CN110129208B - Penicillium oxalicum with broad-spectrum acid production characteristic - Google Patents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09C1/00—Reclamation of contaminated soil
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- 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
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- 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
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Abstract
The invention discloses a penicillium oxalicum capable of producing acid in a broad spectrum. The preservation number of the strain is as follows: CCTCC NO. M2019233, well grows in a solid culture medium which takes calcium phosphate or iron phosphate as a unique phosphorus source, and after 48 hours of culture, a large number of bacterial colonies grow after 3-7 days of culture by using an insoluble phosphorus compound as a growth substance; in a liquid culture medium, the strain can efficiently utilize calcium phosphate or iron phosphate with the maximum concentration of 50g/L, and can produce 2500mg/L of soluble phosphate at most. The strain can be metabolized to produce oxalic acid, formic acid, malic acid, acetic acid and lactic acid in a solid calcium phosphate culture medium, and the maximum secretion amount can reach 215.73 mg/L. The microbial strain resource obtained by first separating from the soil environment has good application value in the aspects of improving the phosphorus of farmland soil, improving the soil fertility, promoting the comprehensive treatment of heavy metal soil pollution and the like.
Description
Technical Field
The invention belongs to the technical field of environmental biology, and particularly relates to a phosphate-solubilizing fungus penicillium oxalicum PSF-4 with broad-spectrum acid production characteristics and application thereof.
Background
Phosphorus is one of the major elements necessary for the growth and development of organisms, and is an important component for forming biological membranes, nucleic acids and a plurality of macromolecules (such as adenosine triphosphate). Phosphorus is the most important nutrient element next to nitrogen for plant growth. Phosphorus is transformed and flows in different compound forms among plants, animals, microorganisms and soil, the animals and the microorganisms can supplement required phosphorus elements through predation or catabolism and the like, and the plants realize phosphorus (mainly comprising primary phosphoric acid H) in a biologically effective state in the soil mainly through plant roots2PO4 -And secondary phosphoric acid HPO4 2-) Is suckedAnd (6) harvesting. Therefore, the content of phosphorus in the soil in a biologically available state is an important environmental factor influencing plant growth, and is also a main component for phosphorus compound conversion and phosphorus element circulation in the soil environment.
The total phosphorus content in the soil is very high, but more than 95 percent of phosphorus exists in the form of indissolvable phosphorus compounds, and the content of phosphorus in a biological effective state is lower, so that the phosphorus deficiency phenomenon of the soil is caused. It is reported that about 67% of the soil in the world is in a phosphorus deficiency state, and about 74% of the cultivated land soil in China is in phosphorus deficiency state. Although the problem of phosphorus deficiency of plants can be solved in a short time by applying the inorganic phosphate fertilizer, the agricultural production cost is increased, and meanwhile, heavy metals (such as lead, zinc and the like) in the phosphate fertilizer are easily introduced into the soil environment, so that the environment pollution is caused.
The phosphate solubilizing microorganism is a microorganism which can convert insoluble phosphorus compounds (including organic phosphorus and inorganic phosphorus compounds) into soluble phosphorus in a soil environment, and can drive the insoluble phosphorus compounds to be decomposed or dissolved into soluble phosphorus for plants to absorb and utilize, so that the conversion rate and the utilization rate of the biological effective phosphorus in the soil are improved, the accumulation of the phosphorus in the soil is reduced, and the plants are promoted to grow in the soil which is deficient in phosphorus and has a pH value which is slightly acidic. The phosphate solubilizing mechanism of phosphate solubilizing microorganisms has not been completely proven at present, but recent researches show that the dissolution of insoluble phosphorus compounds is related to the secretion of organic acids by the phosphate solubilizing microorganisms, and insoluble inorganic phosphorus compounds, such as Ca, can be dissolved outside cells3(PO4)2、FePO4Etc. to thereby convert H therein2PO4 -、HPO4 2-And releasing phosphate radical with equal solubility. Meanwhile, researches in recent years find that various organic acids secreted by the phosphate solubilizing bacteria can also be used as good biological chelating agents for heavy metal contaminated soil to reduce the concentration of heavy metal ions in the soil, so that the aim of effectively repairing the heavy metal contamination is fulfilled. However, at present, few phosphate solubilizing fungi with broad-spectrum organic acid metabolism characteristics are obtained at home and abroad, so that the research on the application of the phosphate solubilizing fungi to soil remediation is very limited.
