WO2024261867A1 - Soil environment simulation device for observing soil microorganism - Google Patents

Abstract

Provided is a soil environment simulation device for observing soil microorganisms, wherein the soil environment simulation device includes a body including an adhesion part and a processing part connected to the adhesion part, a soil simulation structure is formed on an upper surface of the processing part, the soil simulation structure includes a plurality of columnar structures, the columnar structure has an upper surface part at an upper end, and the upper surface part of the columnar structure is included in substantially the same plane as the upper surface of the adhesion part.

Description

A soil environment simulator for observing soil microorganisms

This disclosure relates to a soil environment simulator for observing soil microorganisms.

Soil generally contains 10 ⁹ -10 ¹⁰ microorganisms per gram, with 10 ⁴ -10 ⁵ species. These diverse and abundant microorganisms can coexist in soil because the microenvironment contains voids of a size suitable for the growth of each microorganism, isolating them from each other. Observing the behavior of microorganisms in such soil microenvironments can be important for understanding the functions of microorganisms and developing microbial utilization technologies in agriculture, environmental science, etc. However, many microorganisms are difficult to cultivate in a laboratory environment, and the ecology of many of them is unknown. In addition, soil is optically opaque, and there are limited techniques for observing microorganisms in the soil environment.

One of the major issues regarding soil microorganisms is that most microorganisms that live in soil are difficult to culture. This means that it is difficult to understand the microbial populations present in soil. One solution to this problem is the development of rRNA amplicon analysis, an analytical technique that does not require cultivation, which makes it possible to determine the types and proportions of microorganisms present in the target soil. There is also technology that improves the collection rate of difficult-to-culture microorganisms by culturing microorganisms in the environment using hollow fiber membranes (Non-Patent Document 1).

Yoshiteru Aoi, "Breaking the conventional wisdom of technology: A new strategy for isolating and culturing microorganisms," Waseda University Institute for Advanced Study News, May 6, 2009. https://www.waseda.jp/inst/wias/news/2009/05/06/3442/

Because microorganisms interact with each other, positional information about what other microorganisms these microorganisms have symbiotic relationships with is important in order to clarify the functions of microorganisms in soil. With conventional technology, it is possible to know what microorganisms exist in soil and to collect those microorganisms, but it is not possible to obtain positional information about how microorganisms are distributed in the soil environment. In addition, because soil is optically opaque, it has been difficult to verify the positional relationships between microorganisms, such as microbial segregation and symbiotic relationships, in the diverse microbial communities that exist in natural soil.

This disclosure was made in consideration of the above-mentioned problems, and aims to provide a soil environment simulator for observing soil microorganisms.

In one embodiment of the present disclosure, a soil environment simulator for observing soil microorganisms includes a main body including an adhesive part and a processing part connected to the adhesive part, and a soil simulation structure is formed on the upper surface of the processing part, and the soil simulation structure includes a plurality of columnar structures, each of which has an upper surface at its upper end, and the upper surface of the columnar structure is included in approximately the same plane as the upper surface of the adhesive part.

According to the present disclosure, it is possible to provide a soil environment simulator for observing soil microorganisms.

FIG. 1A is a schematic diagram (top view) showing the whole and a part of a soil environment simulator according to an embodiment. FIG. 1B is a schematic diagram (side view) showing the whole and a part of the soil environment simulator of the embodiment. FIG. 2A is a schematic diagram (top view) showing a cover part of the soil environment simulator of the embodiment. FIG. 2B is a schematic diagram (side view) showing the cover of the soil environment simulator of the embodiment. FIG. 3A is a schematic diagram (top view) showing a soil environment simulator including macro-aggregates according to an embodiment. FIG. 3B is a schematic diagram (side view) showing a soil environment simulator including macro-aggregates according to an embodiment. FIG. 4 is a schematic diagram (side view) showing a soil environment simulator including a plurality of main bodies stacked vertically according to the embodiment.

The following describes an embodiment of the present disclosure with reference to the drawings.

The soil microorganisms in this disclosure are not limited to organisms that live in soil and are of a size that can be accommodated in the soil environment simulator of the embodiment. The soil microorganisms may be a variety of organisms including bacteria, archaea, fungi, animals, plants, or combinations thereof.

　The soil environment simulator of the embodiment allows observation of soil microorganisms. The soil microorganisms can be observed without any special staining treatment, or can be observed after staining (e.g., fluorescent staining) by a method known to those skilled in the art. Not only at the stage immediately prior to observation, but also at the stage of introducing the microorganisms into the device, embodiments are contemplated in which chemically or genetically modified microorganisms are included to facilitate detection or for other purposes.

