CN103614302A - High-efficiency phosphate-solubilizing penicillium oxalicum with heavy metal tolerance characteristic - Google Patents

High-efficiency phosphate-solubilizing penicillium oxalicum with heavy metal tolerance characteristic Download PDF

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CN103614302A
CN103614302A CN201310607201.3A CN201310607201A CN103614302A CN 103614302 A CN103614302 A CN 103614302A CN 201310607201 A CN201310607201 A CN 201310607201A CN 103614302 A CN103614302 A CN 103614302A
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phosphate
penicillium oxalicum
heavy metal
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solubilizing
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CN103614302B (en
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不公告发明人
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Beijing Forestry University
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Beijing Forestry University
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Abstract

The invention discloses a strain penicillium oxalicum with high-efficiency phosphate solubilizing characteristic, which is classified and named as Penicillium oxalicum. The penicillium oxalicum has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number: CGMCC NO.7699. The invention further discloses tolerance of high-efficiency phosphate-solubilizing penicillium oxalicum to heavy metals. The strain has strong dissolving effect on insoluble phosphate tricalcium phosphate, magnesium phosphate and aluminum phosphate under the condition of shake-flask liquid culture condition, the dissolving effect on the tricalcium phosphate and magnesium phosphate can reach 100%, and the dissolving effect on the aluminum phosphate can reach 95.5%. The penicillium oxalicum is respectively inoculated into liquid culture liquor containing heavy metals such as Pb<2+>, Zn<2+>, Cr<2+>, Mn<2+>, Cu<2+>, As<5+> and Cd<2+>, has strong resistance to the heavy metals, especially, the concentration of tolerance to the Pb<2+>, Zn<2+>, Cr<2+> and Mn<2+> can respectively reach 2000mg/L. Therefore, the penicillium oxalicum can provide excellent strain resources for developing heavy metal-resistant high-efficiency phosphate-solubilizing bacteria agents.

