Device for enriching non-culturable microorganisms in organic matter compost based on bidirectional diffusion principle

Abstract

The invention provides a device for enriching non-culturable microorganisms in organic matter compost based on a bidirectional diffusion principle, and belongs to the technical field of environmental microorganism technology and organic solid waste recycling. The problems of high liquid leakage risk, low material exchange efficiency and complicated operation in the existing diffusion chamber technology are solved. The device comprises an inner chamber, an outer chamber, a composite membrane system and a culture medium, the inner chamber and the outer chamber are both of a cylindrical structure, the inner chamber is arranged in the outer chamber, the top of the inner chamber penetrates through the top of the outer chamber and extends to the outer side of the outer chamber, and a plurality of inner chamber holes are evenly distributed in the side wall of the part, located in the outer chamber, of the inner chamber; the composite membrane system comprises a PTFE non-woven fabric composite hydrophobic membrane, the PTFE non-woven fabric composite hydrophobic membrane wraps the outer side of the side wall of the part, located in the outer chamber, of the inner chamber, the culture medium is arranged in the inner chamber, and straw compost is arranged in the outer chamber. The device is mainly used for microbial culture.

Description

Technical Field

[0001] The invention belongs to the field of environmental microbiology technology and organic solid waste resource technology, and particularly relates to a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion. Background Art

[0002] Traditional microbial cultivation techniques have long faced the challenge of isolating unculturable microorganisms. Research has shown that over 99% of microorganisms in nature cannot be isolated and cultured using conventional laboratory methods, severely impacting the development and utilization of these microbial resources. In the field of straw composting, efficient lignin degradation relies on the functional synergy between microbial communities. Existing microbial cultivation techniques primarily optimize culturable strains, but most microorganisms struggle to adapt to conventional culture conditions and remain in an "unculturable" state, leaving their functional potential untapped.

[0003] In recent years, diffusion chamber technology has provided new insights for isolating uncultivable microorganisms by simulating natural environments. However, issues such as high leakage risk, low material exchange efficiency, and complex operation persist. For example, most traditional diffusion chambers utilize a single membrane structure, which cannot meet the requirements of leak prevention and bidirectional diffusion of materials. Therefore, there is an urgent need to develop a highly sealed diffusion chamber device specifically for straw composting. By using a low-nutrient culture medium and based on a bidirectional diffusion mechanism, it is possible to achieve efficient enrichment and functional analysis of uncultivable lignin-degrading bacteria, providing a new path for the development of technologies for exploiting composting microbial resources. Summary of the Invention

[0004] In view of this, the present invention aims to propose a device for enriching non-culturable microorganisms in organic compost based on the principle of bidirectional diffusion, so as to solve the problems of high leakage risk, low material exchange efficiency and complex operation in the existing diffusion chamber technology.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solutions: a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion, which includes an inner chamber, an outer chamber, a composite membrane system and a culture medium, wherein the inner chamber and the outer chamber are both cylindrical structures, the inner chamber is arranged inside the outer chamber, the top of the inner chamber passes through the top of the outer chamber and extends to the outside of the outer chamber, and a plurality of inner chamber holes are evenly distributed on the side wall of the inner chamber located inside the outer chamber, the composite membrane system includes a PTFE non-woven composite hydrophobic membrane, the PTFE non-woven composite hydrophobic membrane is wrapped around the outside of the side wall of the inner chamber located inside the outer chamber, the hydrophobic surface of the PTFE non-woven composite hydrophobic membrane faces outward, and the non-woven surface of the PTFE non-woven composite hydrophobic membrane faces inward, the culture medium is arranged in the inner chamber, and straw compost is arranged in the outer chamber.

[0006] Furthermore, RTV silica gel is provided between the PTFE non-woven composite hydrophobic membrane and the outer wall of the inner and outer chambers.

[0007] Furthermore, the outer side of the PTFE non-woven composite hydrophobic membrane is coated with a nylon mesh protective layer.

[0008] Furthermore, a plurality of silicone elastic rings are provided on the outer side of the nylon mesh protective layer, and the nylon mesh protective layer is tightened by the silicone elastic rings.

[0009] Furthermore, the top of the inner chamber is screwed with an inner chamber screw cover.

[0010] Furthermore, the top of the outer chamber is screwed with an outer chamber screw cover, the center of the outer chamber screw cover is provided with a through hole that matches the inner chamber, and the inner chamber is provided with the through hole.

