CN116286394B - Penicillium chrysogenum, microbial agent and application thereof - Google Patents
Penicillium chrysogenum, microbial agent and application thereof Download PDFInfo
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- CN116286394B CN116286394B CN202310106063.4A CN202310106063A CN116286394B CN 116286394 B CN116286394 B CN 116286394B CN 202310106063 A CN202310106063 A CN 202310106063A CN 116286394 B CN116286394 B CN 116286394B
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- penicillium chrysogenum
- lead
- straw
- microbial agent
- phosphate
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- 241000228150 Penicillium chrysogenum Species 0.000 title claims abstract description 78
- 230000000813 microbial effect Effects 0.000 title claims abstract description 41
- 239000010902 straw Substances 0.000 claims abstract description 52
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 40
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 12
- 239000010452 phosphate Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 16
- 235000021317 phosphate Nutrition 0.000 claims description 16
- 240000008042 Zea mays Species 0.000 claims description 11
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 11
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 11
- 235000005822 corn Nutrition 0.000 claims description 11
- 241000209140 Triticum Species 0.000 claims description 8
- 235000021307 Triticum Nutrition 0.000 claims description 8
- 238000009629 microbiological culture Methods 0.000 claims description 7
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 7
- 239000001506 calcium phosphate Substances 0.000 claims description 5
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 5
- 235000011010 calcium phosphates Nutrition 0.000 claims description 5
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 5
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 claims description 4
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 claims description 4
- 238000005067 remediation Methods 0.000 claims description 4
- 239000005955 Ferric phosphate Substances 0.000 claims description 2
- 229940032958 ferric phosphate Drugs 0.000 claims description 2
- 229910000399 iron(III) phosphate Inorganic materials 0.000 claims description 2
- 238000011109 contamination Methods 0.000 claims 2
- 239000002054 inoculum Substances 0.000 claims 1
- 230000007928 solubilization Effects 0.000 claims 1
- 238000005063 solubilization Methods 0.000 claims 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 abstract description 57
- 235000006408 oxalic acid Nutrition 0.000 abstract description 18
- 239000002689 soil Substances 0.000 abstract description 14
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 8
- 238000004090 dissolution Methods 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 238000001179 sorption measurement Methods 0.000 abstract description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 4
- 239000011707 mineral Substances 0.000 abstract description 4
- 230000028327 secretion Effects 0.000 abstract description 4
- 238000011084 recovery Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- IBIRZFNPWYRWOG-UHFFFAOYSA-N phosphane;phosphoric acid Chemical compound P.OP(O)(O)=O IBIRZFNPWYRWOG-UHFFFAOYSA-N 0.000 abstract description 2
- 239000002686 phosphate fertilizer Substances 0.000 abstract description 2
- 239000002367 phosphate rock Substances 0.000 abstract description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 20
- 239000011574 phosphorus Substances 0.000 description 20
- 229910052698 phosphorus Inorganic materials 0.000 description 20
- 238000011282 treatment Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000002131 composite material Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- RLJMLMKIBZAXJO-UHFFFAOYSA-N lead nitrate Chemical compound [O-][N+](=O)O[Pb]O[N+]([O-])=O RLJMLMKIBZAXJO-UHFFFAOYSA-N 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000003337 fertilizer Substances 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 108091023242 Internal transcribed spacer Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 235000010633 broth Nutrition 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000002068 microbial inoculum Substances 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910000398 iron phosphate Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 229920012266 Poly(ether sulfone) PES Polymers 0.000 description 1
- 244000007853 Sarothamnus scoparius Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- QXDMQSPYEZFLGF-UHFFFAOYSA-L calcium oxalate Chemical compound [Ca+2].[O-]C(=O)C([O-])=O QXDMQSPYEZFLGF-UHFFFAOYSA-L 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000007154 intracellular accumulation Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- XNCMOUSLNOHBKY-UHFFFAOYSA-H iron(3+);trisulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XNCMOUSLNOHBKY-UHFFFAOYSA-H 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
-
- 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
- C12R2001/80—Penicillium
- C12R2001/82—Penicillium chrysogenum
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Pest Control & Pesticides (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Biodiversity & Conservation Biology (AREA)
- Soil Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to penicillium chrysogenum, a microbial agent and application thereof. The penicillium chrysogenum provided by the invention promotes the dissolution of the insoluble phosphate phosphorus through the secretion of oxalic acid, improves the utilization rate of the insoluble phosphate in soil, reduces the application of chemical phosphate fertilizer and reduces the consumption of phosphate rock; in addition, the penicillium chrysogenum provided by the invention has excellent tolerance and adsorption capacity on heavy metal lead, can release a large amount of oxalic acid, fixes lead by forming insoluble lead oxalate minerals, can repair lead pollution, and is beneficial to improving ecological environment; in addition, when the penicillium chrysogenum combined straw is used, the release amount of oxalic acid can be further increased, the effect of fixing lead is better than that of penicillium chrysogenum which is singly used, the lead pollution in the environment is reduced, and the efficient utilization and recovery of straw resources are realized.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to penicillium chrysogenum, a microbial agent and application thereof.
