CN105647839B - A kind of desert Oligotrophic bacteria DOB150 and the application in fixing the sand - Google Patents
A kind of desert Oligotrophic bacteria DOB150 and the application in fixing the sand Download PDFInfo
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- CN105647839B CN105647839B CN201610203913.2A CN201610203913A CN105647839B CN 105647839 B CN105647839 B CN 105647839B CN 201610203913 A CN201610203913 A CN 201610203913A CN 105647839 B CN105647839 B CN 105647839B
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Abstract
The present invention discloses a kind of desert Oligotrophic bacteria DOB150 and the application in fixing the sand.By separation, screening, selection and breeding, domestication in Junggar Basin, Xinjiang, china ancient capital Xi'an, one plant of Oligotrophic bacteria is obtainedBacillus sp.DOB150 CGMCC NO.11925, it is a kind of new strain, after the microbial inoculum prepared by using the Oligotrophic bacteria DOB150 bacterial strains being separated to sprays application in desert, sand fixation can not only be played, and preferable moisture condition may be provided for the sprouting of some slower vegetable seeds of speed of germination, prevent some because soil moisture deficiency obtains growth and development from the plant sprouted；After the microbial inoculum of preparation sprays application in desert, adhesion sand grains, prevention drift sand activation have been not only acted as, moreover it is possible to be effectively increased major microorganisms quantity in soil, improve essential nutrient contents of soil, strengthening part soil enzyme activities, and can obviously restore algae skinning, it is with a wide range of applications.
Description
Technical field
The present invention relates to technical field of microbe application, specifically, the present invention relates to a kind of new Oligotrophic bacterias in life
The technical field applied in object skinning tackling quicksand.
Background technology
Oligotrophic bacteria is a kind of microorganism grown under conditions of nutrient concentrations are very low, it belongs to particular surroundings
Microbe groups, it is also very low to required nutritional requirement of surviving, during diversity is formed with biomass in entire biosphere
All there is larger advantage, thus played an important role in biogeochemical cycle.Since generation in centurial year, oligotrophic
Oligotrophic mechanism of the bacterium in nature or man-made environment, the effect to hungry physiological reaction and in the ecosystem are always
It is one of the Disciplinary Frontiers of microbial ecology research, theory value is with application prospect by various countries microbial ecological scholar
With the extensive attention of environmentalists.
On the surface of desert soil, some is covered with one layer of soil crust formed by biology, they are drilled in promotion soil
It replaces, improve surface soil water situation, prevent from playing an important role in terms of the soil erosion.By filamentous cyanobacteria, especially surface
There are the cyanobacteria type dominance of glue sheath and a large amount of extracellular polymerics of secretion, the extracellular polymer that these algae secrete in metabolic process
Object, that is, exocellular polysaccharide has preferable bonding grains of sand effect, and the stability of desert topsoil can be improved.But it is similar for new discovery
Oligotrophic bacteria is applied to have not been reported in biological breadcrust tackling quicksand.
Currently, since microorganism can live in various extreme environments and barren habitat, there is extensive annidation,
Closely bound up with human lives, existence, development, and mankind's activity approach can have an impact desertification, thus microorganism and
It is closely connected between desertification.Thermophilic, thermophilic cold, acidophilus, thermophilic saline and alkaline and oligotrophic particular surroundings microorganism pass through for a long time certainly
So selection has adapted to the adverse circumstances factor, has stable particular function and gene.Therefore, the species resource of such bacterium and
Molecular biology at home and abroad conducts extensive research, and increasingly attracts people's attention.By document Investigation knot
Fruit shows that in terms of environmental, Oligotrophic bacteria is studied mainly for water environment at present, and resists to dry early Desert Regions barren
The oligotrophic microorganism report of environment, which is not much, to be seen, is also had no that some new discoveries are fixed similar to Oligotrophic bacteria in biological breadcrust and is flowed
The report applied in sand.
Invention content
For being had no in the prior art in relation to that the excellent new Oligotrophic bacteria of screenability and can be used in solid
Husky excellent species report, and the strain of separation screening can not only play sand fixation, and can be that speed of germination is slower
The sprouting of some vegetable seeds provides preferable moisture condition, prevents some because soil moisture deficiency is obtained from the plant sprouted
The state of the art of growth and development, the present invention is intended to provide a kind of desert Oligotrophic bacteria DOB150 and the application in fixing the sand.This
Invention in Junggar Basin, Xinjiang, china ancient capital Xi'an by isolating a collection of microbial strains, by further dividing
From, screening, selection and breeding, domestication, the Bacillus strain that one plant of number is DOB150 is obtained.The Oligotrophic bacteria DOB150 being separated to
Bacterial strain is a kind of typical novel bacterial, using the strain prepare microbial inoculum spray application in desert after, not only acted as adhesion sand grains,
Prevent drift sand activation, moreover it is possible to be effectively increased major microorganisms quantity in soil, improve essential nutrient contents of soil, strengthening part soil
Soil enzyme, and can obviously restore algae skinning.Therefore, new Oligotrophic bacteria DOB150 provided by the invention is a kind of relatively reason
The biomaterial that fixes the sand thought, this provides new method effectively to restore biological breadcrust, repairing impaired sand ground.
