CN107142230A - A kind of M. luteus bacterial strain and application thereof - Google Patents
A kind of M. luteus bacterial strain and application thereof Download PDFInfo
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- CN107142230A CN107142230A CN201710366006.4A CN201710366006A CN107142230A CN 107142230 A CN107142230 A CN 107142230A CN 201710366006 A CN201710366006 A CN 201710366006A CN 107142230 A CN107142230 A CN 107142230A
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Abstract
The invention discloses a kind of M. luteus bacterial strain and application thereof, its morphological feature：Bacterium colony mode of appearance of the strain on LB culture mediums：Golden yellow, concave mesa-shaped is circular, opaque, neat in edge, the smooth moistening in surface, and glossy, bacterium colony size is about 2mm.The bacterium is Gram-positive tetrads.Energy efficient-decomposition high sulfur coal gangue of the invention discharges the micrococcus luteus of phosphorus.
Description
Technical field
The invention belongs to technical field of microbiology, a kind of micrococcus luteus is related in particular to, the bacterium is also related to
Purposes of the strain in dissociation release high sulfur coal gangue in terms of phosphorus.
Background technology
Most rich coal resources, great variety of goods, widely distributed using coal as the country of main energy sources, coal is to economy
Development play very important effect.In coal mining and processing, 20-25% gangue can be produced, only China is useless at present
The gangue abandoned just has billions of tons.According to the height of content sulphur in coal, coal can be divided into low-sulfur coal and sulphur coal, sulfur content exists
The amplitude of change in coal is very big, and up to 8%, minimum 0.12%.The exposure stacking of gangue, after rainwater shower, portion
Divide harmful substance to be dissolved, and soil, surface water body or body of groundwater are flowed into rainwater, cause its serious pollution.It is contaminated
Surface water body in water quality can gradually be acidified, when for cultivating or during irrigating plant, animal husbandry and vegetation can be caused can not
Inverse infringement, and destruction aquatic ecological environment.When leaching water flows into soil with rainwater, the flat of heavy metal in soil is broken
Weighing apparatus, also destroys the nutrient of soil, and influences the metabolic activity of Soil Microorganism, and the presence of these harmful substances, not only
The development and growth of root system of plant are hindered, can also put aside, be detrimental to health by food chain in plant organism.
Containing much abundant nutrient in gangue, necessary to having many thing plant growths.Have in mineral
Microorganism can decompose mineral, so as to dissociate element therein, because these bacterial origins are in the Nature, itself to environment without
Evil, and decomposable process and catabolite are harmless, it is environmentally safe, it is environmental protection.Gangue is decomposed with microbial bacterial,
Effective ingredient therein is dissociateed, the new application of gangue is developed, is the important channel of utilization of coal gangue application.
The content of the invention
It is an object of the invention to one plant for overcoming disadvantages mentioned above and providing energy efficient-decomposition high sulfur coal gangue release phosphorus
Micrococcus luteus.
Another object of the present invention is to provide use of the micrococcus luteus in dissociation release high sulfur coal gangue in terms of phosphorus
On the way.
A kind of micrococcus luteus (Micrococcus luteus) GZU-Mi01 of the present invention, its morphological feature：Strain exists
Bacterium colony mode of appearance on LB culture mediums：Golden yellow, concave mesa-shaped is circular, and opaque, neat in edge, the smooth moistening in surface has
Gloss, bacterium colony size is about 2mm.The bacterium is Gram-positive tetrads.
The bacterial strain bacterium was preserved in China typical culture collection center (CCTCC) on 03 16th, 2017, and preserving number is：
CCTCC NO：M 2017126, title：Micrococcus luteus GZU-Mi01 (Micrococcus luteus GZU-Mi01).
The micrococcus luteus GZU-Mi01 of present invention separation, comprises the following steps：
1. separation screening
1) primary dcreening operation
The sample ore on coal gangue hill is gathered, using dilution method, Phos solid medium flat board is then inoculated in, waits to grow it
Afterwards, by looking first at bacterium colony mode of appearance, then plate streaking separating-purifying method is carried out, finally carries out Gram's staining and carry out primary dcreening operation
Bacterial strain.
2) secondary screening
The obtained bacterial strain of activation primary dcreening operation is simultaneously cultivated, under certain condition, is carried out dissociation high sulfur coal gangue experiment, is passed through and determine
Available phosphorus content after dissociation in gangue, so as to filter out the good bacterial strain of phosphorus decomposing effect.
