An in-situ self-conducting gas enrichment device, enrichment method and application for microorganisms in the vadose zone of contaminated soil

Abstract

This application relates to an in-situ self-aeration enrichment device for microorganisms in the vadose zone of contaminated soil, comprising an in-situ enrichment module, a filling matrix, and a composite nutrient additive. The in-situ enrichment module consists of a cuboid, a smooth frustum, and a long cylindrical air-guiding channel, which are integrally formed from bottom to top. The filling matrix is ​​placed into the enrichment module through a fine-mesh mesh bag. The composite nutrient additive is injected into the enrichment module through the long cylindrical air-guiding channel. This enrichment device improves the enrichment efficiency of uncultured microorganisms in the vadose zone of organically contaminated sites through the combined action of the in-situ enrichment module, the filling matrix, and the composite nutrient additive.

Description

Technical Field

[0001] This application relates to the fields of environmental microbiology technology and contaminated soil remediation technology, specifically to an in-situ self-conducting gas enrichment device, enrichment method and application for microorganisms in the vadose zone of contaminated soil. Background Technology

[0002] Microorganisms play a crucial role in the degradation of organic pollutants due to their broad-spectrum synergistic degradation characteristics. Traditional microbial culture techniques based on "screening culture + plate separation" are essentially ectopic enrichment and separation methods under controlled conditions. While this technique has successfully obtained and applied numerous pure culture strains, it relies heavily on fixed culture media and specific culture conditions. Screening results cannot fully reflect the complete microbial community in polluted habitats, and only a very small percentage of microorganisms can be successfully cultured in the laboratory. The limitations of traditional pure culture methods are becoming increasingly apparent. However, for pure culture strains that cannot currently be obtained under artificial culture conditions, these strains, acting as "microbial dark matter," play a significant role in the removal of pollutants from environmental media and in biodegradation systems.

[0003] Currently, biochemical and molecular biological methods based on non-culture modes such as fluorescence in situ hybridization, construction of bacterial 16S rRNA gene libraries, high-throughput sequencing, and metagenomics have also discovered many previously undiscovered microbial species. However, these methods cannot verify the culturability of the target microorganisms, nor can they investigate the metabolic characteristics and environmental interactions of pure bacterial species.

[0004] In summary, existing standard culture techniques are insufficient to fully explore the microbial community and metabolic functions in the vadose zone of soil, and to tap into the microbial resources in this area. Therefore, there is an urgent need to develop in-situ enrichment methods and devices for microorganisms in the vadose zone of contaminated soil. Summary of the Invention

[0005] This application addresses at least one of the problems of the related technology in the following aspects.

[0006] Therefore, the first aspect of this application proposes an in-situ self-conducting gas enrichment device for microorganisms in the vadose zone of contaminated soil. The device is characterized by comprising an in-situ enrichment module, a filling matrix, and a composite nutrient additive. The in-situ enrichment module is composed of a cuboid, a smooth frustum, and a long cylindrical gas-conducting channel. The cuboid, smooth frustum, and long cylindrical gas-conducting channel are integrally formed from bottom to top. The in-situ enrichment module has a wall thickness of 2 mm and a height of 10-20 cm. The cuboid has a length of (10-20) cm × a width of (6-12) cm × a height of 2 cm. The smooth frustum has a height of... The smooth prism has 48-96 small holes evenly distributed on it, each hole being circular with a diameter of 4-6 mm. The elongated cylindrical air channel has an inner diameter of a square with a side length of 1 cm and a wall thickness of 4 mm. The elongated cylindrical air channel can be stacked and extended, with a base height of 4-8 cm and an extension length of 0-960 cm. The part of the extension that connects with the smooth prism can be bent at a 90-degree angle. The filling matrix is ​​placed into the in-situ enrichment module through a fine mesh bag. The composite nutrient additive is injected into the in-situ enrichment module through the elongated cylindrical air channel.

[0007] In some embodiments, when the enrichment device is placed in the soil for in-situ enrichment, the long cylindrical air-conducting channel extends 0.1-0.3m above the ground, and the above-ground portion of the port is covered with a parafilm membrane to provide low-concentration cascade oxygen to the vadose zone of the soil.

