CN110564624A - high-salt-and-alkali-resistance penicillium chrysogenum and separation method and application thereof - Google Patents
high-salt-and-alkali-resistance penicillium chrysogenum and separation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high saline-alkali resistant penicillium chrysogenum and a separation method and application thereof, wherein 250.0g/L corn straw powder culture medium solid plates containing NaCl with different concentrations are coated with straw powder suspension liquid subjected to gradient dilution, the treatment is repeated for 3 times, a single colony growing well is picked after being cultured for 4-15 days, and the single colony is inoculated on a screening culture medium CMC-Na solid culture medium to generate a transparent ring-shaped strain, so that the penicillium chrysogenum is obtained, and the preservation name is as follows: penicillium chrysogenum W2; the preservation number is: CGMCC NO. 17972; the high-saline-alkali-resistance penicillium chrysogenum has high anti-permeability characteristic, is favorable for improving the degradation capability of straws in a high-saline-alkali environment, can be directly used for decomposing biological substances such as straw fermentation in fields and the like, and has good effect on improvement of secondary salinization or continuous cropping obstacle soil and environmental pollution treatment.
Description
Technical Field
The invention relates to the field of microbiological engineering and technology, in particular to high-saline-alkali-resistance penicillium chrysogenum and a separation method and application thereof.
Background
The straw resources in China are rich and have wide sources, and the sources are the first in the world, particularly the crop straw yield is rapidly increased along with the great increase of the grain yield since the agricultural reform, so that the straw resources become important sources of the straw resources in China. With the continuous development of science and technology, the utilization field of the straws is greatly expanded, for example, the straws can be used as fuel, feed, base materials (seedling culture substrates), industrial raw materials and the like. But the comprehensive utilization rate of the resource of the crop straws is still low at present, the crop straws are mostly treated coarsely and even directly burnt in the field, so that the serious pollution is caused to the environment, the loss of a large amount of fertilizers is also meant, and the wide attention of the whole society is attracted;
The high saline-alkali resistant fungi are microbial resources separated from high-salt, high-alkali or high-saline-alkali coexisting environment, and have important research, application and development values, so that the high saline-alkali resistant fungi have great significance in excavation, development and utilization. At present, people apply the high saline-alkali resistant fungi to environmental management, such as restoration and transformation of saline-alkali soil, purification treatment of sludge and sewage, cyclic utilization of agricultural wastes and the like;
The microbial degradation of straws and returning to the field is an important measure for improving soil, particularly for repairing saline-alkali soil, but most microbial agents on the market can play a good role in normal soil at present, in the saline-alkali soil, due to high-concentration salt and high pH value environment, the strains can hardly play due roles, and in order to efficiently degrade the straws of the saline-alkali soil in a short time and realize fertilizer returning to the field, the screening of high-saline-alkali resistant fungi from the saline-alkali soil is necessary, so that the invention provides the high-saline-alkali resistant penicillium chrysogenum and the separation method and application thereof, and the defects in the prior art are overcome.
Disclosure of Invention
Aiming at the problems, the invention provides high-saline-alkali-resistance penicillium chrysogenum and a separation method and application thereof, and the high-saline-alkali-resistance penicillium chrysogenum has high anti-permeability characteristic, is favorable for improving the degradation capability of straws in a high-saline-alkali environment, can be directly used for decomposing biological substances such as straw fermentation in fields and the like, and has good effects on improvement of secondary salinization or continuous cropping obstacle soil and environmental pollution treatment.
The invention provides high-saline-alkali-resistance Penicillium chrysogenum, which is preserved in the China general microbiological culture Collection center in 2019, 06, 14 th month, and the preservation unit address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation name is: penicillium chrysogenum W2; and (3) classification and naming: penicillium chrysogenum (penicillium chrysogenum); the preservation number is: CGMCC NO. 17972.
A separation method of high saline-alkali resistant penicillium chrysogenum comprises the following steps:
the method comprises the following steps: coating the straw powder suspension subjected to gradient dilution on a corn straw powder culture medium solid plate containing NaCl with different concentrations of 250.0g/L, repeating the treatment on each corn straw powder culture medium solid plate for 3 times, and then culturing for 4-15 days, and then selecting a well-grown single colony for storage and later use;
Step two: inoculating the single colony which is picked in the step one and is preserved for later use on a screening culture medium CMC-Na solid culture medium until a transparent ring-shaped strain is generated;
Step three: and D, preserving the transparent ring-shaped strain generated in the step two, and identifying an ITS sequence to obtain an identification result.
The further improvement lies in that: the corn straw powder culture medium in the first step is composed of 10g of corn straw powder and 4g of (NH)4)2SO4、0.5gMgSO4·7H2O、2gKH2PO41g of peptone, 16g of agar and 1000mL of deionized water.
