CN107384804A - Gibberella NT 1 and its application - Google Patents
Gibberella NT 1 and its application Download PDFInfo
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- CN107384804A CN107384804A CN201710671482.7A CN201710671482A CN107384804A CN 107384804 A CN107384804 A CN 107384804A CN 201710671482 A CN201710671482 A CN 201710671482A CN 107384804 A CN107384804 A CN 107384804A
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- C12R2001/645—Fungi ; Processes using fungi
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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Abstract
The invention belongs to microbial technology field, and in particular to a kind of gibberella (Gibberella sp.NT 1) and its in Cu2+Application in Adsorption.Gibberella (Gibberella sp.NT 1), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on April 17th, 2017：CGMCC No.13888.The bacterium has stronger acidproof, resistance to copper feature and polluted by copper ability, 28 DEG C, pH 5.0, initial Cu2+Concentration is 200mg L‑1When, 5d is interior to Cu in solution2+Eliminating rate of absorption up to 45.6%.The bacterial strain be acidified and heavy metal copper combined contamination soil it is biological prosthetic in there is good application prospect.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of gibberella (Gibberella sp. NT-1) and its
In Cu2+Application in Adsorption.
Background technology
Due to largely applying the copper fungicides such as Bordeaux mixture in fruit production of forestry, cause copper a large amount of in orchard soil
Accumulation, and the improper administration of fertilizer and agricultural chemicals and the caused orchard soil acidifying that raises fruit trees all the year round then can further increase soil
The biological effectiveness of copper in earth, increase fruit tree absorb the risk of enriching Cu, reduce the yield and quality of fruit, and then threaten human body
Health.Orchard, which is acidified in the microorganism remediation technology with cuprum compound polluted soil, primarily to be solved the problems, such as to be screening to combined pollution
Microbial strains with higher patience and adsorption capacity.Although having studied separation identifies some heavy metal coppers with one
Determine the bacterial strain of patience and absorption property, but the patience of these bacterial strains acidity multipair greatly is poor, can not be compound with heavy metal in acidifying
Grown in the orchard soil of pollution.
The content of the invention
Present invention aims at a kind of gibberella (Gibberella sp.NT-1) of offer and its in Cu2+In Adsorption
Using.
To achieve the above object, the present invention use technical scheme for：
A kind of gibberella (Gibberella sp.NT-1), gibberella are the red mould (Gibberella of beading
Moniliformis), it is preserved in on April 17th, 2017 in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart (address：The institute 3 of BeiChen West Road, Chaoyang District, BeiJing City 1, Institute of Microorganism, Academia Sinica), deposit number：CGMCC
No.13888。
The gibberella (Gibberella sp.NT-1) is rapid in potato glucose cultured on solid medium, bacterium colony
It is velvet shape in relatively regular circle, neat in edge, quality, is combined with culture medium more close.Diameter when 3d is cultivated at 28 DEG C
56~58mm is can reach, bacterium colony is in milky white or faint yellow, center slightly projection (Figure 1A)；When culture is to 7d, mycelia is close to be covered with
Whole flat board, and start to produce bronzing pigment, bacterium colony center is also changed into pale red (Figure 1B).
A kind of application of gibberella (Gibberella sp.NT-1), the gibberella (Gibberella sp.NT-1) exist
Remove Cu2+In application.
Specially：Gibberella (Gibberella sp.NT-1) is inoculated in potato dextrose broth, shaken
Culture is swung to logarithmic phase；Accessed to cultivating to the bacteria suspension of exponential phase containing Cu2+Environment in concussion and cultivate, that is, realize inhale
Cu in attached removal environment2+；Bacterial strain NT-1 is to Cu in solution2+Eliminating rate of absorption up to 45.6%.
It is described culture to logarithmic phase gibberella (Gibberella sp.NT-1) access to acidity contain Cu2+Environment in
Concussion and cultivate, that is, realize the Cu in Adsorption environment2+。
The concussion and cultivate condition is 28 DEG C, 150rpm shaken cultivations.The acidity is that pH value is 5.0.
A kind of copper ion remover, remover are the bacteria suspension of gibberella (Gibberella sp.NT-1).
The bacteria suspension of the gibberella (Gibberella sp.NT-1) is by gibberella (Gibberella sp.NT-1)
It is inoculated in potato dextrose broth, shaken cultivation to logarithmic phase.