Disclosure of Invention
In order to solve the defects and shortcomings in the prior art, the invention provides a phosphate-solubilizing microorganism penicillium oxalicum PSF-4 with broad-spectrum acid production characteristics, which is delivered to China Center for Type Culture Collection (CCTCC) for preservation in 4 months and 4 days in 2019, wherein the preservation number is as follows: CCTCC NO: m2019233, category name: penicillium oxalicum PSF-4, address: wuhan, Wuhan university.
The invention also aims to provide application of the penicillium oxalicum PSF-4, and the strain provided by the invention has the fungus with high phosphate solubilizing property and can secrete and produce various organic acid compounds. The strain can grow in solid calcium phosphate and iron phosphate solubilizing culture media under laboratory conditions, PSF-4 can grow within 72 hours, and a remarkable transparent phosphate solubilizing ring is formed. The efficiency of degrading calcium phosphate by PSF-4 is higher than that of iron phosphate; meanwhile, in a solid calcium phosphate liquid shaking flask experiment, PSF-4 can generate a large amount of organic acid, and HPLC (high performance liquid chromatography) detection shows that the PSF-4 can be metabolized to generate 5 organic acids such as oxalic acid, formic acid, malic acid, acetic acid, lactic acid and the like. Has important application prospect in the aspects of improving the phosphorus of farmland soil, improving the soil fertility, promoting the comprehensive treatment of heavy metal soil pollution and the like.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
in a first aspect, the penicillium oxalicum PSF-4 is provided, which is delivered to China Center for Type Culture Collection (CCTCC) for preservation 4 months and 4 days in 2019, and the preservation number is as follows: CCTCC NO: m2019233, category name: penicillium oxalicum PSF-4, address: wuhan, Wuhan university. Penicillium oxalicum PSF-4 CCTCC NO: m2019233 is short for Penicillium oxalicum PSF-4.
In a second aspect, a microbial inoculum is provided, which comprises Penicillium oxalicum PSF-4Penicillium oxalicum PSF-4 with a deposit number of: CCTCC NO: and M2019233.
The growth cycle of the strain in liquid and solid phosphate dissolving culture medium is longer. The liquid fermentation medium reaches logarithmic growth phase within 48 hours of shaking culture, and the phosphate-solubilizing medium is changed from turbid to clear. On a phosphate-solubilizing solid culture medium, fungus colonies are irregular in size, the edges of the fungus colonies are white villiform, the centers of mature colonies are green, aerial hyphae and substrate hyphae are developed, and the fungus colonies are easy to pick. The bacterial colony is irregular round, flat and raised, and is easy to pick up; the colony is large and the edge is irregular. Observed under an optical microscope, the bacterial colony is in a velvet shape, the branches between the bacterial colony are forked, the bacterial colony is mostly in a three-fork shape, and the center is asymmetric and forked; the spore is green, elliptical and spherical, and has a diameter of about 0.10 μm; the spore stems are short and obvious in branching, the branches are acute-angled and asymmetric, and the length of the branches is about 0.25 mu m. The ITS rDNA gene sequence of the strain is subjected to sequencing analysis, the sequence is submitted to a GenBank database, and the strain is identified as Penicillium oxalicum (Penicillium oxalicum) through sequence comparison analysis, wherein the sequence number in the GenBank is MK 720103.