[0008] In some embodiments, the filling matrix comprises, by weight percentage, the following components: bio-activated carbon: 8-75%, vermiculite: 15-90%, foamed silicon carbide: 2-20%, and polyhydroxybutyrate: 5-20%.

[0009] In some embodiments, the compound nutritional adjuvant includes one or more of sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannooligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid. Preferably, the compound nutritional adjuvant includes sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannooligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid in a mass ratio of (2-6): (3-9): (6-12): (0.5-4): (2-5): (0.8-8): (0.1-2), preferably 5:8:10:2:5:5:0.5.

[0010] In some embodiments, the total effective concentration of the compound nutritional supplement is 150 mg / L-4800 mg / L, preferably 800 mg / L-2600 mg / L, and more preferably 1800 mg / L.

[0011] In some embodiments, the preparation method of the filling matrix includes the following steps: pulverizing bio-activated carbon and vermiculite, and sieving to obtain bio-activated carbon powder and vermiculite powder; premixing polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder, then adding vermiculite powder for shear premixing, and finally adding foamed silicon carbide and the remaining bio-activated carbon powder for secondary mixing to obtain the filling matrix.

[0012] In some embodiments, the polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder are added to a high-speed mixer for premixing at a speed of 300-500 rpm for 5 min.

[0013] The second aspect of this application provides a method for in-situ enrichment of microorganisms in the vadose zone of contaminated soil, comprising the following steps: placing the enrichment device according to any one of claims 1-7 in a pre-dug pit; backfilling the enrichment device with soil from the original site during the excavation of the pit, with a long cylindrical air-conducting channel extending 0.1-0.3m above the ground, and the above-ground portion of the long cylindrical air-conducting channel covered with a parafilm membrane; and taking soil samples and analyzing their microbial diversity.

[0014] In some embodiments, the depth of the pit is 0.8-10.0 m; optionally, the microorganism is at least one of Achromobacterium, Rhodococcus, Acinetobacter, Aureobacterium, Neosphingolipids, Diaphorobacter, Microbacterium, and Trichomonas.

[0015] The third aspect of this application proposes the application of the enrichment device described in any embodiment of the first aspect of this application in the in-situ enrichment of microorganisms in contaminated soil.

[0016] The embodiments of this application achieve the following beneficial effects:

[0017] This application designs an in-situ enrichment device for uncultured microorganisms in soil from organically contaminated sites based on the principles of diffusion and directional enrichment. This device breaks through the cultivation limit of microorganisms in the vadose zone. Through the combined action of the in-situ enrichment module, filling matrix, and composite nutrient additives, it improves the enrichment efficiency of uncultured microorganisms in the vadose zone of organically contaminated sites. Through in-situ micro-disturbance enrichment, it utilizes a multi-component filling matrix of foamed silicon carbide, activated carbon, polyhydroxybutyrate, and vermiculite to enhance the material cycle in the in-situ habitat, providing a micro-environment for uncultured microorganisms to attach and proliferate more easily. This simulates and restores the energy transfer and signal exchange in the natural ecosystem. Furthermore, this device has strong application compatibility and can be adapted to the in-situ enrichment and screening of various environmental samples such as soil and river / lake sediments. It is suitable for the directional separation of various organic pollutant-degrading bacteria. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the technology roadmap for this application; Figure 2 This is a three-dimensional schematic diagram of the in-situ enrichment module according to an embodiment of this application; Figure 3 This is a three-dimensional orthogonal top view of the in-situ enrichment module according to an embodiment of this application; Figure 4 This is a three-dimensional orthogonal front view of the in-situ enrichment module according to an embodiment of this application; Figure 5 This is a diagram illustrating the application effect of in-situ screening of microorganisms in the vadose zone of petroleum hydrocarbon-contaminated soil according to an embodiment of this application. Figure 6 This image shows the effect of in-situ screening of microorganisms in the vadose zone of polycyclic aromatic hydrocarbon and aniline contaminated soil according to embodiments of this application. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative and not intended to limit the scope of the invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0021] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] The first aspect of this application discloses an in-situ self-conducting aeration enrichment device for microorganisms in the vadose zone of contaminated soil, comprising an in-situ enrichment module, a filling matrix, and a composite nutrient additive. The in-situ enrichment module is composed of a cuboid, a smooth frustum, and a long cylindrical aeration channel. The cuboid, smooth frustum, and long cylindrical aeration channel are integrally formed from bottom to top. The in-situ enrichment module has a wall thickness of 2 mm and a height of 10-20 cm. The cuboid has a length of (10-20) cm × a width of (6-12) cm × a height of 2 cm. The smooth frustum has a height of 4- The smooth prism has 48-96 small holes evenly distributed on it, each hole being circular with a diameter of 4-6 mm. The elongated cylindrical air channel has an inner diameter of a square with a side length of 1 cm and a wall thickness of 4 mm. The elongated cylindrical air channel can be stacked and extended, with a base height of 4-8 cm and an extension length of 0-960 cm. The part of the extension that connects with the smooth prism can be bent at a 90-degree angle. The filling matrix is ​​placed into the enrichment module through a fine mesh bag. The composite nutrient additive is injected into the enrichment module through the elongated cylindrical air channel.