2. the culture of bacterial strain
Slightly solubility Phos fluid nutrient medium：Glucose 10g, (NH4)SO40.5g, NaCl 0.3g, KCl 0.3, MgSO4·
7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.03g, Ca3(PO4)210g, distilled water 1000mL, pH=7.0-
7.2。
Slightly solubility Phos solid medium：Add agar 15g-20g in slightly solubility Phos fluid nutrient medium.
LB fluid nutrient mediums：Peptone 5g, sodium chloride 5g, beef extract 1g, yeast extract 2g, pH=7.4, distilled water 1L.
LB solid mediums：Add agar 15g-20g in liquid LB fluid nutrient mediums.
3. three kinds of conservation modes
1）The bacterial strain of preservation will be needed, lined on LB solid medium flat boards or in slant tube, 30 DEG C of culture 18-24h are put
In 4 DEG C of refrigerators, preserve one month.
2）The 20% glycerol liquids culture medium that the bacterial strain grown on flat board is sterilized is rinsed, and is then stored in -20
DEG C refrigerator in, can preserve 1 year.
3）Will on flat board well-grown bacterial strain, add 30% defatted milk, sterile ampoule pipe is poured into after mixing
In, the mouth of pipe is lived with sterile absorbent tampon, -20 DEG C of refrigerator pre-freeze 3-4h are put into, then vacuumize, the mouth of pipe is sealed, -80 DEG C of ice is put into
Preserved in case, this method can be preserved for a long time.
The growth curve of 4.GZU-Mi01 bacterial strains
Under suitable condition of culture, the growth and breeding of bacterium will undergo period of delay, logarithmic phase, stationary phase and decline phase.Using
The growth curve of spectrophotometry bacterium, using incubation time as abscissa, using biomass growth rate or the logarithm of bacterium number as
Ordinate draws growth curve.By determining growth curve, growing state of the bacterium in different time sections can be learnt.
The molecular biology identification of 5.GZU-Mi01 bacterial strains
By carrying out 16SrRNA clones to bacterial strain, be then sequenced, then the sequence is compared with NCBI bacteriums storehouse, find out and
The category of its homology recently, and by a series of biochemical tests, finally determine the kind of this plant of bacterium.
Another object of the present invention is to provide use of the micrococcus luteus in dissociation release high sulfur coal gangue in terms of phosphorus
On the way.
The present invention compared with prior art, with obvious beneficial effect, as can be known from the above technical solutions:From discarded coal
Separate, screen in spoil sample, purification obtains the bacterial strain of phosphorus in efficient-decomposition gangue, and the present invention has before application well
Scape.The gangue discarded with bacterial treatment, is a kind of gentle, greenization technique, can effectively alleviate gangue to environment band
The harm come, can also allow gangue to turn waste into wealth, make full use of efficient resource therein.If gangue is prepared into compound
Fertilizer imposes on soil, can not only alleviate the problem of lacking phosphorus in soil, but also the environment brought using chemical fertilizer can be avoided to ask
Topic, such as soil hardening.Compared with current applied chemistry method processing gangue, to more environment-friendly.
Brief description of the drawings
Fig. 1 .GZU-Mi01 strain growth curves
Fig. 2 .GZU-Mi01 bacterial strain bacterium colony outside drawings
Fig. 3 .GZU-Mi01 thalli morphology figures
Fig. 4 .PCR amplification reaction systems and amplification reaction condition
The systematic evolution tree that Fig. 5 .GZU-Mi01 bacterial strains are set up based on part 16S rRNA gene orders
Several bacterium high sulfur coal gangue dissociation results of Fig. 6
Embodiment
Micrococcus luteus GZU-Mi01 separation, comprises the following steps：
1. the separation and screening of function stem GZU-Mi01 bacterial strains
1）Sample collection
（1）Sample is picked up from weathering gangue on certain sulfur-bearing coal gangue hill, and the sample of collection then is loaded into plastic sample and adopted
Collect in bag.
（2）By the sample broke of collection.
（3）Screening, collected the sample of 100 mesh.
（4）The refrigerator that sample is put into 4 DEG C is standby.
2）The screening of function stem
（1）1g samples accurately are weighed in the 250mL of sterilizing conical flask, add 99mL sterilized waters.
（2）Then in 170r/min, 30min is vibrated in 37 DEG C or so of shaking table.
（3）Control group is to add 100mL sterilized waters in 250mL conical flask.