The main technical schemes that the present invention uses：
By the separation screening of microorganism fungus kind, a batch is isolated in Junggar Basin, Xinjiang, china ancient capital Xi'an
Microbial strains, by further detaching, screening, selection and breeding, domestication, obtain one plant number be DOB150 Bacillus strain.
By carrying out morphological feature, physio-biochemical characteristics and 16S rDNA sections determined dna sequences and systematic growth point to obtained bacterial strain
Analysis has primarily determined its classification position, and screening selection and breeding domestication obtains a kind of and common bacillus, and there are significantly different attributes
Oligotrophic bacteria bacterial strain, from its molecular level identification and analysis it is found that being a kind of typical novel bacterial.Separation screening of the present invention
The bacterial strain that number is DOB150 can be grown on oligotrophic culture medium, facultative aerobic；Have the function of fixing the sand simultaneously, can glue
Even sand grains, prevention drift sand activation, are with a wide range of applications.
The present invention specifically provides a kind of new Oligotrophic bacteria bacillus (Bacillus sp.), by the quasi- main officer of Tibet in Xinjiang
Separation, screening, domestication and culture in your basin ancient capital Xi'an, obtain a collection of bacterium microbe bacterial strain, therefrom sieve
The bacterial strain that one plant of number is DOB150 is selected, through microbiological classification and identification, belongs to bacillus (Bacillus sp.) class
Group.
Specifically, the present invention by being detached to the microorganism in Junggar Basin, Xinjiang, china ancient capital Xi'an,
Screening and culture obtain a collection of microbial strains, the bacterial strain that one plant of number is DOB150 are therefrom filtered out, through microbiological classification
With identification, which belongs to bacillus (Bacillus sp.) monoid.The present invention uses strain number for the gemma of DOB150
Bacillus (Bacillus sp.), the bacterial strain were preserved in budapest treaty microorganism International Depository Authority before the applying date：In
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC).Address：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica, postcode：100101.Preservation date is on December 24th, 2015, and preserving number is
CGMCC No.11925.It is accredited as bacillus (Bacillus sp.) through microbiology.The bacterial strain is Gram-positive bacillus,
3-5 μm of 1.0-1.2 μ ms, thalline both ends blunt circle, amphitrichous, no pod membrane, mostly at short catenation.6 are grown on LB culture mediums
Gemma can be formed after hour, gemma is less than thalline transverse diameter, is located at thalline center, can be grown on oligotrophic culture medium,
Facultative aerobic, optimal temperature is 28-35 DEG C.On LB culture mediums can well-grown, bacterium colony is larger, opaque, rough surface
Like ground-glass appearance or, edge has the function of sand consolidation with biologic, secrete exocellular polysaccharide, generate lecithinase, casein often at extension shape
Enzyme, gelatinase, catalase.Reference《Primary Jie Shi systematic bacteriologies identification handbook》8th edition and《Common bacterial system identification
Handbook》It is that DOB150 bacterial strains are identified to number, determines that bacterium numbering is the bacterium of DOB150 in conjunction with Physiology and biochemistry comprehensive detection
Strain is the member in bacillus (Bacillus sp.), is bacillus (Bacillus sp.) from taxology angular divisions.
Further, a kind of Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 of present invention offer are fixing the sand
In application.After the microbial inoculum prepared by using DOB150 bacterial strains sprays application in desert, adhesion sand grains, prevention stream have been not only acted as
Sand activation, moreover it is possible to be effectively increased major microorganisms quantity in soil, improve essential nutrient contents of soil, strengthening part soil enzymatic activities
Property, and can obviously restore algae skinning.Therefore, new Oligotrophic bacteria Bacillus sp.DOB150CGMCC provided by the present application
NO.11925 is a kind of more satisfactory biomaterial that fixes the sand, this is provided effectively to restore biological breadcrust, repairing impaired sand ground
New method.
By implementing the specific invention content of the present invention, following advantageous effect can be reached:
(1) bacterial strain Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 provided by the invention have relatively strong
Growth and breeding ability, growth rate is fast, hereditary capacity stablize.