The soil environment simulator of the embodiment is a device that simulates soil structure, and can advantageously be used by being embedded in soil for at least a certain period of time. By embedding the device in actual soil for a certain period of time, the microbial flora in the soil can expand within the device. The soil environment simulator of the embodiment is a device that reproduces the large and small gaps in soil, which are thought to be important for the differentiation of microorganisms, and by reproducing the soil microstructure with openings through which microorganisms in the soil can flow in and out on the substrate surface, it is possible to observe the distance relationship of the microbial population within the microstructure. Such a soil-embedded device can be embedded in soil for a certain period of time and then retrieved, and the microorganisms within the device can be observed. However, it is also possible to artificially select a microbial population and apply it to the device without embedding it in soil.

The soil simulation device of the embodiment can be composed of a substrate (e.g., glass, polymer, etc.) on whose surface a soil simulation structure is formed, and a cover portion (which can be, for example, glass, polymer, etc., and can be the same material as the substrate or a different material) adhered to the substrate surface, and the soil simulation structure formed on the substrate surface can reproduce three types of microstructure sizes: gaps within microaggregates (e.g., 1-10 μm), gaps between microaggregates (e.g., 10-50 μm), and gaps between macroaggregates (e.g., ≧50 μm). This feature can provide the effect of making it possible to observe the distribution of microorganisms and/or the spatial relationships between microorganisms within the microstructure.

1A and 1B are schematic diagrams showing the whole (lower side of FIG. 1A and FIG. 1B) and a part (upper side of FIG. 1A, which is a partially enlarged view) of an example of a soil environment simulator of an embodiment. The soil environment simulator shown in these figures has a main body 1, which has an adhesive part 2, an upper surface 2' of the adhesive part, a processed part 3, an upper surface 3' of the processed part, a soil simulation structure 4, a columnar structure 5, and an upper surface part 5' at the upper end of the columnar structure, and the columnar structure 5 is clustered to form a micro aggregate 6. FIG. 1A shows the appearance of this soil environment simulator as seen from above, and FIG. 1B shows the appearance as seen from the side. Such a soil environment simulator can be provided in different sizes depending on the individual application, and can be, for example, a microfluidic device-like device with a diameter (or maximum diameter) of the processed part of 10 cm or less or 5 cm or less. The diameter (or maximum diameter) of the processed part is typically 0.5 cm or more or 1 cm or more, but the lower limit is not particularly limited. In a typical example, the processed portion 3 may be 2-6 cm wide by 2-3 cm long (at the boundary line with the adhesive portion 2), and such a processed portion can be fabricated in a substrate having the same dimensions as a normal microscope slide. In this case, the dimensions of the main body 1 may be 7-8 cm wide by 2-3 cm long. The thickness of the main body may be, for example, 0.5-5 mm.

The main body 1 includes an adhesive portion 2 and a processed portion 3 connected thereto. The main body 1 may be formed from a transparent substrate, and examples of materials for the transparent substrate include glass and various synthetic resins. It is preferable that the processed portion 3 of the main body is transparent, and in particular, it is preferable that the area of the processed portion 3 that does not have the columnar structure 5 is transparent. The use of a transparent substrate can facilitate observation of the sample in the device using a microscope or the like. However, an embodiment in which a soil environment simulator made of an opaque substrate is observed using a microscope equipped with, for example, epi-illumination can also be contemplated. The thickness of the main body 1 is not limited, but it is preferable that the thickness is suitable for microscopic observation (for example, 25 μm to 1600 μm).

The adhesive portion 2 typically has an upper surface 2' that is a flat surface. The adhesive portion 2 can be bonded to the cover portion 7 described below or to the underside of the main body 1 of another stacked soil environment simulator via the upper surface 2'. The underside of the cover portion 7 or the underside of the other stacked main body 1 can have a shape (typically a flat surface) corresponding to the upper surface 2' so as to enable bonding (e.g., bonding by close contact) with the upper surface 2' of the adhesive portion 2. The upper surface 2' of the adhesive portion 2 does not necessarily have to be flat all over. For example, although not shown, the upper surface 2' may have a protrusion and the underside of the cover portion 7 or the underside of the other stacked main body 1 may have a hole that engages with the protrusion, or vice versa.

