CN108949582B - High-cadmium-resistance oyster mushroom strain and application thereof - Google Patents
High-cadmium-resistance oyster mushroom strain and application thereof Download PDFInfo
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- CN108949582B CN108949582B CN201810815151.0A CN201810815151A CN108949582B CN 108949582 B CN108949582 B CN 108949582B CN 201810815151 A CN201810815151 A CN 201810815151A CN 108949582 B CN108949582 B CN 108949582B
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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
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
Abstract
The invention discloses a high-cadmium resistant oyster mushroom strain, the preservation number of which is CGMCC NO. 16084. The invention also discloses application of the strain in repairing cadmium-polluted soil, wherein a part of fungus bags of fungus sticks inoculated with the strain are removed and then transplanted into the soil, the part of the removed fungus bags are in the soil, and the part of the removed fungus bags are exposed outside the soil.
Description
Technical Field
The invention relates to the technical field of heavy metal pollution remediation, in particular to a high-cadmium-resistance oyster mushroom strain and application thereof in remediation of cadmium-polluted soil.
Background
With the rapid development of modern industry and agriculture, the condition that soil in China is polluted by heavy metal is more and more serious, wherein cadmium is one of the most main elements in heavy metal pollution. Cadmium is a highly toxic pollutant, and in recent years, many cadmium pollution events have occurred in China, and the health of human beings is seriously influenced.
In recent years, the cadmium-containing soil is repaired by using biotechnology, and the cadmium-containing soil is safer and more effective than chemical and physical repair methods. At present, the biological remediation of the cadmium-containing soil mainly depends on plant varieties, the cadmium content in the soil is reduced by screening and culturing the plant varieties with high cadmium resistance and utilizing the absorption of cadmium elements in the growth process of the plant varieties, so that the remediation of the cadmium-containing soil is realized. Meanwhile, the plant varieties with high-efficiency heavy metal enrichment characteristic and high growth speed are few, the screening and culturing process generally consumes a long time, and the cost is high.
In addition, for alkaline soil in some northern areas, heavy metals generally exist in a chelated state, the metal effectiveness is low, efficient biological removal is difficult, and the restoring capability of plant varieties with cadmium enrichment characteristics to the soil is not ideal.
Disclosure of Invention
Aiming at the technical current situation, the invention provides the oyster mushroom strain with high cadmium resistance, which can overcome the problems in the prior art and efficiently repair cadmium-containing soil, particularly alkaline soil with low metal effectiveness, and further provides the application of the strain in repairing cadmium-containing soil.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-cadmium resistant Pleurotus Ostreatus strain is registered and preserved in China general microbiological culture Collection center (CGMCC) at 7/6.2018 with the preservation number of CGMCC NO. 16084.
The invention also provides application of the oyster mushroom strain in repairing cadmium-polluted soil.
Further, the soil is alkaline soil.
Further, the soil has a pH of 8.5 or more.
The oyster mushroom strain can efficiently repair cadmium-containing soil, particularly alkaline soil with low heavy metal effectiveness, and the oyster mushroom strain can dissociate chelated cadmium for enrichment and absorption, so that the polluted soil can be repaired. When the pH value of the soil is more than 8.5, the heavy metals in the soil are difficult to absorb by common crops, the repairing difficulty is high, and the strain disclosed by the invention also has a good repairing effect on the soil with the pH value of more than 8.5. For acid and neutral soil, the heavy metal effectiveness is higher than that of alkaline soil, and the bacterial strain can also efficiently repair the acid and neutral soil.
The invention also provides a method for repairing cadmium-polluted soil by using the oyster mushroom strain, which comprises the following steps: inoculating the oyster mushroom strain into a PDA culture medium for activation, taking an inoculating block from the activated strain culture medium, inoculating the inoculating block into the PDA culture medium, culturing to obtain a mycelium, inoculating the mycelium into a culture medium for culturing to obtain a culture mushroom stick, performing primordium induction on the mushroom stick, and transferring the mushroom stick into cadmium-polluted soil after the primordium appears for fruiting management.
Wherein, the PDA culture medium is a conventional culture medium in the field, and the preparation method comprises the following steps: boiling peeled potato 200g, filtering to obtain juice, adding glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 0.5g, agar 15g, distilled water to volume of 1000mL, boiling, and autoclaving at 121 deg.C for 30 min.
The culture substrate is also a conventional oyster mushroom culture substrate, and the substrate adopted in the invention comprises:
the solid raw material comprises the following components in percentage by mass: 78% of cottonseed hulls, 20% of bran, 1% of gypsum and 1% of sucrose;
and water in an amount of 1.6 times the mass of the solid raw material.