Description

One strain has the efficient phosphate-solubilizing penicillium oxalicum of tolerance heavy metal characteristic
Technical field
The invention belongs to Environmental Biotechnology field, be specifically related to the penicillium oxalicum that a strain has efficient phosphate-solubilizing and tolerance heavy metal characteristic.
Background technology
Phosphorus is one of essential nutritive element of plant, and there is 74% the scarce phosphorus of arable soil in China.Phosphorus in soil more than 95% is invalid form, and plant is difficult to directly absorb.The phosphate fertilizer this season crop utilization rate applying is 5%~25%, the Ca in most of phosphorus and soil 2+, Fe 3+, Fe 2+, Al 3+in conjunction with, form insoluble phosphate.The utilization ratio that improves phosphorus is the problem that agronomist pays close attention to always.Several factors affects the utilising efficiency of soil phosphorus, and wherein, microorganism is very large to the conversion of soil phosphorus and availability influence.A large amount of results of study prove: in soil, have a large amount of microorganisms, the phosphorus that plant can be difficult to absorb is converted into the form that can absorb, and strengthen the picked-up of plant to Soil Phosphorus element.At present, the research of phosphate solubilizing microorganism is mainly concentrated on to phosphate-solubilizing bacteria aspect, the research of phosphorus decomposing fungi aspect is relatively less.For example: Chinese invention patent (application number: 201110121381.5) disclose strain efficient phosphate-solubilizing penicillium pinophilum of laxus malrel rhizosphere and an application thereof; Chinese invention patent (application number: 201210235182.1) disclose a kind of Pinus massoniana Lamb rhizosphere phosphorus decomposing fungi Aspergillus awamori and application thereof.
Mining causes the large stretch of vegetation in mining area destroyed with arable land, also produces a large amount of mine wastes simultaneously, has a strong impact on the soil texture and structure.A large amount of acid mine waters that mining produces and tailings ore deposit are the one of the main reasons that causes mining area and peripheral region ecosystem heavy metal contamination 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, wherein screening and the cultivation of suitable raw good pioneer plant kind are a kind of effective methods.But land reclamation in mining area ground is except existing the problem of heavy metal contamination, and the fertility of reclaimed land soil is low, especially the low problem of available phosphorus content, is the important factor of restriction land reclamation in mining area ground revegetation.
The strain efficient phosphate-solubilizing penicillium oxalicum that the Pb-Zn tailings district of the Xiangxi Huayuan County, bacterial classification Shi Cong Hunan Province that this patent relates to separates, and this bacterial strain is to Pb 2+, Zn 2+, Cr 2+, Mn 2+in heavy metal, there is very strong resistance.Research about efficient phosphate-solubilizing penicillium oxalicum and resistance to heavy metal aspect thereof at present yet there are no relevant report at home.Therefore, from mining area, impurely screen the efficient phosphate-solubilizing fungi of tolerance heavy metal, 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
The object of this invention is to provide a kind of efficient phosphate-solubilizing penicillium oxalicum that can tolerate heavy metal.
In order to realize the object of foregoing invention, the technical solution used in the present invention is as follows:
The bacterial strain of the efficient phosphate-solubilizing that the present invention adopts, tolerance heavy metal out, is numbered TJ2 by our laboratory separation screening from the plumbous zinc ore topsoil of Xiangxi Huayuan County, Hunan Province, Classification And Nomenclature be penicillium oxalicum ( penicillium oxalicum), be preserved in (address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No. 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 and is vase-like, the normal chain-like growth of conidium, spherical to oval, colourless to painted, smooth surface in branch top.This bacterium colony edge on potato dextrose agar (PDA) flat board is white, light green to deep green after sporulation.
The 18SrDNA ITS sequence of this bacterial strain is passed through to pcr amplification, the amplified production that obtains 600bp left and right length, amplified production carries out sequencing through order-checking company, check order row is carried out to BLAST with the sequence in GenBank database and compare, result shows, this bacterial strain and Penicillium ( penicillium) homology is all very high, with penicillium oxalicum ( p. oxalicum) similarity be more than 99%.Combining form feature, cultural characteristic and 18SrDNA ITS sequential analysis, this bacterial strain be defined as penicillium oxalicum ( penicillium oxalicum).
The effect of above-mentioned bacterial strains in dissolving insoluble inorganic phosphate: in liquid shaking bottle experiment, this bacterial strain is to insoluble phosphate tricalcium phosphate (Ca 3(PO 4) 2), trimagnesium phosphate (Mg 3(PO 4) 2) and aluminum phosphate (AlPO 4) there is extremely strong solute effect.Wherein, the solute effect of tricalcium phosphate and trimagnesium phosphate is reached to 100%, the solute effect of aluminum phosphate is reached to 95.5%.