Background
Phosphorus is one of the essential nutrient elements for plant growth and production, and soluble phosphorus in soil is an important way for plants to obtain phosphorus. However, most of the phosphorus in the soil exists in the form of insoluble inorganic phosphate and is difficult to be absorbed and utilized by plants. Although the application of chemical phosphate fertilizers can increase crop yield, the applied phosphate fertilizers still have a significant portion fixed by soil particles, adsorbed or precipitated as insoluble phosphates, subject to the low mobility of phosphorus in the soil.
Lead is a very common heavy metal pollution element in the environment, and is mainly characterized by strong toxicity, long pollution latency, difficult degradation and the like. Lead pollution can directly harm the growth and development of crops and microorganisms, and can enter human bodies and livestock bodies in a food chain enrichment mode. Adsorption is an effective and economical lead removal technique, some microorganisms are capable of immobilizing heavy metals by intracellular accumulation and extracellular precipitation, etc., and these microorganisms are also capable of promoting the release of phosphorus in poorly soluble phosphates and the degradation of straw by the secretion of organic acids. However, the effect of the microorganism reported in the prior art on dissolving phosphorus and fixing lead is not remarkable.
Disclosure of Invention
In order to solve the problems, the invention provides penicillium chrysogenum, a microbial agent and application thereof. The penicillium chrysogenum (Penicillium Chrysogenum) provided by the invention can not only effectively promote the dissolution of insoluble phosphate, but also fix heavy metal lead, can treat a solution with the lead concentration of 1000mg/L, has a strong lead fixing capability for lead removal rate up to 97.2%, is beneficial to improving ecological environment, improves the utilization rate of phosphorus in soil, and reduces fertilizer application.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides penicillium chrysogenum (Penicillium Chrysogenum), which is preserved in China general microbiological culture collection center with a preservation number of CGMCC No.23271.
The invention also provides a microbial agent, which comprises the penicillium chrysogenum according to the technical scheme.
Preferably, the spore concentration of the penicillium chrysogenum in the microbial agent is 1 multiplied by 10 7 ～9×10 7 CFU/mL。
The invention also provides application of the penicillium chrysogenum or the microbial agent in the technical scheme in promoting the dissolution of insoluble phosphate.
Preferably, the poorly soluble phosphate salt includes one or more of iron phosphate, aluminum phosphate, and calcium phosphate.
The invention also provides the application of the penicillium chrysogenum or the microbial agent in the technical scheme in repairing lead pollution.
The invention also provides application of the penicillium chrysogenum or the microbial agent combined straw in the technical scheme in lead pollution remediation.
Preferably, the straw comprises wheat straw and/or corn straw.
The invention also provides a method for repairing lead pollution, which comprises the following steps:
applying the microbial agent according to the technical scheme to the area to be repaired;
or the microbial agent and the straw in the technical scheme are applied to the area to be repaired.