(2) bacterial strain Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 provided by the invention are gram
Positive bacillus, 3-5 μm of 1.0-1.2 μ ms, thalline both ends blunt circle, amphitrichous, no pod membrane, mostly at short catenation.It is cultivated in LB
Gemma can be formed after being grown on base 6 hours, gemma is less than thalline transverse diameter, is located at thalline center, can be in oligotrophic culture
It is grown on base, facultative aerobic, optimal temperature is 28-35 DEG C.On LB can well-grown, bacterium colony is larger, opaque, and surface is thick
It is rough like ground-glass appearance or, edge has the function of sand consolidation with biologic often at extension shape, secrete exocellular polysaccharide, generation lecithinase, junket egg
White enzyme, gelatinase, catalase, by Physiology and biochemistry, molecular level comprehensive identification it is found that the number that the present invention screens is
The bacterial strain of DOB150 is a kind of new Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925.
(3) bacterium solution prepared by the Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 that the present invention screens makes
Sand fixation can not only be played for desert surface, and may be carried for the sprouting of some slower vegetable seeds of speed of germination
For preferable moisture condition, prevent some because soil moisture deficiency obtains growth and development from the plant sprouted.
(4) the microbial inoculum spray that prepared by Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 provided by the invention
After being applied in desert, adhesion sand grains, prevention drift sand activation have been not only acted as, moreover it is possible to be effectively increased major microorganisms number in soil
Amount improves essential nutrient contents of soil, strengthening part soil enzyme activities, and can obviously restore algae skinning.Therefore, Oligotrophic bacteria
Bacillus sp.DOB150CGMCC NO.11925 are a kind of more satisfactory biomaterials that fixes the sand, this is effectively recovery biology
Skinning, reparation are damaged sand ground and provide new method.
Description of the drawings
Fig. 1 is shown as the phyletic evolution development tree of Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925
Figure.
Specific implementation mode
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
All raw and auxiliary materials selected in the present invention, and the Spawn incubation method selected all are well known in the art selection
, the present invention involved in % be all weight percentage, unless otherwise indicated except.
Embodiment one：Separation, the screening and identification of Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925
1, the separation and screening of strain
Desert Oligotrophic bacteria used in the present invention is sampled from Junggar Basin, Xinjiang, china ancient capital Xi'an and is detached,
According to strain Analysis on action mechanism, preliminary screening goes out the bacterial strain that can be grown on LB culture mediums.Utilize traditional tablet culture
Method isolates the microorganism in Junggar Basin, Xinjiang, china ancient capital Xi'an, and screening strain is carried out using primary dcreening operation and secondary screening,
Plate streak purifying bacterial strain filters out a collection of well-grown by comparing growth and breeding situation of the strain on LB culture mediums
Microbial strains, therefrom preferably go out the desert Oligotrophic bacteria bacterial strain that one plant of good combination property number is DOB 150.
Separating step：The gradient dilution method detached using microorganism obtains a series of gradient dilution liquid, uses sterilized straw
I0 is drawn respectively-1-10-5The Mixed Microbes dilution 0.lmL of dilution is moved be put on the tablet of LB solid mediums respectively,
It is uniform with sterile glass rod coating, it is then inverted in 37 DEG C of constant incubator and cultivates to single bacterium colony is grown, in sterile working
The separation of the flat lining out equipped with LB solid mediums is inoculated into the single bacterium colony grown on aseptic inoculation ring picking tablet in platform
It after growing single bacterium colony, is transferred to again on sterilizing test tubes inclined-plane, every plant of bacterium is cooked three repetitions every time, is cultivated under the conditions of being placed in 37 DEG C
3-5 days.Using the consistency of the visible light bacterium colony through light, reflected light and dark background, smear staining is seen with light microscope
The consistency for examining thalline, it is spare after bacterium colony and individual uniformity through repeatedly isolating and purifying repeatedly.By bacterial strain after purification
Using the modes preservation such as lyophilized products ampoul tube, glycerol tube and liquid nitrogen, a part is stored in 4 DEG C and is directly used in subsequently grinds a part
Study carefully.
The LB culture mediums:Peptone 10g, yeast extract 5g, Nacl 10g, 10g~15g agar powder distill l000mL,
PH7.0,121 DEG C of sterilizing 20min.
Bacterial strain DOB150 provided by the invention can be increased in the solid medium or fluid nutrient medium for being suitble to its proliferation
Grow culture.Using the culture of Conventional solid inclined-plane culture, the method for low-temperature preservation, passage every time can preservation 3 months or more；With drying
The long term storage strain that freezing is manufactured, can preservation 1 year or more；Or long term storage is carried out with glycerol tube.
2, the Physiology and biochemistry identification of bacterial strain DOB150
Morphological feature：The strain is separately cultured through LB agar mediums, and the bacterium colony progress of the doubtful bacillus of picking is micro-
It is tentatively defined Bacillus strain by sem observation；The bacterial strain cross on LB culture medium flat plates culture 6 hours after can be with shape
At gemma, gemma is less than thalline transverse diameter, is located at thalline center.
