Microbial strain cultivation method for environment-friendly fertilizer

Abstract

The invention belongs to the technical field of microbial strain cultivation methods, and discloses a microbial strain cultivation method for an environment-friendly fertilizer, which comprises the following steps: step 1, selecting a proper microbial strain; 2, preparing a culture medium; step 3, inoculation and culture; 4, growth monitoring and adjustment; 5, preparing a fermentation culture medium; step 6, inoculation and fermentation: inoculating the cultured trichoderma harzianum strain into a fermentation culture medium, wherein the inoculation amount is 5%-10% of the volume of the culture medium; putting the inoculated fermentation culture medium into a fermentation tank, and carrying out fermentation culture; the fermentation conditions are set as follows: the temperature is 25-30 DEG C, the humidity is kept at 70-85%, and the ventilation volume is 0.5-1.0 vvm; the fermentation time is 70-90 hours; step 7, fermentation liquor treatment; the invention can achieve the beneficial effects of environmental protection, sustainability, promotion of plant growth and development, improvement of soil structure, wide adaptability and the like, and brings significant ecological benefits and economic benefits to agricultural production.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial strain cultivation methods, and specifically relates to a microbial strain cultivation method for environmentally friendly fertilizers. Background Art

[0002] In agricultural production, soil-borne diseases are one of the important factors affecting crop yield and quality. Although traditional chemical pesticide control methods can control the occurrence of diseases to a certain extent, long-term use will lead to environmental pollution, ecological balance destruction, and increased resistance of pests and diseases. Therefore, it is particularly important to find an environmentally friendly, efficient, and sustainable disease control method.

[0003] As a pure biological agent, Trichoderma harzianum has a wide antibacterial spectrum and significant disease prevention effect. It can effectively prevent and control diseases caused by various pathogens such as Fusarium, Pythium, and Rhizoctonia, and has good preventive and therapeutic effects on fungal diseases of crops. However, the cultivation process of Trichoderma harzianum is relatively complicated, and the culture conditions need to be strictly controlled to ensure its growth, reproduction and disease prevention effect. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for cultivating microbial strains for environmentally friendly fertilizers, which effectively solves the problems raised by the background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for cultivating microbial strains for environmentally friendly fertilizers, comprising the following steps:

[0006] Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 25-30°C, and culture for 3-5 days until obvious colonies grow;

[0007] Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.0-6.5; sterilize the medium with high pressure steam at 120° C. for 1 hour;

[0008] Step 3, inoculation and cultivation: under sterile conditions, inoculate the activated Trichoderma harzianum strains onto the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set at 25-30°C, the humidity is maintained at 70-85%, and the culture time is 7-10 days;

[0009] Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time.

[0010] Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 6.0-7.0; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour;

[0011] Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 5%-10% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 25-30° C., a humidity of 70-85%, a ventilation volume of 0.5-1.0 vvm; and a fermentation time of 70-90 hours;

[0012] Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

[0013] Preferably, the culture temperature in step 1 is set to 28° C. and the culture time is 4 days.

[0014] Preferably, the pH value in step 2 is 6.3.

[0015] Preferably, the inoculation amount in step 3 is 0.1 ml per dish; the culture temperature is set to 28° C., the humidity is maintained at 80%, and the culture time is 8 days.

[0016] Preferably, the fermentation medium in step 5 comprises glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements.

[0017] Preferably, the fermentation conditions in step six are set as a temperature of 28° C., a humidity of 80%, a ventilation volume of 0.8 vvm (volume air flow / volume fermentation liquid); and a fermentation time of 80 hours.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Environmentally friendly and sustainable: This method avoids the use of chemical pesticides, reduces the risk of environmental pollution and ecological balance damage, and is in line with the green and sustainable development trend of modern agriculture. As a biological agent, Trichoderma harzianum is non-toxic and harmless, and will not have a negative impact on soil, water sources and ecosystems;

[0020] Promote plant growth and development: After colonizing the plant root system, Trichoderma harzianum can secrete a variety of growth regulators to promote plant growth and development, and improve crop stress resistance and yield. At the same time, it can also improve the soil environment and provide more suitable growth conditions for plants;

