CN114437941A - Saline-alkali-resistant penicillium oxalicum agent, and preparation method and application thereof - Google Patents
Saline-alkali-resistant penicillium oxalicum agent, and preparation method and application thereof Download PDFInfo
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- 241000985513 Penicillium oxalicum Species 0.000 title claims abstract description 91
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- 239000000843 powder Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 239000002361 compost Substances 0.000 claims description 14
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- 241001474374 Blennius Species 0.000 claims description 13
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- 238000002386 leaching Methods 0.000 claims description 7
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- 229910052698 phosphorus Inorganic materials 0.000 description 4
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- 108010059892 Cellulase Proteins 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
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- 230000008520 organization Effects 0.000 description 2
- ZFRKQXVRDFCRJG-UHFFFAOYSA-N skatole Chemical compound C1=CC=C2C(C)=CNC2=C1 ZFRKQXVRDFCRJG-UHFFFAOYSA-N 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
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- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- JQJCSZOEVBFDKO-UHFFFAOYSA-N lead zinc Chemical compound [Zn].[Pb] JQJCSZOEVBFDKO-UHFFFAOYSA-N 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
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- 235000005985 organic acids Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000009260 qiming Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 229940074386 skatole Drugs 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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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
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- 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
- C12N1/36—Adaptation or attenuation of cells
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- C—CHEMISTRY; METALLURGY
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- C09K2109/00—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE pH regulation
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Abstract
The invention provides a saline-alkali-resistant penicillium oxalicum agent, a preparation method and application thereof. The preparation method of the saline-alkali resistant penicillium oxalicum agent comprises the following steps: s1: domesticating penicillium oxalicum with the preservation number of CGMCC No. 12628; s2: fermenting the domesticated penicillium oxalicum under a high saline-alkali condition; wherein, the high saline-alkali conditions comprise: the salt concentration is more than 10% and the pH value is more than 11. The penicillium oxalicum CGMCC No.12628 is domesticated, and can ferment and produce spores under the high saline-alkali condition, and the prepared saline-alkali tolerant penicillium oxalicum agent has an obvious growth promoting effect, can be applied to extreme severe saline-alkali soil environments, can still keep a high survival rate under severe natural conditions, and effectively improves the pH environment of soil.
Description
Technical Field
The invention relates to the technical field of environmental biology, in particular to a saline-alkali-resistant penicillium oxalicum agent, and a preparation method and application thereof.
Background
CN 103695317A discloses a production method of a penicillium oxalicum agent with a characteristic of heavy metal tolerance, the high-efficiency phosphate-dissolving penicillium oxalicum is separated and screened from lead-zinc ore surface soil in Huayuan county of Hunan province, and has the characteristic of multiple heavy metal tolerance; the production method mainly takes sweet potato starch wastewater and mushroom bran waste residue as main culture medium raw materials, adopts a liquid-solid combined fermentation method for culture, and has a pH close to neutral in the culture process.
CN 110499256A discloses a penicillium oxalicum capable of degrading organic acid odor and an amplification culture method thereof, wherein a sample is collected from stacked cow breeding feces, the sample is inoculated in a screening culture medium, a plurality of antibiotics, a plurality of organic acids, indole and skatole are added in the screening culture medium to perform induced domestication culture and separation purification on bacteria in the sample, and penicillium oxalicum DH-1 is obtained, the treatment temperature of the penicillium oxalicum DH-1 is 25-40 ℃, the pH value is 4.0-9.5, and the penicillium oxalicum DH-1 grows at the fastest speed and has the highest degradation efficiency under the conditions of the temperature and the pH value.
CN 110484452A discloses penicillium oxalicum capable of degrading straw to produce 2-phenylethyl alcohol and application thereof, wherein penicillium oxalicum T1 has good cellulose degradation capability, can produce cellulase, has strong endoglucanase activity, filter paper enzyme activity, exonuclease activity and beta-glucosidase activity, can degrade straw stalks to produce 2-phenylethyl alcohol, the pH of a screening culture medium of a cellulose degradation strain is 7.0, the pH of a seed culture medium is 6.0, and the pH of a fermentation enzyme production culture medium is natural.