Preferably, the mass volume ratio of the microbial agent to the straw is 0.5-2 mL: 1-2 g.
The beneficial effects are that:
the invention provides a penicillium chrysogenum which is preserved in China general microbiological culture collection center with a preservation number of CGMCC No.23271. The penicillium chrysogenum provided by the invention promotes the dissolution of the insoluble phosphate phosphorus through the secretion of oxalic acid, improves the utilization rate of the insoluble phosphate in soil, reduces the application of chemical phosphate fertilizer and reduces the consumption of phosphate rock; in addition, the penicillium chrysogenum provided by the invention has excellent tolerance and adsorption capacity on heavy metal lead, can release a large amount of oxalic acid, fixes lead by forming insoluble lead oxalate minerals, can repair lead pollution, and is beneficial to improving ecological environment; in addition, when the penicillium chrysogenum combined straw is used, the release amount of oxalic acid can be further increased, the effect of fixing lead is better than that of penicillium chrysogenum which is singly used, the lead pollution in the environment is reduced, and the efficient utilization and recovery of straw resources are realized.
Description of biological preservation
Penicillium chrysogenum, latin is Penicillium Chrysogenum and is preserved in China general microbiological culture Collection center (CGMCC) at 30/09/2021 at the site of North Chen West Lu No. 1/3 in the Korean area of Beijing, and at the site of China academy of sciences microbiological study (CGMCC No. 23271).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a colony chart of Penicillium chrysogenum provided by the invention;
FIG. 2 is a microscope image of Penicillium chrysogenum provided by the present invention;
FIG. 3 is a phylogenetic tree analysis chart of Penicillium chrysogenum provided by the invention.
Detailed Description
The invention provides penicillium chrysogenum (Penicillium Chrysogenum), which is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 23271.
The colony center of the penicillium chrysogenum is blue-green, the edge of the colony is provided with white hypha, the colony is velvet-shaped and has more spores, and the colony surface is granular and is provided with concentric rings (see figure 1); the strain has long conidiophore single branch, enlarged top end of the conidiophore, broom shape, and spherical or elliptic single conidiophore (see figure 2).
In the present invention, the ITS sequence of P.chrysogenum is preferably shown in SEQ ID NO. 1.
The penicillium chrysogenum provided by the invention can promote the dissolution of phosphorus in insoluble phosphate by secreting oxalic acid, reduce the bioavailability of lead in the environment, be favorable for improving ecological environment, improve the utilization rate of phosphorus in soil and reduce fertilizer application.
The invention also provides a microbial agent, which comprises the penicillium chrysogenum according to the technical scheme.
In the present invention, the spore concentration of Penicillium chrysogenum in the microbial agent is preferably 1X 10 7 ～9×10 7 CFU/mL, more preferably 1X 10 7 CFU/mL。
In the present invention, the medium for preparing the microbial agent preferably includes PDA medium; the culture temperature for preparing the microbial agent is preferably 28 to 30 ℃, more preferably 30 ℃.
The invention also provides application of the penicillium chrysogenum or the microbial agent in the technical scheme in promoting the dissolution of insoluble phosphate.
In the present invention, the poorly soluble phosphate salt preferably includes one or more of iron phosphate, aluminum phosphate, and calcium phosphate.
The invention also provides the application of the penicillium chrysogenum or the microbial agent in the technical scheme in repairing lead pollution. The penicillium chrysogenum provided by the invention has excellent tolerance and adsorption capacity on heavy metal lead, can release a large amount of oxalic acid, fixes lead by forming insoluble lead oxalate minerals, can repair lead pollution, reduces the bioavailability of lead in the environment, and is beneficial to improving ecological environment. The penicillium chrysogenum provided by the invention can treat a solution with the lead concentration of 1000mg/L, has the lead removal rate of 97.2%, and has stronger lead fixing capability.
The invention also provides application of the penicillium chrysogenum or the microbial agent combined straw in the technical scheme in lead pollution remediation.