Preferably, the primordial induction is: the opening is formed in the opening of the fungus bag, directional primordium induction is carried out at the opening, sporophores grow directionally from the opening, growth vigor is better, and the enrichment rate of cadmium is higher.
Preferably, when the fungus stick is moved into cadmium-containing soil, removing part of the fungus bags, burying the part, which is used for removing the fungus bags, in the soil, exposing the part, which is not used for removing the fungus bags, out of the soil, further preferably, the height ratio of the removed fungus bags to the parts, which are not used for removing the fungus bags, is 1-3: 1, and efficient enrichment and absorption of cadmium are realized on the premise of ensuring normal fruiting.
The oyster mushroom strain has high cadmium tolerance, can grow mushroom normally in soil with high cadmium content, can enrich and absorb cadmium in the growth process, reduces the cadmium content in the soil, has the theoretical cadmium extraction efficiency of fruit bodies and mushroom sticks of 255 g/mu/year, realizes the remediation of cadmium-polluted soil, particularly has good remediation effect on alkaline soil with low heavy metal effectiveness, can also obviously reduce the pH value of the soil, and provides conditions for the remediation of soil with multiple varieties of crops. The oyster mushroom strains screened by the method are popular varieties, have wide culture conditions, and have the advantages of low cost and good effect when being used for repairing high-cadmium soil.
The method for restoring the cadmium-polluted soil by using the bacterial strain is simple and easy to operate, primordium induction is carried out on the opening at the pricked position of the bacterial bag, the bacterial stick is directly transplanted into the soil after the primordium is generated, efficient enrichment is also realized in the bacterial stick through the growth activity of the bacterial strain, and the soil restoration efficiency is further improved.
Drawings
FIG. 1 is the effect of the strains of the invention on soil pH;
FIG. 2 is a graph of the concentration of cadmium enriched neutron entities at different harvest tide numbers;
FIG. 3 is a graph of the aggregate cadmium enrichment of neutron entities at different harvest tide numbers;
FIG. 4 is a graph of cadmium concentration in soil harvest plugs for different cadmium treatment concentrations;
FIG. 5 is a variation trend of cadmium concentration in soil during fruiting process;
FIG. 6 shows the fruiting in soil with different cadmium treatment concentrations.
Detailed Description
In order to make the purpose, technical scheme and advantages of the invention more clear and clear, the cadmium enrichment performance of the strain of the invention is illustrated below by taking the remediation of cadmium-contaminated soil as an example. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A test material
1 test strains
The oyster mushroom strain CGMCC NO. 16084.
2 test soil sampling
Soil for covering soil is collected from a test field in the institute of agricultural resources, institute of genetics and developmental biology, academy of sciences of China.
Two test methods
1 soil treatment
1.1 soil preparation
Air drying the soil, grinding, sieving with a 2mm sieve, and removing impurities from the soil.
1.2 soil pH determination
5g of dry soil is taken in a 50mL centrifuge tube, 25mL of ultrapure water is added, the mixture is inverted from top to bottom for several times and then is kept stand for 1h, and when the solution is clear, a pH meter is used for measuring the pH value in a supernatant.
1.3 maximum Water holding Capacity determination
50g of the prepared soil were taken in a funnel with filter paper, a hose was added under the funnel, and the hose was held by a clip. 50mL of water was poured into the soil, and after the water was equilibrated in the soil for 1 hour, the clamp was opened to allow the water to naturally flow out, and collected. And finishing water collection when the water does not drip out. Maximum water holding capacity of soil (initial water addition volume-final water collection volume)/initial soil mass.
1.4 soil Cd treatment
Accurately weighing a certain amount of soil, adding Cd solution, and fully mixing to ensure that the Cd content of the soil is 0, 10 and 150mg-1. And (5) after the soil is balanced for 30 days, performing pH determination on the soil with each Cd concentration for later use.
2 preparation of Strain
2.1 preparation of PDA Medium (1L)
Peeling Potato (Potato)200g, boiling, filtering to obtain juice, adding glucose (Dextrose)20g, and potassium dihydrogen phosphate (KH)2PO4)2g magnesium sulfate (MgSO)4)0.5g, 15g of Agar (Agar), and distilled water to a constant volume of 1000mL, boiling, and autoclaving at 121 ℃ for 30 min.
2.2 Strain activation: inoculating the strain to the center of PDA culture medium, and culturing at 28 deg.C in dark for 5 days.
2.3 mycelium yield: inoculating 0.5cm diameter inoculating block in PDA culture medium, and culturing at 28 deg.C in dark for 7 days to obtain mycelium material.
