CN105586292B - One bacillus and its application - Google Patents
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- CN105586292B CN105586292B CN201510995205.2A CN201510995205A CN105586292B CN 105586292 B CN105586292 B CN 105586292B CN 201510995205 A CN201510995205 A CN 201510995205A CN 105586292 B CN105586292 B CN 105586292B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/07—Bacillus
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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Abstract
The invention discloses a bacillusBacillus luteaGSS08 and its application.The bacterial strain is preserved in China typical culture collection center on December 11st, 2015, and preserving number is CCTCC M 2015739.The bacillus of the present invention, provides a kind of new gram positive bacterial strain for liquid humid acid fertilizer, is with a wide range of applications in soil remediation, soil improvement etc..
Description
Technical field
The invention belongs to field of environment microorganism, and in particular to a bacillus and its application.
Background technology
The substance cycle of nature can be summarized as two aspects：First, the organic matter of inorganic matter, i.e. biosynthesis act on；
The other is the inanimate matter of organic matter, i.e. mineralization or decomposition.It is existing studies have shown that microorganism organic matter mine
It plays a decisive role in change, the mineralising of 90% or more organic matter is completed by microorganism such as bacterium and fungi on the earth.Leather is blue
Family name's positive bacteria is highly important microbe groups, and superiority is occupied in anaerobism soil, soil nutrient is utilized, pollution
Object reparation etc. plays an important roll.Bacillus（Bacillus）Representative object of the bacterium as gram-positive bacteria in bacterium
Kind, strong to extraneous injurious factor resistance, quantity is more in the natural environment, distribution is wide, is widely used to soil/water pollution
It repairs.
The breathing of humus respiration, also known as quinone refers to microorganism using humus as terminal electron acceptor, passes through and aoxidizes electronics confession
Body is coupled liquid humid acid fertilizer, and the energy of vital movement is stored during this.Humus respiration is the new of discovered in recent years
Type microorganism energetic supersession mode.Humus respiration is in key element geochemical cycle process, remediation contaminated soil and organic
It plays a significant role in the cyclic utilization of waste, for studying the micro-interface process and its function of soil typical material conversion and cycle,
With important scientific value.Traditional view thinks that humus respiration bacterium is generally Gram-negative bacteria, and gram-positive bacteria is special
It is not that bacillus had once been mistaken as not having real humus respiration function, their depositing in humus respiration environment
System balancing and complementary effect are maintained only serving.In recent years, researcher has found Corynebacterium humireducens, gemma bar successively
The gram-positive bacterias such as bacterium have the characteristic of humus respiration.Gram-positive humus respiration bacterium especially bacillus has both
Extracellular respiration capability and the strong advantage of resistance, in terms of clean energy resource production and contaminated soil remediation and waste utilization increasingly
It is taken seriously.
Early in nineteen ninety-five, Boone et al., which has just been successfully separated first plant, has humus respiration capabilityB. infernus.Later, and there are more plantsBacillusThe liquid humid acid fertilizer bacterium of category is found successively.However, pure due to what is reported
CultureBacillusCategory humus respiration bacterium is strict anaerobes, and extracellular electron transmission efficiency is low, and culture is inconvenient, progress
Slowly.
Invention content
The purpose of the present invention is to provide a bacillus and its applications.
The technical solution used in the present invention is：
Bacillus, Classification And Nomenclature areBacillus luteaGSS08 has been preserved in China typical culture collection
The heart, preserving number are CCTCC M 2015739.
A kind of environmental pollution reparation microorganism formulation, the microorganism formulation above-mentioned bacillus in containingBacillus luteaGSS08。
BacillusBacillus luteaApplications of the GSS08 in microorganism formulation.
Preferably, the microorganism formulation is that microorganism formulation is repaired in environmental pollution.
The beneficial effects of the invention are as follows：
The present invention provides a kind of new bacillus with liquid humid acid fertilizer abilityB. luteaGSS08 is humic
Matter reduction provides a kind of new gram positive bacterial strain, before soil remediation, soil improvement etc. have a wide range of applications
Scape.
The microorganism formulation of the present invention, can be advantageously applied to soil remediation, soil improvement.
B. luteaGSS08 amphimicrobians, can be under neutral and alkaline condition（PH is up to 9.5）It is grown and is increased
It grows, can carry out liquid humid acid fertilizer with acetic acid, glucose under anaerobic.With existing similar Strain comparison, the strain culturing
Convenient, the range of adaptation is wider.
Description of the drawings
Fig. 1 present inventionB. luteaStereoscan photographs of the GSS08 under 20000 times；
Fig. 2 present inventionB. luteaThe phylogenetic tree of GSS08；
Fig. 3 present inventionB. luteaGSS08 restores AQDS situations using different electron donors.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated, and however, it is not limited to this.