[0023] In some embodiments, the in-situ self-conducting gas enrichment device for microorganisms in the vadose zone of contaminated soil includes an in-situ enrichment module, a filling matrix, and a composite nutrient additive. The in-situ enrichment module is a combination of a cuboid, a smooth frustum, and a long cylindrical gas guiding channel. Small holes are arranged on the surface of the smooth frustum, and there is a snap-on base at the bottom and a long cylindrical gas guiding channel at the top. In a specific embodiment of this application, the in-situ enrichment module has an overall height of 20cm and is divided into three parts: a cuboid, a smooth frustum, and a long cylindrical air guide channel. The cuboid is 2cm high, the smooth frustum is 10cm high, and the two together are 12cm high. The long cylindrical air guide channel has a basic height of 8cm and can be extended to 0-960cm. The extended part is made of stainless steel. This channel can be stacked and extended. The part connected to the smooth frustum can be bent at 90 degrees. The inner diameter of the long cylindrical air guide channel is a square with a side length of 1cm and a wall thickness of 4mm. The enrichment module has an overall wall thickness of 2mm. The bottom is a cuboid (20cm long × 12cm wide × 2cm high). There are 96 small holes evenly distributed on the smooth frustum. The small holes are circular and have a diameter of 6mm.

[0024] In some embodiments, the in-situ enrichment module can be simultaneously enlarged or reduced. If manual installation is used for in-situ enrichment, the in-situ enrichment module with the dimensions described above can be used. If an SH-30 impact drill is used for drilling before installing the in-situ enrichment module, the module size can be proportionally reduced. The module is 10cm high and consists of three parts: a cuboid, a smooth frustum, and a long cylindrical air channel. The cuboid is 2cm high, the smooth frustum is 4cm high (totaling 6cm), and the long cylindrical air channel has a base height of 4cm, which can be extended from 0-960cm. The extended portion is made of stainless steel. The material is such that the channel can be stacked and extended, and the part that connects with the smooth prism can be bent at 90 degrees. The inner diameter of the long cylindrical air-conducting channel is a square with a side length of 1cm and a wall thickness of 4mm. This long cylindrical air-conducting channel can supply low-concentration cascade oxygen to the vadose zone of the soil through the Parafilm membrane exposed on the ground, moderately regulate the gas composition of the vadose zone region of the soil, and effectively enhance the activity of facultative anaerobic microorganisms. The overall wall thickness of the in-situ enrichment module is 2mm, and the bottom is a cuboid (length 10cm × width 6cm × height 2cm). There are 48 small holes evenly distributed on the smooth prism. The small holes are circular and have a diameter of 4mm.