[0011] Furthermore, both the inner chamber screw cover and the outer chamber screw cover are provided with ventilation holes, and the ventilation holes are covered with Parafilm membranes.

[0012] Furthermore, the Parafilm is in a pleated shape.

[0013] Furthermore, the culture medium is a low-nutrient lignin culture medium, and the components and proportions of the low-nutrient lignin culture medium are as follows in terms of weight-volume ratio: 1.0% straw powder, 0.2% microcrystalline cellulose, 0.05% peptone, 10% compost extract, 0.1% agar, the balance is filled with sterile water, and the pH value is adjusted to 6.5-7 with phosphate buffer.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention discloses a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion. The device breaks through the cultivation limit and improves the enrichment efficiency of uncultivable microorganisms in straw compost by synergistically regulating low-nutrient culture medium and adding compost leachate. 2. The present invention discloses a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion. Through precise environmental simulation, bidirectional diffusion of PTFE non-woven composite hydrophobic membrane is used to restore the material circulation and signal exchange in the natural ecology, thereby improving the survival rate of uncultivable microorganisms and meeting the requirements of leak prevention and bidirectional diffusion of materials. In addition, the application compatibility of this device is strong, and it can be adapted to a variety of samples such as soil and sludge, and is suitable for the directional separation of lignin-degrading bacteria, organic pollutant-degrading bacteria, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings: Figure 1 This is a schematic structural diagram of a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to the present invention; Figure 2 This is a schematic diagram of part of the structure of a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to the present invention; Figure 3 This is a schematic structural diagram of the inner chamber of a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to the present invention; Figure 4 This is a schematic structural diagram of the outer chamber of a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to the present invention.

[0016] 1-inner chamber, 2-outer chamber, 3-straw compost, 4-culture medium, 5-inner chamber hole, 6-silicone elastic ring, 7-Parafilm membrane, 8-vent, 9-nylon mesh protective layer, 10-PTFE non-woven composite hydrophobic membrane. DETAILED DESCRIPTION

[0017] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely explain the technical solutions in the embodiments of the present invention. It should be noted that the embodiments of the present invention and the features therein can be combined with each other in the absence of conflict, and the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0018] See also Figure 1-4 This embodiment describes a device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion, which includes an inner chamber 1, an outer chamber 2, a composite membrane system and a culture medium 4. The inner chamber 1 and the outer chamber 2 are both cylindrical structures. The inner chamber 1 is arranged inside the outer chamber 2, and the top of the inner chamber 1 passes through the top of the outer chamber 2 and extends to the outside of the outer chamber 2. A number of inner chamber holes 5 are evenly distributed on the side wall of the part of the inner chamber 1 located in the outer chamber 2. The composite membrane system includes a PTFE non-woven composite hydrophobic membrane 10, which is wrapped around the outside of the side wall of the part of the inner chamber 1 located in the outer chamber 2, the hydrophobic surface of the PTFE non-woven composite hydrophobic membrane 10 faces outward, and the non-woven surface of the PTFE non-woven composite hydrophobic membrane 10 faces inward. The culture medium 4 is arranged in the inner chamber 1, and straw compost 3 is arranged in the outer chamber 2.

[0019] The inner chamber 1 described in this embodiment serves as an oligotrophic culture unit, accommodating a culture medium 4 and providing an enrichment environment for uncultivable microorganisms. The inner chamber 1 described in this embodiment is a cylindrical structure, and the culture medium 4 is a low-nutrient lignin culture medium. The components and proportions of the low-nutrient lignin culture medium are as follows in terms of weight-volume ratio: 1.0% straw powder, 0.2% microcrystalline cellulose, 0.05% peptone, 10% compost extract, 0.1% agar, and the balance is filled with sterile water, and the pH value is adjusted to 6.5-7 with phosphate buffer.

[0020] The inner chamber pores 5 in this embodiment allow bidirectional diffusion of small molecules between the inner and outer chambers, thereby blocking the migration of microbial cells.

[0021] The outer chamber 2 described in this embodiment is filled with fresh straw compost 3 raw materials to simulate the natural compost microecosystem and maintain the in situ microbial community structure. The outer chamber 2 described in this embodiment is a cylindrical structure. The straw compost 3 serves as the outer chamber reaction matrix and continuously releases quorum sensing signal molecules as a material exchange carrier. The straw compost 3 is fresh, undecomposed straw compost with a moisture content of 60%.