Advantage for present invention：The present invention is from long-term plantation apple and sprays the orchard soil of cupric agricultural chemicals Bordeaux mixture
Separated in earth, filter out gibberella NT-1 (Gibberella sp.NT-1), the bacterial strain has stronger acidproof, resistance to copper and Cu2+
Adsorption ability, the bacterium is in acid condition to Cu2+The effect of Adsorption, for further research and development acidifying and the compound dirt of copper
Dye the biological prosthetic of orchard soil provides new resources and new approaches, and be widely used potentiality.
Brief description of the drawings
Fig. 1 is NT-1 colony morphology characteristics provided in an embodiment of the present invention, wherein：a：Cultivate to 3 d；b：Cultivate to 7d；
Fig. 2 is the light micrograph of NT-1 mycelia provided in an embodiment of the present invention and spore, wherein： a：Object lens are 40
Times；b：Object lens are 100 times；
Fig. 3 is bacterial strain NT-1 provided in an embodiment of the present invention phylogenetic tree；
Fig. 4 is bacterial strain NT-1 provided in an embodiment of the present invention growth curve；
Fig. 5 is influence of the different factors provided in an embodiment of the present invention to polluted by copper clearance；Wherein： a：PH influence；
b：The influence of temperature；c：The influence of inoculum concentration；d：Initial Cu2+The influence of concentration.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and examples.
Bacterial strain of the present invention has stronger acidproof, resistance to copper feature and polluted by copper ability, 28 DEG C, pH 5.0, initial Cu2+
Concentration is 200mg L-1When, 5d is interior to Cu in solution2+Eliminating rate of absorption up to 45.6%.The bacterial strain is in acidifying and heavy metal
Cuprum compound polluted soil it is biological prosthetic in there is good application prospect.
Embodiment 1：The bacterial strain of resistance to copper NT-1 separation and identification
(1) bacterial strain of resistance to copper NT-1 separation and screening
The bacterial strain NT-1 of the present invention is divided from the long-term orchard soil planted apple and spray cupric agricultural chemicals Bordeaux mixture
From, filter out.Culture medium (potato dextrose medium) and copper Selective agar medium (will based on the culture medium of separation
CuSO4·5H2O is made into Cu2+Concentration is 10g L-1Stock solution, be separately added into beef extract-peptone basal medium and potato
In glucosyl group basal culture medium, adjust to required copper concentration).Solid medium is that 20g L are added in liquid medium within-1Fine jade
Fat.
Separation screening step：Take 5g fresh soil samples to vibrate 20min in the triangular flask equipped with 50mL sterilized waters, soil is made
Leaching liquor.Leaching liquor is diluted to 10 step by step-3、10-4、10-5Afterwards, it is 200mg to take 0.1mL to be coated on copper ions concentration respectively
L-1Selective agar medium, 28 DEG C culture 2d after, picking individual colonies rule again separation after continue to cultivate, finally choose the speed of growth
Relatively fast, the typical bacterium of colony characteristicses is further purified.By the preferable bacterial strain of the growing way filtered out continuously add cupric from
Sub- concentration is 200mg L-1Copper Selective agar medium in, cultivate centrifuging and taking supernatant after 5d, measure wherein content of copper ion, compare
Its copper removal rate, to screen the bacterial strain that copper tolerance is high, copper removal rate is high, as purpose bacterial strain.
By described separation and screening operation, one plant of speed of growth of acquisition is fast, copper tolerance is high, high to copper removal rate
Bacterial strain NT-1, has a good application prospect.