Oxygen and carbon dioxide utilizes experiment：It sterilizes after agar medium is packed into test tube, is carried out when being cooled to 45-50 DEG C
Label, the bacteria suspension that bacterial strain is drawn in sterile working are added in test tube, and the quick rubbing test tube of both hands avoids oscillation from making excessive air
It is mixed into culture medium, after strain is uniformly distributed in culture medium, test tube is placed in ice bath, agar quick solidification is made, its is quiet
It is placed in after 37 DEG C of constant incubators heat preservation 48h to start to be carried out continuously and observe and record, until result is clear.The result shows that：It should
Bacterium is facultative aerobe.
Optimum growth temperature is tested：The bacteria suspension that bacterial strain is drawn with sterile working is added in fluid nutrient medium, observation experiment
Growing state of the bacterial strain at 10 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, the increment of thalline with
The size of the OD value measured at Du800 type spectrophotometers 680nm indicates.If encountered when cannot grow, at least
Can observation 15-20 days, be grown with affirming fully it.Each temperature is repeated 3 times.The result shows that Oligotrophic bacteria DOB150
Optimal temperature is 28-35 DEG C
By above-mentioned thalli morphology for strain Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925,
Cultural characteristic is observed and Determination of Physiological And Biochemical Indices, i.e., is surveyed by thalli morphology observation, strain culturing observation of characteristics, growth temperature
The experiments such as fixed, resistance test, compared with common bacillus, reference《Primary Jie Shi systematic bacteriologies identification handbook》8th edition and
《Common bacterial system identification handbook》Method classify, from bacterium classification angle by bacterium numbering be DOB150 bacterial strain it is comprehensive
Conjunction is accredited as bacillus (Bacillus sp).
Specifically, the present invention by being detached to the microorganism in Junggar Basin, Xinjiang, china ancient capital Xi'an,
Screening and culture obtain a collection of microbial strains, the bacterial strain that one plant of number is DOB150 are therefrom filtered out, through microbiological classification
With identification, which belongs to Bacillus sp.The present invention uses strain number for the bacillus (Bacillus of DOB150
Sp.), which was preserved in budapest treaty microorganism International Depository Authority before the applying date：Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center (CGMCC).Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, postcode：100101.Preservation date is on December 24th, 2015, and preserving number is CGMCC No.11925.Through
Microbiology is accredited as bacillus (Bacillus sp).
Bacterial strain can well be grown on oligotrophic culture medium R2A culture mediums and 1/5TSA culture mediums, also can be in common bacterium
Culture medium is such as grown in LB culture mediums.Wherein, using 30 DEG C of constant temperature incubations of R2A culture medium flat plates, bacterium colony is rounded, edge
Irregular, slightly raised, faint yellow, translucent, thin easy picking.Morphology microexamination, thalline are in rod-shaped, 1.0~1.2 μ ms
3~5 μm, thalline both ends blunt circle, amphitrichous, no pod membrane, mostly at short catenation.It can after being grown 6 hours on LB culture mediums
To form gemma, gemma is less than thalline transverse diameter, is located at thalline center, can be grown on oligotrophic culture medium, facultative aerobic, most
Preference temperature is 28-35 DEG C.On LB can well-grown, bacterium colony is larger, opaque, and rough surface is like ground-glass appearance or, edge
Often at extension shape, have the function of sand consolidation with biologic, secrete exocellular polysaccharide, generates lecithinase, casease, gelatinase, peroxidating
Hydrogen enzyme.Reference《Primary Jie Shi systematic bacteriologies identification handbook》8th edition and《Common bacterial system identification handbook》It is to number
DOB150 bacterial strains are identified, determine that the bacterial strain that bacterium numbering is DOB150 is bacillus in conjunction with Physiology and biochemistry comprehensive detection
Member in (Bacillus sp.) is bacillus (Bacillus sp.) from taxology angular divisions, still, passes through synthesis
The bacterial strain of DOB150 known to analysis is the member in a kind of new bacillus (Bacillus sp.).
R2A culture mediums：Yeast extract powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g are solvable
Property starch 0.5, dipotassium hydrogen phosphate 0.3g, K2HPO40.3g, MgSO40.024g, Sodium Pyruvate 0.3g, agar 15.0, distilled water
1000mL, pH value 7.2 or so.
1/5TSA culture mediums：Tryptone 5.0g, soya peptone 1.0g, NaCl 10g 15.0g, agar 15.0, distilled water
1000mL, pH value 7.2 or so.
LB culture mediums：Tryptone 10g, yeast extract 5g, NaCl 10g, agar 15.0, distilled water 1000mL, pH value
7.2 left and right.
Embodiment two：The molecular level of Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 is identified
1, the DNA sequence dna of PCR amplification Oligotrophic bacteria DOB150 and its sequencing
The single bacterium colony of a small amount of Oligotrophic bacteria DOB150 bacterial strains of picking is put into the EP pipes for filling 25 μ L sterile waters, 100 DEG C
8-10min is boiled, is put into 5min in mixture of ice and water rapidly afterwards.10000r/min, 5min, 4 DEG C of preservations are centrifuged, the used time takes
Clearly.
