CN103614302B - One strain has the efficient phosphate-solubilizing penicillium oxalicum of heavy metal tolerance characteristic - Google Patents
One strain has the efficient phosphate-solubilizing penicillium oxalicum of heavy metal tolerance characteristic Download PDFInfo
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Abstract
The invention discloses a strain and there is the bacterial strain penicillium oxalicum of efficient phosphate-solubilizing characteristic, categorized called after penicillium oxalicum (<i>penicillium</i><i>oxalicum</i>), it being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No. 7699.The invention also discloses the patience of above-mentioned efficient phosphate-solubilizing bacterial strain penicillium oxalicum heavy metal.Insoluble phosphate tricalcium phosphate, magnesium phosphate and aluminum phosphate, in liquid submerged culture situation, are had extremely strong solute effect by bacterial strain of the present invention, and the solute effect of tricalcium phosphate and magnesium phosphate is reached 100%, and the solute effect of aluminum phosphate is reached 95.5%.This penicillium oxalicum is inoculated in respectively containing Pb2+、Zn2+、Cr2+、Mn2+、Cu2+、As5+、Cd2+Etc. in the liquid medium of heavy metal, above-mentioned heavy metal is respectively provided with stronger resistance, particularly to Pb2+、Zn2+、Cr2+、Mn2+Tolerable concentration all do not reach 2000mg/L.Therefore, the present invention can provide excellent strain resource for exploitation preventing from heavy metal efficient phosphate-solubilizing bacteria agent in the future.
Description
Technical field
The invention belongs to field of environmental biotechnology, be specifically related to a strain and there is efficient phosphate-solubilizing and the penicillium oxalicum of heavy metal tolerance characteristic.
Background technology
Phosphorus is one of required nutrient of plant, and there is the arable soil phosphorus deficiency of 74% in China.In soil, the phosphorus of more than 95% is invalid form, and plant is difficult to directly absorb.The phosphate fertilizer this season crop utilization rate applied is 5%～25%, the Ca in major part phosphorus and soil2+、Fe3+、Fe2+、Al3+In conjunction with, form insoluble phosphate.The utilization rate improving phosphorus is always up the problem that agronomist pays close attention to.Several factors affects the utilization ratio of soil phophorus, and wherein, microorganism is very big to conversion and the availability influence of soil phophorus.Substantial amounts of result of study proves: there is substantial amounts of microorganism in soil, it is possible to the phosphorus that plant is difficult to absorb is converted into the form that can absorb, and strengthens the plant picked-up to Soil Phosphorus element.At present, the research of phosphate solubilizing microorganism being concentrated mainly on phosphate-solubilizing bacteria aspect, the research of phosphorus decomposing fungus aspect is relatively fewer.Such as: Chinese invention patent (application number: 201110121381.5) discloses strain efficient phosphate-solubilizing penicillium pinophilum of laxus malrel rhizosphere and an application thereof；Chinese invention patent (application number: 201210235182.1) discloses a kind of Pinus massoniana Lamb rhizosphere phosphorus decomposing fungus aspergillus awamori and application thereof.
Mining causes mining area sheet vegetation and ploughs destroyed, also produces a large amount of mine wastes simultaneously, has a strong impact on the soil texture and structure.The produced a large amount of acid mine waters of mining and tailings ore deposit are the one of the main reasons causing mining area and surrounding area ecosystem heavy metal pollution thereof, become the primary pollution source of Heavy Metals In Environment.In recent years, very active about the research of land reclamation in mining area and ecological reconstruction both at home and abroad, screening and the cultivation of wherein fitting raw excellent pionner kind are a kind of effective methods.But land reclamation in mining area ground is except the problem that there is heavy metal pollution, and the fertility of reclaimed land soil is low, the problem that especially available phosphorus content is low, it it is the key factor of restriction land reclamation in mining area ground revegetation.