In a solid calcium phosphate and iron phosphate solubilizing culture medium, PSF-4 can grow within 72 hours and form a remarkable transparent phosphate solubilizing ring. PSF-4 can grow in a liquid phosphate dissolving culture medium with solid calcium phosphate and iron phosphate concentration of 5-50g/L, and can completely degrade the calcium phosphate and the iron phosphate at the concentration of 5 g/L. The efficiency of degrading calcium phosphate by PSF-4 is higher than that of iron phosphate, and the maximum phosphorus dissolving amount exceeds 2400 mg/L. By changing the experimental conditions such as culture temperature, fungus inoculation concentration, rotating speed and the like, the PSF-4 has little change on the degradation efficiency of the solid calcium phosphate, can realize 100 percent degradation at most, and has the maximum phosphorus dissolving amount exceeding 3000 mg/L.
In a solid calcium phosphate liquid shake flask experiment, PSF-4 can generate a large amount of organic acid, HPLC detection shows that the PSF-4 can be metabolized to generate 5 kinds of organic acid such as oxalic acid, formic acid, malic acid, acetic acid, lactic acid and the like, secretion of the organic acid can dissolve a part of insoluble phosphate in soil, so that bioavailable effective phosphorus is released, the content of the available phosphorus in the soil is improved, and the maximum secretion amount can reach 215.7 mg/L.
In a third aspect, the application of the penicillium oxalicum PSF-4 or the microbial inoculum in degrading the slightly soluble inorganic phosphate is provided.
Preferably, the inorganic phosphate is calcium phosphate or iron phosphate, preferably calcium phosphate.
In a fourth aspect, the application of the penicillium oxalicum PSF-4 or the microbial inoculum in the preparation of products for degrading insoluble inorganic phosphate is provided.
In the fifth aspect, a method for improving farmland soil phosphorus is provided, wherein the soil is treated by the penicillium oxalicum PSF-4 or a microbial inoculum so as to degrade insoluble inorganic phosphate in the soil.
The strain of the invention is prepared from the following most common insoluble phosphorus compounds in soil: calcium phosphate and iron phosphate grow well in a solid culture medium which is the only phosphorus source, and after 48 hours of culture, a large number of biological colonies can be grown after 3-7 days of culture by using an insoluble phosphorus compound as a growth substance. In a liquid culture medium, the bacterial strain can efficiently utilize calcium phosphate and iron phosphate with the maximum concentration of 50g/L, and can generate 2500mg/L of soluble phosphate at most. The invention also discloses the broad-spectrum organic acid metabolism characteristic of the fungus, 5 organic acids such as oxalic acid, formic acid, malic acid, acetic acid, lactic acid and the like can be metabolized and generated in a culture medium added with solid calcium phosphate, and the maximum amount of secretion can reach 215.7 mg/L. Because the phosphorus-dissolving fungi with broad-spectrum organic acid metabolism characteristics obtained at home and abroad are few at present, the strain is a microbial strain resource obtained by the first separation of an inventor from a soil environment, and has good application value in the aspects of improving farmland soil phosphorus, improving soil fertility, promoting comprehensive treatment of heavy metal soil pollution and the like.