The structure of 16S r DNA gene sequencings and its systematic evolution tree：Oligotrophic bacteria is extracted according to a conventional method
Dilution universal primer is carried out the PCR amplification of 16S r DNA sections with deionized water by the total DNA of DOB150 bacterial strains.
The reaction system of 50 μ l contains：10 × PCR buffer solutions 5 μ l, each 20pmol of primer, template DNA 1 μ l, TaqTM
8 μ l of (TaKaRa companies) 0.5U, dNTP.PCR amplification condition：95 DEG C of pre-degeneration 5min, 94 DEG C of 45sec, 55 DEG C of 1min, 72 DEG C
1min is recycled 30 times, and 72 DEG C of 10min, which are repaired, to be extended, and 4 DEG C terminate reaction.The purifying of PCR product：PCR product carries out 1% agarose
Electrophoresis in gel recycles target fragment with TaKaRa PCR FragmentRecoveryKit from glue, is dissolved in the high-purity of 30 μ l
In water.It send to sequencing company and carries out the measurement of 16S r DNA section sequences, the gene order ginseng of strain Oligotrophic bacteria DOB150
See attached gene order table.
AGAGTTTGATCCTGGTTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATT
AAGAGCTTGTTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGTCCATAAGACTGGGATAACT
CCGGGAAGCCGGGGCTAGTACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACT
TATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAG
AGTTTGATCGGCCACACTGGGACTGAGCCACGGCCCAGACTCCTACGGGAGGCACCAGTAGCCAATCTTCCGCAATG
GACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGCCAAGAAC
AAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCG
GTAATACGTAGTTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGTGCGCAGGTGGTTTCTTAAGTCTGATGTG
AAAGCCCACGGCTCAACCGTGCAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATG
TGTAGCCGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAG
GCGCGAAAGGGTGGGGAGCAAACAGGATTAGATACCCTGGTTGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGA
GGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGTCTGGGGAGTACGGCCGCAAGGCTGAAACTCA
AAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTATAATTCGAAGCAACGCGAAGAACCTTACCAGGTC
TTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCA
CCTCGTGTCGTGAGATGGTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCACTATTAAGTTGGGC
ACTCTAAGGTGATTGACGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGAACTGGG
CTACACACGTGCTACAATGAACGGTACAAAGAGCTGTAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCA
GTTCGGATTGTACGCTGCAACTCGCCTACATGAAGCTGCAATCGCTAGTGATCGCGGATCAGCTTGCCGCGGTGAAT
ACGTTCCCGGGCCTTGTACACACCGCCCCTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTT
TGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGCGTGAAGTCGTAACAAGGTAGCCGAATCGGAAGGTGCGGCTG
GATCACCTCCTT
2, the comparison of 16S r DNA gene orders and Phylogenetic Analysis
The 16S r DNA sequence dnas that sequencing is obtained carry out BLAST analyses with the nucleotide sequence in GenBank databases,
Therefrom obtain similar 16S r DNA sequence dnas, by the 16S r DNA sequence dnas of Oligotrophic bacteria DOB150 in ncbi database into
After row BLAST analyses, systematic evolution tree is built, shown in attached drawing 1.
By 16S rRNA gene sequencings and comparison, and establish phylogenetic tree analysis, the results showed that bacterial strain belongs to
Bacillus (Bacillus.sp), it is homologous with NCBI report bacterial strain Bacillus cereus WJL-063 (FJ527559)
Property highest, highest similitude be 97.6%, be respectively 97.5% with the category type strain sequence alignment highest homology
(Bacillus cereusATCC 14579T), 97.4% (Bacillus anthracisATCC 14578T), 97.4%
(Bacillus thuringiensisATCC 10792T), 97.4% (Bacillus toyonensis BCT-7112T) and
97.1% (Bacillus pseudomycoides DSM 12442T), tentatively predicate the potential novel species of the category.With
The GEN III of BiologMicrostation systems measure card and are tested, D-Maltose, D- trehaloses, D- turanoses, water
Threose, α-D- lactose, melibiose, N- acetyl-d-glucosamine N- acetyl-β-D-MANNOSE amine, α-D- glucose, D-MANNOSE, D-
Fructose, D- galactolipin 3- formyl glucose, D-Fructose, PEARLITOL 25C, D-arabitol, inositol, glycerine, D- Robison esters, D-
Fructose-6-phosphate, D-Ser, glycyl-L-PROLINE, Serine, pectin, L- galacturonic acid lactones, D- glucose
Acid, D- glucuronic acids, glucuronamide, glactaric acid, mucic acid, quininic acid, saccharic acid, L MALIC ACID, gamma-amino-butyric acid, α-hydroxyl
Base-butyric acid, beta-hydroxy-D, L butyric acid acetoacetate, formic acid etc. are determined as that bacillus one is potential using being positive findings
Novel species, the biology which suggests are named as Bacillus sp.DOB150.