The processing unit 3 has a soil simulation structure 4 formed on its upper surface 3'. By storing soil microorganisms in the processing unit 3 on which the soil simulation structure 4 is formed, the behavior of the soil microorganisms in the simulated soil structure can be observed. As described later, the processing unit 3 can simulate soil structures of different sizes, including one or more of columnar structures 5, microaggregates 6, and macroaggregates 8. Each columnar structure 5 can mimic individual soil or sand grains in nature. The microaggregates 6 and macroaggregates 8 in the device of the embodiment can mimic aggregates in nature. In nature, aggregates are larger particles of soil or sand that have been consolidated, and are classified into microaggregates (diameter less than 250 μm) and macroaggregates (diameter 250 μm or more) according to size. Microaggregates in nature are typically soil grains that have been consolidated by bacterial secretions, and macroaggregates are typically formed by the entanglement of microaggregates, fungal hyphae, and plant roots. In this specification, the term aggregates used in the context of the device is used to mean a structure that mimics aggregates found in nature.

In an embodiment, the processed portion 3 forms a flow path to allow microorganisms to flow in from the environment and to mimic the gaps between particles in real soil. The entire surface of the processed portion 3 is soil-simulating processed, and at least one side (two opposing sides in FIG. 1A) is connected to the outside of the device, allowing microorganisms to flow in from the environment into the device. Meanwhile, the area of the main body 1 that is left adjacent to (or connected to) the processed portion 3 without the soil-simulating process can form a "glue" or adhesive portion 2 for adhering the cover portion 7 to the main body 1.

In an embodiment, for example, when the substrate is glass, the surface may be subjected to a charge imparting treatment such as poly-L-lysine coating or other surface modification treatments known to those skilled in the art to adjust the adsorption of microorganisms.

The soil simulation structure 4 is a structure that mimics a natural soil structure, formed as a group of the upper surface 3' of the processed part 3 and the columnar structures 5 arranged thereon. The method for forming the soil simulation structure 4 can be selected appropriately by a person skilled in the art based on his/her general knowledge and is not particularly limited. For example, the soil simulation structure 4 may be formed by directly engraving the substrate, or by using a laser processing method known to those skilled in the art. In addition, when a glass substrate is used, a glass etching method known to those skilled in the art can be used. When the substrate is a silicone-based or non-silicone-based resin such as polydimethylsiloxane (PDMS), the soil simulation structure 4 can be formed by a molding method such as PDMS molding known to those skilled in the art. A separately manufactured columnar structure 5 may be bonded to the upper surface 3' of the processed part, in which case the material of the columnar structure 5 may be the same as or different from the other parts of the processed part 3.

The soil simulation structure 4 includes a plurality of columnar structures 5. Each column in the columnar structure 5 corresponds to an individual soil grain in the soil. The cross-sectional shape of the column may be circular, but is not limited thereto, and may be any shape, including, for example, elliptical, polygonal, and irregular. In order to close the upper side by adhering the cover part 7 to form a flow path in the processing part 3, the upper surface part 5', which is the upper end of the columnar structure 5 forming the soil simulation structure, is formed so as to be included in approximately the same plane as the upper surface 2' of the adhesive part. This means that when the cover part 7 is adhered to the adhesive part 2 of the main body 1, the upper surface part 5' of the columnar structure 5 in the processing part 3 can also contact the cover part 7. There are gaps between adjacent columnar structures 5 so that liquid and, in some cases, microorganisms can flow, and these gaps form the above-mentioned flow path. The height of the upper surface part of the columnar structure can be, for example, 1 to 150 μm from the bottom surface (upper surface 3') of the processing part. This height may be varied according to the position within the soil simulation structure 4 by varying the depth of the bottom surface (upper surface 3') of the processed portion, as described below.

As shown in Figures 1A and 1B, the soil-simulating structure 4 may include a plurality of micro-aggregates 6. Each of the plurality of micro-aggregates 6 is a cluster with a diameter of 250 μm or less formed by a plurality of the columnar structures (5) densely packed together. The term "cluster" here means that a plurality of columnar structures are densely packed together at a density higher than the columnar structure density of the soil-simulating structure 4 as a whole, forming a visible outline in the soil-simulating structure 4, or a bundle of columns (or in some cases, a bundle of bundles of columns, like macro-aggregates described later). In the lower part of Figure 1A, the outline of the micro-aggregate 6 is drawn with a solid line to aid understanding, but this does not mean that the micro-aggregate 6 needs to be defined by some kind of outline structure, and the outline of the micro-aggregate 6 may simply be provided by the outer edge of the bundle of columns itself. The distance between adjacent columnar structures (5) in a cluster of micro-aggregates (6) may preferably be 1 to 10 μm. The distance between adjacent columnar structures (5) in a cluster of micro-aggregates (6) may also be expressed as the width of the "inter-micro-aggregate gap". The distance between adjacent microaggregates (6) may preferably be 10 to 50 μm, or 10 to 30 μm.