[0022] In this embodiment, RTV silicone is provided between the PTFE non-woven composite hydrophobic membrane 10 and the outer wall of the inner chamber 1 , and the RTV silicone is used for sealing.

[0023] In this embodiment, the outer side of the PTFE non-woven composite hydrophobic membrane 10 is covered with a nylon mesh protective layer 9. The nylon mesh protective layer 9 is used to protect the PTFE non-woven composite hydrophobic membrane 10 from physical damage and filter large particles of impurities.

[0024] In this embodiment, a plurality of silicone elastic rings 6 are provided on the outside of the nylon mesh protective layer 9. The nylon mesh protective layer 9 is tightened by the silicone elastic rings 6. The silicone elastic rings 6 serve as an annular fastening component to fix the nylon mesh protective layer 9 and the PTFE non-woven composite hydrophobic membrane 10 to ensure the stability of the membrane interface structure.

[0025] In this embodiment, the top of the inner chamber 1 is screwed with an inner chamber screw cover, and the top of the outer chamber 2 is screwed with an outer chamber screw cover. A through hole matching the inner chamber 1 is opened in the center of the outer chamber screw cover, and the inner chamber 1 is set through the through hole.

[0026] In this embodiment, both the inner chamber screw cover and the outer chamber screw cover are provided with a vent 8, which is used to maintain aerobic conditions and regulate gas exchange. In this embodiment, the vent 8 is covered with a Parafilm membrane 7, which is pleated. The water supply of the culture system is carried out through the preset pleated structure.

[0027] The bidirectional diffusion mechanism of the device for enriching unculturable microorganisms in organic compost based on the bidirectional diffusion principle described in this embodiment is as follows: outer chamber 2 diffuses into inner chamber 1: providing volatile compounds, siderophores, and vitamin B12, thereby activating dormant cells and providing essential growth factors required by unculturable microorganisms; inner chamber 1 diffuses into outer chamber 2: providing nutrients (such as lignin degradation products) and signal molecules (such as quorum sensing factors) that attract some symbiotic bacteria in the outer chamber to produce feedback metabolites, forming a bidirectional interactive cycle.

[0028] The preparation and cultivation method of the present invention comprises the following steps: Step 1: Use a marker to mark the locations where holes need to be drilled on the sidewall of inner chamber 1. Use a micro-rotary hand drill to drill inner chamber holes 5 according to the marks. Sand the edges of the holes to remove burrs. Use a micro-rotary hand drill to evenly drill two vent holes 8 in the center of the inner chamber screw cap. Attach Parafilm membrane 7 to the vent holes 8, leaving a wrinkled portion for ventilation. Inject 200 mL of methylene blue solution into inner chamber 1 and let it stand for 24 hours to observe for leakage. Step 2: Use a compass cutter to cut a through hole in the center of the outer chamber screw cap of the outer chamber 2, and use a micro rotary hand drill to evenly punch four vent holes 8 on the edge, and stick the Parafilm film 7 on the vent holes 8, leaving a folded part for ventilation; Step 3: Apply RTV silicone evenly to the outer wall of the inner chamber 1, apply the PTFE non-woven composite hydrophobic membrane 10, leaving 2 cm extra at the edge, and cover the starting end of the film to form an overlapping seal. Use a roller to squeeze out the bubbles to ensure a complete seal. Then use RTV silicone to seal along the edge and cure for 24 hours. Cover the outside of the membrane with a nylon mesh protective layer 9 to ensure that the compost particles do not stick to the membrane holes, and use silicone elastic rings 6 to evenly fix and tighten at fixed points. Step 4: Preparation of culture medium 4: per liter of culture medium 4, 10 g of straw powder (passed through a 100-mesh sieve), 2 g of microcrystalline cellulose, 0.5 g of peptone, 100 mL of compost extract, and 1 g of agar, with the balance filled with sterile water. The pH was adjusted to 6.8 using phosphate buffer (0.1 M) and sterilized. The compost extract was prepared by mixing 1 kg of fresh straw compost with 2 L of sterile water, shaking at 25°C for 24 h, centrifuging at 4000 rpm for 10 min, and filtering the supernatant through a 0.22 μm filter membrane for sterilization. In this embodiment, agar was added to culture medium 4 to form a semi-solid system to prevent leakage. Step 5: Enrichment of uncultivable microorganisms: Pour fresh, undecomposed straw compost 3 into a basin and add water until no water overflows when held by hand and the basin remains loose (moisture content 60%). Insert the inner chamber 1 into the center of the outer chamber 2, filling the annular gap with straw compost 3. Gently press until there are no gaps. Fill the inner chamber 1 with 200 mL of sterilized culture medium 4. Tighten the inner chamber screw cap with a vent, leaving the Parafilm film wrinkled. Screw the inner chamber 1 to the outer chamber 2 through the outer chamber screw cap. Step 6: Incubate the device at 25-30°C in the dark for 42 days. Every two weeks, use a sterile syringe to pierce the inner chamber 1 through the fold of Parafilm 7 at an angle, add 2 mL of sterile water, and wipe the puncture point with an alcohol pad. Once a week, use a sterile syringe to insert the outer chamber through the vent to the middle of the compost and slowly add water to 60% moisture content.

