White rot fungus amplification culture device and method

Abstract

The invention provides a white rot fungus amplification culture device and method, the device comprises a sterile room provided with a door, the device also comprises a sterilization system, the sterilization system comprises an ozone generator, the ozone generator is communicated with the interior of the sterile room through a pipeline, and the top of the sterile room is also provided with an ultraviolet lamp; the microorganism culture system comprises a sealed culture tank, an air blower communicated with the culture tank is arranged outside the culture tank, a sulfuric acid sterilization tank is communicated with an air blowing pipe between the air blower and the culture tank, the concentration of sulfuric acid in the sulfuric acid sterilization tank is greater than 20%, and the air blowing pipe is communicated with an aeration pipe arranged at the bottom of the culture tank; the top of the culture tank is provided with a bacterial liquid adding bottle, a nutrient solution adding bottle and a one-way exhaust valve, the culture tank is internally provided with filler and a heating ring, the top of the culture tank is provided with a sterile water inlet pipe, and the bottom of the culture tank is provided with a waste liquid discharge pipe. The production efficiency of the white rot fungi can be remarkably improved, and the white rot fungi are easily kneaded and stripped from the filler after being taken out.

Description

Technical Field

[0001] The invention belongs to the technical field of white rot fungus cultivation, and relates to a device and method for expanding and cultivating white rot fungi. Background Art

[0002] White-rot fungi, filamentous fungi belonging to the higher basidiomycetes, are ubiquitous in nature. They saprophytically grow on trees or wood, decomposing wood fibers and ultimately forming a white, spongy substance. Among the over a thousand identified white-rot fungi, Phanerochaete chrysosporium has been particularly well-studied. These fungi are renowned for their remarkable environmental purification abilities: they can degrade environmentally hazardous substances such as halogenated compounds, organochlorine pesticides, polycyclic aromatic hydrocarbons, azo dyes, and polychlorinated biphenyls (PCBs); they exhibit significant biosorption capacity for heavy metal ions and synthetic dyes; and they possess a broad range of phenolic compound degradation capabilities and unique resistance to biotoxins. Therefore, white-rot fungi hold great potential for treating toxic and recalcitrant industrial wastewater. Despite this, research in my country on white-rot fungi wastewater treatment technology remains in its early stages, and large-scale application faces numerous challenges: unstable treatment results, prolonged treatment cycles, immature bioreactor technology for continuous cultivation of white-rot fungi, and difficulty in strain selection. These issues urgently require further research and improvement. Summary of the Invention

[0003] The purpose of the present invention is to provide a white rot fungus expansion culture device and method to improve the efficiency of white rot fungus large-scale culture in response to the defects of the existing technology.

[0004] A first aspect of the present invention provides a white rot fungus expansion culture device, comprising a sterile chamber in a closed structure, the sterile chamber having a door, an air filter provided on the side wall of the sterile chamber, and a negative pressure suction pump provided at the bottom of the sterile chamber, and further comprising: A sterilization system comprising an ozone generator connected to the sterile chamber via a pipeline, and an ultraviolet lamp is also provided on the top of the sterile chamber; A microbial culture system provided in a sterile room includes a sealed culture tank, a blower provided on the outside of the culture tank and connected to the culture tank, a blast pipe between the blower and the culture tank is connected to a sulfuric acid sterilizer, the sulfuric acid concentration in the sulfuric acid sterilizer is greater than 20%, and the blast pipe is connected to an aeration pipe arranged at the bottom of the culture tank; The top of the culture tank is equipped with a bacterial solution adding bottle, a nutrient solution adding bottle and a one-way exhaust valve, fillers are arranged inside the culture tank, a heating ring is provided inside the culture tank, a sterile water inlet pipe is provided on the top of the culture tank, and a waste liquid discharge pipe is provided at the bottom.

[0005] Preferably, the filler is made of polyurethane material.

[0006] Preferably, the heating ring is arranged at the bottom of the culture tank.

[0007] Preferably, the pore size of the filler is greater than 20 ppi, the thickness of a single sheet is less than 0.5 cm, and the interval between two adjacent sheets is greater than 1 cm.

[0008] Preferably, the aeration tube has an opening rate of 30% and a pore diameter less than 2 mm.

[0009] Preferably, the air filter uses a cotton filter element with a filtration accuracy of 0.22μm and 0.45μm.

