CN109628317A - A kind of mould and its application with degradation slightly solubility Phos function - Google Patents
A kind of mould and its application with degradation slightly solubility Phos function Download PDFInfo
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- CN109628317A CN109628317A CN201810637649.2A CN201810637649A CN109628317A CN 109628317 A CN109628317 A CN 109628317A CN 201810637649 A CN201810637649 A CN 201810637649A CN 109628317 A CN109628317 A CN 109628317A
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- mould
- phos
- slightly solubility
- phosphorus
- degradation
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/645—Fungi ; Processes using fungi
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
Abstract
The present invention provides a kind of mould with degradation slightly solubility Phos function and applications, belong to technical field of bioengineering.The Paecilomyces lilacinus bacterial strain screening of the degradation Phos function is from soil near Guizhou Province's Weng'an biota tailing.Bacterial strain of the present invention is preserved in Wuhan City, Hubei Province Wuhan University China typical culture collection center, and deposit number is CCTCC NO:M2018239.
Description
Technical field
The present invention relates to field of biotechnology is belonged to, has the function of insoluble inorganic phosphorus of solving problem in particular to one plant
Separation, identification and the phosphorus decomposing performance of mould.
Background technique
To generate a large amount of phosphorite tailings in phosphorus ore recovery process, most of tailing using abandon or inventory by the way of into
Row disposition.It is deposited in Tailings Dam for a long time, can affect, be mainly manifested in environment: when tailing is corroded
Or certain transportable elements can cause serious pollution to atmosphere and water and soil, lead to soil degradation, vegetation when chemical transport occurs
Destroy the existence for even directly threatening people and animals；The dust that mining tailing wasteland surface generates can deteriorate the sanitary condition of surrounding area；
Related ingredient and remaining dressing agent in tailing can cause damages to periphery ecological environment.In fact, the group of phosphorite tailings
Dtex sign is that w (P2O5)≤10%, w (MgO) >=15% can accomplish to turn waste into wealth if being suitably applied.China's phosphorus ore tail
The comprehensive utilizating research of mine is started late, and less than 40%, utilization ways mainly include the comprehensive utilization ratio of phosphorite tailings at this stage
4 points below:
(1) the direct application of phosphorus mine tailing.
(2) valuable mineral is extracted from phosphorus tailing using technologies such as chemical flotation, magnetic separation or electric separations.
(3) compound fertilizer is prepared using acid hydrolyzation.
(4) construction material is prepared.
Above method or because its processing cost is excessively high or because it is extremely low to effective phosphorus utilization, and phosphorite tailings are not implemented
Scale application.
With Applied Biotechnology and microbiological development, the conversion of slightly solubility Phos is realized using phosphate solubilizing microorganism
As research hotspot, phosphate solubilizing microorganism has significant conversion capability to slightly solubility Phos, can promote plant growth and development.?
In existing patent and document, it includes bacterium, fungi and actinomyces etc. that is be currently known, which has the microorganism of solution Phos ability,
Wherein the value volume and range of product of phosphate-solubilizing bacteria is more, mainly there is Alcaligenes (Alcaligenes), bacillus
(Bacillus), pseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), Erwinia
(Erwinia), Salmonella (Salmonella), Serratia (Serratia), more Thiobacillus
(Thiobacillus) etc..And reported Mycophyta mainly has Penicillium (Penicillium), aspergillus
(Aspergillus) and rhizopus (Rhizopus) etc..
Paecilomyces lilacinus (Paecilomyces lilacinus) is a kind of endoparasitism Nematophagous fungi, not only to root
The ovum of tie lines worm and cyst roundworm has stronger parasitic ability, moreover it is possible to infect larva and adult, make root-knot nematode and cyst roundworm
Dieback, it is considered to be most promising nematode biocontrol fungi, therefore often by as biocontrol microorganisms.But also there is research to send out
Existing, Paecilomyces lilacinus can promote the dissolution of difficultly-soluble phosphates, have certain environmental protection effect.