3 preparation of the fungus stick
3.1 formula and preparation of culture medium
Uniformly mixing 78% of cottonseed hulls, 20% of bran, 1% of gypsum and 1% of cane sugar to obtain a solid raw material, adding water which is 1.6 times of the solid raw material, fully mixing uniformly, naturally standing for 30min to enable water to fully permeate into a matrix, drying a small amount of mixed matrix, measuring the content of Cd, putting the rest of mixed matrix into polypropylene dog-ear bags of 130 × 240 × 0.045.045 mm, compacting as much as possible, punching the center of the matrix, fastening the bag opening, enabling the wet weight of each bag of matrix to be 350 +/-5 g, and autoclaving at 121 ℃ for 100 min.
3.2 inoculation and culture of the fungus sticks
And (3) after the substrate is cooled, inoculating the cultured inoculation block containing the oyster mushroom mycelia into the substrate, tying the mushroom bag, and culturing at the constant temperature of 28 ℃ in a dark place for 25 days to obtain the cultured mushroom sticks.
4 potted plant
4.1 preparation of the fungus sticks
The fungus sticks full of the fungus are moved into a mushroom house, the temperature of the mushroom house is 18 ℃, the humidity is 80-85%, and the mushroom house is suitable for 2 days. Opening the fungus stick at the tying position for primordium induction. The temperature of the primordium induction is 18 ℃, the humidity is 80-85%, the primordium is ventilated for 1 hour in the morning and evening every day, and the temperature is 5-8 ℃ and the humidity is about 50% during ventilation. And (5) performing potting treatment when the primordium appears.
4.2 potting treatment
Selecting bacteria sticks with consistent primordium size, cutting 2/3 bacteria bags, transferring into a flowerpot with the inner diameter of 210 × 160mm, placing in the center of the flowerpot, filling Cd in the rest part of the flowerpot to treat balanced soil, adopting a semi-underground cultivation mode for the bacteria sticks, exposing the part without removing the bacteria bags outside, adding 1080g of balanced dry soil into each pot, adding water to reach 65% of the maximum water holding capacity, inserting a soil water collector into the soil 1cm away from the bacteria sticks, inserting 1 bacteria stick into each pot, harvesting the fruiting bodies from the first day when the bacteria sticks enter the soil to the third crop, and taking 5mL of soil water solution every 7d for measuring the Cd content and organic carbon of the soil water solution, wherein each Cd treatment concentration is set to be 3 times.
4.3 potted plant culture
And (4) after the fungus sticks are buried, performing fruiting management. Keeping the temperature of the mushroom growing room at 18 ℃ and the humidity of 85-95 percent, and ventilating once in the morning and evening, each time for 0.5 h. And (4) after harvesting the fruiting bodies of each tide, numbering the fruiting bodies, placing the numbering on the fruiting bodies in an oven, drying, weighing, and simultaneously performing primordium induction and fruiting management on the next tide of mushrooms. After harvesting the three-tide sporocarp, taking out the fungus stick from the soil and drying the fungus stick for measuring the content of Cd; and collecting and drying soil in each basin, and measuring pH.
Analysis of 5Cd content
And (3) taking 0.3g of each dried fruiting body for processing each harvested stubble, the culture medium before inoculation and the culture medium after harvesting, transferring the fruiting bodies, the culture medium before inoculation and the culture medium after harvesting into a digestion tube, adding 8mL of 65% nitric acid (premium grade purity), soaking overnight, placing on an electric heating plate for digestion, adding a funnel for digestion for 24 hours, then dispelling acid at high temperature, supplementing 5mL of 65% nitric acid (premium grade purity), and continuously heating until the mixture is evaporated to dryness. Dissolved in 5mL of ultrapure water. Each sample was digested in 3 replicates. Analysis of Cd content was measured by flame method using Zeenit 700P atomic absorption spectrophotometer (Analytik Jena, Germany).
6 data analysis
Data analysis was performed using SPSS 16.0. Data comparisons were performed using one-way anova with a test standard P < 0.05.
Results of three tests
1 maximum water holding capacity of soil and growth of fruiting body
The maximum water holding capacity of the soil was determined to be 45%.
After the fungus sticks enter the fruiting chamber, 7 days are needed for each tide of fruiting body primordium induction, and 7 days are needed for fruiting body growth. The first tide fruiting body is moved into the soil after the primordium is shown, and the third tide fruiting body is subjected to the total culture of the fungus stick in the soil for 35 d.