Drawings
FIG. 1: growth of fungus PSF-4 on solid phosphate solubilizing plates of calcium phosphate and iron phosphate;
a is a calcium phosphate solid phosphate dissolving plate, and B is an iron phosphate solid phosphate dissolving plate;
FIG. 2: spore stem of fungus PSF-4 under optical microscope and molecular biological phylogenetic tree thereof;
a is the spore stem of the fungus PSF-4 under an optical microscope, and B is the molecular biology phylogenetic tree of the fungus PSF-4;
FIG. 3: detecting the broad-spectrum organic acid metabolism characteristic of PSF-4 by HPLC;
biological characterization of the deposited fungi:
the fungus is preserved in China center for type culture Collection with a preservation number of CCTCC NO: and M2019233. Address: china, wuhan university, latin academic name: penicillium oxalicum PSF-4, with a Chinese name: penicillium oxalicum PSF-4, which was deposited at 4 months and 4 days in 2019.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
[ EXAMPLE 1 ] screening of phosphate solubilizing fungi
The experimental soil sample is collected from activated sludge of a sewage treatment plant in a Yueyang Yunxi district industrial park in Hunan province, is immediately and naturally dried for 48 hours, after redundant impurities are removed, the soil sample is ground through a 20-mesh soil sieve, and the redundant soil sample is stored in a refrigerator at 4 ℃.10 g of the sieved soil sample was added to 100mL of sterilized liquid enrichment medium (containing sterile glass beads) (enrichment medium components: peptone 10g/L, NaCl 1.0g/L, KH)2PO31.0g/L, pH 7), shaking at 150rpm for 7 days. After 1 week of culture, 1mL of the culture medium in the enrichment medium was collected, and 1mL of the supernatant was diluted to 10 by the dilution plating method-5Taking out 10 of it-4,10-5,10-6200 mul of solution under 3 dilution gradients are respectively put in phosphate dissolving culture medium (the components of the phosphate dissolving culture medium are 10g/L of sucrose and Ca) which takes calcium phosphate and ferric phosphate as the only phosphorus sources3(PO4)2Or FePO4 10g/L，NaCl 0.3g/L,KCl 0.3g/L,MgS04·7H 20 0.3g/L,FeS04 0.03g/L,MnS04 0.03g/L,(NH4)2S040.5g/L, yeast extract 0.5g/L, phosphate solubilizing liquid medium of the same composition and solids but not containing agar) were coated, 3 plates were repeated for each treatment, the plates were then incubated in an incubator at 28 ℃ for 5 days, the number of strains on each plate was recorded, the colony characteristics of the microorganisms on the solid plates were observed, and pure phosphate solubilizing microorganisms were obtained by streaking based on the phosphate solubilizing circles formed by the respective strains on the phosphate solubilizing medium.
As can be seen from the growth of FIG. 1, PSF-4 grew normally on phosphate-dissolving plates of calcium phosphate and iron phosphate and had a very high degradation capacity.
[ example 2 ] identification of the Strain
The purified strain was inoculated into a solid PDA medium (composition: potato 200g/L, glucose 20g/L, agar 20g/L, pH 7) and cultured for 5 days, then the colony morphology of the strain on a solid plate was observed, then a few colonies were picked up and the microstructure of the strain was observed under an optical microscope, and the morphology of the strain was recorded: the colony is in a velvet shape, branches among the colony are branched, the colony is mostly in a three-fork shape, and the center is asymmetric; the spore is green, elliptical and spherical, and has a diameter of about 0.10 μm; the spore stems are short and obvious in branching, the branches are acute-angled and asymmetric, and the length of the branches is about 0.25 mu m.
Fungal ITS rDNA sequencing includes strain DNA extraction, ITS rDNA in vitro amplification (using ITS universal primers ITS1 and ITS4), and gene sequencing. Sequence results obtained from gene companies sequencing were analyzed, combined and brought into the NCBI database for BLAST gene sequence alignment by software. According to the morphological observation and the constructed phylogenetic tree result of FIG. 2, the strain is identified as Penicillium oxalicum (Penicillium oxalicum)
[ example 3 ] analysis of degradation characteristics of the strains to calcium phosphate and iron phosphate
The final concentrations of calcium phosphate and iron phosphate added to the liquid phosphate solubilizing medium were set to 5g/L and 2g/L, respectively. Before measurement, the strain is inoculated in a phosphate-solubilizing slant culture medium for culture, activated and cultured at 28 ℃ for 72 hours, then 1mL of sterile water is taken and added into a slant, spores and hyphae of the slant fungi are scraped into the sterile water to form spore suspension, and the spore suspension is added into the culture medium according to the volume ratio of 5%. All cultures were subjected to shaking culture for 14 days on a shaker at 150rpm and 28 ℃ and the pH value of the medium and the soluble phosphorus content were measured, respectively. Finally, the phosphorus dissolution amount of PSF-4 on 5g/L calcium phosphate is 2071mg/L, the phosphorus dissolution rate is 100%, and the phosphorus dissolution amount on 2g/L iron phosphate is 198mg/L, and the phosphorus dissolution rate is 48.5%.