1 bacillus of embodimentB. luteaCulture, separation, screening, identification and the preservation of GSS08
Culture, the separation of 1.1 bacterial strains
The bacillus of the present inventionB. luteaGSS08 is by matured compost enrichment culture isolate and purify
It arrives.
Specific isolation and purification method includes the following steps：
（1）Take 5.0 g matured composts（Municipal sludge and crop material）The liquid that pH value is 7.2 is inoculated in be separately cultured
In base；The composition of the liquid separation culture medium is：Contain 0.6 g acetic acid, 0.6 g NaH in every liter of liquid separation culture medium2PO4,
0.25 g NH4Cl, 0.1 g KCl, 2.5 g NaHCO3, 0.2 g yeast powders, 10.0 mL vitamin solutions, 10.0 mL are micro
Element Solution, 1.0 mM AQDS.Wherein, vitamin solution is to contain 2.0 mg biotins, 2.0 mg leaves in every liter of deionized water
Acid, 10.0 mg vitamin Bs6, 5.0 mg thiamines, 5.0 mg riboflavin, 5.0 mg niacin, 5.0 mg calcium pantothenates, 0.1 mg dimensions
Raw element B12, 5.0 mg p-aminobenzoic acid, 5.0 mg lipoic acids；Trace element solution is to contain 1.5 g ammonia in every liter of deionized water
Triacetic acid, 3.0 g MgSO4•7H2O, 0.5 g MnSO4•H2O, 1.0 g NaCl, 0.1 g FeSO4•7H2O, 0.1 g
CoCl2•6H2O, 0.1 g CaCl2, 0.1 g ZnSO4•7H2O, 0.01 g CuSO4•5H2O, 0.01 g AlK (SO4)2•
12H2O, 0.01g H3BO3, 0.01g Na2MoO4•2H2O。
（2）Into the liquid separation culture medium for be inoculated with matured compost, drum fills high-purity mixed gas and is no less than 30 minutes, institute
It states high-purity mixed gas and N can be used2And CO2According to 80：The mixed gas of 20 volume ratio, air-blowing is finished to be sealed with aluminium lid, is placed in
Anaerobism work station（N2And CO2According to 80：The mixing of 20 volume ratio）, 30 DEG C of stationary cultures observe the color change feelings of culture solution
Condition.
（3）After cultivating 10d, culture solution color from it is colourless become buff when, culture solution is transferred with 10% inoculum concentration
To another fresh liquid separation culture medium, drum fills high-purity mixed gas and is no less than 30 minutes, and air-blowing is finished to be sealed with aluminium lid, is set
In 30 DEG C of stationary cultures of anaerobism work station.Such enrichment culture is three times.
（4）Culture solution after forth generation is reacted is diluted to 10 using dilution-plate method-5-10-6, by the culture after dilution
0.1 mL of liquid is spread evenly across on LB solid mediums, is placed in anaerobism work station（N2/CO2=80/20）30 DEG C of 48 h of culture, picking
Single bacterium colony carries out isolation and purification.The composition of the LB solid mediums is：Peptone 10 g/L, yeast extract 5g/L,
NaCl is 10 g/L, 20 g/L of agar powder, pH value 7.2.
Thalli morphology characteristic
Using conventional bacterium electron microscope observation, which is gram-positive bacteria, rodlike, and thalline size is 1.3-
3.5 0.6-1.8 μm of μ ms have motility.See Fig. 1.
Colonial morphology characteristic
On LB Solid agar culture tablets after 24 h of aerobic culture, bacterium colony is round, yellow, slightly protrusion, opaque, bacterium
Fall a diameter of 1-2 mm.
Physiological and biochemical property
To bacterial strainB. luteaGSS08 carries out the identification of physiological and biochemical property, and is compared with several nearly source bacterial strains,
It the results are shown in Table 1.
1. bacterial strain Physiology and biochemistry qualification result of table
（In table：+ it is expressed as the positive, W is expressed as weakly positive ,-it is expressed as feminine gender）
By the result identified（Table 1）Known to：Bacterial strainB. luteaGSS08 amphimicrobians, can be in the culture solution of 0-4% NaCl
Middle growth, and most suitable NaCl is 1%；Growth temperature is 25-45 DEG C, and optimum growth temperature is 37 DEG C；PH value is 6.5-9.5, most suitable
PH is 7.5；Contact enzyme positive, oxidase negative；With aesculin hydrolysing activity, Gelatinolytic activity.Bacterial strainB. luteaGSS08 can utilize rhamnose, adipic acid, L- trehaloses, D-MANNOSE, L-arabinose, D-Glucose, D- sweet dews
Alcohol is as carbon source；G+C contents are 49.5%.