The strain that this patent relates to is the strain efficient phosphate-solubilizing penicillium oxalicum that the Pb-Zn tailings district from Xiangxi Huayuan County, Hunan Province separates, and this bacterial strain is to Pb2+、Zn2+、Cr2+、Mn2+Etc. heavy metal, there is very strong resistance.Research in efficient phosphate-solubilizing penicillium oxalicum and resistance to heavy metal thereof at present yet there are no relevant report at home.Therefore, impurely screen the efficient phosphate-solubilizing fungus of heavy metal tolerance from mining area, to regulating soil phosphorus imbalance between supply and demand, improve land reclamation in mining area ground soil fertility, promote that local vegetation growth is significant.
Summary of the invention
It is an object of the invention to provide a kind of can the efficient phosphate-solubilizing penicillium oxalicum of heavy metal tolerance.
In order to realize the purpose of foregoing invention, the technical solution used in the present invention is as follows:
Efficient phosphate-solubilizing that the present invention adopts, the bacterial strain of heavy metal tolerance are that from the Pb-Zn deposits topsoil of Xiangxi Huayuan County, Hunan Province, separation screening is out by our laboratory, it is numbered TJ2, Classification And Nomenclature is penicillium oxalicum (Penicilliumoxalicum), it is preserved in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode: 100101), deposit number CGMCCNo.7699, preservation date is on June 14th, 2013.
The main biological property of this bacterial strain: conidiophore is upright, colourless, have separation, have penicillus and for doing the broom shape form of tertiary branching, on penicillus, stigma grows thickly in branch top is vase-like, the normal chain-like growth of conidium, spherical to oval, colourless to painted, smooth surface.This bacterium colony edge on potato dextrose agar (PDA) flat board is white, light green to bottle green after Sporulation.
The 18SrDNAITS sequence of this bacterial strain is passed through pcr amplification, obtain the amplified production of about 600bp length, amplified production carries out sequencing through order-checking company, by being checked order, row carry out BLAST comparison with the sequence in GenBank data base, result shows, this bacterial strain is all significantly high with Penicillium (Penicillium) homology, is more than 99% with the similarity of penicillium oxalicum (P.oxalicum).Combining form feature, cultural characteristic and 18SrDNAITS sequence analysis, this bacterial strain is defined as penicillium oxalicum (Penicilliumoxalicum).
Above-mentioned bacterial strains effect in dissolving slightly solubility inorganic phosphate: in liquid shaking bottle is tested, this bacterial strain is to insoluble phosphate tricalcium phosphate (Ca3(PO4)2), magnesium phosphate (Mg3(PO4)2) and aluminum phosphate (AlPO4) there is extremely strong solute effect.Wherein, the solute effect of tricalcium phosphate and magnesium phosphate is reached 100%, the solute effect of aluminum phosphate is reached 95.5%.
The effect of above-mentioned bacterial strains heavy metal patience aspect: various heavy, except having very strong inorganic salt phosphorus decomposing characteristic, is also had patience by bacterial strain of the present invention.This bacterial strain is inoculated in respectively containing Pb2+、Zn2+、Cr2+、Mn2+、Cu2+、As5+、Cd2+Etc. in the liquid medium of heavy metal, above-mentioned heavy metal is respectively provided with stronger resistance, particularly to Pb2+、Zn2+、Cr6+、Mn2+Tolerable concentration all do not reach more than 2000mg/L.Therefore, this bacterium has wide application potential in the biological restoration of the mining soil of heavy metal pollution.
Accompanying drawing explanation
Fig. 1 be penicillium oxalicum TJ2 bacterial strain when cultivating in shaking flask dissolving P capacity and pH value with dynamic variation diagram.
Detailed description of the invention
According to example below, it is possible to make those skilled in the art be more fully understood that the present invention.It is merely to illustrate the present invention described by embodiment, but not as the restriction to the scope of the present invention.