The effect of above-mentioned bacterial strains to Heavy Metal Tolerance aspect: bacterial strain of the present invention, except having very strong inorganic salt phosphorus decomposing characteristic, also has patience to various heavy.This bacterial strain is inoculated in respectively and contains Pb 2+, Zn 2+, Cr 2+, Mn 2+, Cu 2+, As 5+, Cd 2+in liquid medium in heavy metal, above-mentioned heavy metal is all had to stronger resistance, particularly to Pb 2+, Zn 2+, Cr 6+, Mn 2+tolerance concentration all do not reach 2000mg/L more than.Therefore, this bacterium has wide application potential in the biological restoration of the mining soil of heavy metal contamination.
Accompanying drawing explanation
Fig. 1 be penicillium oxalicum TJ2 bacterial strain while cultivating in shaking flask phosphorus decomposing ability and pH value with dynamic variation diagram.
Embodiment
According to embodiment below, can make those skilled in the art understand better the present invention.Embodiment is described only for the present invention is described, but not as the restriction to the scope of the present invention.
Embodiment 1: the screening and identification of phosphorus decomposing penicillium oxalicum
Separation screening is out from the plumbous zinc ore topsoil of Xiangxi Huayuan County, Hunan Province for the penicillium oxalicum bacterial strain of present embodiment.Screening is carried out according to the following steps: the soil of collection is taken back to laboratory immediately, and air-dry, grind, sieve, the soil after getting 10.0g wherein and sieving is put in the 100ml sterilized water with granulated glass sphere, makes soil supension, and this suspension concentration is 10 -1.This suspension is put on constant temperature oscillator and fully shakes 30min with 150 turn/min, standing a little after taking-up, from this sample bacteria suspension, to get in the sterilized water that 10ml joins 90ml, the concentration of making bacteria suspension is 10 -2, then carry out 10 times of gradient dilutions with this method, until the concentration of diluent is 10 -4till.Get 10 -4the bacteria suspension 0.2ml of concentration, is placed on and optionally improves (substratum composition (g/L): (NH on Pikovskaya culture medium flat plate 4) 2sO 40.5g, MgSO 47H 2o 0.3g, NaCl 0.3g, KCl 0.3g, FeSO 40.03g, MnSO 4h 2o 0.03g, yeast powder 0.5g, glucose 10.0g, Ca 3(PO 4) 225g, agar 15g, the distilled water l000ml of pH7.0), being placed in 28 ℃ of incubators and cultivating 5d, the bacterium colony that picking phosphorus decomposing scope is larger is made further screening and bacterial strain purifying.The phosphate solubilizing bacteria obtaining, through the ITS of morphology, cultural characteristic and 18SrDNA sequence sequencing analysis, identifies that this bacterial strain is penicillium oxalicum, called after penicillium oxalicum ( penicillium oxalicum), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No. 7699.
Embodiment 2: the phosphorus decomposing test of phosphorus decomposing penicillium oxalicum to insoluble phosphate
Select five kinds of insoluble phosphates, be made into following substratum
Phosphorus decomposing substratum 1: glucose 10.0g, (NH 4) 2sO 40.5g, MgSO 47H 2o 0.3g, NaCl 0.3g, KCl 0.3g, FeSO 40.03g, MnSO 4h 2o 0.03g, yeast powder 0.5g, Ca 3(PO 4) 210g, distilled water l000ml, pH7.0.
Phosphorus decomposing substratum 2: with secondary magnesium phosphate (MgHPO 4) replace the Ca in phosphorus decomposing substratum 1 3(PO 4) 2, other composition and content are constant.
Phosphorus decomposing substratum 3: with zinc phosphate (Zn 3(PO 4) 2replace the Ca in phosphorus decomposing substratum 1 3(PO 4) 2, other composition and content are constant.
Phosphorus decomposing substratum 4: with aluminum phosphate (AlPO 4) replace the Ca in phosphorus decomposing substratum 1 3(PO 4) 2, other composition and content are constant.
Phosphorus decomposing substratum 5: with tertiary iron phosphate (FePO 4) replace the Ca in phosphorus decomposing substratum 1 3(PO 4) 2, other composition and content are constant.
Above-mentioned bacterial strains is inoculated on PDA flat board, cultivates after 5d, with sterilized water, prepare spore suspension, by blood cell plate counting process, determine that spore suspension concentration is 1.6 * 10 7individual spore/mL, is inoculated into respectively in the 100mL triangular flask that 50mL phosphorus decomposing substratum 1,2,3,4 and 5 are housed by 1mL/ bottle inoculum size, take and adds the phosphorus decomposing substratum of same volume not inoculate as contrast (CK), and each processes three bottles of repetitions.Be placed in 28 ℃ of shaking tables, 150rpm shaking culture is cultivated, and cultivates after one week, and fermented liquid is in 4 ℃, and the centrifugal 10min of 10000r/min, measures titanium pigment content in fermented liquid with molybdenum antimony resistance colorimetric method, the results are shown in Table shown in 1.
Wherein, phosphorus content * 100 that add in phosphorus decomposing rate (%)=(inoculation process in titanium pigment content-contrast titanium pigment content)/substratum.
The dissolving power of table 1 penicillium oxalicum to five kinds of insoluble phosphates