[0021] Improve soil structure: Through the cultivation and application of Trichoderma harzianum, the soil microecological environment can be significantly improved, the soil organic matter content can be increased, the soil aggregate structure can be improved, the soil permeability and water and fertilizer retention capacity can be improved, and a better soil environment can be provided for the growth of crops;

[0022] Wide adaptability: The Trichoderma harzianum obtained by this cultivation method has strong adaptability and can be applied to different types of soil and climatic conditions, meeting the needs of different regions and crops, and providing more flexible and diverse disease prevention and control methods for agricultural production;

[0023] In summary, it can achieve beneficial effects such as environmental sustainability, promoting plant growth and development, improving soil structure, and wide adaptability, bringing significant ecological and economic benefits to agricultural production. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] The present invention relates to a method for cultivating microbial strains for environmentally friendly fertilizers, comprising the following steps:

[0026] Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 25-30°C, and culture for 3-5 days until obvious colonies grow;

[0027] Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.0-6.5; sterilize the medium with high pressure steam at 120° C. for 1 hour;

[0028] Step 3, inoculation and cultivation: under sterile conditions, inoculate the activated Trichoderma harzianum strains onto the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set at 25-30°C, the humidity is maintained at 70-85%, and the culture time is 7-10 days;

[0029] Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time.

[0030] Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 6.0-7.0; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour;

[0031] Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 5%-10% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 25-30° C., a humidity of 70-85%, a ventilation volume of 0.5-1.0 vvm; and a fermentation time of 70-90 hours;

[0032] Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

[0033] The culture temperature in step 1 was set to 28°C and the culture time was 4 days.

[0034] The pH value in step 2 is 6.3.

[0035] The inoculation volume in step 3 is 0.1 ml per dish; the culture temperature is set to 28° C., the humidity is maintained at 80%, and the culture time is 8 days.

[0036] The fermentation medium in step five includes glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements; specifically, the trace elements are easily added at 1:1000, and the rest is distilled water.

[0037] The fermentation conditions in step six were set as a temperature of 28° C., a humidity of 80%, a ventilation volume of 0.8 vvm (volume air flow / volume fermentation liquid); and a fermentation time of 80 hours.

[0038] Embodiment 1:

[0039] This embodiment relates to a method for cultivating microbial strains for environmentally friendly fertilizers, comprising the following steps:

[0040] Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 25°C, and culture for 3 days until obvious colonies grow;

[0041] Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.0; sterilize the medium with high pressure steam at 120° C. for 1 hour;

[0042] Step 3, inoculation and cultivation: under sterile conditions, the activated Trichoderma harzianum strains are inoculated onto the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set at 25°C, the humidity is maintained at 70%, and the culture time is 7 days;

[0043] Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time.

[0044] Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 6.0; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour;

[0045] Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 5%-10% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 25° C., a humidity of 70%, a ventilation volume of 0.5 vvm; and a fermentation time of 70 hours;

[0046] Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

[0047] The inoculum volume in step 3 is 0.1 ml per dish;

[0048] The fermentation medium in step 5 includes glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements.

[0049] Embodiment 2:

[0050] This embodiment relates to a method for cultivating microbial strains for environmentally friendly fertilizers, comprising the following steps:

[0051] Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 30°C, and culture for 5 days until obvious colonies grow;

[0052] Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.5; sterilize the medium with high pressure steam at 120° C. for 1 hour;

[0053] Step 3, inoculation and cultivation: under sterile conditions, the activated Trichoderma harzianum strains are inoculated into the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set to 30°C, the humidity is maintained at 85%, and the culture time is 10 days;

[0054] Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time.

[0055] Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 7.0; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour;

[0056] Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 5%-10% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 30° C., a humidity of 85%, a ventilation volume of 1.0 vvm; and a fermentation time of 90 hours;

[0057] Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

[0058] The inoculum volume in step 3 is 0.1 ml per dish;

[0059] The fermentation medium in step 5 includes glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements.

