits on phenols, an improvement using the same, evolution.39:783-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 inventionTCompared with the existing azospirillum, the azospirillum has a plurality of obvious and remarkable different characteristics, including phenotype, physiology, biochemistry, cytochemistry and the like. 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 inventionTRepresents 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-5-1THas 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 (10)
1. Azospirillum oleraceum RWY-5-1TThe preservation number of the strain in China general microbiological culture Collection center is CGMCC No. 17324.
2. A microbial preparation comprising Azospirillum oleaginous (RWY-5-1) of claim 1T。
3. The Azospirillum olysospira (Azospirillum oleuisticicum) RWY-5-1 of claim 1TOr the use of a microbial preparation according to claim 2 for the degradation of petroleum or crude oil.
4. Use according to claim 3, characterized in that: the petroleum is heavy petroleum; the crude oil is heavy crude oil.
5. The Azospirillum olysospira (Azospirillum oleuisticicum) RWY-5-1 of claim 1TOr the use of a microbial preparation according to claim 2 in microbial oil recovery.
6. The Azospirillum olysospira (Azospirillum oleuisticicum) RWY-5-1 of claim 1TOr the use of a microbial preparation according to claim 2 for biological nitrogen fixation.
7. The Azospirillum olysospira (Azospirillum oleuisticicum) RWY-5-1 of claim 1TUse of a microbial preparation according to claim 2 for soil remediation and/or soil improvement。
8. The Azospirillum olysospira (Azospirillum oleuisticicum) RWY-5-1 of claim 1TOr the use of a microbial preparation according to claim 2 for promoting plant growth.
9. The application is characterized in that: the application is the application of Azospirillum oleraceum or a microbial preparation containing the Azospirillum oleraceum in any one of the following:
(a1) degrading petroleum or crude oil;
(a2) microbial oil recovery;
(a3) biological nitrogen fixation;
(a4) soil remediation and/or soil reclamation;
(a5) promoting the growth of plants;
the Azospirillum oleraceum (Azospirillum oleraceum) is a gram-negative rod-shaped bacterium, and its 16S rRNA gene sequence has 98.7% or more homology with SEQ ID No. 1.
10. Use according to claim 9, characterized in that: the petroleum is heavy petroleum; the crude oil is heavy crude oil.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN117815160B (en) * | 2023-11-08 | 2024-05-28 | 首都医科大学附属北京潞河医院 | Preparation of astragalus membranaceus powder decoction pieces and application of astragalus membranaceus powder decoction pieces in treatment of chronic renal insufficiency |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN117815160B (en) * | 2023-11-08 | 2024-05-28 | 首都医科大学附属北京潞河医院 | Preparation of astragalus membranaceus powder decoction pieces and application of astragalus membranaceus powder decoction pieces in treatment of chronic renal insufficiency |
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