2 the test soil was alkaline soil with a pH of 8.9. As shown in FIG. 1, the cultivation of the fruiting body of Pleurotus ostreatus lowers the pH of the soil. The pH value of the soil before Cd treatment and after Cd treatment is not obviously different, but the pH value of the soil after fruiting is obviously reduced, so that the growth process of the strain can reduce the pH value of the soil, and the effect of restoring alkaline soil is achieved.
3 the enrichment capacity of the fruiting bodies is increased along with the increase of the harvest tide number and is increased along with the increase of the concentration of Cd in soil, the first tide of mushrooms almost has no Cd enrichment, the measured value is extremely small and can be ignored, and the enrichment conditions of the fruiting bodies of the second tide of mushrooms and the third tide of mushrooms are shown in fig. 2 and fig. 3. When the soil cadmium treatment concentration is 10 mg/kg: the cadmium enrichment concentration of the fruiting bodies of the second tide mushrooms is 0.745mg/kg, the total cadmium enrichment amount of the fruiting bodies produced by a single mushroom stick is 0.005mg, the cadmium enrichment concentration of the fruiting bodies of the third tide mushrooms is about 10mg/kg, and the total cadmium enrichment amount of the fruiting bodies produced by the single mushroom stick is 0.004 mg. When the soil cadmium treatment concentration is 150 mg/kg: the cadmium enrichment concentration of the fruiting bodies of the second tide mushrooms is 3.2mg/kg, the total cadmium enrichment amount of the fruiting bodies produced by a single mushroom stick is 0.012mg, the cadmium enrichment concentration of the fruiting bodies of the third tide mushrooms is about 4.5mg/kg, and the total cadmium enrichment amount of the fruiting bodies produced by the single mushroom stick is 0.02 mg.
The enrichment capacity of the 4 bacteria sticks is increased along with the increase of the content of Cd in the soil, when the treatment concentration of Cd in the soil is 150mg/kg, the concentration of enriched Cd in a single bacteria stick is 6mg/kg within 35 days, and the total enrichment capacity of Cd is 656.6 mu g, as shown in figure 4.
5 the concentration of Cd in the soil aqueous solution showed a tendency of rising first and then falling with the increase of the number of days of cultivation, and reached the maximum at 28d, as shown in FIG. 5. The Cd in the soil aqueous solution is in a dissociation state, which shows that the growth activity of the sporocarps can dissociate the chelated Cd in the soil so as to enrich in the bacteria stick and the sporocarps, and the concentration of the Cd in the soil aqueous solution is reduced due to factors such as enrichment and absorption of the bacteria stick and the sporocarps after the Cd is dissociated to a certain degree.
6 fruiting status
The fruiting conditions of the strain in the soil with cadmium contents of 0mg/kg, 10mg/kg and 150mg/kg are shown in FIG. 6, and the strain can still keep a good growth state in the soil with high cadmium content.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A high-cadmium-resistance oyster mushroom strain is characterized in that: the strain is registered and preserved in China general microbiological culture Collection center (CGMCC) at 7 and 6 months in 2018, and the preservation number is CGMCC NO. 16084.
2. Use of the oyster mushroom strain according to claim 1 for remediating cadmium contaminated soil.
3. Use according to claim 2, characterized in that: the soil is alkaline soil.
4. Use according to claim 3, characterized in that: the pH of the soil is above 8.5.
5. The method for remediating cadmium-contaminated soil using the oyster mushroom strain of claim 1, comprising: inoculating the oyster mushroom strains into a culture medium for culture to obtain culture mushroom sticks, performing primordial induction on the mushroom sticks, and transferring the mushroom sticks into cadmium-polluted soil for fruiting management after the primordial appears.
6. The method for remediating cadmium-contaminated soil as set forth in claim 5, wherein: the primordia induction is as follows: opening the opening at the tying position of the fungus bag, and performing directional primordium induction at the opening position.
7. The method for remediating cadmium-contaminated soil as set forth in claim 5, wherein: when the fungus stick is moved into the cadmium-containing soil, part of the fungus bag is removed, the part where the fungus bag is removed is buried in the soil, and the part where the fungus bag is not removed is exposed out of the soil.
8. The method for remediating cadmium-contaminated soil as set forth in claim 7, wherein: when the fungus stick is moved into cadmium-containing soil, the height ratio of the removed fungus bag part to the reserved fungus bag part is 1-3: 1.
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| CN112080436B (en) * | 2020-09-29 | 2022-01-04 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Large fungus strain with high cadmium resistance and application thereof |
| CN113263049A (en) * | 2021-07-13 | 2021-08-17 | 河南农业大学 | Application of calcium chloride in enhancing cadmium reduction capability of fungal soil and method |
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