And detecting the content of soluble phosphorus in the supernatant by using a phosphorus-antimony-molybdenum blue spectrophotometry. First, KH is used2PO4The phosphorus standard curve is drawn, and the fitting degree of the drawn curve should exceed 0.995. When measuring the soluble phosphorus content in the experimental group, 50. mu.L of supernatant was accurately aspirated every day by using a pipette gun and added to the containerCentrifuging at 12000rpm for 1min in a centrifuge tube containing 950 μ L of distilled water, carefully adding the supernatant in the centrifuge tube into a test tube with scales, adding 4mL of molybdenum-antimony anti-reagent, metering to 25mL, standing at room temperature for 30min, placing the sample at 700nm for measurement, finally calculating the content of soluble phosphorus according to a standard curve value, and calculating the degradation characteristic.
Calcium phosphate of 5g/L, 10g/L, 20g/L, 25 g/L, and iron phosphate of 1g/L, 2g/L, 4 g/L, 5g/L, and 10g/L were designed to study the degradation capability of PSF-4, and in addition, temperature gradients of 24 ℃ and 28 ℃ and 32 ℃ were designed with the calcium phosphate of 5g/L and the iron phosphate of 2g/L as substrate concentrations, respectively, and 10 c was designed6、107、108Initial inoculation spore concentration, 100, 150, 200rpm rotation speed to study phosphate solubilizing activity.
Table 1: research on degradation characteristics of phosphate solubilizing fungi PSF-4 on calcium phosphate and iron phosphate
The data and experimental phenomena in the table 1 show that PSF-4 has very strong degradation capability on calcium phosphate, can realize a degradation concentration of more than 50g/L at most, has a degradation rate of more than 19%, can realize complete degradation on calcium phosphate under a low concentration condition, and can realize complete degradation on calcium phosphate of 5g/L under different experimental conditions; the degradation capability to the ferric phosphate is weak, the concentration of completely degrading the ferric phosphate can only be 1g/L within 14 days of culture, and the degradation efficiency of the ferric phosphate is greatly influenced by the change of culture conditions.
Example 4 detection assay for PSF-4 metabolizing organic acids
Calcium phosphate (5g/L, 10 g/L) added previously was purified by high performance liquid chromatography (Agilent 1260Infinity)7150 rpm/mL), iron phosphate (2g/L, 10)7/mL, 150rpm), a certain amount of the culture medium liquid is extracted, centrifuged at 12000rpm for 2min, collected in a liquid phase bottle through a 0.22 μm filter head, and the organic acid content is measured on the day. The detector was an ultraviolet detector and the column was a Dimmaplastic ODS C18 column (4.6 mm. times.250 mm, 5 μm, Platisil Co., USA). Mobile phase: phase A is 0.01mol/L, pH 2.9 ammonium dihydrogen phosphate solution, and phase B is acetonitrile. Sample introduction amount: 20 μ L, column temperature: 30 ℃, flow rate: 0.5 mL/min. Firstly, drawing a plurality of organic acid standard curves, wherein the fitting degree of the drawn curves exceeds 0.995, when the concentration of the organic acid is measured, firstly, passing a sample through a filter membrane of 0.22 mu m, then, detecting on a computer, and comparing the peak area of the sample with the standard curve to obtain the concentration of the detected organic acid.