[0010] A second aspect of the present invention provides a method for expanding and culturing white rot fungi, comprising the following steps: Close the door, turn on the ultraviolet lamp to sterilize the sterile room, turn on the ozone generator after sterilization for no less than 30 minutes, use ozone to sterilize the sterile room and internal equipment for a second time, turn on the blower, and turn it on for no less than 1 hour before turning off the ozone generator, turning off the blower, and turning on the negative pressure suction pump; Inject sterile water into the culture tank through the sterile water inlet pipe; The temperature of the culture tank was controlled to 28~30℃ by the heating ring; After the temperature reaches the set value, open the valve on the nutrient solution dosing bottle and add the sterilized nutrient solution into the culture tank. Then put the bacterial solution in the bacterial solution dosing bottle into the culture tank to start the culture reaction. During this period, turn on the ultraviolet light for disinfection every 6 hours, and the disinfection time should be no less than 30 minutes. After the bacterial solution is added, turn on the blower and control the air volume to 0.5~1m3 / m3h to disturb the culture solution and supply oxygen; White rot fungi grow rapidly on the surface of the filler, and the culture is terminated after 5 to 7 days of continuous culture.

[0011] Preferably, the UV light intensity is greater than 70 μW / cm².

[0012] Preferably, when ozone is used for secondary sterilization of the sterile room and internal equipment, the ozone concentration in the sterile room needs to be greater than 60 mg / m³.

[0013] Preferably, when the ultraviolet lamp is turned on to sterilize the sterile room, the sterilization time is preferably 30 minutes to 60 minutes.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses a disinfection room and an aerobic reactor to replace the traditional shaking table to culture white rot fungi, which is more efficient and suitable for expanding the growth of white rot fungi.

[0015] 2. The present invention adopts a four-stage filtration and sterilization of air by filter components, ultraviolet, ozone and sulfuric acid, ensuring that no foreign bacteria enter the reactor, and providing a stable and reliable breeding environment for the efficient reproduction of white rot fungi.

[0016] 3. The present invention uses porous polyurethane fillers to assist the growth of white rot fungi, which can significantly improve the production efficiency of white rot fungi, and the fungi can be easily peeled off from the fillers after being taken out. 4. The production device of the present invention has a high degree of automation in the operation process, is efficient and reliable, and can culture the next batch of white rot fungi in a short auxiliary time after completing one culture, greatly shortening the auxiliary time in the batch culture process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the system of the present invention.

[0018] In the above drawings: 1. Sterile chamber; 11. Door; 12. Air filter; 13. Negative pressure suction pump; 2. Ozone generator; 21. UV lamp; 211. Pipeline; 3. Culture tank; 31. Blower; 311. Sulfuric acid sterilization tank; 312. Blower pipe; 313. Sterile water inlet pipe; 314. Waste liquid discharge pipe; 32. Bacteria solution dosing bottle; 321. Valve; 33. Nutrient solution dosing bottle; 34. One-way exhaust valve; 35. Filler; 36. Heating coil; DETAILED DESCRIPTION In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described below in conjunction with specific embodiments. However, it should be understood that the drawings are only for illustrative purposes and cannot be understood as limiting this patent; in order to better illustrate this embodiment, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted; the positional relationships described in the drawings are only for illustrative purposes and cannot be understood as limiting this patent.

[0019] Traditional white rot fungi are cultured using a shaking table, but the culture scale is small and cannot meet the needs of large-scale culture.

[0020] As a preferred embodiment of the present invention, refer to Figure 1 This embodiment provides a white rot fungus expansion culture device, comprising a sterile chamber 1 with a closed structure, the sterile chamber 1 having a door 11, an air filter 12 provided on the side wall of the sterile chamber 1, a negative pressure suction pump 13 provided at the bottom of the sterile chamber 1, and further comprising: The sterilization system includes an ozone generator 2, which is connected to the sterile chamber 1 through a pipe 211. An ultraviolet lamp 21 is also provided on the top of the sterile chamber 1; The microbial culture system provided in the sterile room 1 includes a sealed culture tank 3, a blower 31 connected to the culture tank 3 is provided on the outside of the culture tank 3, and a blast pipe 312 between the blower 31 and the culture tank 3 is connected to a sulfuric acid sterilization tank 311. The sulfuric acid concentration in the sulfuric acid sterilization tank 311 is greater than 20%. The blast pipe 312 is connected to an aeration pipe arranged at the bottom of the culture tank 3. The sterilization method described above can completely kill miscellaneous bacteria and spores in the culture medium, prevent miscellaneous bacterial contamination, and ensure the purity of the bacterial culture and the reliability of the experimental results. A bacterial solution adding bottle 32, a nutrient solution adding bottle 33 and a one-way exhaust valve 34 are provided on the top of the culture tank 3. A filler 35 is arranged inside the culture tank 3. A heating ring 36 is provided inside the culture tank 3. A sterile water inlet pipe 313 is provided on the top of the culture tank 3, and a waste liquid discharge pipe 314 is provided at the bottom.