[0025] In some embodiments, the filling matrix that is used in conjunction with the in-situ enrichment module can serve as a carrier for microbial enrichment. The specific carrier components include bio-activated carbon, vermiculite, foamed silicon carbide, and polyhydroxybutyrate, with a mass composition ratio of 8-75:15-90:2-20:5-20.

[0026] In some embodiments, the compound nutritional adjuvant includes one or more of sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannooligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid. In some embodiments, the compound nutritional adjuvant comprises sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannooligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid in a mass ratio of (2-6): (3-9): (6-12): (0.5-4): (2-5): (0.8-8): (0.1-2), preferably 5:8:10:2:5:5:0.5, at which the in-situ enrichment effect is best. In some embodiments, the total effective concentration of the compound nutritional adjuvant is 150 mg / L-4800 mg / L, preferably 800 mg / L-2600 mg / L, and more preferably 1800 mg / L.

[0027] In some embodiments, the preparation method of the filling matrix includes the following steps: pulverizing bio-activated carbon and vermiculite, and sieving to obtain bio-activated carbon powder and vermiculite powder; premixing polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder, then adding vermiculite powder for shear premixing, and finally adding foamed silicon carbide and the remaining bio-activated carbon powder for secondary mixing to obtain the filling matrix. In a specific embodiment of this application, the bio-activated carbon is first pulverized and sieved to obtain a uniform powder (e.g., 20-50 mesh), and dried in a 105°C drying oven to remove moisture; the vermiculite is ground and sieved through a 20-mesh sieve for later use; commercially available 20-mesh polyhydroxybutyrate particles are selected, and the polyhydroxybutyrate and an equal amount of bio-activated carbon powder are first premixed in a high-speed mixer (e.g., a V-type mixer, a three-dimensional motion mixer), then the sieved vermiculite is added for shear premixing, and finally the foamed silicon carbide and the remaining bio-activated carbon powder are added for secondary mixing to obtain the filling matrix. In some embodiments, the polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder are added to a high-speed mixer for premixing at a speed of 300–500 rpm for 5 minutes. The filling matrix is ​​contained in a fine-mesh bag made of polyhydroxybutyrate and nylon, and the bag is tied tightly with a nylon rope and then extended along the long cylindrical air channel to the exposed part of the long cylindrical air channel on the ground.

[0028] In some embodiments, the assembly sequence of the enrichment device is as follows: first, the filling matrix is ​​placed in a fine mesh bag and then placed into the enrichment module, and then the rectangular cover at the bottom of the enrichment module is fastened to ensure that there is no spillage or leakage.

[0029] The second aspect of this application proposes a method for in-situ enrichment of microorganisms in the vadose zone of contaminated soil, comprising the following steps: placing the enrichment device described in any embodiment of the first aspect of this application in a pre-dug pit; backfilling the enrichment device with the in-situ soil used to dig the pit, with the long cylindrical air-conducting channel extending 0.1-0.3m above the ground, and the above-ground portion of the long cylindrical air-conducting channel covered with a parafilm membrane; and taking soil samples and analyzing their microbial diversity. In a specific embodiment of this application, the operation procedure for the enrichment device to enter the soil for in-situ enrichment is as follows: If manual excavation is used, a pit approximately 5 cm larger than the size of the enrichment device can be dug around the pre-buried soil using a handheld soil-digging device, based on the size of the sampling enrichment device. The pit depth is 0.8-10.0 m, preferably 3.2 m. Sterile water is sprinkled into the pit, and then the assembled enrichment device is buried, placed at the bottom of the pit, and backfilled with soil from the original location where the pit was dug. The long cylindrical air-conducting channel extends approximately 0.1-0.3 m above the ground. The above-ground ends of the cylindrical air-conducting channel are covered with parafilm; alternatively, an SH-30 drilling rig, a truck-mounted drill, or a Geoprobe drilling rig is used to excavate holes to a depth of 0.8-10.0m, preferably 4.5m. The assembled enrichment device is then placed at the bottom of the hole, and the enrichment device is backfilled with soil in situ. The long cylindrical air-conducting channel extends 0.1-0.3m above the ground. After backfilling, 0.5L-2L of sterile water is injected through the long cylindrical air-conducting channel, and the above-ground ends of the long cylindrical air-conducting channel are covered with parafilm. Soil samples are taken at predetermined depths after 60 days and 90 days after both manual and mechanical hole excavation methods, and the microbial diversity is analyzed.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0032] Example 1: Application of the enrichment device in in-situ screening of microorganisms in the vadose zone of petroleum hydrocarbon contaminated soil 1.1 Soil selection and drilling operations Typical petroleum hydrocarbon-contaminated soil was selected as the test site, and vertical drilling was carried out using a Rhino handheld hydraulic soil sampling drill (model HPD60). The drilling depth was set at 3.2 m, and the hole diameter was designed to allow for the easy placement of the enrichment device, which had dimensions of 20 cm in length, 12 cm in width, and 12 cm in height. If hard soil layers or obstacles were encountered during drilling, manual excavation and trimming were used to ensure a smooth hole wall and flat bottom.