In the present invention, the straw preferably comprises wheat straw and/or corn straw, more preferably corn straw. Cellulose contained in the agricultural waste straw can effectively adsorb and complex heavy metal ions, and the bioavailability of the heavy metal is reduced. The penicillium chrysogenum combined straw provided by the invention has better lead pollution restoration effect than that of the penicillium chrysogenum and the independent straw, and the penicillium chrysogenum combined corn straw restoration effect is better than that of the penicillium chrysogenum combined wheat straw.
The invention also provides a method for repairing lead pollution, which comprises the following steps:
applying the microbial agent according to the technical scheme to the area to be repaired;
or the microbial agent and the straw in the technical scheme are applied to the area to be repaired.
In the invention, the volume mass ratio of the microbial agent to the straw is preferably 0.5-2 mL:1 to 2g, more preferably 1mL:1g; the straw preferably comprises wheat straw and/or corn straw, more preferably corn straw; the length of the straw is preferably 1-2 cm.
In the present invention, the area to be repaired preferably comprises lead-contaminated soil or water.
In the water body, the microbial inoculum is preferably used for 0.5-2 mL of the water body inoculation microbial inoculum with the dosage of 100-200 mL, and the straw is preferably used for 1-2 g of the water body with the dosage of 100-200 mL; the inoculation amount of the soil is preferably 100-200 g, the inoculation amount of the air-dried soil inoculant is 10-20 mL, the inoculation amount of the straw is 20-40 g, namely, the quantity of bacteria in the soil is preferably maintained to be more than the power of 10, and the mass ratio of the straw to the soil is preferably maintained to be more than 1:20.
For further explanation of the present invention, a strain of Penicillium chrysogenum, a microbial agent and applications thereof provided by the present invention are described in detail below with reference to examples and drawings, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Isolation of strains
Penicillium chrysogenum strain isolation material is from corn rhizosphere of North Anhui test station in Anhui agricultural university, and PVK solid culture medium is used for isolating Penicillium chrysogenum. Weighing 10g of soil sample, placing into 90mL of sterile water, shaking with 180rpm at 25deg.C for 30min, collecting supernatant, and mixing with water -2 、10 -3 、10 -4 And 10 -5 Gradually diluting, respectively taking 0.1mL of diluted supernatant liquid, coating on PVK solid culture medium, carrying out 3 times of parallel repeated experiments on each concentration, culturing for 3-5 days at 28 ℃, picking single bacterial colony with obvious phosphate dissolving ring, and streaking and culturing on PDA solid culture medium. Through the screening, a strain which can degrade insoluble phosphate is obtained and named AH-F-1-1.
PVK solid medium: 10g of glucose, 5g of calcium phosphate, 0.2g of sodium chloride, 0.25g of magnesium sulfate heptahydrate, 0.03g of ferric sulfate heptahydrate, 0.03g of manganese sulfate tetrahydrate, 0.2g of potassium chloride, 0.5g of ammonium sulfate and 15g of agar, and the deionized water is added to 1000mL.
PDA solid medium: 200g of potato, 20g of glucose and 20g of agar, and the deionized water is added to 1000mL.
(2) Identification of strains
The purified AH-F-1-1 strain was cultured on PDA solid medium at 28℃to observe colony morphology and microscopic features. The colony structure is shown in figure 1, the center of the colony is blue-green, white hypha is arranged at the edge, the colony has a velvet shape and more spores, and the colony surface is granular and has concentric ring grains; the microscopic image is shown in figure 2, the single branch of the conidiophore of the strain is longer, the top end of the conidiophore is enlarged, the conidiophore is broom-shaped, and the single conidiophore is spherical or elliptic. Extracting genome DNA by using a fungus DNA extraction kit under a sterile condition, performing agarose gel electrophoresis detection on an extracted product after PCR amplification, and sequencing after gel product cutting, recovery and purification, wherein the ITS sequence of the AH-F-1-1 strain is shown as SEQ ID NO. 1: 5'-AGGG TCTCTGGGTCACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTCCGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAA-3';
the PCR amplification primers are ITS1 and ITS4; the sequence of ITS1 is shown as SEQ ID NO. 2: 5'-TCCGTAGGTGAACCTGCGG-3'; the sequence of ITS4 is shown as SEQ ID NO. 3: 5'-TCCTCCGCTTATTGATATGC-3'.