CN 101914450B discloses a compost biological dehydration fungus agent and a preparation method thereof, the fungus agent takes a penicillium oxalicum strain F12 CGMCC No.3682 as a strain, the water content of the fungus agent can be reduced by 10-20% after the fungus agent is inoculated into livestock and poultry manure with high water content and fermented for 10-15 days, and the pH value of a culture medium of a production fermentation tank is 4-6. The Penicillium strain F12 has the optimum enzyme-producing pH value of 5-6, the CMCase and FPA of the strain are basically consistent, the CMCase and FPA both have a tendency to decrease with the increase or decrease of the pH value beyond the pH value of 5-6, and the enzyme activity is hardly detected when the pH value reaches 9.
The existing penicillium oxalicum has obvious phosphate solubilizing effect, but can only be used in neutral, slightly acidic or slightly alkaline environmentCan grow and reproduce to play a role, and is difficult to grow in an alkaline environment. According to incomplete statistics of textbook organization of the world United nations and food and agricultural organization of the world United nations, the global saline-alkali land occupies 10 hundred million hm of the world area2The land occupation area of the saline-alkali land of China is 1 hundred million hm2And the third position. As agriculture and large population countries, how to take effective measures to prevent, control and improve saline-alkali soil so as to effectively utilize land resources is a problem to be solved urgently.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a saline-alkali-resistant penicillium oxalicum agent, a preparation method and an application thereof.
The invention provides a preparation method of a saline-alkali-resistant penicillium oxalicum agent, which comprises the following steps:
s1: domesticating penicillium oxalicum with the preservation number of CGMCC No. 12628;
s2: fermenting the domesticated penicillium oxalicum under the condition of high salt and alkali.
The penicillium oxalicum is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of institute of academy of sciences, China in 2016 (6.23.6.2016), with the preservation number of CGMCC No. 12628.
The high-saline-alkali condition refers to a condition with higher salt content and alkali content, and the high-saline-alkali condition is not strictly limited; specifically, the high saline-alkaline conditions may include: the salt concentration is more than 10% and the pH value is more than 11.
In the present invention, the acclimation includes saline-alkali tolerant acclimation; specifically, the saline-alkali tolerant acclimation may include: domesticating and culturing penicillium oxalicum by adopting culture media with different salt concentration gradients and alkali concentration gradients; during the domestication culture, the culture can be carried out step by step from a low concentration gradient to a high concentration gradient. The salt concentration gradient and the base concentration gradient are not strictly limited, and the salt concentration gradient may be set, for example, as: NaCl with the mass content of 0%, 2%, 5%, 7% and 10% in sequence; the alkali concentration gradient may be set, for example, as: the pH values are, in order, pH6, pH8, pH9, pH10 and pH 11.
Further, the domestication can also comprise saline-alkali tolerant directional domestication; the directional saline-alkali tolerant domestication comprises the step of continuously domesticating and culturing the saline-alkali tolerant domesticated penicillium oxalicum by adopting a culture medium added with a high saline-alkali material leaching solution. The high saline-alkali materials in the high saline-alkali material leaching solution are not strictly limited and can be selected according to the actual application requirements; specifically, the high saline-alkali material may include at least one of monosodium glutamate slag, alkaline hydrolyzed seaweed slag, and kitchen waste compost fermentation material, wherein: the alkaline hydrolysis seaweed residues are seaweed residues generated by an alkaline hydrolysis process, and the kitchen waste compost fermentation materials are kitchen waste compost fermented materials.
In one embodiment, the high-saline-alkali material can comprise a material A and a material B, wherein the material A is monosodium glutamate residue, and the material B is alkaline hydrolysis seaweed residue or kitchen garbage compost fermentation material; the mass ratio of the material A to the material B in the high-saline-alkali material can be (1.5-2.5): 1, preferably 2: 1.
the high saline-alkali material leachate refers to a leachate of a high saline-alkali material, and the preparation method of the leachate is not strictly limited, and may include, for example: mixing the high-saline-alkali material with water, stirring, standing, and taking supernatant to obtain high-saline-alkali material leachate; wherein, the mass volume ratio of the high saline-alkali material to the water can be 1 g: (1.5-2.5) mL; the concentration of the high saline-alkaline material leach solution in the culture medium may be in the range 15% to 25%, for example 20%.