Belong to gemma bar from its ownership in conjunction with the Morphologic Characteristics and physio-biochemical characteristics of Oligotrophic bacteria DOB150
Pseudomonas monoid.Measured 16S r DNA sequence dnas are inputted into Genbank, tetraploid rice is carried out with Blast programs, finds it
It is maximum with the similitude of the 16S r DNA sequence dnas of Bacillus sp HN002, it is a kind of typical new Bacillus.
In conjunction with the Morphologic Characteristics and physio-biochemical characteristics of Oligotrophic bacteria DOB150, determines that it is bacterium numbering and integrated for DOB150
It is named as bacillus (Bacillus sp.).
Embodiment three：The characteristic research and application of Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925
1, experimentation
After scribing line activates repeatedly on LB culture mediums by Oligotrophic bacteria DOB150, it to be used for the preparation of liquid bacterial agent, inoculation
37 DEG C of shaking flask culture 60h of postposition.
(1) fixation experiment
The disk 2 that long 30cm × wide 20cm × high 5cm is filled with drift sand, drift sand is fitted into disk, by shaking table concussion
Training, the Oligotrophic bacteria DOB150 after supporting are sprayed at the drift sand surface of one of sand table, another sand table sprays oneself of equivalent
Water is as a contrast.
(2) water conservation experiment
Oneself is taken to know that (wherein 10 water sprays compare, and 10 spray Oligotrophic bacteria DOB150's for 20, the crucible of drying constant weight
Bacterium solution) it weighs, it is denoted as W1, claim 30g sands to be fitted into this 20 crucibles respectively, 10 husky crucibles of dress respectively spray 6g widow battalion thereto
The bacterium solution of bacterium DOB150 is supported as test group, 6g is respectively sprayed water as a control group to the other 10 husky crucibles of dress, is denoted as W2.It will
This 20 crucibles are placed in constant incubator, on day 2, the 4th day, the 6th day, the 8th day and the 10th day, take out respectively test group and
Control combination 2, is placed in 105 DEG C of baking ovens and dries 2-3 hours, weighs after cooling, is denoted as W3, its soil is measured using baking weight method and is contained
Water.
(3) keep a full stand of seedings experiment
Equivalent sand is filled in 10 flowerpots, equivalent water is sprayed, uniformly sows seeds into each flowerpot, Glycyrrhiza Uralensis,
Each 50 of epheday intermedia, then equal earthing 40g, being placed in windowsill makes it grow naturally, and after 2-3 days to be grown, Radix Glycyrrhizae, Chinese ephedra are unearthed
After having remembered number, Oligotrophic bacteria DOB150 bacterium solutions are sprayed.Setting various concentration gradient sprays the equal 30ml of bacterium solution into flowerpot, is arranged
1 group of control and 4 groups of bacterial concentration gradients 25%, 50%, 75%, 100%, every group is done three repetitions, records Average Survival daily
Seedling number, until the 7th day, it can tentatively find out whether bacterium solution has effect of keeping a full stand of seedings.
2, experimental result
(1) fixation experiment result
The control sand table surface of sprinkling tap water is formed without skinning, sprays the experiment sand table of Oligotrophic bacteria DOB150 bacterium solutions
It places surface after a few days and forms skinning cortex, which shows to be sprayed at drift sand table using Oligotrophic bacteria DOB150 bacterium solutions
Face can play the role of fixing the sand, prevent drift sand surface active, this will provide scientific theory foundation for biological carpet sand control engineering
And species resource, therefore check winds and fix drifting sand improve the ecological environment aspect will have great application value.
(2) it is retained test effect
Oligotrophic bacteria DOB150 bacteria preparations are sprayed at the sand table on drift sand surface and the control sand table of sprinkling tap water in perseverance
After being placed 2,4,6,8,10 days under conditions of temperature, its soil moisture content is measured, the results showed that, although experimental group and control group have
The loss of moisture, but as the speed ratio control group of the loss of the extension test group moisture of time is slow, it is right after especially 8 days
It is increased rapidly according to a group water loss rate, therefore there is certain guarantor using Oligotrophic bacteria DOB150 bacterium solutions sprinkling drift sand surface
Water acts on.