Embodiment 1: the screening of phosphorus decomposing penicillium oxalicum and qualification
Separation screening is out from the Pb-Zn deposits topsoil of Xiangxi Huayuan County, Hunan Province for the penicillium oxalicum bacterial strain of present embodiment.Screening sequentially includes the following steps: the soil of collection is taken back laboratory immediately, air-dry, grind, sieve, and takes the soil after wherein 10.0g sieves and is put in the 100ml sterilized water with bead, makes soil supension, and this suspension concentration is 10-1,.Being put on constant temperature oscillator by this suspension fully to shake 30min with 150 turns/min, somewhat stand after taking-up, take in the 10ml sterilized water joining 90ml from this sample bacteria suspension, the concentration making bacteria suspension is 10-2, then carry out 10 times of gradient dilutions in this approach, until the concentration of diluent is 10-4Till.Take 10-4The bacteria suspension 0.2ml of concentration, is placed on (culture medium composition (g/L): (NH on selective improvement Pikovskaya culture medium flat plate4)2SO40.5g, MgSO4·7H2O0.3g, NaCl0.3g, KCl0.3g, FeSO40.03g, MnSO4·H2O0.03g, yeast powder 0.5g, glucose 10.0g, Ca3(PO4)225g, the distilled water l000ml of agar 15g, pH7.0), it is placed in 28 DEG C of incubators and cultivates 5d, the bacterium colony that picking phosphorus decomposing scope is bigger makes screening and bacterial strain purification further.The phosphate solubilizing bacteria obtained is through the ITS sequence sequencing analysis of morphology, cultural characteristic and 18SrDNA, identify that this bacterial strain is penicillium oxalicum, called after penicillium oxalicum (Penicilliumoxalicum), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.7699.
Embodiment 2: the phosphorus decomposing of insoluble phosphate is tested by phosphorus decomposing penicillium oxalicum
Select five kinds of insoluble phosphates, be made into following culture medium
Phosphorus decomposing culture medium 1: glucose 10.0g, (NH4)2SO40.5g, MgSO4·7H2O0.3g, NaCl0.3g, KCl0.3g, FeSO40.03g, MnSO4·H2O0.03g, yeast powder 0.5g, Ca3(PO4)210g, distilled water l000ml, pH7.0.
Phosphorus decomposing culture medium 2: with magnesium hydrogen phosphate (MgHPO4) replace the Ca in phosphorus decomposing culture medium 13(PO4)2, other composition and content are constant.
Phosphorus decomposing culture medium 3: with zinc phosphate (Zn3(PO4)2Replace the Ca in phosphorus decomposing culture medium 13(PO4)2, other composition and content are constant.
Phosphorus decomposing culture medium 4: with aluminum phosphate (AlPO4) replace the Ca in phosphorus decomposing culture medium 13(PO4)2, other composition and content are constant.
Phosphorus decomposing culture medium 5: with iron phosphate (FePO4) replace the Ca in phosphorus decomposing culture medium 13(PO4)2, other composition and content are constant.
Above-mentioned bacterial strains is inoculated on PDA plate, after cultivating 5d, prepares spore suspension with sterilized water, determine that spore suspension concentration is 1.6 × 10 by blood cell plate counting method7Individual spore/mL, is inoculated in the 100mL triangular flask equipped with 50mL phosphorus decomposing culture medium 1,2,3,4 and 5 by 1mL/ bottle inoculum concentration respectively, does not inoculate as comparison (CK), each process three bottles repetition with the phosphorus decomposing culture medium adding same volume.Being placed in 28 DEG C of shaking tables, 150rpm shaken cultivation is cultivated, and after cultivating one week, fermentation liquid is in 4 DEG C, and 10000r/min is centrifuged 10min, measures titanium pigment content in fermentation liquid with molybdenum antimony resistance colorimetric method, and result is shown in Table 1.
Wherein, phosphorus content × 100 added in phosphorus decomposing rate (%)=(in inoculation process titanium pigment content in titanium pigment content-comparison)/culture medium.