The further improvement lies in that: the straw powder suspension in the first step is a straw soil surface layer soil sample with the thickness of 4-6cm and containing a straw sample, and the straw powder suspension is obtained after the straw soil surface layer soil sample is stored at 4 ℃, diluted and supernatant liquid is taken.
The further improvement lies in that: the pH value of the corn straw powder culture medium in the first step is 5.0-10.0.
the further improvement lies in that: before ITS sequence identification, morphological identification is required for the transparent ring-shaped strain in the third step, and the specific process is as follows: and (4) inoculating the transparent ring-shaped strain obtained by screening in the step two to a corn straw powder culture medium containing 5% of NaCL, culturing for 5 days at the ambient temperature of 30 ℃, and observing the colony morphology.
The further improvement lies in that: the concrete process of carrying out ITS sequence identification on the transparent circled strain in the third step is as follows: and submitting the amplified ITS sequence data to GenBank, and performing homology comparison by using online software Blast on an NCBI website to obtain an identification result.
The further improvement lies in that: after the ITS sequence identification is carried out on the transparent ring-shaped strain in the third step, the identification of high salt resistance and optimal salt concentration, the measurement of the growth PH of the strain, the measurement of the cellulase production activity of the strain and the measurement of the straw degradation effect of the strain are also required to be carried out on the strain.
An application of high-saline-alkali-resistance penicillium chrysogenum in saline-alkali soil reformation.
An application of high-saline-alkali-resistance penicillium chrysogenum in degradation of straws in environmental pollution treatment.
The invention has the beneficial effects that: the high-saline-alkali-resistance penicillium chrysogenum has high anti-permeability characteristic, is favorable for improving the degradation capability of straws in a high-saline-alkali environment, can be directly used for decomposing biological substances such as straw fermentation in fields and the like, and has good effect on improvement of secondary salinization or continuous cropping obstacle soil and environmental pollution treatment.
drawings
FIG. 1 is a diagram showing morphological characteristics of a Penicillium chrysogenum colony in example II of the present invention.
FIG. 2 is a schematic diagram of conidiophores and conidia according to the second embodiment of the present invention
FIG. 3 is a schematic diagram of straw degradation effect of the strain in the fifth embodiment of the present invention.
Detailed Description
In order to further understand the present invention, the following detailed description will be made with reference to the following examples, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example one
A separation method of high saline-alkali resistant penicillium chrysogenum comprises the following steps:
The method comprises the following steps: coating a straw powder suspension subjected to gradient dilution on a 250.0g/L corn straw powder culture medium solid plate containing NaCl with different concentrations, wherein the corn straw powder culture medium comprises 10g of corn straw powder and 4g of (NH)4)2SO4、0.5gMgSO4·7H2O、2gKH2PO4The culture medium comprises 1g of peptone, 16g of agar and 1000mL of deionized water, wherein the pH value of a rice straw powder culture medium is 6.5, a straw powder suspension is a straw soil surface layer soil sample which is 5cm thick and contains a straw sample, the straw soil surface layer soil sample is stored and diluted at 4 ℃, a supernatant is taken to obtain the straw powder suspension, the treatment on each corn straw powder culture medium solid plate is repeated for 3 times, and then a well-grown single bacterial colony is selected and stored for later use after being cultured for 4 days;
step two: inoculating the single colony which is picked in the step one and is preserved for later use on a screening culture medium CMC-Na solid culture medium until a transparent ring-shaped strain is generated;
step three: preserving the transparent ring-shaped strain generated in the step two, and then carrying out ITS sequence identification to obtain an identification result which is penicillium chrysogenum;
the ITS sequence identification specific process is as follows: the amplified ITS sequence data is submitted to GenBank, homology comparison is carried out by using online software Blast on NCBI website, the homology of the strain and Aspergillus strain MG647847.1 reaches 99%, a phylogenetic tree is constructed by using Maximum likelihood method in bioinformatics analysis software MEGA 7.0, and the strain is penicillium chrysogenum.