[0021] In some preferred embodiments, filler 35 is made of polyurethane. Polyurethane has a porous structure, providing a large specific surface area, which facilitates the attachment and growth of white-rot fungi. The porous structure of polyurethane promotes the uniform distribution of oxygen and nutrients, improving mass transfer efficiency. By improving mass transfer conditions, polyurethane fillers can promote the growth and metabolism of white-rot fungi and shorten the culture cycle. Furthermore, polyurethane has excellent mechanical strength and chemical stability, maintaining structural integrity during the culture process. By providing stable physical support, polyurethane fillers can reduce structural collapse and bacterial loss during the culture process, ensuring continuous and stable culture. Furthermore, polyurethane fillers have a long service life and are easily regenerated, reducing replacement and maintenance frequency.

[0022] In some embodiments, the heating ring 36 is provided at the bottom of the culture tank 3, and the ambient temperature can be quickly adjusted by utilizing the characteristic of upward flow of hot air.

[0023] In some embodiments, the filler pore size is greater than 20ppi, the thickness of a single sheet is less than 0.5cm, and the interval between two adjacent sheets is greater than 1cm. This embodiment design can increase the permeability of the filler, which is conducive to the uniform distribution of oxygen and nutrients inside the filler. In some preferred embodiments, the aeration tube has an opening rate of 30% and a pore diameter of less than 2 mm, which reduces the possibility of filler clogging and ensures smooth flow of culture medium and gas; the larger pore diameter and appropriate sheet spacing can provide more attachment space for white rot fungi and promote the uniform distribution of bacteria on the filler surface. By improving the efficiency of bacteria attachment, this filler design can increase the formation rate and stability of the biofilm.

[0024] In some preferred embodiments, the air filter 12 uses a cotton filter element with a filtration accuracy of 0.22 μm and 0.45 μm. By using a combination of 0.22 μm and 0.45 μm cotton filter elements, bacteria and tiny particles in the culture medium can be effectively removed, ensuring the sterility of the culture environment, thereby improving the success rate and purity of bacterial culture.

[0025] As another preferred embodiment of the present invention, this embodiment provides a method for expanding and culturing white rot fungi, using a white rot fungi expansion and culturing device as described in any of the above embodiments, specifically comprising the following steps: Close the door 11, turn on the ultraviolet lamp 21 to sterilize the sterile room 1, turn on the ozone generator 2 after sterilization for at least 30 minutes, use ozone to perform secondary sterilization on the sterile room 1 and the internal equipment, turn on the blower 31, and turn on the ozone generator 2 and blower 31 after continuous operation for at least 1 hour, and turn on the negative pressure suction pump 13; Inject sterile water into the culture tank 3 through the sterile water inlet pipe 313; The temperature of the culture tank is controlled to 28-30°C by the heating coil 36; After the temperature reaches the set value, open the valve 321 on the nutrient solution addition bottle 33 and add the sterilized nutrient solution into the culture tank 3. Then, add the bacterial solution in the bacterial solution addition bottle 32 into the culture tank 3 to start the culture reaction. During this period, turn on the ultraviolet light for disinfection every 6 hours, and the disinfection time is not less than 30 minutes. After the bacterial solution is added, the blower 31 is turned on and the air volume is controlled to 0.5~1m3 / m3h to disturb the culture solution and supply oxygen; White rot fungi grow rapidly on the surface of the filler, and the culture is terminated after 5 to 7 days of continuous culture.