[0033] 1.2 Preparation of the filling matrix for the enrichment device (1) Raw material pretreatment: The bio-activated carbon is pulverized by a pulverizer and passed through a 20-mesh standard sieve. The uniform powder passing through the sieve is collected and dried in a constant temperature drying oven at 105°C until constant weight to remove residual moisture. Vermiculite is ground and passed through a 20-mesh sieve for later use. Polyhydroxybutyrate is directly selected from commercially available 20-mesh granules without further processing. Foamed silicon carbide is of commercial specifications.

[0034] (2) Material ratio: The components are weighed accurately according to the mass ratio of biological activated carbon, vermiculite, foamed silicon carbide and polyhydroxybutyrate of 75:60:12:6.

[0035] (3) Mixing process: First, add polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder to a high-speed mixer for premixing. The speed is controlled at 300-500 rpm and the mixing time is 5 min. Then, add sieved vermiculite, switch to shear mixing mode, and continue mixing for 8 min. Finally, add foamed silicon carbide and the remaining bio-activated carbon powder, and mix for 10 min to ensure that the components are fully dispersed and uniform.

[0036] (4) Filling treatment: The mixed matrix is ​​rinsed three times with sterile water and air-dried naturally until the moisture content is about 20-30%; the semi-wet matrix is ​​put into a fine polypropylene mesh bag, sealed and placed into the enrichment module cavity, and the bottom rectangular sealing cap is fastened to ensure that the filling matrix is ​​leak-free and loose.

[0037] 1.3 Underground Layout of Enrichment Devices The assembled enrichment device is gently placed at the bottom center of the drilled soil hole, and its posture is adjusted to keep it upright, with the bottom surface in close contact with the soil at the bottom of the hole, so as to ensure effective contact between the substrate and soil microorganisms and pollutants.

[0038] 1.4 Backfilling of Soil Holes and Deployment of Air Pipes The stacked, compressed, long-tube gas venting system at the top of the enrichment device was stretched upwards section by section until its venting port was 0.2 m above ground level. In-situ contaminated soil collected during drilling (i.e., the original sampling soil) was used as backfill material, and the boreholes were backfilled in layers. Every 10 cm of backfill was moderately compacted with a wooden mallet or compaction rod to simulate the natural bulk density of the soil. Throughout the backfilling process, the gas venting system was kept vertically centered, with only its venting port exposed above the ground surface to ensure unimpeded gas exchange between the inside and outside of the device.

[0039] 1.5 Ventilation end sealing and physical protection After backfilling is completed, immediately wrap the exposed air duct port with Parafilm sealing film tightly for 2-3 layers to form a waterproof and breathable sealing layer, preventing natural precipitation evaporation and the intrusion of soil particles, insects, and other foreign objects. Then, take a clean 50 mL centrifuge tube, invert it and place it over the outside of the sealing film, and press it down slightly to make it form a stable support with the surface soil, acting as a mechanical protective cover to prevent damage to the device from external impacts or trampling.