(3) keep a full stand of seedings test effect
Oligotrophic bacteria DOB150 bacterium solutions are sprayed at the test effect of keeping a full stand of seedings on drift sand surface.It waits emerging, sprays various concentration
The 7th day after bacterium solution, oneself was slowly die due to arid again through unearthed seedling is most of.But, it is apparent that spray
The seedling survival number of days of watering is most short, dies an death most soon, and the seedling survival number of days for spraying Oligotrophic bacteria DOB150 bacterium solutions is longer,
Die an death relatively slow, but to be not the longer therefore suitable bacterial concentration of number of days of the higher seedling survival of bacterial concentration can play one
Fixed seedling protecting effect.
As it can be seen that in the Oligotrophic bacteria Bacillus sp.DOB150CGMCC provided by the invention of drift sand surface sprinkling
The bacterium solution of NO.11925 forms skinning, compared with the control of sprinkling equivalent water, soil moisture evaporation rate can be made to slow down, and extends
The soil water retention time.So if Oligotrophic bacteria DOB150 bacterium solutions, which are used in desert surface, can not only play the work that fixes the sand
With, and preferable moisture condition may be provided for the sprouting of some slower vegetable seeds of speed of germination, make some because of soil
The plant that earth water deficient cannot be sprouted obtains growth and development.
Example IV:Oligotrophic bacteria Bacillus sp.DOB150CGMCC NO.11925 are to ancient capital Xi'an
The influence of soil environment
1, experimentation
After scribing line activates repeatedly on LB culture mediums by Oligotrophic bacteria DOB150, it to be used for the preparation of liquid bacterial agent, inoculation
37 DEG C of shaking flask culture 60h of postposition, prepare Oligotrophic bacteria DOB150 microbial inoculums.
It selects the shifting sand area of 2 5m × 5m as test plot on the innerland of ancient capital Xi'an, is arranged at 2
Reason is sprayed Oligotrophic bacteria DOB150 microbial inoculums and is not sprayed, the latter is control.Acquired respectively after 1 month each sample prescription 0-2cm and
2-5cm soil samples carry out microbial populations in soil quantity, soil enzyme activities, the measurement of cryptogam, and each soil sample is all provided with 3 weights
It is multiple.
2, experimental result
(1) influence that sprinkling Oligotrophic bacteria DOB150 microbial inoculums are distributed different soil major microorganisms
The application of Oligotrophic bacteria DOB150 microbial inoculums, affects the quantative attribute of the original microbe groups of soil.Control is each
Soil layer, after spraying Oligotrophic bacteria microbial inoculum, all kinds of micro organism quantities have obvious increase.For bacterium, control sample
0-2cm layers of bacterial number is sprayed significantly less than 2-5cm soil layers in the sample ground of Oligotrophic bacteria DOB150 microbial inoculums, bacterium
The regularity of distribution shows as surface layer and is more than time surface layer, and 0-2cm layers of bacterial number is significantly higher than control.Spray Oligotrophic bacteria
Afterwards, the fungi of each soil layer and Population of Actinomycetes increased.Oligotrophic bacteria in Oligotrophic bacteria DOB150 microbial inoculums
After DOB150 enters soil, certain facilitation is played to the growth and breeding of other microorganisms, to soil microbial activities
Raising have certain help.
(2) influence of the sprinkling Oligotrophic bacteria DOB150 microbial inoculums to different soil soil enzyme activities
It is shown in Table 1, compared with the control, the application of Oligotrophic bacteria DOB150 is different to 5 kinds of enzymatic activitys in testing soil
The influence of degree.Improve the catalase and polyphenol oxidase activity of 0-2cm and 2-5cm soil layers.The variation of urease activity
It is in opposite trend in 2 soil layers, shows as upper layer urease activity and increase, and lower layer's activity reduces.The different responses of enzymatic activity
It may be related with the change of soil nutrient.
The soil enzyme activities on 1 different experiments sample ground of table
(3) influence that sprinkling Oligotrophic bacteria DOB150 microbial inoculums form biological breadcrust
Cyanobacteria is the sociales in algae skinning, and very important effect is played to the early formation and recovery of biological breadcrust.
The study show that being shown in Table 2, after spraying sprinkling Oligotrophic bacteria DOB150 microbial inoculums to the areas Luo Sha, cyanobacteria quantity obviously increases (200-
300/g soil), and check plot cyanobacteria is few (10-20/g soil), shows that Oligotrophic bacteria DOB150 has and promotes algae skinning shape
At effect, but do not find lichens and moss crust in 2 sample ground.
Under 2 different disposal of table in soil main cryptogam monoid variation
| Title | Cyanobacteria | Lichens | Moss |
| Spray Oligotrophic bacteria DOB150 microbial inoculums | 200-300 | 0 | 0 |
| Sprinkling Oligotrophic bacteria DOB150 microbial inoculums are not sprayed | 10-20 | 0 | 0 |
Oligotrophic bacteria Bacillus of the present invention using voluntarily screening separation domestication is can be seen that from above-mentioned evaluation
After sp.DOB150CGMCC NO.11925 are applied to sandy soil, adhesion sand grains, prevention drift sand activation have been not only acted as, moreover it is possible to effectively
Increase major microorganisms quantity in soil, improves essential nutrient contents of soil, strengthening part soil enzyme activities, and can obviously restore
Algae skinning.Therefore, Oligotrophic bacteria DOB150 is a kind of more satisfactory biomaterial that fixes the sand, this is tied effectively to restore biology
Skin, reparation are damaged sand ground and provide new method.