(2) morphology and molecular biology identification of bacterial strain
The resistance to copper fungi screened is inoculated in potato glucose flat board, is placed in 28 DEG C of constant incubators and cultivates 1 week,
The growth characteristics of fungus colony are observed, the form of mycelia and its conidial life side are observed using inserted sheet method under the microscope
Formula.The genomic DNA for the bacterial strain of resistance to copper is extracted, primer is designed and synthesized according to conserved sequence in fungi ITS1-5.8S-ITS4 regions
(forward primer ITS1： 5'-TCCGTAGGTGAACCTGCGG-3'；Reverse primer ITS4：5'-
TCCTCCGCTTATTGATATGC-3' performing PCR amplification) is entered.PCR amplification system (50 μ L) condition is：The μ L, Mix (2 of template DNA 5
×) 25 μ L, primer I TS1 2 μ L, primer I TS4 2 μ L, ddH2O 16μL.PCR reaction conditions：94 DEG C of 2.5min, 94 DEG C of 30s；
57 ℃1min；72 DEG C of 1.5min, circulate 35 times；72 DEG C of extension 3min.Electrophoresis detection is carried out to amplified production, commission Shanghai is beautiful
Lucky biological medicine Science and Technology Ltd. is sequenced.Existing ITS gene orders in the sequence of gained and ncbi database are carried out
BLAST is analyzed, and chooses bacterial strain similar in homology, using the software building phylogenetic trees of MEGA 6.0.
NT-1 is rapid in potato glucose cultured on solid medium, and bacterium colony is in relatively regular circle, neat in edge, quality
For velvet shape, combined with culture medium more close.Diameter can reach 56-58mm when 3d is cultivated at 28 DEG C, bacterium colony be in it is milky white or
It is faint yellow, center slightly projection (Fig. 1 a)；When culture is to 7d, mycelia is close to be covered with whole flat board, and starts to produce bronzing pigment,
Bacterium colony center is also changed into pale red (Fig. 1 b).
Observe the upgrowth situation of mycelia under an optical microscope with inserted sheet method, the results showed that, mycelia is elongated branch, and is in
Asymmetric branch；Conidiophore betides matrix.Conidium has two kinds of forms, and microconidia is oval, structure
It is compact, it is born on single raw bottle stalk, is polymerized to pelletizing on bottle stalk top, color is relatively deep (Fig. 2 a)；Macroconidium is spindle,
Quantity is more, is born in mycelia bifurcation (Fig. 2 b).
NT-1 sequencing result is subjected to BLAST in ncbi database and compares analysis, it is found that bacterial strain NT-1 and beading are red
Mould Gibberella moniliformis (Genbank accession number JF499676.1) similitude is 100%, with reference to NT-1
The morphological feature of bacterial strain, bacterial strain NT-1 is tentatively accredited as gibberella category fungi (Gibberella sp.NT-1).Fig. 3 bacterium
The phylogenetic tree of strain.
Embodiment 2：The measure of the bacterial strain of resistance to copper NT-1 growth curves
Using dry weight method, the fresh bacterial strain seed liquors of 1mL are drawn, are inoculated in containing 50mL potato glucose Liquid Cultures
In the triangular flask of liquid, 28 DEG C, 150rpm it is incubated, every 8h take out 3 parallel samples filtered, by filtered bacterium
Silk, which is placed in 80 DEG C of baking oven, to be dried to constant weight and weighs, and draws growth curve.
Bacterial strain NT-1 growth curve is as shown in Figure 4.Under conditions of 28 DEG C, 150rpm, it is put into after NT-1 inoculations 8h
Exponential phase, and enter stationary phase after 96h.
Embodiment 3：The patience characteristic and physiological responses that bacterial strain NT-1 is coerced various concentrations copper
The ring NT-1 mycelium inoculations of picking one are in containing Cu2+Concentration is respectively 100,200,300,400,500 and 600mg L-1
Potato glucose solid medium on, be placed in 28 DEG C of permanent incubator and cultivate 5d, observation mycelia on solid plate
Growing state, measure and record colony diameter.
Bacterial strain NT-1 seed liquor is inoculated in containing Cu2+Concentration is respectively 100,200,300,400,500 and 600mg L-1Potato dextrose broth in, be placed in 28 DEG C, cultivate 5d in 150rpm constant temperature oscillator, observation bacterial strain is in liquid
Growing state in body culture medium, bacterium solution is centrifuged and is dried to constant weight after 80 DEG C, measures and records hypha biomass dry weight.
As shown in Table 1, when cultivating 5d, NT-1 is containing Cu2+Concentration is more than 300mg L-1Growth on flat board is significantly pressed down
System, with the increase of copper concentration, colony growth radius further reduces, containing Cu2+Concentration is more than 400mg L-1Flat board on it is several
It can not grow.
1 different Cu of table2+The lower NT-1 of concentration stress upgrowth situation
"-" represents that bacterium colony can not grow.