The soil environment simulator of this embodiment may have a cover part 7 that is attached to the adhesive part 2 and covers the processing part 3.

FIGS. 2A and 2B are schematic diagrams showing the cover part 7 of the soil environment simulator of the embodiment. FIG. 2A shows the appearance of the soil environment simulator with the cover part 7 attached as seen from above, with the outlines of the micro-aggregates and adhesive part 2 visible through the transparent cover part 7 shown by dashed lines. FIG. 2B shows the appearance of the device as seen from the side. The portion of FIG. 2B excluding the cover part 7 corresponds to FIG. 1B.

The cover portion 7 may be adhered to the upper surface 2' of the adhesive portion 2. The cover portion 7 may also be in contact with or adhered to the upper surface portion 5' of the columnar structure 5.

The cover part 7 can be bonded by a bonding method known to those skilled in the art. For example, when the base material of the main body 1 is glass, bonding can be performed by pressure bonding (see, for example, Funano et al., Lab Chip, 2021, 21, 2244-2254). More specifically, for example, bonding can be achieved by dropping a neutral detergent diluted with water onto the glass adhesive part 2, covering it with a glass cover part, firmly fastening it with a clip, and leaving it for 1 to 24 hours, preferably 8 to 12 hours, and more preferably about 10 hours. When the base material of the main body 1 is resin, bonding can be achieved by a bonding method using plasma treatment known to those skilled in the art. An adhesive known to those skilled in the art may be used for bonding.

Referring to FIG. 3, the soil environment simulation device of the embodiment may include macro-aggregates 8. The macro-aggregates 8 are clusters with diameters of more than 250 μm formed by the close gathering of a plurality of the micro-aggregates 6. When there are a plurality of macro-aggregates 8, the distance between the macro-aggregates 8 may be, for example, 10 to 50 μm or 50 μm or more. In addition to the macro-aggregates 8, the soil simulation structure 4 of the device may further include isolated micro-aggregates 6 and/or isolated columnar structures 5 that are not involved in the formation of the macro-aggregates.

FIGS. 3A and 3B are schematic diagrams showing a soil environment simulation device including macro aggregates 8 according to an embodiment. FIG. 3A shows the appearance of the soil environment simulation device including macro aggregates 8 as seen from above, and FIG. 3B shows the appearance of the device as seen from the side.

Between the micro-aggregates 6 contained in the macro-aggregates 8, there are intra-macro-aggregate gaps 9. The intra-macro-aggregate gaps 9 are more generally understood to be one aspect of the "inter-micro-aggregate gaps". Outside the clusters of macro-aggregates, there are extra-macro-aggregate gaps 10. Two or more of the extra-macro-aggregate gaps 10, the intra-macro-aggregate gaps 9 (or the inter-micro-aggregate gaps), and the intra-micro-aggregate gaps described above may have different depths. In this context, "depth" refers to the distance from the bottom surface (the upper surface 3' of the processed portion) of the gap to the position of the upper surface 5' of the adjacent columnar structure 5. In the embodiment illustrated in FIG. 3B, the depth of the extra-macro-aggregate gaps 10 is smaller than the depth of the intra-macro-aggregate gaps 9. In this way, it is possible to provide imitation of various soil environments not only by the difference in gap width but also by the difference in gap depth. The depth of the inter-macroaggregate gaps may be, for example, 50 to 1600 μm, preferably 50 to 150 μm or 50 to 60 μm. The depth of the inter-microaggregate gaps, with or without the presence of macroaggregates, may be, for example, 1 to 1600 μm, preferably 1 to 150 μm or 1 to 5 μm.

The soil environment simulator of the embodiment may include multiple main bodies 1 stacked one on top of the other. Figure 4 shows such an embodiment.

The soil environment simulator of the embodiment illustrated in FIG. 4 includes a plurality of bodies 1 stacked one on top of the other, with the upper surface 2' of the adhesive portion 2 of a first body of the plurality of bodies 1 being adhered to the lower surface 2'' of a second body stacked directly above the first body. The terms "first" and "second" used here are terms of convenience to describe any two bodies 1 directly stacked, and are not meant to specify, for example, the bottom two of the plurality of stacked bodies 1. As shown in FIG. 4, the processing portion 3 of the first body can be closed by the lower surface of the second body instead of the cover portion 7.

The soil environment simulator, which includes multiple bodies 1 stacked one on top of the other in the embodiment, allows the stacked bodies 1 to be separated and dyed without compromising their condition after recovery from being buried in soil. For example, a neutral detergent diluted with water is dropped onto the adhesive part 2 of a glass body 1, another body 1 is placed on top of it, and the laminate of bodies 1 that has been bonded by firmly fastening and pressing with clips can be peeled off using a razor blade.