?by table 1, can be drawn, respectively with insoluble phosphate calcium phosphate (Ca 3(PO 4) 2), secondary magnesium phosphate (MgHPO 4), zinc phosphate (Zn 3(PO 4) 2, aluminum phosphate (AlPO 4) and tertiary iron phosphate (FePO 4) be unique phosphorus source, to cultivate after 7d, in this bacterial strain fermentation liquor, titanium pigment content is all higher than contrast, but between different treatment, there is some difference.Wherein, this bacterial strain is to insoluble phosphate calcium phosphate (Ca 3(PO 4) 2), secondary magnesium phosphate (MgHPO 4) and aluminum phosphate (AlPO 4) phosphorus decomposing very capable, all reach more than 95%, to tertiary iron phosphate (FePO 4) there is certain phosphorus decomposing, but to zinc phosphate (Zn 3(PO 4) 2phosphorus decomposing ability a little less than.
Embodiment 3: the dynamic change of penicillium oxalicum phosphorus decomposing
Above-mentioned bacterial strains is inoculated on PDA flat board, cultivates after 5d, with sterilized water, prepare spore suspension, by blood cell plate counting process, determine that spore suspension concentration is 1.6 * 10 7individual spore/mL, is inoculated in 50ml improvement Meng Jinna liquid nutrient medium by 1mL/ bottle inoculum size, is made as blank group with what do not connect bacterium simultaneously, adds 1ml sterilized water in each bottle blank group, and each processes three bottles of repetitions.Be placed in 28 ℃ of shaking tables, 150rpm shaking culture, the content of titanium pigment and the pH value of substratum that every 24 hours, detect in supernatant liquor, METHOD FOR CONTINUOUS DETERMINATION 14d.The results are shown in Figure shown in 1.
As seen from Figure 1, above-mentioned bacterial strains phosphorus decomposing ability, through the mensuration of 14d, except rigidly connecting the first day of planting, all presents very high phosphorus decomposing ability in other 2-14d.Wherein, at inoculation above-mentioned bacterial strains, in the time of the 7th day, phosphorus decomposing amount reaches the highest, and other times also all show very high phosphorus decomposing ability in the phase, and the phosphorus decomposing amount in each time period is all over 1500mg/L.From the variation of pH value, postvaccinal the 2nd day pH fast-descending, reached Schwellenwert by the 4th day, rising afterwards, but after inoculation in 2-14 days, in substratum, pH value is all the time lower than 4.0.
Embodiment 4: heavy metal is to penicillium oxalicum phosphorus decomposing effect
The improvement Meng Jinna liquid nutrient medium that preparation contains lead, zinc, manganese and four kinds of different concns heavy metals of chromium respectively, heavy metal concentration is from 100mg/L, be followed successively by 100mg/L, 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L, with the processing that do not add heavy metal in contrast.
Above-mentioned bacterial strains is inoculated on PDA flat board, cultivates after 5d, with sterilized water, prepare spore suspension, by blood cell plate counting process, determine that spore suspension concentration is 1.6 * 10 7individual spore/mL, is inoculated in respectively by 1mL/ bottle inoculum size in the liquid nutrient medium that contains different sorts, different concns heavy metal, and each processes inoculation 1mL.Be placed in 28 ℃ of shaking tables, 150r/min shaking culture 10 days, measure the variation of this bacterium titanium pigment content in containing the liquid nutrient medium of various types of, different concns heavy metal, the results are shown in Table 2.
The phosphorus decomposing of table 2 penicillium oxalicum under different heavy metal concentrations measured
Note: phosphorus decomposing amount unit: mg/L
By table 2, can be drawn, in the liquid nutrient medium that contains heavy metal lead, zinc, manganese, chromium, along with the rising of heavy metal concentration, the phosphorus decomposing amount of this bacterial strain is the trend reducing gradually.Wherein, this bacterial strain is the strongest to the resistance performance of heavy metal zinc, in the substratum that contains 2000mg/L heavy metal zinc, it is 2068.2mg/L corresponding to the phosphorus decomposing amount that does not add the contrast of heavy metal that the phosphorus decomposing amount of this bacterial strain still reaches 1953.7mg/L(), phosphorus decomposing amount reaches 94.46% of contrast.In the liquid nutrient medium that contains heavy metal lead, manganese and chromium, when each heavy metal content is 2000mg/L, their phosphorus decomposing amount reaches respectively 593.6mg/L, 424.6mg/L and 107.3mg/L, is respectively 28.70%, 15.83% and 5.19% of contrast phosphorus decomposing amount.Observation this strain growth amount in containing the liquid nutrient medium of each concentration heavy metal is found, this bacterium is managed all extraordinary growths of energy in substratum throughout, even thalli growth amount compared with the control in the substratum that contains 2000mg/L heavy metal, still there is no too big difference.Also can illustrate thus, above-mentioned bacterial strains is all very strong to the resistance of heavy metal lead, zinc, manganese and chromium, and in phosphorus decomposing amount, this bacterium is the strongest to the resistance of heavy metal zinc.