In cupric fluid nutrient medium, with Cu2+The continuous improvement of concentration, the growth of bacterial strain are also gradually suppressed.When
Cu2+Concentration reaches 500mg L-1When, strain growth speed substantially slows down, and is mutually wound in nutrient solution bottom, forms diameter
2mm or so mycelium pellet.Work as Cu2+Concentration reaches 600mg L-1When, the growth of bacterial strain is substantially suppressed, but by 5d training
Support, still it is observed that a certain amount of mycelial growth, illustrates the bacterial strain under solution condition to Cu2+Patience ratio in solid culture bar
It is strong under part.
Embodiment 4：Gibberella (Gibberella sp.NT-1) bacterial strain is to Cu in solution2+Adsorption effect
Influence of the different factors to bacterial strain absorbing copper
Experiment condition：The NT-1 seed liquors that 5mL is in exponential phase are seeded to 50mL and contain Cu2+Concentration is 200mg
L-1Potato dextrose broth in, the constant-temperature shaking culture 5d under conditions of 5.0,28 DEG C of pH, 150rpm.
To investigate different factors to NT-1 Adsorption of Cu2+The influence of concentration, change following parameter respectively by above-mentioned experiment condition：
(1) initial Cu2+The influence of concentration：Cu is prepared respectively2+Concentration be 100,200,300,400,500,600mg L-1Nutrient solution；
(2) pH influence：Using 1mol L-1HCl and NaOH solution regulation culture medium pH be respectively 3.0,4.0,5.0,6.0,
7.0、8.0；(3) influence of temperature：The cultivation temperature of constant temperature oscillator is respectively set to 20 DEG C, 28 DEG C and 37 DEG C；(4) it is inoculated with
The influence of amount：Respectively the NT-1 seed liquors of exponential phase are inoculated with by 5%, 10% and 20% concentration (volume ratio).
The processing that above-mentioned all experiments are all provided with not connecing bacterium is repeated 3 times as control, each processing.After culture terminates, by bacterium
Liquid is transferred to centrifuge tube, in 4000r min-115min is centrifuged, the filter membrane for taking supernatant to cross 0.45 μm is former using flame after dilution
Cu in sub- absorption spectrophotometer (TAS-990, Beijing Pu Xi General Corporations) measure solution2+Content.
Data processing method：Clearance (%)=(Cu in solution2+Initial concentration-solution in Cu2+Final concentration it is dense
Degree) Cu in/solution2+Initial concentration × 100%.
(1) NT-1 is inoculated into different pH cupric culture medium and cultivates 5d, its clearance on copper is influenceed such as Fig. 5 a institutes
Show.Bacterial strain NT-1 can grow in the range of pH 3.0-8.0, show that the bacterium has stronger adaptability.At the initial pH of solution
When 3.0-5.0, bacterial strain significantly increases to the clearance of copper with pH increase, and reaches maximum when pH=5.0
24.4%；When the initial pH of solution is in 5.0-8.0, NT-1 bacterial strains gradually reduce to the clearance of copper with pH increase, show
Bacterial strain NT-1 has stronger acid-resistant property, and this is also the spy screened from acidifying and cuprum compound polluted orchard soil with NT-1 bacterial strains
Sign is consistent.
(2) from Fig. 5 b, in the range of 20-28 DEG C, NT-1 is raised to the clearance of copper and increased with temperature, at 28 DEG C
When reach maximum.Hereafter, as the rise of temperature, NT-1 gradually reduce to the clearance of copper.This result shows, bacterial strain
NT-1 has wide adaptability to growth temperature, can be grown in the range of 20-37 DEG C.
(3) from Fig. 5 c, with the increase of inoculum concentration, NT-1 significantly improves to the clearance of copper.But when inoculum concentration from
When 10% raising is to 20%, the clearance of copper does not have the proportional increase with the increase of inoculum concentration, thalline absorption knot in solution
Site utilization rate is closed to decline.Therefore, consider from the cost of microorganism remediation with effect, inoculum concentration is advisable with 10%.