The research of biological and ecological methods to prevent plant disease, pests, and erosion function, slightly solubility Phos of degrading to it are mostly focused on to the report of Paecilomyces lilacinus at present
Though function refers to, temporarily this is probed into detail without related article or patent.
Summary of the invention
In view of this, it is an object of the invention to: provide it is a kind of with degradation slightly solubility Phos function mould and its
Using to solve the above problems.
The present invention provides a kind of mould with degradation slightly solubility Phos function, deposit number are as follows: CCTCC NO:
M2018239。
The bacterial strain cultivates 7d, colony diameter 4.5-5.5cm in PDA culture medium at 30 DEG C, and bacterium colony is in regular circle shapes,
Bacterium colony outer ring color is white, and median rise part is lavender, and the time is longer, and color is deeper；Bacterium colony is not easy to provoke, opaque,
Scanning electron microscopic observation conidiophore is in ampuliform, obstructs single raw or is gathered into coremium, there is colyliform branch, the nearly cylinder in bottle metulae portion
Shape, top are elongated；Conidium is colourless, smooth, not of uniform size, and ellipse, is concatenated by 2-8 × 2-4 μm, and base portion forms diverging
Long spore chain；
The present invention also provides a kind of application of above-mentioned Paecilomyces lilacinus in degradation slightly solubility Phos.
It can fast and effeciently degrade slightly solubility Phos compared with the prior art, the advantages of the present invention are as follows: the mould,
In terms of degradation recycling and reusing applied to phosphorite tailings, recycling cost can be greatly reduced, realizes phosphorite tailings
Scale application avoids a large amount of wastes of resource of tailings.
Detailed description of the invention
Fig. 1 is bacterial strain GZP5 morphological feature, note: A: culture medium bacterium colony front；B: the culture medium bacterium colony back side；C:400X mirror
Inspection figure；D:1000X microscopy figure；
Fig. 2 is the ITS sequence PCR amplification electropherogram of GZP5, note: M:Marker；Y: negative control；
Fig. 3 is the phylogenetic tree of the phosphorus decomposing fungus G ZP5 based on ITS sequence；
Fig. 4 is the phosphorus decomposing loop graph of GZP5；
Fig. 5 is the change curve of amount of phosphorus dissolved in GZP5 Liquid Culture；
Fig. 6 is phosphorus mine tailing scanning electron microscope (SEM) photograph, and note: A: phosphorus mine tailing under natural conditions, B: C: blank control adds
Phosphorus mine tailing after GZP5 effect.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below with reference to embodiment to of the invention preferred real
Scheme is applied further to be described in detail, described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Embodiment:
The present embodiment provides a kind of Paecilomyces lilacinus, and morphological feature: the bacterial strain is trained at 30 DEG C in PDA culture medium
7d, colony diameter 4.5-5.5cm are supported, bacterium colony is in regular circle shapes, and bacterium colony outer ring color is white, and median rise part is pale purple
Color, the time is longer, and color is deeper.Bacterium colony is not easy to provoke, opaque, and scanning electron microscopic observation conidiophore is in ampuliform, obstruct it is single raw or
It is gathered into coremium, has colyliform branch, bottle metulae portion is closely cylindrical, and top is elongated.Conidium is colourless, smooth, not of uniform size,
Ellipse, is concatenated by 2-8 × 2-4 μm, and base portion forms the long spore chain of diverging.The bacterial strain is deposited in China on April 27th, 2018
Type Tissue Collection (address: Wuhan, China Wuhan University), deposit number: CCTCC NO:M2018239 is entitled pale purple
Paecilomycerol GZP5 (Paecilomyces lilacinus GZP5).