In the invention, the fermentation can adopt a solid state fermentation mode; the solid fermentation raw material comprises at least one of monosodium glutamate residue, alkaline hydrolysis seaweed residue and kitchen waste compost fermentation material. Specifically, the solid fermentation raw material comprises a material A and a material B, wherein the material A is monosodium glutamate residue, and the material B is alkaline hydrolysis seaweed residue or kitchen garbage compost fermentation material; the mass ratio of the material A to the material B in the solid fermentation raw material is (1.5-2.5): 1.
the solid state fermentation process is not critical and may include:
culturing the seedsAdding the liquid culture into solid fermentation raw material, wherein the inoculation amount is not less than 1 × 104cfu/g material, and culturing at the constant temperature of 28-30 ℃ for 7-10 days to obtain the saline-alkali resistant penicillium oxalicum agent; wherein, the inoculation amount of the seed liquid culture can be 15-25% (m/m).
The preparation method of the seed liquid culture can comprise the following steps:
mixing activated spore powder of domesticated penicillium oxalicum, a dispersing agent and sterile water to prepare a spore bacterium suspension;
inoculating the spore bacteria suspension into a sterilized liquid seed culture medium, and performing shake culture at constant temperature of 28-30 deg.C for 60-68h to obtain seed liquid culture.
Specifically, the dispersant may be glycerol or tween 80; spore concentration in spore bacterial suspension is not less than 3 × 107cfu/mL; the inoculum size of the spore bacterial suspension can be 2-4% (V/V).
Further, the preparation method of the saline-alkali resistant penicillium oxalicum agent of the invention further comprises the step S3: crushing, drying and screening the fermentation product to obtain a saline-alkali-resistant penicillium oxalicum agent; the effective viable count of the saline-alkali resistant penicillium oxalicum agent is not less than 2 multiplied by 109cfu/g, moisture content not higher than 15%.
The invention also provides a saline-alkali-resistant penicillium oxalicum agent prepared by the preparation method.
The invention also provides application of the saline-alkali resistant penicillium oxalicum agent in crop planting or saline-alkali soil improvement.
The implementation of the invention has at least the following advantages:
1. the invention domesticates the penicillium oxalicum CGMCC No.12628, and the salt and alkali resistant penicillium oxalicum obtained by domestication can still normally ferment to produce spores under the extreme environmental conditions of salt concentration of 10% and pH 12, breaks through the pH limit range of the growth of the penicillium oxalicum, enables high salt and alkali materials to be directly utilized as main fermentation raw materials under the conditions of no desalination and acid regulation, and solves the problem of difficult utilization of high salt and alkali waste materials;
2. the saline-alkali resistant penicillium oxalicum agent disclosed by the invention has the characteristic of high saline-alkali resistance, can be normally propagated and metabolized in an extreme saline-alkali environment, widens the limitation of penicillium oxalicum on application conditions, can be well applied to the improvement of extreme severe saline-alkali soil, and can reduce the pH value by more than 4.75% by singly applying the saline-alkali resistant penicillium oxalicum agent.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 test for domesticating salt and alkali resistance of Penicillium oxalicum
1. Domestication of saline-alkali tolerance of strain in plate culture
1) Preparation of culture medium for domestication of saline-alkali tolerance
The solid activation medium is: 200g of potato is cooked, filtered and clear juice is obtained, 20g of cane sugar and 20g of agar are obtained, the pH value is 11, and water is added to the clear juice to reach a constant volume of 1L.
Adding NaCl of 0%, 2%, 5%, 7% and 10% into the solid activation culture medium respectively, corresponding to pH conditions of 6, 8, 9, 10 and 11, obtaining solid culture medium with different saline-alkali concentration gradients, and sterilizing at 121 deg.C for 30min for later use.
2) Plate domestication for saline-alkali tolerance
Taking the penicillium oxalicum CGMCC No.12628 spore powder which is activated in advance, streaking in solid activated culture media with different saline-alkali concentration gradients, and culturing at constant temperature of 28 ℃ for 3-7 days. Taking a flat plate with 7% NaCl-pH10 and 10% NaCl-pH11 salt concentration as main observation objects, selecting mycelium or spore powder, continuously carrying out continuous passage for more than 50 times in a solid flat culture medium with the same concentration, and as the passage times increase, on a 10% NaCl-pH11 gradient flat plate, the initial mycelium grows slowly, cannot produce spores, the mycelium growth density increases in the later period, and the spore production time is obviously shortened, which indicates that the strain has obvious saline-alkali resistance after acclimation, collecting the flat plate spore powder subjected to final acclimation, and carrying out the next test for later use.