PCR amplification System (25. Mu.l): 12.5. Mu.l PCR Master mix, 1.0. Mu.l ITS1, 1.0. Mu.l ITS4, 2. Mu.l DNA fragment and 8.5. Mu.l dH 2 O。
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 90s are used as a cycle, and the cycle is carried out for 35 times; extending at 72 ℃ for 5min, and preserving heat at 4 ℃ for 30min.
In GenBank database, the ITS sequence of AH-F-1-1 strain is searched by BLAST, the homology with penicillium chrysogenum is higher, the strain with the homology higher than 99% is selected for systematic development tree construction, and the result shows that the AH-F-1-1 strain and the penicillium chrysogenum MK267448.1:24-609 are gathered on the same branch and have a relatively close genetic distance, and the penicillium chrysogenum is identified as penicillium (shown in figure 3).
The AH-F-1-1 strain obtained by screening is identified as penicillium chrysogenum, the classification is named as penicillium chrysogenum (Penicillium Chrysogenum), the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 9 months and 30 days in 2021, the preservation address is China center for China general microbiological culture Collection, national institute of sciences of China, no.3, the North Chen West road No.1, the Korean region of Beijing, and the preservation number is CGMCC No.23271.
Example 2
A microbial agent is prepared by the following method:
inoculating the penicillium chrysogenum strain screened in the example 1 into a PDA solid culture medium, wherein the inoculation amount is one loop, and culturing for 5 days at the constant temperature of 30 ℃; collecting the spores formed to a containerIn a conical flask of sterile water, and filtering with three layers of sterile gauze to obtain a concentration of 1×10 7 CFU/mL of Penicillium chrysogenum spore suspension.
Application example 1 effect of Penicillium chrysogenum on the Release ability of phosphorus from poorly soluble phosphates
Shake flask culture assay: the experiment is divided into four treatments, namely PCH, PCH+Fe-P, PCH +Al-P and PCH+Ca-P, wherein each treatment is provided with 3 conical flasks which are respectively cultivated for 1, 3 and 5 days, and each conical flask is subjected to 3 parallel repeated experiments; the method comprises the following steps:
PCH: 50mL of PDB liquid culture medium is filled in a 150mL conical flask, 1mL of the microbial agent prepared in the example 2 is inoculated, and shaking culture is carried out at 28 ℃ and 180rpm after sealing;
pch+fe-P: 50mL of PDB liquid culture medium is filled in a 150mL conical flask, 0.5g of ferric phosphate is added as an initial phosphorus source, 1mL of the microbial agent prepared in the example 2 is inoculated, and shaking culture is carried out at 28 ℃ and 180rpm after sealing;
pch+al-P: similar to PCH+Fe-P, the only difference is that the initial phosphorus source is aluminum phosphate;
pch+ca-P: similar to PCH+Fe-P, the only difference is that the initial phosphorus source is calcium phosphate;
the formula of the PDB liquid culture medium is as follows: 200g of potato and 20g of glucose, and the deionized water is added to 1000mL.
The effective phosphorus content in each of the treated culture solutions was measured by ICP-OES, and the measurement results are shown in Table 1.
Oxalic acid content was measured in each of the treated culture solutions by HPLC, and the measurement results are shown in Table 2.
TABLE 1 content of available phosphorus (mg/L) in the culture broths of different treatment groups
Note that: a, b, c, d represent significant differences between treatments (p < 0.05)
TABLE 2 oxalic acid content (mg/L) in different treatment group cultures
Note that: a, b, c, d represent significant differences between treatments (p < 0.05)
As can be seen from tables 1 and 2, the effective phosphorus concentration and oxalic acid content in all treatments after 1, 3 and 5 days of culture are in an ascending trend (oxalic acid and calcium ions in PCH+Ca-P treatment form stable calcium oxalate minerals, so that the oxalic acid content in the culture solution is low), and the penicillium chrysogenum can be obviously stimulated to secrete oxalic acid in the presence of Fe-P, so that the penicillium chrysogenum can effectively promote the release of phosphorus in insoluble phosphates through the secretion of oxalic acid.