(1) separating step of Paecilomyces lilacinus GZP5 (Paecilomyces lilacinus GZP5):
From sample is acquired in soil near the Wengfu phosphorus tailing stack-up-yard of Guizhou, the pedotheque 5g of preservation is weighed, is added and contains
In the conical flask for having 45mL sterile saline, it is 10 that dilution, which is made,-1Soil supension, be placed in 30 DEG C of shaking table and vibrate
30min.Soil supension is made 10 by 10 times of gradients-2To 10-6, the soil supension 0.2mL of above-mentioned gradient is taken to be uniformly coated on nothing
On machine phosphorus Selective solid culture medium, each dilution is repeated 3 times.It is inverted in 30 DEG C of constant incubators and cultivates 5-7d,
With oese picking there is the single colonie of Soluble phosphorus circle to be isolated and purified on potato glucose solid medium.It will after purification
Strain put be connected on Phos Selective solid culture medium respectively, observation Soluble phosphorus circle generates after 5-7d is cultivated under the conditions of 30 DEG C
Situation filters out the bacterial strain with obvious Soluble phosphorus circle and is transferred on potato glucose slant medium, is placed at 4 DEG C and saves.
(2) culture medium that culture Paecilomyces lilacinus uses
Separation screening culture medium
Phos Selective solid culture medium: glucose 10.0g, Ca3(PO4)25.0g, (NH4)2SO40.5 g, NaCl
0.3g, KCl 0.3g, MgSO47H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, agar powder 18g steam
Distilled water 1000mL, pH 7.0-7.5
Storage medium
Potato glucose solid medium: potato leaching powder 5.0g, glucose 20.0g, agar 14.0g, distilled water
1000mL, pH 5.8-6.2
Fermentation medium
Phos fluid nutrient medium: glucose 10.0g, Ca3 (PO4) 25.0g, (NH4) 2SO40.5g, NaCl 0.3g,
KCl 0.3g, MgSO47H2O 0.3g, FeSO47H2O 0.03g, MnSO44H2O 0.03g, distilled water 1000mL, pH
7.0-7.5
(3) identification of Paecilomyces lilacinus GZP5
(1) Morphological Identification
By the phosphorus decomposing bacterial strain dibbling isolated and purified on potato glucose solid medium, 30 DEG C of culture 5-7d, observation
Single colonie feature records the features such as its color, shape, transparency, surface gloss, edge feature, protuberance degree.Picking is a little
Spore observes conidium and conidiophore form under 400X and 1000X.
(2) molecular biology identification
By the strain access after activation equipped in the triangular flask of 100mL potato dextrose broth, 30 DEG C are placed in,
Shaken cultivation in the shaking table of 160r/min collects mycelium pellet until with filtered through gauze after growing mycelium pellet, then is inhaled with aseptic filter paper
Solid carbon dioxide point, with liquid nitrogen grinding at powder.Fungi is extracted according to fungal genomic DNA extracts kit (BIOMIGA company, the U.S.)
DNA.DNA sequencing sequencing result is inputted into National Center for Biotechnology Information NCBI (National Center of
Biotechnology Information) database is compared, and it chooses the higher strain sequence of homology and is used as reference, use
5.05 software building bacterial strain phylogenetic tree of MEGA carries out strain idenfication.By phosphorus decomposing bacterial strain GZP5 with its belong to affiliation compared with
Close type strain carries out homology analysis and phylogenetic tree construction, GZP5 and Paecilomyces lilacinus (Paecilomyces
Lilacinus) bacterial strain gathers in same branch, sequence homology 99%.
Dissolving P capacity measures under the conditions of one, solid culture of experimental example
The strain isolated and purified point is connected on Phos Selective solid culture medium plate, is seen after 30 DEG C of culture 7d
Phosphorus decomposing circle production is examined, by crossing method measurement phosphorus decomposing loop diameter (D) and colony diameter (d), and calculates dissolution phosphorus circle
The ratio (D/d) of diameter and colony diameter.
Test proves that (D/d) value of GZP5 bacterial strain is 1.15 ± 0.003.
Dissolving P capacity measures under two, liquid culture condi of experimental example
The sterilized triangle equipped with 100mL Phos selected liq culture medium is added in 1mLGZP5 bacterial strain seed liquor
In bottle.Inoculation is placed on 30 DEG C, cultivates 7d in 160r/min shaking table.1mL sterile water is accessed simultaneously as blank control, setting three
Group is parallel.Per sampling for 24 hours, OD700 value is measured with molybdenum antimony resistance colorimetric method, is calculated in supernatant according to phosphorus content standard curve
The content of available phosphorus.