2. Domestication of saline-alkali tolerance of strain in shake flask
1) Preparation of liquid culture medium for domestication of saline-alkali tolerance
Preparing a liquid domestication culture medium: 10g of sucrose, 5g of yeast extract, 3g of magnesium sulfate, 200g of material C leachate, pH 12, and adding water to a constant volume of 1L;
preparing a solid domestication culture medium: 10g of sucrose, 5g of yeast extract, 3g of magnesium sulfate, 200g of material C leachate, 20g of agar powder and pH value of 11, and adding water to a constant volume of 1L;
preparing a material C leaching solution: the material A (monosodium glutamate slag) and the material B (kitchen garbage compost fermentation material) are mixed according to the weight ratio of 2:1(m/m) to obtain a material C; mixing the material C and water according to the proportion of 1: 2(m/v), fully stirring for 10-20min, standing overnight at 4-10 ℃, and sucking supernatant to obtain material C leachate for storage and later use;
the above culture medium was sterilized at 121 ℃ for 30 min.
2) Shake flask domestication experiment for saline-alkali tolerance
Inoculating a ring of spore powder preserved after the plate acclimation in the step 1 into a liquid culture medium, performing shake culture at 28 ℃ for 2-5 days until mycelium pellets are precipitated, inoculating the mycelium pellets into a solid acclimation culture medium plate, performing standing culture at 28 ℃ for 2-7 days until spores are produced in the plate, transferring the mycelium pellets into the liquid acclimation culture medium for culture, circulating the steps for more than 20 times, finishing the acclimation experiment, and preserving the finally obtained spore powder, namely the salt and alkali resistant penicillium oxalicum strain with the salt and alkali resistant property.
Example 2 method for producing saline-alkali-resistant penicillium oxalicum agent by fermentation under high saline-alkali condition
1. Activation of fermentation Strain
Strain solid activation medium: cooking 200g of potato, filtering to obtain clear juice, 20g of sucrose, 20g of agar, pH of the clear juice is 11, and adding water to a constant volume of 1L;
taking 1 ring of domesticated penicillium oxalicum spore powder, inoculating the domesticated penicillium oxalicum spore powder into a solid activation culture medium, and standing and culturing for 2-4 days at 28 ℃ to obtain the spore powder, namely the activator.
2. Liquid seed culture
Liquid seed culture medium: 10g of sucrose, 5g of yeast extract, 3g of magnesium sulfate, 200g of material C leachate, pH 12, and adding water to a constant volume of 1L;
preparing a material C leaching solution: the material A (the refined slag of the taste) and the material B (the kitchen garbage compost fermentation material) are mixed according to the proportion of 2:1(m/m) is mixed uniformly to form a material C, and the material C and water are mixed according to the proportion of 1: 2(m/v), fully stirring for 10-20min, standing overnight at 4-10 ℃, and taking supernatant as leachate of the material C;
preparing spore powder activated in the step 2 into spore suspension with sterile water, and counting the spore concentration by plate is not less than 3 × 108cfu/ml, inoculating in sterilized liquid seed culture medium according to 3% (V/V), and shaking at 28 deg.C for about 64h to obtain seed liquid.
3. Solid fermentation
Mixing a material A (monosodium glutamate slag) and a material B (kitchen garbage compost fermentation material) in a proportion of 2:1(m/m) to obtain a material C as a main raw material for solid fermentation, adding the cultured seed liquid culture into the material C according to the amount of 20% (m/m), and culturing at the constant temperature of 28-30 ℃ for 5-7 days to obtain the solid penicillium oxalicum agent fermentation product.
4. Fermentation treatment
Pulverizing the solid Penicillium oxalicum fermentation product, drying, and sieving to obtain Penicillium oxalicum effective viable count with water content not higher than 15%3×109cfu/g saline-alkali resistant penicillium oxalicum agent.