Table 2: research on content of organic acid secreted by calcium phosphate and iron phosphate by phosphate solubilizing fungi PSF-4
Table 3: research on content of organic acid secreted by calcium phosphate for one week by phosphate solubilizing fungi PSF-4
Claims (7)
1. The penicillium oxalicum PSF-4 strain is characterized by being named after classification: penicillium oxalicum (B)Penicillium oxalicum) PSF-4, which is preserved in China center for type culture Collection with the preservation date as follows: 4 months and 4 days in 2019, the preservation number is: CCTCC NO: m2019233, the deposit address is: wuhan, Wuhan university.
2. A microbial preparation comprising the Penicillium oxalicum (Penicillium) of claim 1Penicillium oxalicum） PSF-4。
3. Use of the penicillium oxalicum PSF-4 according to claim 1 or the microbial agent according to claim 2 for degrading a sparingly soluble inorganic phosphate.
4. Use according to claim 3, characterized in that the poorly soluble inorganic phosphate is calcium phosphate or iron phosphate.
5. Use according to claim 4, characterized in that the poorly soluble inorganic phosphate is calcium phosphate.
6. Use of the bacterial agent of claim 1 or 2 or PSF-4 in the preparation of a product for degrading poorly soluble inorganic phosphate.
7. A method for improving farmland soil phosphorus, which is characterized in that the soil is treated by the fungicide of claim 1 or 2, so as to degrade the insoluble inorganic phosphate in the soil.
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| CN110951625B (en) * | 2019-12-27 | 2022-05-31 | 滨州学院 | Penicillium phosphate solubilizing PSF and application thereof |
| CN111924982B (en) * | 2020-08-12 | 2021-09-21 | 中国科学院武汉岩土力学研究所 | Biological permeable reactive barrier for composite contaminated site and preparation method thereof |
| CN111979159A (en) * | 2020-09-03 | 2020-11-24 | 中南大学 | Phosphate solubilizing bacterium agent and preparation method and application thereof |
| CN113333459B (en) * | 2021-06-08 | 2022-06-03 | 湘潭大学 | Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same |
| CN114854600B (en) * | 2022-03-21 | 2023-05-16 | 中国科学院南京土壤研究所 | Penicillium oxalate and application thereof |
| CN114540208B (en) * | 2022-03-28 | 2023-06-20 | 山东省林业科学研究院 | Absidia with high-efficiency phosphate-dissolving effect and application thereof |
| CN116410870B (en) * | 2023-02-14 | 2024-02-27 | 安徽农业大学 | Penicillium oxalate, composite microbial inoculum, combined repairing agent and application thereof |
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| JPS5241293A (en) * | 1975-09-29 | 1977-03-30 | Koichi Ogata | Method of manufacturing glucose-1-phosphate and glucose-6-phosphate |
| CN101643704A (en) * | 2008-08-07 | 2010-02-10 | 中国农业科学院农业资源与农业区划研究所 | Phosphorus dissolvable penicillium oxalicum P8 |
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| CN103614302B (en) * | 2013-11-27 | 2016-07-06 | 北京林业大学 | One strain has the efficient phosphate-solubilizing penicillium oxalicum of heavy metal tolerance characteristic |
| CN103695317B (en) * | 2013-12-12 | 2016-11-23 | 北京林业大学 | There is the production method of the efficient phosphate-solubilizing penicillium oxalicum microbial inoculum of heavy metal tolerance characteristic |
| CN103789214B (en) * | 2014-01-03 | 2015-10-28 | 南京林业大学 | A kind of Pinus massoniana Lamb rhizosphere phosphorus decomposing fungi Gua Na Karst mould JP-NJ2 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5241293A (en) * | 1975-09-29 | 1977-03-30 | Koichi Ogata | Method of manufacturing glucose-1-phosphate and glucose-6-phosphate |
| CN101643704A (en) * | 2008-08-07 | 2010-02-10 | 中国农业科学院农业资源与农业区划研究所 | Phosphorus dissolvable penicillium oxalicum P8 |
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