[0026] In some embodiments, the UV light intensity is greater than 70 μW / cm²; when ozone is used for secondary sterilization of the sterile chamber 1 and its internal equipment, the ozone concentration in the sterile chamber 1 must be greater than 60 mg / m³. This design achieves efficient sterilization, prevents contamination by foreign bacteria, reduces the use of chemical sterilants, and prolongs the duration of the sterile environment. It also overcomes the limitations of UV light, removes residual contaminants, and improves sterilization efficiency. These measures work together to ensure high cleanliness in the sterile chamber, increase the success rate of white rot fungus cultivation, and enhance the reliability of experimental results.

[0027] When comparing the traditional culture method and the culture method of the present invention, it was found that the culture method of the present invention has obvious advantages in single culture scale and culture cycle. The statistical results of the experimental comparison are shown in Table 1 below: Table 1 Comparison between traditional shaker culture and culture of the present invention

[0028] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A white rot fungus expansion culture device, characterized in that: The invention comprises a sterile room with a closed structure, wherein the sterile room is provided with a door, an air filter is provided on the side wall of the sterile room, and a negative pressure suction pump is provided at the bottom of the sterile room, and further comprises: A sterilization system comprising an ozone generator connected to the sterile chamber via a pipeline, and an ultraviolet lamp is also provided on the top of the sterile chamber; A microbial culture system provided in a sterile room includes a sealed culture tank, a blower provided on the outside of the culture tank and connected to the culture tank, a blast pipe between the blower and the culture tank is connected to a sulfuric acid sterilizer, the sulfuric acid concentration in the sulfuric acid sterilizer is greater than 20%, and the blast pipe is connected to an aeration pipe arranged at the bottom of the culture tank; The top of the culture tank is equipped with a bacterial solution adding bottle, a nutrient solution adding bottle and a one-way exhaust valve, fillers are arranged inside the culture tank, a heating ring is provided inside the culture tank, a sterile water inlet pipe is provided on the top of the culture tank, and a waste liquid discharge pipe is provided at the bottom.

2. A white rot fungus expansion culture device according to claim 1, characterized in that: The filling is made of polyurethane material.

3. The white rot fungus expansion culture device according to claim 1, characterized in that: The heating ring is arranged at the bottom of the culture tank.

4. The device for expanding and culturing white rot fungi according to claim 1, wherein: The pore size of the filler is greater than 20ppi, the thickness of a single piece is less than 0.5cm, and the interval between two adjacent pieces is greater than 1cm.

5. The device and method for expanding and culturing white rot fungi according to claim 1, wherein: The aeration tube has an opening rate of 30% and a pore diameter of less than 2 mm.

6. The device for expanding and culturing white rot fungi according to claim 1, wherein: The air filter adopts a cotton cloth filter element with a filtration accuracy of 0.22μm and 0.45μm.

7. A method for expanding and culturing white rot fungi according to claim 1, characterized in that: The device for expanding and culturing white rot fungi according to any one of claims 1 to 6 comprises the following steps: Close the door, turn on the ultraviolet lamp to sterilize the sterile room, turn on the ozone generator after sterilization for no less than 30 minutes, use ozone to sterilize the sterile room and internal equipment for a second time, turn on the blower, and turn it on for no less than 1 hour before turning off the ozone generator, turning off the blower, and turning on the negative pressure suction pump; Inject sterile water into the culture tank through the sterile water inlet pipe; The temperature of the culture tank was controlled to 28~30℃ by the heating ring; After the temperature reaches the set value, open the valve on the nutrient solution dosing bottle and add the sterilized nutrient solution into the culture tank. Then put the bacterial solution in the bacterial solution dosing bottle into the culture tank to start the culture reaction. During this period, turn on the ultraviolet light for disinfection every 6 hours, and the disinfection time should be no less than 30 minutes. After the bacterial solution is added, turn on the blower and control the air volume to 0.5~1m3 / m3h to disturb the culture solution and supply oxygen; White rot fungi grow rapidly on the surface of the filler, and the culture is terminated after 5 to 7 days of continuous culture.

8. A method for expanding and culturing white rot fungi according to claim 7, characterized in that: The UV light intensity must be greater than 70μW / cm².

9. The device and method for expanding and culturing white rot fungi according to claim 7, wherein: When using ozone for secondary sterilization of the sterile room and internal equipment, the ozone concentration in the sterile room must be greater than 60mg / m³.

10. The device and method for expanding and culturing white rot fungi according to claim 7, characterized in that: When the ultraviolet lamp is turned on to sterilize the sterile room, the sterilization time is preferably 30 minutes to 60 minutes.

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