Application example 2 determination of lead-fixing ability of Penicillium chrysogenum
Shake flask culture assay: adding lead nitrate (Pb (NO) 3 ) 2 ) The method is used for simulating lead pollutants in a real environment, and the concentration of the used lead is set to be 1000mg/L. 100mL of PDB liquid culture medium is filled in a 250mL conical flask, 1mL of the microbial agent prepared in the example 2 is inoculated, the microbial agent is subjected to shaking culture for 10 days at the temperature of 28 ℃ and the speed of 180rpm after sealing, and three parallel repeated experiments are carried out. The lead content in the culture broth was measured by ICP-OES, and the results are shown in Table 3.
TABLE 3 lead content in culture broth (mg/L)
As shown in Table 3, the concentration of lead in the culture solution is reduced to 28.3mg/L, the lead removal rate is 97.2%, and the result shows that the penicillium chrysogenum screened in example 1 has stronger lead fixing capability.
Application example 3 capability of penicillium chrysogenum composite straw to lead pollution repair
Shake flask culture assay: the test is divided into five treatments, namely PCH+Pb, WST+Pb, MST+Pb, PCH+WST+Pb and PCH+MST+Pb, wherein each treatment is provided with 3 conical flasks which are respectively cultivated for 2, 4 and 6 days, and each conical flask is subjected to 3 parallel repeated experiments; the method comprises the following steps:
pch+pb: 0.1599g of lead nitrate (Pb) was added to a 250mL Erlenmeyer flask containing 100mL of PDB liquid medium (Pb concentration: 1000 mg/L), 1mL of the microbial agent prepared in example 2 was inoculated, and after sealing, the mixture was cultured at 28℃and 180 rpm;
WST+Pb: 0.1599g of lead nitrate is added into a 250mL conical flask filled with 100mL of PDB liquid culture medium, 1g of wheat straw is added, and after sealing, the mixture is cultured at 28 ℃ and 180 rpm;
mst+pb: 0.1599g of lead nitrate is added into a 250mL conical flask filled with 100mL of PDB liquid culture medium, 1g of corn stalk is added, and the mixture is cultivated at 28 ℃ and 180rpm after sealing;
pch+wst+pb: 0.1599g of lead nitrate is added into a 250mL conical flask filled with 100mL of PDB liquid medium, 1mL of the microbial agent prepared in the example 2 and 1g of wheat straw are added, and after sealing, the mixture is cultured at 28 ℃ and 180 rpm;
pch+mst+pb: 0.1599g of lead nitrate was added to a 250mL Erlenmeyer flask containing 100mL of PDB liquid medium, 1mL of the microbial agent prepared in example 2 and 1g of corn stalk were added, and after sealing, the mixture was cultured at 28℃and 180 rpm.
After the completion of the cultivation, the resultant was filtered with a qualitative filter paper, the filtrate was collected and the filtrate was removed, and the resultant was dried at 65℃for 24 hours.
Oxalic acid content in each of the treated culture solutions was measured by HPLC, the measurement results are shown in Table 4, lead content in each of the treated filtrate was measured by ICP-OES, and the measurement results are shown in Table 5.
The lead content in the precipitate is leached by adopting a TCLP method, and the method is concretely as follows: 2g of the filtrate-removed product was taken and added with 40mL of an extractant, and the mixture was shaken at 28℃for 18 hours at 180rpm, and left to stand, and the supernatant was filtered through a 0.45 μm Polyethersulfone (PES) filter membrane and then measured for lead content by ICP-OES, and the measurement results are shown in Table 6.