（4）After half an hour, supernatant is taken to be diluted to 10 step by step-3, 10-4, 10-5, 10-6, 10-7。
（5）Every kind of concentration takes 100uL to be coated on 3 Phos solid mediums respectively, and blank is coating sterilized water
Flat board.
（6）Flat board is put in 30 DEG C or so of constant incubator 3-4 days.
（7）By the different single bacterium colony streak inoculation of external appearance characteristic in Phos solid culture.
（8）After growing, observed by preliminary mode of appearance and microscopic morphology observation, whether observation bacterial strain purifies, do not have
There are purifying then more to carry out several times, until it becomes single bacterial strain, while distinguishing different bacterial strains.
（9）By the bacterial strain of purifying, Gram's staining is carried out.
（10）The bacterium of the primary dcreening operation of purifying is cultivated, high sulfur coal gangue is being processed to, therein is had by determining
Phosphorus content is imitated, the discomposing effect of the more high then bacterium of content is better, so as to filter out the good bacterial strain of phosphorus decomposing effect.
2. the drafting of the growth curve of function stem
1）The preparation of seed liquor.Take the oese of bacterial strain one to be inoculated in LB fluid nutrient mediums, under the conditions of 30 DEG C, cultivate to right
The number phase.
2）Inoculation.Take the 10mL of above-mentioned seed liquor to be inoculated in the LB liquid medium equipped with 200mL, taken respectively after mixing
5mL mixed liquors are loaded in upper markd 18 sterile test tubes respectively.
3）Culture.Vaccinated test tube is put in 37 DEG C, 170r/min shaking table and cultivated, cultivating the corresponding time respectively takes
Go out.
4）Measurement.Blank is done with nonvaccinated LB fluid nutrient mediums, photoelectricity is carried out together with treating test sample under 600nm wavelength
Than turbid.
Measurement result such as Fig. 1, it can be seen that 0-10h is lag phase, 10-28h is logarithmic phase, and 28h-38h is stable
It is decline phase after phase, 38h.
3. the identification of function stem
1）Bacterium colony mode of appearance
Such as Fig. 2, golden yellow, concave mesa-shaped circle, opaque, edge is presented in the bacterial strain on LB solid mediums as we know from the figure
Neatly, the smooth moistening in surface, glossy, and bacterium colony size is about 2mm.
2）Gram's staining, thalline microscopic morphology
Such as Fig. 3, the bacterial strain is Gram-positive tetrads as we know from the figure.
3）Bacterial strain is identified
（1）The extraction of DNA of bacteria
The single bacterium of the bacterial strain of picking purifying is dropped down onto is cultivated in the conical flask equipped with LB fluid nutrient mediums, obtains pure culture fermentation
After liquid, DNA is extracted with bacterial genomes extracts kit (centrifugation column type, Tiangeng biochemical technology Beijing Co., Ltd), made with it
PCR (Polymerase chain reaction, PCR) is carried out for masterplate, is expanded using Taq DNA polymer
Increase 16SrRNA, with 2F (5 '-AGAGTTTGATCCTGGCTCAGGATGA-3 '), 1492R (5 '-TACGGCTACCT
TGTTACGACTTAGC-3 ') universal primer expanded, pcr amplification reaction system and amplification reaction condition such as Fig. 4 institutes
Show.
1.0% Ago-Gel is prepared with 1 × tbe buffer liquid, 40 μ LPCR amplified productions, plus 8 μ are added
L2000bpDNAMarker, in voltage 110V, electric current 70mA, the min of electrophoresis 45, observes gel imaging result.
（2）Sequence analysis
By PCR amplification product through Gel Extraction kit, sequencing company (the vertical luxuriant and rich with fragrance biological skill in Shanghai is delivered
Art Co., Ltd) sequencing, the sequence measured is utilized into Standard on NCBI (US National Biotechnology Information center)
Nucleotide Blast are compared, and are based on adjacent method using softwares such as ClustalW, MEGA5.05
(NeighborJoining Method) carries out Phylogenetic Analysis, phylogenetic tree construction（Fig. 5）, to obtain the classification of strain
Information.It can be seen that GZU-Mi01 bacterium gathers with Micrococcus sp.Japan-TUT1210 (AB188213.1)
In same branch, sequence homology is accredited as Micrococcus (Micrococcus sp.) up to 99%, GZU-Mi01.
（3）Bio-chemical characteristics
Pass through a series of biochemical tests, reference《Primary Jie Shi Bacteria Identifications handbook》（8th edition）With《Common bacteria system identification hand
Volume》（Eastern show pearl etc., 1999）, the identified bacterial strain is micrococcus luteus.