Table 1 penicillium oxalicum solvability to five kinds of insoluble phosphates
Can be drawn by table 1, respectively with insoluble phosphate calcium phosphate (Ca3(PO4)2), magnesium hydrogen phosphate (MgHPO4), zinc phosphate (Zn3(PO4)2, aluminum phosphate (AlPO4) and iron phosphate (FePO4) for unique phosphorus source, after cultivating 7d, in this bacterial strain fermentation liquor, titanium pigment content is above comparison, but between different disposal, there is some difference.Wherein, this bacterial strain is to insoluble phosphate calcium phosphate (Ca3(PO4)2), magnesium hydrogen phosphate (MgHPO4) and aluminum phosphate (AlPO4) dissolving P capacity very strong, all reach more than 95%, to iron phosphate (FePO4) there is certain phosphorus decomposing, but to zinc phosphate (Zn3(PO4)2Dissolving P capacity more weak.
Embodiment 3: the dynamic change of penicillium oxalicum phosphorus decomposing
Above-mentioned bacterial strains is inoculated on PDA plate, after cultivating 5d, prepares spore suspension with sterilized water, determine that spore suspension concentration is 1.6 × 10 by blood cell plate counting method7Individual spore/mL, is inoculated into 50ml by 1mL/ bottle inoculum concentration and improves in Meng Jinna fluid medium, is set to blank group with what do not connect bacterium simultaneously, adds 1ml sterilized water, each process three bottles repetition in each bottle blank group.It is placed in 28 DEG C of shaking tables, 150rpm shaken cultivation, the content of the titanium pigment detected in supernatant every 24 hours and the pH value of culture medium, METHOD FOR CONTINUOUS DETERMINATION 14d.Result is as shown in Figure 1.
As seen from Figure 1, above-mentioned bacterial strains dissolving P capacity, through the mensuration of 14d, except just inoculated first beyond the highest heavens, all presents significantly high dissolving P capacity in other 2-14d.Wherein, when inoculating above-mentioned bacterial strains the 7th day, phosphorus decomposing amount reaches the highest, also all shows significantly high dissolving P capacity in the other times phase, and the phosphorus decomposing amount in each time period is all more than 1500mg/L.From the change of pH value, postvaccinal 2nd day pH rapid decrease, reached minimum by the 4th day, slowly rise afterwards, but after inoculation in 2-14 days, in culture medium, pH value is consistently lower than 4.0.
Embodiment 4: heavy metal is to penicillium oxalicum phosphorus decomposing effect
The preparation improvement Meng Jinna fluid medium containing lead, zinc, manganese and four kinds of variable concentrations heavy metals of chromium respectively, heavy metal concentration is from 100mg/L, it is followed successively by 100mg/L, 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L, to be not added with the process of heavy metal as comparison.
Above-mentioned bacterial strains is inoculated on PDA plate, after cultivating 5d, prepares spore suspension with sterilized water, determine that spore suspension concentration is 1.6 × 10 by blood cell plate counting method7Individual spore/mL, by 1mL/ bottle inoculum concentration be inoculated in respectively containing variety classes, variable concentrations heavy metal fluid medium in, each process inoculation 1mL.Being placed in 28 DEG C of shaking tables, 150r/min shaken cultivation 10 days, measure the change of titanium pigment content in the fluid medium containing various types of, variable concentrations heavy metal of this bacterium, result is in Table 2.