[0029] The embodiments of the present invention disclosed above are intended only to illustrate the present invention. The embodiments do not describe all details in detail, nor do they limit the present invention to the specific embodiments described. Numerous modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A device for enriching non-culturable microorganisms in organic compost based on the principle of bidirectional diffusion, characterized by: It comprises an inner chamber (1), an outer chamber (2), a composite membrane system and a culture medium (4), wherein the inner chamber (1) and the outer chamber (2) are both cylindrical structures, the inner chamber (1) is arranged inside the outer chamber (2), the top of the inner chamber (1) passes through the top of the outer chamber (2) and extends to the outside of the outer chamber (2), a plurality of inner chamber holes (5) are uniformly distributed on the side wall of the portion of the inner chamber (1) located inside the outer chamber (2), the composite membrane system comprises a PTFE non-woven composite hydrophobic membrane (10), the PTFE non-woven composite hydrophobic membrane (10) is wrapped around the outside of the side wall of the portion of the inner chamber (1) located inside the outer chamber (2), the membrane hydrophobic surface of the PTFE non-woven composite hydrophobic membrane (10) faces outward, and the non-woven surface of the PTFE non-woven composite hydrophobic membrane (10) faces inward, the culture medium (4) is arranged in the inner chamber (1), and straw compost (3) is arranged in the outer chamber (2).

2. The device for enriching uncultivable microorganisms in organic compost based on the bidirectional diffusion principle according to claim 1, characterized in that: RTV silica gel is provided between the PTFE non-woven composite hydrophobic membrane (10) and the outer wall of the inner chamber (1).

3. The device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to claim 1, characterized in that: The outer side of the PTFE non-woven composite hydrophobic membrane (10) is coated with a nylon mesh protective layer (9).

4. The device for enriching uncultivable microorganisms in organic compost based on the bidirectional diffusion principle according to claim 2, characterized in that: A plurality of silicone elastic rings (6) are provided on the outside of the nylon mesh protective layer (9), and the nylon mesh protective layer (9) is tightened by the silicone elastic rings (6).

5. The device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to claim 1, characterized in that: The top of the inner chamber (1) is screwed with an inner chamber screw cover.

6. The device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to claim 5, characterized in that: The top of the outer chamber (2) is screwed with an outer chamber screw cover, and a through hole matching the inner chamber (1) is provided at the center of the outer chamber screw cover, and the inner chamber (1) is provided through the through hole.

7. The device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to claim 6, characterized in that: The inner chamber screw cover and the outer chamber screw cover are both provided with ventilation holes (8), and the ventilation holes (8) are covered with a Parafilm membrane (7).

8. The device for enriching uncultivable microorganisms in organic compost based on the principle of bidirectional diffusion according to claim 7, characterized in that: The Parafilm membrane (7) is in a wrinkled shape.

9. The device for enriching uncultivable microorganisms in organic compost based on the bidirectional diffusion principle according to claim 1, characterized in that: The culture medium (4) is a low-nutrient lignin culture medium, and the components and proportions of the low-nutrient lignin culture medium are as follows in terms of weight-volume ratio: 1.0% straw powder, 0.2% microcrystalline cellulose, 0.05% peptone, 10% compost extract, 0.1% agar, the balance is filled with sterile water, and the pH value is adjusted to 6.5-7 with phosphate buffer.

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