Claims (3)

1. the Penicillium notatum that a strain can high-efficiency dissolution inorganic phosphate, is characterized in that: this bacterial strain be a strain penicillium oxalicum ( penicillium oxalicum), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No. 7699.
2. penicillium oxalicum claimed in claim 1, it is characterized in that: 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 and in branch top, is vase-like, the normal chain-like growth of conidium, spherical to oval, colourless to painted, smooth surface; On potato dextrose agar (PDA) flat board, colony edge is white, light green to deep green after sporulation; The 18S rDNA ITS gene order of bacterial strain has been carried out sequencing analysis, and sequence has been delivered to GenBank database.
3. penicillium oxalicum claimed in claim 1, is characterized in that, can be in the situation that heavy metal exist, and high-efficiency dissolution insoluble inorganic phosphorus.
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Cited By (9)

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CN104560738A (en) * 2015-01-14 2015-04-29 浙江大学 Penicillium oxalicum for reducing hexavalent chromium and screening method thereof
CN105296363A (en) * 2015-10-28 2016-02-03 南京农业大学 Oxalic acid penicillium strain NJDL-03 and application thereof
CN105647817A (en) * 2016-01-26 2016-06-08 华中农业大学 Penicillium janthinellum for decomposing hard-soluble aluminum phosphate in acid soil and application thereof
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
CN106929433A (en) * 2017-03-29 2017-07-07 山东农业大学 Phosphorus decomposing Penicillium notatum and its application
CN110129208A (en) * 2019-04-28 2019-08-16 湘潭大学 One plant of phosphorus decomposing penicillium oxalicum with wide spectrum characteristic of lactic acid production
CN110951626A (en) * 2019-12-27 2020-04-03 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN111304096A (en) * 2020-03-11 2020-06-19 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

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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

Patent Citations (2)

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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

Cited By (15)

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Publication number Priority date Publication date Assignee Title
CN104560738A (en) * 2015-01-14 2015-04-29 浙江大学 Penicillium oxalicum for reducing hexavalent chromium and screening method thereof
CN104560738B (en) * 2015-01-14 2017-03-22 浙江大学 Penicillium oxalicum for reducing hexavalent chromium and screening method thereof
CN105296363A (en) * 2015-10-28 2016-02-03 南京农业大学 Oxalic acid penicillium strain NJDL-03 and application 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
CN105647817A (en) * 2016-01-26 2016-06-08 华中农业大学 Penicillium janthinellum for decomposing hard-soluble aluminum phosphate in acid soil and application thereof
CN106929433A (en) * 2017-03-29 2017-07-07 山东农业大学 Phosphorus decomposing Penicillium notatum and its application
CN106929433B (en) * 2017-03-29 2020-07-31 山东农业大学 Phosphate solubilizing penicillium and application thereof
CN110129208A (en) * 2019-04-28 2019-08-16 湘潭大学 One plant of phosphorus decomposing penicillium oxalicum with wide spectrum characteristic of lactic acid production
CN110951626A (en) * 2019-12-27 2020-04-03 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN110951626B (en) * 2019-12-27 2021-05-25 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN111304096A (en) * 2020-03-11 2020-06-19 大连理工大学 Penicillium oxalicum and culture method and application thereof
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

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