Before the TCLP method is adopted for extraction, the pH value of the filtrate is measured, and different extracting agents are selected according to the different pH values of the filtrate: (1) when the pH is less than 5, selecting the extractant 1; the extractant 1 is prepared by the following method: dissolving 5.7mL of glacial acetic acid in 500mL of deionized water, adding 64.3mL of NaOH solution with the concentration of 1mol/L, fixing the volume to 1L, and using 1mol/L of HNO 3 Or NaOH of 1mol/L is used for regulating the pH value of the solution, and the pH value of the solution is kept within the range of 4.93+/-0.05; (2) when the pH is more than 5, selecting the extractant 2; the extractant 2 is prepared by the following method: dissolving 5.7mL of glacial acetic acid in deionized water, fixing the volume to 1L, and using 1mol/L HNO 3 Or 1mol/L NaOH to adjust the pH value of the solution, and keeping the pH value of the solution within the range of 2.88+/-0.05.
TABLE 4 influence of Penicillium chrysogenum composite straw on oxalic acid concentration in culture solution (mg/L)
Note that: a, b, c, d, e represent significant differences between treatments (p < 0.05).
TABLE 5 influence of Penicillium chrysogenum composite straw on lead concentration in culture solution (mg/L)
Note that: a, b, c, d, e represent significant differences between treatments (p < 0.05).
TABLE 6 influence of Penicillium chrysogenum composite straw on effective lead content (mg/L) in the post-cultivation product
Note that: a, b, c, d, e represent significant differences between treatments (p < 0.05).
As can be seen from tables 4 to 6, the composite straw of penicillium chrysogenum can effectively promote the release of oxalic acid, the composite straw of penicillium chrysogenum can more effectively reduce the lead concentration in the culture solution and the effective lead content in the cultured product than the single penicillium chrysogenum and the single straw (the straw has a certain adsorptivity, can rapidly adsorb lead in the solution, but has the defects of limited adsorption capacity, and the adsorbed lead is easily released again and unstable, so that the lead concentration of the culture solution in the single straw in table 5 shows a trend of firstly reducing and then rising), the repair effect of the composite straw of penicillium chrysogenum on lead pollution is better, and the repair effect of the composite corn straw of penicillium chrysogenum is better than that of the composite wheat straw of penicillium chrysogenum.
In conclusion, the penicillium chrysogenum provided by the invention can promote the dissolution of the phosphorus in the insoluble phosphate, has stronger fixing capability on heavy metal lead ions, and plays an important role in improving the utilization rate of phosphorus in soil, reducing fertilizer application and improving ecological environment.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
1. Penicillium chrysogenum strainPenicillium Chrysogenum) The method is characterized in that the penicillium chrysogenum is preserved in China general microbiological culture collection center (CGMCC) No.23271.
2. A microbial agent comprising penicillium chrysogenum according to claim 1.
3. The microbial agent according to claim 2, wherein the spore concentration of penicillium chrysogenum in the microbial agent is 1 x 10 7 ~9×10 7 CFU/mL。
4. Use of penicillium chrysogenum according to claim 1 or the microbial agent of claim 2 or 3 to promote solubilization of poorly soluble phosphates; the indissolvable phosphate is one or more of ferric phosphate, aluminum phosphate and calcium phosphate.
5. Use of penicillium chrysogenum according to claim 1 or the microbial agent according to claim 2 or 3 for remediation of lead contamination.
6. The use of penicillium chrysogenum combined straw as claimed in claim 1 or the microbial inoculant combined straw as claimed in claim 2 or 3 in the remediation of lead pollution.
7. The use according to claim 6, characterized in that the straw is wheat straw and/or corn straw.
8. A method of repairing lead contamination comprising the steps of:
applying the microbial agent of claim 2 or 3 into an area to be repaired;
alternatively, the microbial agent of claim 2 or 3 and straw are applied to the area to be repaired.
9. The method of claim 8, wherein the volume to mass ratio of the microbial agent to the straw is 0.5-2 ml: 1-2 g.
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