Example two
according to FIGS. 1 and 2, the present example proposes the morphological identification of the transparent circle-shaped strain:
Inoculating the screened transparent ring-shaped strain to a corn straw powder culture medium containing 5% NaCL, culturing for 5 days at the environment temperature of 30 ℃, and observing the colony morphology, wherein the specific process comprises the following steps: clamping a sterilized cover glass by using tweezers, obliquely inserting the cover glass into the middle of a corn straw powder culture medium containing 5% NaCL, inoculating a bacterial transparent ring-shaped strain with the diameter of 7mm (the hypha surface of the strain faces downwards) in front of the cover glass, then placing a flat plate in a constant-temperature incubator at 30 ℃, regularly observing the growth condition of the hypha, cutting the culture medium connected with the cover glass after the hypha climbs on the cover glass, taking out the cover glass, observing the shapes of the hypha and spores under a microscope, after culturing for 9 days on the corn straw powder culture medium containing 5% NaCL at 30 ℃, ensuring that the diameter of a circular bacterial colony is about 55.56mm, the mycelium at the edge is white, a large amount of grey-green spores are generated, the structure of the bacterial colony is felty, the edge is ciliated, the reverse side of the bacterial colony is light yellow brown, and the color of the corn straw powder culture medium is unchanged in the culture process (as shown in figure;
Microscopic observation results show that: conidiophores are generally brood-shaped and three-wheeled, the top of the conidiophores is slightly enlarged, the phialides are in the shape of ampoules, the conidiophores are (nearly) spherical, (nearly) elliptical, light green and smooth, the conidiophores are slightly forked into loose columns (as shown in figure 2), and the conidiophores are morphologically identified as penicillium chrysogenum.
EXAMPLE III
This example provides the high salt resistance and optimum salt concentration identification of Penicillium chrysogenum W2: preparing a corn straw powder culture medium with gradient NaCl content (the PH is 7.0), increasing the salt concentration (mass fraction) from 0% to 31% (saturation concentration), increasing each level by 2%, inoculating equivalent strains at the center of the culture medium, and culturing for 1 week at 30 ℃, and finding that the strains can grow in the corn straw powder culture medium with 0% -31% NaCl at the temperature of 30 ℃, but the growth of the strains is obviously inhibited along with the increase of the salt concentration, and the strains grow relatively slowly at the concentration of 1% -5%; the growth of the strain is accelerated under the concentration of 5-11%, the growth of the strain is rapidly slowed down under the concentration of 15-25%, and the thallus basically does not grow under the concentration of 27-31%, so that the strain has the salt resistance and belongs to fungi with high salt resistance;
Culturing the strain with serial salt gradient PD (potato liquid culture medium without agar and with pH of 7.0) used in the identification process of high salt resistance, shaking at 25 deg.C for one week, centrifuging to collect mycelium, repeatedly rinsing with deionized water for 4 times, oven drying at 105 deg.C to determine dry weight of the mycelium, and comparing to find that the dry weight of the strain is maximum at 5-10% concentration and the salt concentration for optimum growth is 5-10%.
Example four
this example presents a strain growth PH assay: preparing an acid-base gradient corn straw powder culture medium with the pH value of 1.0-13.0, adding NaCl until the final concentration (mass fraction) is 5%, then inoculating the strain, culturing at 30 deg.C, observing to find that the growth time of the strain on corn straw powder culture medium with different pH values is different, culturing for 1 day, hypha growth can be seen on a corn straw powder culture medium with the pH value of 3.0-8.0, hypha growth can be seen in the corn straw powder culture medium with the pH value of 9.0 after 2 days of culture, hypha growth can be seen in the corn straw powder culture medium with the pH value of 10.0 after 5 days of culture, hypha growth can be seen only in the corn straw powder culture medium with the pH value of 11 after 7 days of culture, in a corn straw powder culture medium with the pH value of 1.0, 2.0, 12.0 and 13.0, hypha growth is not seen all the time, so that the growth pH range of the strain is 3.0-11.0;
in order to determine the optimum pH range for the growth of the strain, the strain was further cultured in PD broth using the above pH gradient, after shaking at 30 ℃ for 1 week, mycelia were collected by centrifugation, washed repeatedly with deionized water 4 times, and then dried at 105 ℃ to compare the dry weight of the mycelia, and the results showed that the strain had the largest dry weight of the cells in the pH range of 4.0 to 9.0, and thus the optimum pH range of the strain was 4.0 to 9.0.
EXAMPLE five
According to FIG. 3, this example proposes the determination of cellulase production activity and straw degradation effect of strains: an equal amount of the strain was inoculated into an enzyme-producing medium (composed of 5.0g/L of CMC-Na, 0.5g/L of yeast extract powder, 0.5g/L of enzymatically hydrolyzed casein, 1.0g/L of Ca (NO)3)2Acid hydrolysis casein of 0.5g/L, K of 0.2g/L2HPO40.25g/L MgSO4·7H2O0.25g/L and pH 7.0), shake culturing at 30 deg.C for 4 days, and determining cellulase production activity by DNA method, the result shows that 0% NaCl is used to produce cellulaseCulturing for 4 days by using an enzyme culture medium, wherein the enzyme activity reaches 43.73U/mL, and culturing for 4 days by using a 5% NaCl enzyme production culture medium, wherein the enzyme activity reaches 82.67U/mL;
Placing 0.6g of crushed straw (about 5mm in length) in 50mL conical flask, sterilizing at 121 deg.C for 20min, inoculating 3mL spore suspension into each conical flask, and adjusting the concentration of spore suspension to 1 × 105-1×107Culturing in 30 ℃ incubator, observing growth condition periodically, and after culturing for 5 days, finding that the strain can grow on the straw, which shows that the strain can degrade the straw (the result is shown in figure 3).