As a result such as Fig. 5, test proves that available phosphorus content rises to highest in bacterial strain GZP5 culture solution in 72h, for 98.09 μ
g/mL。
Degradation of three, of experimental example to phosphorus mine tailing
It is to be placed in 30 DEG C in the Phos selected liq culture medium of unique phosphorus source with phosphorus mine tailing that GZP5, which is accessed,
Phosphoric residue mine tailing is obtained by filtration after cultivating 3d in 160r/min shaking table, while bacterial strain is not added as blank control.It is right respectively
Phosphorus mine tailing after drying, the blank control phosphorus mine tailing of phosphorus mine tailing and access sterile water effect after bacterial strain effect 3 days
Electronic Speculum detection and analysis are scanned, as a result see Fig. 3.
The ground phosphate rock surface (Fig. 6-A) under natural conditions is in more apparent angular, is in typical mineral state, phosphorus ore
The broken little particle of powder is more, the feature with detrital grain.And most of little particle phosphorus tailing powder after GZP5 is decomposed and utilized
It completely disappears, apparent cellular status is also presented in the phosphorus tailing surface of bulky grain, and has mycelia.And never add strain effect
It can be seen that the concussion because of shaking table in blank control figure (Fig. 6-B), be hardly visible the phosphorus tailing of bulky grain, be little particle, and
It is paved with entire interface, is disappeared without phosphorus tailing powder.The phosphorus tailing surface of bulky grain remains to see also without apparent cellular status
Typical mineral characteristic out.As it can be seen that bacterial strain GZP5 has stronger solvability to insoluble phosphorus miberal powder.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (3)
1. a kind of mould with degradation slightly solubility Phos function, it is characterised in that: its deposit number are as follows: CCTCC NO:
M2018239。
2. a kind of mould with degradation slightly solubility Phos function according to claim 1, it is characterised in that: the bacterial strain exists
In PDA culture medium, 7d, colony diameter 4.5-5.5cm are cultivated at 30 DEG C, bacterium colony is in regular circle shapes, and bacterium colony outer ring color is white
Color, median rise part are lavender, and the time is longer, and color is deeper；Bacterium colony is not easy to provoke, opaque, and scanning electron microscopic observation is mitogenetic
Sporophore is in ampuliform, obstructs single raw or is gathered into coremium, has colyliform branch, bottle metulae portion is closely cylindrical, and top is elongated；Mitogenetic spore
Son is colourless, smooth, not of uniform size, and ellipse, is concatenated by 2-8 × 2-4 μm, and base portion forms the long spore chain of diverging.
3. a kind of application of mould as described in claim 1 in degradation slightly solubility Phos.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940946A (en) * | 2021-01-27 | 2021-06-11 | 贵州大学 | Method for preparing paecilomyces lilacinus from marigold straws to prevent and control tobacco root-knot nematodes |
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| WO2017131971A1 (en) * | 2016-01-28 | 2017-08-03 | Novozymes Bioag A/S | Phosphate-solubilzing fungal strains |
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| WO2017131971A1 (en) * | 2016-01-28 | 2017-08-03 | Novozymes Bioag A/S | Phosphate-solubilzing fungal strains |
| CN107488594A (en) * | 2016-08-15 | 2017-12-19 | 郑毅男 | One plant of new Penicillium notatum and its metabolite pacify him and intend sour A |
| CN106434391A (en) * | 2016-12-21 | 2017-02-22 | 中国科学院微生物研究所 | Efficiently phosphorus-dissolving Aspergillus tubingensis strain and application thereof |
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| HERNANDEZ-LEAL等: "In vitro PHOSPHATE SOLUBILIZATION BY A STRAIN OF Paecilomyces lilacinus (Thom) Samson", 《AGROCIENCIA》 * |
| 李芳等: "淡紫拟青霉对辛硫磷的降解效应", 《应用与环境生物学报》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940946A (en) * | 2021-01-27 | 2021-06-11 | 贵州大学 | Method for preparing paecilomyces lilacinus from marigold straws to prevent and control tobacco root-knot nematodes |
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Application publication date: 20190416 |