4. bacterial strain is to the measure of the dissociation capability of high sulfur coal gangue
Using same source high sulfur coal gangue, make GZU-Mi01 bacterial strains and two plants of traditional bacterial strain-bacillus megateriums and silicate
Bacterium contrast solution separating test, compares their phosphorus decomposing effect.First in four principal elements of influence gangue dissociation：Particle diameter is big
Small, system pH solves the incubation time of bacterium amount and system, makees single factor experiment, and optimum condition is found respectively, then by design just
Experiment is handed over to find GZU-Mi01, bacillus megaterium, silicate bacteria in most preferably dissociation gangue condition, and comparing to sulphur coal
The dissociation effect of spoil, as a result such as Fig. 6.
From fig. 6 it can be seen that the effect of GZU-Mi01 dissociation high sulfur coal gangues is substantially in bacillus megaterium and silicic acid
Salt bacterium.
Sequence table
<110>Guizhou University
<120>It is a kind of to dissociate the bacterial strain of phosphorus in release high sulfur coal gangue
<130> 1
<160> 1
<170>Patentln version 3.3
<210> 1
<211> 1400
<212> DNA
<213>Micrococcus luteus（Micrococcus luteus）
<400> 1
catgcaagtcgaacgatgaagcccagcttgctgggtggattagtggcgaacgggtgagta 60
acacgtgagtaacctgcccttaactctgggataagcctgggaaactgggtctaataccgg 120
ataggagcgccgaccgcatggtgggtgttggaaagatttatcggttttggatggactcgc 180
ggcctatcagcttgttggtgaggtaatggctcaccaaggcgacgacgggtagccggcctg 240
agagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcag 300
tggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacgg 360
ccttcgggttgtaaacctctttcagtagggaagaagcgaaagtgacggtacctgcagaag 420
aagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcgagcgttatccg 480
gaattattgggcgtaaagagctcgtaggcggtttgtcgcgtctgtcgtgaaagtccgggg 540
cttaaccccggatctgcggtgggtacgggcagactagagtgcagtaggggagactggaat 600
tcctggtgtagcggtggaatgcgcagatatcaggaggaacaccgatggcgaaggcaggtc 660
tctgggctgtaactgacgctgaggagcgaaagcatggggagcgaacaggattagataccc 720
tggtagtccatgccgtaaacgttgggcactaggtgtggggaccattccacggtttccgcg 780
ccgcagctaacgcattaagtgccccgcctggggagtacggccgcaaggctaaaactcaaa 840
ggaattgacgggggcccgcacaagcggcggagcatgcggattaattcgatgcaacgcgaa 900
gaaccttaccaaggcttgacatgttctcgatcgccgtagagatacggtttcccctttggg 960
gcgggttcacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagt 1020
cccgcaacgagcgcaaccctcgttccatgttgccagcacgtaatggtggggactcatggg 1080
agactgccggggtcaactcggaggaaggtgaggacgacgtcaaatcatcatgccccttat 1140
gtcttgggcttcacgcatgctacaatggccggtacaatgggttgcgatactgtgaggtgg 1200
agctaatcccaaaaagccggtctcagttcggattggggtctgcaactcgaccccatgaag 1260
tcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgt 1320
acacaccgcccgtcaagtcacgaaagtcggtaacacccgaagccggtggcctaacccttg 1380
tggggagccgtcgaaggtga
Claims (2)
1. a kind of micrococcus luteus (Micrococcus luteus) GZU-Mi01, it is characterised in that the preserving number of the bacterial strain
For：CCTCC NO： M 2017126.
2. micrococcus luteus according to claim 1 (Micrococcus luteus) GZU-Mi01 dissociation discharge high-sulfur
Purposes in gangue in terms of phosphorus.
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Cited By (2)
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| CN110342965A (en) * | 2019-01-31 | 2019-10-18 | 贵州大学 | A kind of production method of microbial manure |
| CN110343630A (en) * | 2019-01-31 | 2019-10-18 | 贵州大学 | A kind of micrococcus luteus and its application |
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| CN110343630A (en) * | 2019-01-31 | 2019-10-18 | 贵州大学 | A kind of micrococcus luteus and its application |
| CN110342965B (en) * | 2019-01-31 | 2022-01-25 | 贵州大学 | Method for preparing microbial fertilizer |
| CN110343630B (en) * | 2019-01-31 | 2022-05-31 | 贵州大学 | Micrococcus luteus and application thereof |
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