By comparing,B. luteaGSS08 bacterial strain amphimicrobians, the carbon source that can be utilized is wider, andB. humi DSM
16318、B. andreesenii DSM 23948、B. subtilis10 Hes of DSMB. timonensisDSM 25372 is
Micro- aerobic or strict anaerobes, the carbon source kind that can be utilized are then extremely limited.In additionB. luteaThe G+C contents of GSS08 are bright
It is aobvious to be higher than nearly source bacterial strain.
Molecular biological characteristics
Using SDS- Proteinase Ks, chloroform-isoamyl alcohol（Volume ratio 24：1）The method of extracting, 0.6 volume isopropanol precipitation carries
Take bacteria total DNA.The 16S rRNA that bacterium is expanded using 16S rRNA universal primers F27 and R1492 are returned pcr amplification product
It is sequenced after receipts.Sequencing result is shown in SEQ ID NO.1.Again by obtained base sequence in the international nucleic acid sequence such as GenBank
Homologous sequence search (Blast search) is carried out in database, finds out the bacterial strain and the highest pattern bacterium of homology in database
Strain or the bacterial strain for being preserved in the worlds ATCC HuoDSMDeng Culture Collection Center.B. luteaThe phylogenetic tree result of GSS08 is shown in figure
2。
It is found according to comparison result, the bacterial strain and bacillusB. humiThe 16S rRNA sequences tool of DSM 16318 is most
High homology, similarity 97.5%；Molecule results of hybridization shows, the bacterial strain and bacillusB. humi DSM 16318DNA-
DNA correlations are 50.8%.
In conclusion the comparison of 16S rRNA homologous sequences can determine bacterial strain of the present inventionB.luteaGSS08 belongs to gemma
Bacillus.But due to bacterial strain of the present inventionB. luteaGSS08 have it is many can easily with existing Bacillus phase region
Other phenotypic characteristic, in addition DNA-DNA hybridization and Molecular taxonomic analysis are the result shows that bacterial strain of the present invention is not belonging to bacillus
Existing kind belonged to.Therefore, according to available data, it is possible to determine that bacterial strain of the present invention is a novel species for bacillus.It is named asBacillus luteaGSS08 is abbreviated asB. luteaGSS08 was deposited in Chinese Typical Representative culture on December 11st, 2015
Collection（Wuhan）, preserving number is CCTCC M 2015739.
2 bacillus of embodimentB. luteaGSS08 tests liquid humid acid fertilizer
Liquid Culture based formulas：Contain 0.6 g NaH in every liter of deionized water2PO4, 0.25 g NH4Cl, 0.1 gKCl, 2.5
g NaHCO3, 0.2 g yeast powders, 10.0 mL vitamin solutions, 10.0 mL trace element solutions, 1.0 mM AQDS（Wherein tie up
Raw element solution and trace element solution ingredient are the same as embodiment 1）.It is 7.5 to adjust pH with 0.5mol/L NaOH, in 121 DEG C of sterilizings 20
Minute, glucose, acetic acid, pyruvic acid and the lactic acid solution of sterilizing are added after sterilizing, are uniformly mixed, several substances of above-mentioned addition
Final concentration is 5.0 mmol/L.
From culturedB. lutea2 ring of GSS08 culture solution pickings lawn is seeded in LB liquid medium, in 37 DEG C
Culture, makes bacterial number reach 108（CFU/mL）The order of magnitude.With 10% inoculum concentration inoculation activation bacterium solution in aforesaid liquid culture
In base, anaerobism work station（N2/CO2=80/20）Lower 37 DEG C of stationary cultures；Setting is not added with the control of bacterium simultaneously, observes culture solution
Color change situation.4mL culture solutions are taken every 1 d, reduction-state is measured at 450nm using ultraviolet-visible spectrophotometer
AQDS（AH2DS）Content, with AH2A concentration of indexs of DS, verification bacterial strain restore AQDS abilities by electron donor of lactic acid.As a result see
Fig. 3.
It was found from Fig. 3 results：BacillusB. luteaIt is generated when GSS08 is using glucose, acetic acid as electron donor
AH2DS concentration constantly rises, and in 9 d, AQDS reduction rates respectively reach 77.27% and 58.32%, illustrate B. luteaGSS08 exists
System pH can utilize glucose, acetic acid to restore AQDS when being 7.5.