The above embodiment is merely an example for clearly illustrating the present invention, and does not limit the embodiments.
For those of ordinary skill in the art, other various forms of variations can also be made on the basis of the above description
Or it changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation thus extended
Or it changes and is still in the protection scope of this invention.
SEQUENCE LISTING
<110>Xinjiang University；Mao Peihong
<120>A kind of desert Oligotrophic bacteria DOB150 and the application in fixing the sand
<130> DOB150
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1544
<212> DNA
<213> DOB150
<220>
<221> DOB150
<222> (1)..(1544)
<400> 1
agagtttgat cctggttcag gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
gaatggatta agagcttgtt cttatgaagt tagcggcgga cgggtgagta acacgtgggt 120
aacctgtcca taagactggg ataactccgg gaagccgggg ctagtaccgg ataacatttt 180
gaaccgcatg gttcgaaatt gaaaggcggc ttcggctgtc acttatggat ggacccgcgt 240
cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag 300
agtttgatcg gccacactgg gactgagcca cggcccagac tcctacggga ggcaccagta 360
gccaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct 420
ttcgggtcgt aaaactctgt tgttagccaa gaacaagtgc tagttgaata agctggcacc 480
ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 540
agttggcaag cgttatccgg aattattggg cgtaaagcgt gcgcaggtgg tttcttaagt 600
ctgatgtgaa agcccacggc tcaaccgtgc agggtcattg gaaactggga gacttgagtg 660
cagaagagga aagtggaatt ccatgtgtag ccgtgaaatg cgtagagata tggaggaaca 720
ccagtggcga aggcgacttt ctggtctgta actgacactg aggcgcgaaa gggtggggag 780
caaacaggat tagataccct ggttgtccac gccgtaaacg atgagtgcta agtgttagag 840
ggtttccgcc ctttagtgct gaagttaacg cattaagcac tccgtctggg gagtacggcc 900
gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggtat 960
aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaaaa ccctagagat 1020
agggcttctc cttcgggagc agagtgacag gtggtgcatg gttgtcgtca cctcgtgtcg 1080
tgagatggtg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg ccactattaa 1140
gttgggcact ctaaggtgat tgacggtgac aaaccggagg aaggtgggga tgacgtcaaa 1200
tcatcatgcc ccttatgaac tgggctacac acgtgctaca atgaacggta caaagagctg 1260
taagaccgcg aggtggagct aatctcataa aaccgttctc agttcggatt gtacgctgca 1320
actcgcctac atgaagctgc aatcgctagt gatcgcggat cagcttgccg cggtgaatac 1380
gttcccgggc cttgtacaca ccgcccctca caccacgaga gtttgtaaca cccgaagtcg 1440
gtggggtaac ctttttggag ccagccgcct aaggtgggac agatgattgg cgtgaagtcg 1500
taacaaggta gccgaatcgg aaggtgcggc tggatcacct cctt 1544
Claims (3)
1. a kind of Oligotrophic bacteria Bacillus sp. DOB150, which is characterized in that the Oligotrophic bacteria Bacillus
Sp. the deposit number of DOB150 is CGMCC NO.11925.
2. Oligotrophic bacteria Bacillus sp. DOB150 as described in claim 1, which is characterized in that the Oligotrophic bacteria
The 16s rDNA gene orders of Bacillus sp. DOB150 are as shown in SEQ ID NO.1.
3. applications of the Oligotrophic bacteria Bacillus sp. DOB150 as described in claim 1 in fixing the sand.
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| CN106759248B (en) * | 2016-12-29 | 2018-09-21 | 河海大学 | Barren green desert earth's surface windproof structure |
| CN106638339B (en) * | 2017-01-18 | 2022-04-08 | 甘肃省治沙研究所 | Algae blanket and preparation method thereof |
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| CN108865889A (en) * | 2018-07-04 | 2018-11-23 | 内蒙古工业大学 | Method of the desert from the separation method of source mineralising bacterial strain, the verification method and consolidation sand of mineralization ability |
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| Bacillus cereus strain WJL-063 16S ribosomal RNA gene，partial sequence;FJ527559.1;《Genbank》;20090114;全文 * |
| 科尔沁沙地固沙群落土壤可培养微生物生理类群组成及优势细菌鉴定;耿莉;《中国优秀硕士学位论文全文数据库农业科技辑（电子期刊）》;20150515;摘要，第4章 * |
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