[0060] Embodiment 3:

[0061] This embodiment relates to a method for cultivating microbial strains for environmentally friendly fertilizers, comprising the following steps:

[0062] Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 28°C, and culture for 4 days until obvious colonies grow;

[0063] Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.3; sterilize the medium with high pressure steam at 120° C. for 1 hour;

[0064] Step 3, inoculation and cultivation: under sterile conditions, the activated Trichoderma harzianum strains are inoculated into the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set to 28°C, the humidity is maintained at 80%, and the culture time is 8 days;

[0065] Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time.

[0066] Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 6.3; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour;

[0067] Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 8% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 28° C., a humidity of 80%, a ventilation volume of 0.8 vvm; and a fermentation time of 80 hours;

[0068] Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

[0069] The inoculum volume in step 3 is 0.1 ml per dish;

[0070] The fermentation medium in step 5 includes glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements.

[0071] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0072] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for cultivating microbial strains for environmentally friendly fertilizers, characterized in that: The following steps are involved: Step 1, select a suitable microbial strain: select T-E5 Trichoderma harzianum strain, activate the strain on a suitable solid culture medium, set the culture temperature to 25-30°C, and culture for 3-5 days until obvious colonies grow; Step 2, medium preparation: prepare potato glucose agar medium as a growth medium for Trichoderma harzianum, peel, cut, boil and filter potatoes, add glucose and agar, and adjust the pH value to 6.0-6.5; sterilize the medium with high pressure steam at 120° C. for 1 hour; Step 3, inoculation and cultivation: under sterile conditions, inoculate the activated Trichoderma harzianum strains onto the PDA medium; the inoculation amount can be adjusted according to the strain concentration and the size of the medium, and the inoculated medium is placed in a constant temperature incubator, the culture temperature is set at 25-30°C, the humidity is maintained at 70-85%, and the culture time is 7-10 days; Step 4: Growth monitoring and adjustment: Regularly check the growth of colonies on the culture medium and observe the colony morphology, color and other characteristics; if the colonies are found to be growing poorly or contaminated, the culture medium should be replaced and re-inoculated in time. Step 5, preparation of fermentation medium: preparing fermentation medium; adjusting the pH value to 6.0-7.0; sterilizing the fermentation medium by high pressure steam at 120° C. for 1 hour; Step 6, inoculation and fermentation: inoculate the cultured Trichoderma harzianum into the fermentation medium, the inoculation amount is 5%-10% of the volume of the medium; place the inoculated fermentation medium in a fermenter for fermentation culture; the fermentation conditions are set at a temperature of 25-30° C., a humidity of 70-85%, a ventilation volume of 0.5-1.0 vvm; and a fermentation time of 70-90 hours; Step 7: Fermentation liquid treatment: After the fermentation is completed, the fermentation liquid is centrifuged or filtered to separate the bacteria and the fermentation liquid; then the bacteria are dried to obtain Trichoderma harzianum powder.

2. The method for cultivating microbial strains for environmentally friendly fertilizers according to claim 1, characterized in that: The culture temperature in step 1 is set to 28° C. and the culture time is 4 days.

3. The method for cultivating microbial strains for environmentally friendly fertilizers according to claim 1, characterized in that: The pH value in the step 2 is 6.

3.

4. The method for cultivating microbial strains for environmentally friendly fertilizers according to claim 1, characterized in that: The inoculation amount in step 3 is 0.1 ml per dish; the culture temperature is set at 28° C., the humidity is maintained at 80%, and the culture time is 8 days.

5. The method for cultivating microbial strains for environmentally friendly fertilizers according to claim 1, characterized in that: The fermentation medium in step 5 includes glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and distilled water; the ratio (% w / v) between yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate is 1.5:1.0:0.3:0.2:0.05, and the rest is distilled water and trace elements.

6. The method for cultivating microbial strains for environmentally friendly fertilizers according to claim 1, characterized in that: The fermentation conditions in step 6 are set as a temperature of 28° C., a humidity of 80%, a ventilation volume of 0.8 vvm (volume air flow / volume fermentation liquid); and a fermentation time of 80 hours.