The preparation of 3 micro-organisms bacillus preparation of embodiment
Specific method is：
（1）Shaking flask culture：FromB. luteaThe access of one ring of picking lawn is equipped with LB liquid medium on the inclined-planes GSS08
In conical flask, 37 DEG C, 200 rpm/min shaking table shake culture 12-20 h；
（2）Ferment tank：It will be above-mentioned culturedB. luteaGSS08 zymotic fluids access in 10L fermentation tanks, 37 DEG C,
200 rpm/min fermented and cultured 12-24 h；
（3）Solid state fermentation：It is cooling by solid fermentation medium sterilization, above-mentioned zymotic fluid is accessed by the inoculum concentration of 5-15%,
It is cultivated 3-4 days in 25-30 DEG C；
（4）Fermentation post-processing：Solid medium after above-mentioned fermentation by drying, air-flow crushing, sieving, as gemma bar
Bacteria microorganism preparation.
（5）Product quality inspection：Product quality inspection method is according to agricultural microbial agent standard（GB 20287-2006）
It is detected.Testing result shows living bacteria count >=2 × 10 in above-mentioned preparation8CFU/g, product are qualified.
The LB liquid medium formula is (g/L)：Yeast extract 5g；Tryptone 10g；NaCl5g；1000 mL of water
；pH 7.2.
The solid fermentation culture medium is the mixed of common culture medium, including wheat bran, analysis for soybean powder of existing solid state fermentation etc.
Material is closed, it is 7.0 to adjust pH, moisture content 45-60%.
In conclusion the present inventionB. luteaGSS08 and the existing bacillus with liquid humid acid fertilizer characteristic are same
Class Strain comparison,B. luteaGSS08 can be enlarged culture and fermentation by aerobic condition and conventional medium, and can
Obtain the micro-organisms bacillus preparation for meeting living bacteria count condition, product qualification.So the present inventionB. lutea
Range applicable GSS08 is wider, has good commercial value and social value.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
<110>Pingtan comprehensive test region municipal administration gardens Co., Ltd
<120>One bacillus and its application
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1453
<212> DNA
<213>Bacillus（Bacillus lutea）
<400> 1
aggagtgcgg cagctataca tgcagtcgag cgaatctaag ggagcttgct cccaaaagat 60
tagcggcgga cgggtgagta acacgtgggc aacctacctg taagactggg ataacttcgg 120
gaaaccggag ctaataccgg ataacatatg agaccacatg gtcttatatt aaaagatggc 180
ttcttgctat cacttacgga tgggcccgcg gcgcattagc tagttggtga ggtaacggct 240
caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag cgaagaaggt cttcggatcg taaagctctg ttgttaggga 420
agaacaagta tcgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540
gcgtaaagcg cgcgcaggcg gtcctttaag tctgatgtga aagcccacgg ctcaaccgtg 600
gagggtcatt ggaaactggg ggacttgagt gcagaagagg agagcggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcggctc tctggtctgt 720
aactgacgct gaggcgcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgaagtcaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca ctcctagaga taggacgttc ccttcgggga cagagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggatggt acaaagggca gcgaaaccgc gaggttaagc caatcccata 1260
aaaccattct cagttcggat tgcaggctgc aactcgcctg catgaagccg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtggggtaa ccgtaaggag ccagccgcct 1440
aagttgaccg agt 1453
Claims (4)
1. bacillus (Bacillus lutea) GSS08, is preserved in China typical culture collection center, preserving number is
CCTCC NO：M 2015739.
2. microorganism formulation is repaired in a kind of environmental pollution, which is characterized in that containing described in claim 1 in the microorganism formulation
Bacillus GSS08.
3. applications of the bacillus GSS08 described in claim 1 in preparing microorganism formulation.
4. application according to claim 3, it is characterised in that：The microorganism formulation is that microorganism system is repaired in environmental pollution
Agent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337236A (en) * | 2011-09-01 | 2012-02-01 | 广东省生态环境与土壤研究所 | Alkaline Bacilluspseudofirmus MC02 and application thereof |
| CN104560816A (en) * | 2014-12-16 | 2015-04-29 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis with biomass hydrolase activity and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337236A (en) * | 2011-09-01 | 2012-02-01 | 广东省生态环境与土壤研究所 | Alkaline Bacilluspseudofirmus MC02 and application thereof |
| CN104560816A (en) * | 2014-12-16 | 2015-04-29 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis with biomass hydrolase activity and application thereof |
Non-Patent Citations (1)
| Title |
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| Slobodan Glišic等.Synthesis, characterization and antimicrobial activity of carboxymethyl dextrane stabilized silver nanoparticles.《Journal of Molecular Structure》.2014,第1084卷第 345-351页. * |
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