Comparative example 1
The same procedures as in examples 1 and 2 were repeated except that the material C was monosodium glutamate, thereby obtaining a salt and alkali resistant Penicillium oxalicum preparation of this comparative example.
Comparative example 2
The same procedure as in examples 1 and 2 was repeated except that the material C was fermented by composting with kitchen waste, to obtain a salt and alkali resistant Penicillium oxalicum preparation of this comparative example.
Test example 1 application Effect of salt and alkali resistant Penicillium oxalicum agent
The saline-alkali-resistant penicillium oxalicum agent of examples 1-2 and comparative examples 1-2 was applied to a rice pot, wherein the amount of the base fertilizer was 0.2g/kg of nitrogen and phosphorus (P)2O5)0.15g/kg of soil, potassium (K)2O)0.2g/kg soil, 5 multiplied by 10 of saline-alkali resistant penicillium oxalicum agent5The addition amount of cfu/g soil is directly stirred into the pot soil.
The experimental design is shown in table 1.
TABLE 1 Rice potting experiment design
| Grouping | Fertilization scheme |
| Control | Base fertilizer |
| Process 1 | Base fertilizer + existing Penicillium oxalicum agent (Hubei Qiming biological Co., Ltd.) |
| Treatment 2 | Base fertilizer + salt and alkali resistant Penicillium oxalicum agent of example 2 |
| Treatment 3 | Base fertilizer + saline-alkali-resistant penicillium oxalicum agent of comparative example 1 |
| Treatment 4 | Base fertilizer + saline-alkali-resistant penicillium oxalicum agent of control example 2 |
The detection items are as follows:
and (3) mature period: the plant total nitrogen, phosphorus, potassium and silicon contents; tillering number, root weight and yield.
The results are shown in tables 2 and 3.
TABLE 2 detection results of total nitrogen, phosphorus, potassium and silicon contents of rice plants in mature period
| Grouping | Nitrogen g/kg | Phosphorus g/kg | Potassium g/kg | Silicon g/kg |
| Control | 1.97 | 2.77 | 42.17 | 58.76 |
| Process 1 | 2.27 | 2.91 | 42.89 | 59.28 |
| Treatment 2 | 2.35 | 3.23 | 43.03 | 64.71 |
| Treatment 3 | 2.30 | 2.99 | 43 | 61.24 |
| Treatment 4 | 2.31 | 2.89 | 42.91 | 60.74 |
TABLE 3 tillering number, root weight, yield of rice plants at mature period
| Grouping | Root (dry weight) g | Tillering number (one/one) | Ear of grain (dry weight) g |
| Control | 8.2 | 2.35 | 15.93 |
| Process 1 | 10.7 | 2.52 | 17.77 |
| Treatment 2 | 12.05 | 2.87 | 18.92 |
| Treatment 3 | 11.5 | 2.63 | 17.91 |
| Treatment 4 | 11.24 | 2.74 | 18.25 |
Through comparison of application tests of rice, the saline-alkali-resistant penicillium oxalicum agent produced by the invention has an obvious growth promoting effect, and has obvious advantages compared with commercially available penicillium oxalicum agents.
Experimental example 2 application of salt and alkali resistant penicillium oxalicum agent in severe saline-alkali soil
1. Activation of fermentation Strain
Strain solid activation medium: cooking 200g of potato, filtering to obtain clear juice, 20g of cane sugar, 20g of agar, and 11 of pH, and adding water to a constant volume of 1L;
taking 1 ring of domesticated penicillium oxalicum spore powder, inoculating the domesticated penicillium oxalicum spore powder into a solid activation culture medium, and standing and culturing for 2-4 days at 28 ℃ to obtain the spore powder, namely the activator.
2. Liquid seed culture
Liquid seed culture medium: 10g of sucrose, 5g of yeast extract, 3g of magnesium sulfate, 200g of material C leachate, pH 12, and adding water to a constant volume of 1L;
preparing a material C leaching solution: the monosodium glutamate residue and the alkaline hydrolysis seaweed residue are mixed in a ratio of 2:1(m/m) is mixed uniformly to form a material C, and the material C and water are mixed according to the proportion of 1: 2(m/v), fully stirring for 10-20min, standing overnight at 4-10 ℃, and taking the supernatant as the leaching solution of the material C;
preparing spore powder activated in the step 2 into spore suspension with sterile water, and counting the spore concentration by plate is not less than 3 × 108cfu/ml, inoculating in sterilized liquid seed culture medium according to 3% (V/V), and shaking at 28 deg.C for about 64h to obtain seed liquid.