[0040] 1.6 In-situ enrichment culture and sampling After the above setup is completed, the mixture is left to stand in a natural environment for 60 days without any additional disturbance or irrigation. At the end of the enrichment period, carefully remove the centrifuge tube protective cover and sealing film. Use a handheld soil extractor to remove the soil backfilled at the beginning of the experiment. Slowly retract the gas delivery tube and pull the entire enrichment device out, transferring it to the laboratory. Remove the filling substrate from the nylon mesh bag and place it in a beaker. Wash it three times with sterile water and sonicate it for 5 minutes. Then pass it through a 30-mesh stainless steel sieve. Use a powerful soil DNA extraction kit to extract DNA from the sieved mud according to the operating procedures and test the purity of the extracted total DNA. Send the qualified DNA extract to a sequencing company for 16S rDNA-based microbial community analysis.

[0041] Comparative Example 1 To eliminate the interference of soil background effects and environmental fluctuations on the experimental results, control soil holes were drilled at the same depth (3.2 m) within a 1.0 m radius of the test soil holes. Except for the absence of enrichment devices, all other operations in the control holes—including drilling and backfilling—were strictly consistent with those in the test holes, serving as a blank control. After 60 days, soil at a fixed depth of 3.2 m was selected as the microbial community control group.

[0042] Comparison of in-situ enrichment effects The samples from the experimental and control groups were collected and analyzed using the method described above. The test results are as follows: Figure 5 As shown. Figure 5A direct comparison was made between the experimental group and the control group 60 days after the enrichment device was deployed, showing the differences in key indicators such as the relative abundance of petroleum hydrocarbon-degrading bacteria. The relative abundance of Achromobacterium, Rhodococcus, Acinetobacter, and Acanthocephala increased, and the percentage increase in relative abundance was the enrichment efficiency. The in-situ enrichment device could increase the enrichment efficiency of Achromobacterium, Rhodococcus, Acinetobacter, and Acanthocephala by 57.1%, 77.8%, 80%, and 45.5%, respectively.

[0043] Example 2: Application of the enrichment device in in-situ screening of microorganisms in the vadose zone of aromatic hydrocarbon-contaminated soil 2.1 Soil selection and drilling operations Typical soils contaminated with polycyclic aromatic hydrocarbons (PAHs) and aniline were selected as the test site, and vertical drilling operations were carried out using an SH-30 drilling rig. The drilling depth was set at 4.5 m, and the borehole diameter was designed to allow for the easy placement of the enrichment device, which had dimensions of 20 cm in length, 12 cm in width, and 12 cm in height. If hard soil layers or obstacles were encountered during drilling, manual excavation and trimming were employed to ensure a smooth borehole wall and flat bottom.

[0044] 2.2 Preparation of the filling matrix for the enrichment device (1) Raw material pretreatment: The bio-activated carbon is pulverized by a pulverizer and passed through a 20-mesh standard sieve. The uniform powder passing through the sieve is collected and dried in a constant temperature drying oven at 105°C until constant weight to remove residual moisture. Vermiculite is ground and passed through a 20-mesh sieve for later use. Polyhydroxybutyrate is directly selected from commercially available 20-mesh granules without further processing. Foamed silicon carbide is of commercial specifications.

[0045] (2) Material ratio: The components are weighed accurately according to the mass ratio of biological activated carbon, vermiculite, foamed silicon carbide and polyhydroxybutyrate of 75:60:12:6.

[0046] (3) Mixing process: First, add polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder to a high-speed mixer for premixing. The speed is controlled at 300-500 rpm and the mixing time is 5 min. Then, add sieved vermiculite, switch to shear mixing mode, and continue mixing for 8 min. Finally, add foamed silicon carbide and the remaining bio-activated carbon powder, and mix for 10 min to ensure that the components are fully dispersed and uniform.

[0047] (4) Filling treatment: The mixed matrix is ​​rinsed three times with sterile water and air-dried naturally until the moisture content is about 20-30%; the semi-wet matrix is ​​put into a fine polypropylene mesh bag, sealed and placed into the enrichment module cavity, and the bottom rectangular sealing cap is fastened to ensure that the filling matrix is ​​leak-free and loose.