(4) from Fig. 5 d, with Cu in system2+The gradual increase of concentration, bacterial strain NT-1 is to Cu2+Clearance manifest
Downward trend is write, works as Cu2+Concentration is in 100-300mg L-1In the range of when, NT-1 is to Cu2+Clearance up to more than 20%；
Work as Cu2+Concentration>300mg L-1When, NT-1 is to Cu2+Clearance significantly reduce, show the Cu of high concentration2+Growth to thalline
And the functional group of cell membrane surface generates stronger poisonous effect, bacterial strain NT-1 growth is significantly inhibited, and then pressed down
The growth of mycelia is made and its to Cu2+Adsorption capacity.
Claims (8)
- A kind of 1. gibberella (Gibberella sp.NT-1), it is characterised in that：Gibberella (Gibberella sp.NT-1), China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are preserved on April 17th, 2017： CGMCC No.13888。
- A kind of 2. application of the gibberella (Gibberella sp.NT-1) described in claim 1, it is characterised in that：It is described red mould Bacterium (Gibberella sp.NT-1) is removing Cu2+In application.
- 3. the application of the gibberella (Gibberella sp.NT-1) as described in claim 2, it is characterised in that：By gibberella (Gibberella sp.NT-1) is inoculated in potato dextrose broth, shaken cultivation to logarithmic phase；It will cultivate extremely The bacteria suspension of exponential phase is accessed to containing Cu2+Environment in concussion and cultivate, that is, realize the Cu in Adsorption environment2+。
- 4. the application of the gibberella (Gibberella sp.NT-1) as described in claim 3, it is characterised in that：The culture is extremely Logarithmic phase gibberella (Gibberella sp.NT-1) access to acidity contain Cu2+Environment in concussion and cultivate, that is, realize absorption Remove the Cu in environment2+。
- 5. the application of the gibberella (Gibberella sp.NT-1) as described in claim 3 or 4, it is characterised in that：The shake It is 28 DEG C, 150rpm shaken cultivations to swing condition of culture.
- 6. the application of the gibberella (Gibberella sp.NT-1) as described in claim 5, it is characterised in that：The acidity is PH value is 5.0.
- A kind of 7. copper ion remover, it is characterised in that：Remover is the bacteria suspension of gibberella (Gibberella sp.NT-1).
- 8. the copper ion remover as described in claim 7, it is characterised in that：The gibberella (Gibberella sp.NT-1) Bacteria suspension for gibberella (Gibberella sp.NT-1) is inoculated in potato dextrose broth, shaken cultivation To logarithmic phase.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974413A (en) * | 2018-01-18 | 2018-05-01 | 四川龙蟒福生科技有限责任公司 | The preparation method of gibberellic acid seed liquor |
| CN114075514A (en) * | 2021-11-11 | 2022-02-22 | 合肥工业大学 | Geotrichum candidum MF5 with heavy metal ion removing effect, microbial inoculum and application thereof |
| CN114381377A (en) * | 2021-11-11 | 2022-04-22 | 合肥工业大学 | Aspergillus MF1 for removing heavy metal ions, microbial inoculum and preparation method and application thereof |
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| CN103642703A (en) * | 2013-12-12 | 2014-03-19 | 北京林业大学 | Production method of effective phosphate solubilizing aspergillus japonicus agent with characteristic of tolerance to heavy metals |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103642703A (en) * | 2013-12-12 | 2014-03-19 | 北京林业大学 | Production method of effective phosphate solubilizing aspergillus japonicus agent with characteristic of tolerance to heavy metals |
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| MICHAEL SCOTT PRICE: "Characterization of Aspergillus Niger for Removal of Copper and Zinc from Swine Wastewater", 《HTTP://WWW.LIB.NCSU.EDU/RESOLVER/1840.16/1161》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974413A (en) * | 2018-01-18 | 2018-05-01 | 四川龙蟒福生科技有限责任公司 | The preparation method of gibberellic acid seed liquor |
| CN114075514A (en) * | 2021-11-11 | 2022-02-22 | 合肥工业大学 | Geotrichum candidum MF5 with heavy metal ion removing effect, microbial inoculum and application thereof |
| CN114381377A (en) * | 2021-11-11 | 2022-04-22 | 合肥工业大学 | Aspergillus MF1 for removing heavy metal ions, microbial inoculum and preparation method and application thereof |
| CN114381377B (en) * | 2021-11-11 | 2024-01-30 | 合肥工业大学 | Aspergillus MF1 for removing heavy metal ions, microbial inoculum, preparation method and application thereof |
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