3. Solid fermentation
Taking material C (monosodium glutamate residue and alkaline hydrolysis seaweed residue mixed uniformly at a ratio of 2:1 (m/m)) as main raw material, adding cultured seed liquid culture at an amount of 20% (m/m) into material C, culturing at constant temperature of 28-30 deg.C and pH of 11.5 for 7-10 days to obtain solid penicillium oxalicum fermentation product with effective viable count of 2.9 × 109cfu/g, 2.7 × 10 by shaking after drying and air draft spore collection method10cfu/g of spore powder, namely the saline-alkali resistant penicillium oxalicum agent 1.
In the seed liquid culture and the solid fermentation, the material C only adopts monosodium glutamate slag to prepare the saline-alkali resistant penicillium oxalicum agent 2; in the seed liquid culture and solid fermentation, the material C only adopts alkaline hydrolysis seaweed residues to prepare the saline-alkali-resistant penicillium oxalicum agent 3.
4. Application of penicillium oxalicum agent
1) Test soil
The soil is taken from Dalatex region of inner Mongolia Ordos, the region has high south, high north and low terrain, the altitude is 1500m, the region belongs to temperate continental climate, the annual precipitation amount is 350mm, the region is concentrated in 6-9 months, the evaporation amount is larger, the annual evaporation amount reaches 2400mm, the humidity is 0.1-0.2, the heat is sufficient, the sunshine time is long, the annual sunshine is 3000h, the frost-free period is 135-The texture is silt loam, and the volume weight of the soil is 1.49g/cm3The field water retention rate is 22.3 percent (mass water content), and the soil belongs to severe saline-alkali soil.
2) Test method
Filling test soil into a beaker, wherein the concentration of the test soil is 800 g/beaker, and the total quantity is 15; the experimental design is shown in table 4.
Table 4 experimental design
| Grouping | Processing method |
| Control | Regular irrigation clear water (3) |
| Process 1 | Clean water + existing Penicillium oxalicum agent (3 pieces) |
| Treatment 2 | Clear water + salt and alkali resistant penicillium oxalicum agent 1 (3) |
| Treatment 3 | Clear water + salt and alkali resistant penicillium oxalicum agent 2 (3) |
| Treatment 4 | Clear water + salt and alkali resistant penicillium oxalicum agent 3 (3) |
The treatment 1 to 4 are respectively added with the existing penicillium oxalicum agent, the penicillium oxalicum agent 1 with saline-alkali resistance, the penicillium oxalicum agent 2 with saline-alkali resistance and the penicillium oxalicum agent 3 with saline-alkali resistance and are mixed evenly, and the effective bacteria number is not less than5×105cfu/g soil, clear water was poured once every 7 days, 150ml each time, and after 2 months, the change in the number of bacteria, the volume weight of soil, and the pH were measured, and the results are shown in Table 5.
TABLE 5 application results of salt and alkali resistant penicillium oxalicum agent in severe saline-alkali soil
The comparison shows that the existing penicillium oxalicum agent is difficult to survive and play a role in an extreme saline-alkaline soil environment; the survival rate of the saline-alkali-resistant penicillium oxalicum agent with the saline-alkali-resistant characteristic is obviously improved, the pH value is obviously reduced, the pH value can be reduced by 4.75% by using a single agent, and the effect of the modifying agent can be achieved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of a saline-alkali-resistant penicillium oxalicum agent is characterized by comprising the following steps:
s1: domesticating penicillium oxalicum with the preservation number of CGMCC No. 12628;
s2: fermenting the domesticated penicillium oxalicum under a high saline-alkali condition;
preferably, the high saline alkali conditions include: the salt concentration is more than 10% and the pH value is more than 11.