[0048] 2.3 Underground Layout of Enrichment Devices The assembled enrichment device is gently placed at the bottom center of the drilled soil hole, and its posture is adjusted to keep it upright, with the bottom surface in close contact with the soil at the bottom of the hole, so as to ensure effective contact between the substrate and soil microorganisms and pollutants.

[0049] 2.4 Backfilling of Soil Holes and Deployment of Air Pipes The stacked, compressed, long-tube gas venting system at the top of the enrichment device was stretched upwards section by section until its venting port was 0.2 m above ground level. In-situ contaminated soil collected during drilling (i.e., the original sampling soil) was used as backfill material, and the boreholes were backfilled in layers. Every 10 cm of backfill was moderately compacted with a wooden mallet or compaction rod to simulate the natural bulk density of the soil. Throughout the backfilling process, the gas venting system was kept vertically centered, with only its venting port exposed above the ground surface to ensure unimpeded gas exchange between the inside and outside of the device.

[0050] 2.5 Ventilation end sealing and physical protection After backfilling is completed, immediately wrap the exposed air duct port with Parafilm sealing film tightly for 2-3 layers to form a waterproof and breathable sealing layer, preventing natural precipitation evaporation and the intrusion of soil particles, insects, and other foreign objects. Then, take a clean 50 mL centrifuge tube, invert it and place it over the outside of the sealing film, and press it down slightly to make it form a stable support with the surface soil, acting as a mechanical protective cover to prevent damage to the device from external impacts or trampling.

[0051] 2.6 In-situ enrichment culture and sampling After the above setup was completed, the cells were statically cultured under natural environmental conditions for 90 days. During this period, a compound nutrient supplement was added on day 45. The specific components of the nutrient supplement were: sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannan oligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid, with a corresponding mass ratio of 5:8:10:2:5:5:0.5. The total effective concentration of the nutrient supplement was 1800 mg / L. The supplement was injected into the in-situ enrichment device in three separate injections along a long cylindrical gas delivery channel, 500 ml each time. After injection, the device was resealed with film, and a 50 mL centrifuge tube was placed upside down outside the film for protection. At the end of the 90-day enrichment period, carefully remove the protective cover and sealing film of the centrifuge tube. Use a handheld soil sampler to remove the soil backfilled at the beginning of the experiment. Slowly retract the gas delivery tube and pull out the entire enrichment device. Transfer it to the laboratory. Remove the filling substrate from the nylon mesh bag and place it in a beaker. Wash it three times with sterile water and use ultrasonic vibration for 5 minutes. Then pass it through a 30-mesh stainless steel sieve. Use a powerful soil DNA extraction kit to extract DNA from the sieved mud according to the operating procedure and test the purity of the extracted total DNA. Send the qualified DNA extract to a sequencing company for microbial community analysis based on 16S rDNA.

[0052] Comparative Example 2 To eliminate the interference of soil background effects and environmental fluctuations on the experimental results, control soil holes were drilled at the same depth (4.5 m) within a 1.0 m radius of the test soil holes. Except for the absence of enrichment devices, all other operations in the control holes—including drilling and backfilling—were strictly consistent with those in the test holes, serving as a blank control. After 90 days, soil at a fixed depth of 4.5 m was selected as the microbial community control group.

[0053] Comparison of in-situ enrichment effects The samples from the experimental and control groups were collected and analyzed using the method described above. The test results are as follows: Figure 6 As shown. Figure 6 A direct comparison was made between the experimental group and the control group 90 days after the enrichment device was deployed, showing the differences in key indicators such as the relative abundance of aromatic hydrocarbon-degrading bacteria. The relative abundance of *Neosphophytum*, *Diaphorobacter*, *Microbacterium*, and *Trichomonas* increased, and the percentage increase in relative abundance was the enrichment efficiency. The in-situ enrichment device could increase the enrichment efficiency of *Neosphophytum*, *Diaphorobacter*, *Microbacterium*, and *Trichomonas* by 120%, 65%, 85%, and 71.4%, respectively.