The phosphorus decomposing under different heavy metal concentrations of table 2 penicillium oxalicum measures
Note: phosphorus decomposing amount unit: mg/L
Can be drawn by table 2, containing heavy metal lead, zinc, manganese, chromium fluid medium in, along with the rising of heavy metal concentration, the phosphorus decomposing amount of this bacterial strain is in the trend being gradually lowered.Wherein, the Resistant expression of this bacterial strain heavy metal zinc is the strongest, in the culture medium containing 2000mg/L heavy metal zinc, it is 2068.2mg/L that the phosphorus decomposing amount of this bacterial strain still reaches the phosphorus decomposing amount of the 1953.7mg/L(comparison corresponding to not adding heavy metal), phosphorus decomposing amount reaches the 94.46% of comparison.In the fluid medium containing heavy metal lead, manganese and chromium, when each content of beary metal is 2000mg/L, their phosphorus decomposing amount respectively reaches 593.6mg/L, 424.6mg/L and 107.3mg/L, respectively comparison the 28.70% of phosphorus decomposing amount, 15.83% and 5.19%.This strain growth amount in the fluid medium containing each concentration heavy metal of observing finds, this bacterium manages all extraordinary growths of energy in culture medium throughout, even if thalli growth amount is compared with the control in the culture medium containing 2000mg/L heavy metal, remain without too big difference.Thus can also illustrating, the resistance of above-mentioned bacterial strains heavy metal lead, zinc, manganese and chromium is all very strong, and in phosphorus decomposing amount, the resistance of this bacterium heavy metal zinc is the strongest.
Claims (1)
1. a strain can the penicillium of high-efficiency dissolution inorganic phosphate, it is characterized in that: this bacterial strain is a strain penicillium oxalicum (Penicilliumoxalicum), China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.7699 it are preserved in.
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| CN104560738B (en) * | 2015-01-14 | 2017-03-22 | 浙江大学 | Penicillium oxalicum for reducing hexavalent chromium and screening method thereof |
| CN105296363B (en) * | 2015-10-28 | 2019-06-04 | 南京农业大学 | A kind of penicillium oxalicum NJDL-03 bacterial strain and its application |
| CN105733954A (en) * | 2015-12-18 | 2016-07-06 | 沈阳农业大学 | Penicillium oxalicum (fungus) capable of efficiently dissolving phosphorus, culture method and applications thereof |
| CN105647817B (en) * | 2016-01-26 | 2019-04-26 | 华中农业大学 | The penicillium janthinellum of one plant of soil slightly solubility aluminum phosphate that reduces sourness and its application |
| CN106929433B (en) * | 2017-03-29 | 2020-07-31 | 山东农业大学 | Phosphate solubilizing penicillium and application thereof |
| CN110129208B (en) * | 2019-04-28 | 2021-05-04 | 湘潭大学 | Penicillium oxalicum with broad-spectrum acid production characteristic |
| CN110951626B (en) * | 2019-12-27 | 2021-05-25 | 北京林业大学 | Penicillium oxalicum capable of decomposing coal gangue |
| CN111304096B (en) * | 2020-03-11 | 2022-06-07 | 大连理工大学 | Penicillium oxalicum and culture method and application thereof |
| CN111979159A (en) * | 2020-09-03 | 2020-11-24 | 中南大学 | Phosphate solubilizing bacterium agent and preparation method and application thereof |
| CN114561299B (en) * | 2022-03-21 | 2023-11-28 | 广西大学 | Penicillium oxalate and application thereof in manganese leaching |
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| CN1524949A (en) * | 2003-02-24 | 2004-09-01 | 唐文华 | Oxalic acid penicillium, bacterial preparation thereof and its preparation method, and the application of the preparation in dephosphorization, prophylaxis and soil fertility enhancement |
| CN102174412A (en) * | 2010-12-15 | 2011-09-07 | 云南大学 | Phosphorus-solubilizing fungi and application thereof to preparation of biological fungus fertilizer |
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| CN1524949A (en) * | 2003-02-24 | 2004-09-01 | 唐文华 | Oxalic acid penicillium, bacterial preparation thereof and its preparation method, and the application of the preparation in dephosphorization, prophylaxis and soil fertility enhancement |
| CN102174412A (en) * | 2010-12-15 | 2011-09-07 | 云南大学 | Phosphorus-solubilizing fungi and application thereof to preparation of biological fungus fertilizer |
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