2. The method of claim 1, wherein the acclimation comprises saline-alkali tolerant acclimation; the saline-alkali tolerant domestication comprises the following steps: adopting culture mediums with different salt concentration gradients and alkali concentration gradients to carry out domestication culture on the penicillium oxalicum;
preferably, the salt concentration gradient is 0%, 2%, 5%, 7%, 10% NaCl; the alkali concentration gradient was pH6, pH8, pH9, pH10, pH 11.
3. The method of claim 2, wherein the acclimatization further comprises directional acclimatization with salt and alkali tolerance; the saline-alkali tolerant directional domestication comprises domestication culture of penicillium oxalicum by using a culture medium added with a high saline-alkali material leachate, wherein the high saline-alkali material comprises at least one of monosodium glutamate residue, alkaline hydrolysis seaweed residue and kitchen garbage compost fermentation material;
preferably, the high-saline-alkali material comprises a material A and a material B, wherein the material A is monosodium glutamate slag, and the material B is alkaline hydrolysis seaweed slag or kitchen garbage compost fermentation material;
preferably, the mass ratio of the material A to the material B in the high-saline-alkali material is (1.5-2.5): 1.
4. a process according to claim 3, wherein the high salinity and alkalinity feed leachate is produced by a process comprising: mixing the high-saline-alkali material with water, stirring, standing, and taking supernatant to obtain high-saline-alkali material leachate;
preferably, the mass volume ratio of the high saline-alkali material to the water is 1 g: (1.5-2.5) mL;
preferably, the concentration of the high saline-alkali material leaching solution in the culture medium is 15-25%.
5. The method according to claim 1, wherein the fermentation is a solid state fermentation; the solid fermentation raw material comprises at least one of monosodium glutamate residue, alkaline hydrolysis seaweed residue and kitchen waste compost fermentation material;
preferably, the solid fermentation raw material comprises a material A and a material B, wherein the material A is monosodium glutamate residue, and the material B is alkaline hydrolysis seaweed residue or kitchen garbage compost fermentation material;
preferably, the mass ratio of the material A to the material B in the solid fermentation raw material is (1.5-2.5): 1.
6. the method of claim 5, wherein the solid state fermentation comprises:
adding the cultured seed liquid culture into solid fermentation raw material, wherein the inoculation amount is not less than 1 × 104cfu/g material, and culturing at the constant temperature of 28-30 ℃ for 7-10 days to obtain the saline-alkali resistant penicillium oxalicum agent.
7. The method according to claim 6, wherein the method for preparing a liquid culture of seeds comprises:
mixing activated spore powder of domesticated penicillium oxalicum, a dispersing agent and sterile water to prepare a spore bacterium suspension;
inoculating the spore bacteria suspension into a sterilized liquid seed culture medium, and performing shake culture at a constant temperature of 28-30 ℃ for 60-68h to obtain a seed liquid culture;
preferably, the dispersant is glycerol or tween 80;
preferably, the spore concentration in the spore bacterial suspension is not less than 3X 107cfu/mL。
8. The method according to claim 1, further comprising step S3: crushing, drying and screening the fermentation product to obtain a saline-alkali-resistant penicillium oxalicum agent;
preferably, the effective viable count of the saline-alkali-resistant penicillium oxalicum agent is not less than 2 multiplied by 109cfu/g；
Preferably, the saline-alkali-resistant penicillium oxalicum agent has a moisture content not higher than 15%.
9. A salt and alkali resistant penicillium oxalicum agent, characterized by being prepared by the preparation method according to any one of claims 1 to 8.
10. The use of the saline-alkali tolerant penicillium oxalicum agent of claim 9 in crop planting or saline-alkali soil improvement.
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| CN106434389A (en) * | 2016-12-06 | 2017-02-22 | 领先生物农业股份有限公司 | Penicillium sp. LX-ZJ-2 and application thereof |
| WO2021196693A1 (en) * | 2020-04-03 | 2021-10-07 | 河北科技大学 | Penicillium oxalicum sdf-25 and use thereof |
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| CN106434389A (en) * | 2016-12-06 | 2017-02-22 | 领先生物农业股份有限公司 | Penicillium sp. LX-ZJ-2 and application thereof |
| WO2021196693A1 (en) * | 2020-04-03 | 2021-10-07 | 河北科技大学 | Penicillium oxalicum sdf-25 and use thereof |
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