[0054] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A device for in-situ self-conducting gas enrichment of microorganisms in the vadose zone of contaminated soil, characterized in that, The invention includes an in-situ enrichment module, a filling matrix, and a composite nutrient additive. The in-situ enrichment module consists of a cuboid, a smooth frustum, and a long cylindrical air channel. The cuboid, smooth frustum, and long cylindrical air channel are integrally formed from bottom to top. The in-situ enrichment module has a wall thickness of 2 mm and a height of 10-20 cm. The cuboid has a length of (10-20) cm × a width of (6-12) cm × a height of 2 cm. The smooth frustum has a height of 4-10 cm and is evenly distributed with 48-96 circular holes with a diameter of 4-6 mm. The long cylindrical air channel has an inner diameter of a square with a side length of 1 cm and a wall thickness of 4 mm. The long cylindrical air channel can be stacked and extended, with a base height of 4-8 cm and an extension length of 0-960 cm. The extension part that connects with the smooth frustum can be bent at a 90-degree angle. The filling matrix is ​​placed into the in-situ enrichment module by being placed in a fine mesh bag; The compound nutrient additive is injected into the in-situ enrichment module through the long cylindrical air channel.

2. The enrichment device according to claim 1, characterized in that, When the enrichment device is placed in the soil for in-situ enrichment, the long cylindrical air-conducting channel extends 0.1-0.3m above the ground, and the above-ground portion of the port is covered with a parafilm membrane to provide low-concentration cascade oxygen to the vadose zone of the soil.

3. The enrichment device according to claim 1 or 2, characterized in that, The filling matrix comprises the following components by weight percentage: bio-activated carbon: 8-75%, vermiculite: 15-90%, foamed silicon carbide: 2-20%, and polyhydroxybutyrate: 5-20%.

4. The enrichment apparatus according to any one of claims 1-3, characterized in that, The compound nutritional adjuvant includes one or more of sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannan oligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid. Preferably, the compound nutritional adjuvant includes sodium pyruvate, polylactic acid, monoethylenediamine β-cyclodextrin, mannan oligosaccharide, isobutylidene diurea, fumaric acid, and methyl jasmonic acid in a mass ratio of (2-6): (3-9): (6-12): (0.5-4): (2-5): (0.8-8): (0.1-2), preferably 5:8:10:2:5:5:0.

5.

5. The enrichment device according to claim 4, characterized in that, The total effective concentration of the compound nutritional supplement is 150 mg / L-4800 mg / L, preferably 800 mg / L-2600 mg / L, and more preferably 1800 mg / L.

6. The enrichment apparatus according to any one of claims 1-5, characterized in that, The method for preparing the filling matrix includes the following steps: The bio-activated carbon and vermiculite were pulverized and sieved to obtain bio-activated carbon powder and vermiculite powder; The filling matrix is ​​obtained by premixing polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder, then adding vermiculite powder for shear premixing, and finally adding foamed silicon carbide and the remaining bio-activated carbon powder for secondary mixing.

7. The enrichment apparatus according to claim 6, characterized in that, The polyhydroxybutyrate particles and an equal amount of bio-activated carbon powder were added to a high-speed mixer for premixing at a speed of 300-500 rpm for 5 min.

8. A method for in-situ enrichment of microorganisms in the vadose zone of contaminated soil, characterized in that, Includes the following steps: Place the enrichment device according to any one of claims 1-7 in a pre-dug pit; The enrichment device is backfilled with soil from the original location where the pit was dug, and the long cylindrical air-conducting channel extends 0.1-0.3m above the ground. The above-ground portion of the long cylindrical air-conducting channel is covered with a parafilm membrane. Soil samples were taken and their microbial diversity was analyzed.

9. The method according to claim 8, characterized in that, The depth of the pit is 0.8-10.0m; Optionally, the microorganism is at least one of the genera Achromobacterium, Rhodococcus, Acinetobacter, Aureobacterium, Neosphingolipids, Diaphorobacter, Microbacterium, and Trichomonas.

10. The application of the enrichment device according to any one of claims 1-7 in the in-situ enrichment of microorganisms in contaminated soil.

Images