In the soil environment simulator including multiple main bodies 1 stacked vertically in the embodiment, for example when the main bodies 1 are made of resin, it is possible to observe not only on the intact soil environment simulator, but also after processing the sample, such as by sectioning with a microtome. For example, such a soil environment simulator can be used for preparing thin sections for a scanning electron microscope (SEM) and staining serial sections.

<Additional Notes>
The present disclosure includes the following embodiments.
(Appendix 1)
A soil environment simulator for observing soil microorganisms, comprising:
The soil environment simulator includes a main body including an adhesive portion and a processing portion connected to the adhesive portion,
A soil simulating structure is formed on the upper surface of the processed portion,
The soil-simulating structure includes a plurality of columnar structures;
The columnar structure has a top surface at an upper end,
A soil environment simulator, wherein an upper surface of the columnar structure is included in approximately the same plane as an upper surface of the adhesive portion.
(Appendix 2)
The soil environment simulator according to claim 1, wherein the soil simulation structure includes a plurality of micro aggregates, each of the plurality of micro aggregates being a cluster having a diameter of 250 μm or less formed by a plurality of the columnar structures densely packed together.
(Appendix 3)
The soil environment simulator according to claim 1 or 2, wherein the distance between adjacent columnar structures within the cluster of microaggregates is 1 to 10 μm.
(Appendix 4)
The soil environment simulator according to any one of claims 1 to 3, wherein the distance between adjacent clusters among the plurality of clusters which are the plurality of micro aggregates is 10 to 50 μm.
(Appendix 5)
The soil environment simulator according to any one of claims 1 to 4, wherein the height of the upper surface of the columnar structure is 1 to 150 μm from the bottom surface of the processed portion.
(Appendix 6)
The soil environment simulator according to any one of claims 1 to 5, further comprising a cover portion attached to the adhesive portion and covering the processing portion.
(Appendix 7)
The soil environment simulator according to any one of claims 1 to 6, wherein the soil simulation structure includes macro aggregates, and the macro aggregates are clusters having a diameter of more than 250 μm formed by the dense gathering of a plurality of the micro aggregates.
(Appendix 8)
The soil environment simulator includes a plurality of the bodies stacked vertically, and an upper surface of an adhesive portion of a first body among the plurality of the bodies is adhered to a lower surface of a second body stacked directly above the first body. The soil environment simulator described in any one of appendixes 1 to 7.

1 Main body 2 Adhesive portion 2' Upper surface of adhesive portion 2'' Lower surface of adhesive portion 3 Processed portion 3' Upper surface of processed portion 4 Soil simulation structure 5 Columnar structure 5' Upper surface portion of columnar structure 6 Micro aggregate 7 Cover portion 8 Macro aggregate 9 Gaps within macro aggregate 10 Gaps outside macro aggregate

Claims (8)

A soil environment simulator for observing soil microorganisms, comprising:
The soil environment simulator includes a main body including an adhesive portion and a processing portion connected to the adhesive portion,
A soil simulating structure is formed on the upper surface of the processed portion,
The soil-simulating structure includes a plurality of columnar structures;
The columnar structure has a top surface at an upper end,
A soil environment simulator, wherein an upper surface of the columnar structure is included in approximately the same plane as an upper surface of the adhesive portion. The soil environment simulator according to claim 1, wherein the soil simulation structure includes a plurality of micro-aggregates, each of which is a cluster having a diameter of 250 μm or less formed by a plurality of the columnar structures densely packed together. The soil environment simulator according to claim 2, wherein the distance between adjacent columnar structures within the cluster of microaggregates is 1 to 10 μm. The soil environment simulator according to claim 2 or 3, wherein the distance between adjacent clusters among the plurality of clusters that are the plurality of microaggregates is 10 to 50 μm. The soil environment simulator according to claim 1 or 2, wherein the height of the top surface of the columnar structure is 1 to 150 μm from the bottom surface of the processed portion. The soil environment simulator according to claim 1 or 2 further comprises a cover part that is attached to the adhesive part and covers the processing part. The soil environment simulator according to claim 2 or 3, wherein the soil simulation structure includes macro-aggregates, and the macro-aggregates are clusters with a diameter of more than 250 μm formed by the dense gathering of multiple micro-aggregates. The soil environment simulator according to claim 1 or 2, wherein the soil environment simulator includes a plurality of the bodies stacked vertically, and the upper surface of the adhesive portion of a first body among the plurality of the bodies is adhered to the lower surface of a second body stacked directly above the first body.

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