the invention has the beneficial effects that: the high-saline-alkali-resistance penicillium chrysogenum has high anti-permeability characteristic, is favorable for improving the degradation capability of straws in a high-saline-alkali environment, can be directly used for decomposing biological substances such as straw fermentation in fields and the like, and has good effect on improvement of secondary salinization or continuous cropping obstacle soil and environmental pollution treatment.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The high-salt-alkali-resistance penicillium chrysogenum is characterized in that: the high-saline-alkali-resistance penicillium chrysogenum is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms in 2019, in 06, 14 months, and the preservation unit address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation name is: penicillium chrysogenum W2; and (3) classification and naming: penicillium chrysogenum (penicillium chrysogenum); the preservation number is: CGMCC NO. 17972.
2. The method for separating the high saline-alkali resistance penicillium chrysogenum applied to the claim 1 is characterized in that: the method comprises the following steps:
The method comprises the following steps: coating the straw powder suspension subjected to gradient dilution on a corn straw powder culture medium solid plate containing NaCl with different concentrations of 250.0g/L, repeating the treatment on each corn straw powder culture medium solid plate for 3 times, and then culturing for 4-15 days, and then selecting a well-grown single colony for storage and later use;
Step two: inoculating the single colony which is picked in the step one and is preserved for later use on a screening culture medium CMC-Na solid culture medium until a transparent ring-shaped strain is generated;
step three: and D, preserving the transparent ring-shaped strain generated in the step two, and identifying an ITS sequence to obtain an identification result.
3. The method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: the corn straw powder culture medium in the first step is composed of 10g of corn straw powder and 4g of (NH)4)2SO4、0.5gMgSO4·7H2O、2gKH2PO41g of peptone, 16g of agar and 1000mL of deionized water.
4. The method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: the straw powder suspension in the first step is a straw soil surface layer soil sample with the thickness of 4-6cm and containing a straw sample, and the straw powder suspension is obtained after the straw soil surface layer soil sample is stored at 4 ℃, diluted and supernatant liquid is taken.
5. the method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: the pH value of the corn straw powder culture medium in the first step is 5.0-10.0.
6. The method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: before ITS sequence identification, morphological identification is required for the transparent ring-shaped strain in the third step, and the specific process is as follows: and (4) inoculating the transparent ring-shaped strain obtained by screening in the step two to a corn straw powder culture medium containing 5% of NaCL, culturing for 5 days at the ambient temperature of 30 ℃, and observing the colony morphology.
7. The method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: the concrete process of carrying out ITS sequence identification on the transparent circled strain in the third step is as follows: and submitting the amplified ITS sequence data to GenBank, and performing homology comparison by using online software Blast on an NCBI website to obtain an identification result.
8. The method for separating high saline-alkali resistant penicillium chrysogenum according to claim 2, wherein: after the ITS sequence identification is carried out on the transparent ring-shaped strain in the third step, the identification of high salt resistance and optimal salt concentration, the measurement of the growth PH of the strain, the measurement of the cellulase production activity of the strain and the measurement of the straw degradation effect of the strain are also required to be carried out on the strain.
9. The use of the high saline-alkali resistance Penicillium chrysogenum of claim 1 in saline-alkali land improvement.
10. The use of the high saline-alkali resistant penicillium chrysogenum of claim 1 for straw degradation in environmental pollution treatment.
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| CN114381378A (en) * | 2021-12-27 | 2022-04-22 | 农业农村部环境保护科研监测所 | Penicillium chrysogenum capable of degrading lignin and application thereof |
| CN116286394A (en) * | 2023-02-13 | 2023-06-23 | 安徽农业大学 | Penicillium chrysogenum, microbial agent and application thereof |
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| CN110564625A (en) * | 2019-08-13 | 2019-12-13 | 内蒙古世洪农业科技有限公司 | Saline-alkali resistant aspergillus flavus and separation method and application thereof |
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| CN114381378A (en) * | 2021-12-27 | 2022-04-22 | 农业农村部环境保护科研监测所 | Penicillium chrysogenum capable of degrading lignin and application thereof |
| CN114381378B (en) * | 2021-12-27 | 2023-05-26 | 农业农村部环境保护科研监测所 | Penicillium chrysogenum for degrading lignin and application thereof |
| CN116286394A (en) * | 2023-02-13 | 2023-06-23 | 安徽农业大学 | Penicillium chrysogenum, microbial agent and application thereof |
| CN116286394B (en) * | 2023-02-13 | 2023-12-15 | 安徽农业大学 | Penicillium chrysogenum, microbial agent and application thereof |
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