Soil observation device and method of manufacturing the same

Abstract

To provide a soil observation device capable of controlling a physical structure and chemical properties of soil.SOLUTION: A soil observation device includes: a chamber part that holds soil particles and allows a flow of liquid; and a liquid introduction part that introduces the liquid to the chamber part. There is also provided a method of manufacturing the soil observation device that includes the steps of: introducing soil particles and a photocurable resin to the chamber part of a transparent substrate that has the chamber part and is subjected to mask alignment; and exposing the chamber part through the mask and curing the photocurable resin in a region that is not masked by the mask to form the resin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a soil observation apparatus and a manufacturing method thereof. The observation apparatus is a device that reproduces a soil environment, and is useful for analyzing the relationship between microbial activity and soil aggregate structure, for example. [Background technology]

[0002] Soils found in forests, grasslands, farmlands, etc. often have a three-dimensional structure called aggregate structure, which is related to fertility in farmland, for example. The moisture and organic matter contained in the soil cause soil particles to aggregate into larger aggregates. Different soils are formed by mixing aggregates of different sizes and shapes in different proportions and distributions. In addition, the chemical properties of the soil particles themselves, such as hydrophobicity and solubility, can affect the dynamics of microorganisms, nutrients, toxic substances, etc. in the soil. It is known that soil aggregate structure affects microbial activity, water retention and drainage, and carbon sequestration in soil. If the relationship between the physical structure of soil and microbial activity is clarified, it will be possible to evaluate or predict microbial activity by evaluating the physical structure of a given soil. However, because visible light does not pass through soil, it is difficult to directly observe the effect of soil aggregate structure on microbial activity, and it is difficult to determine the causal relationship.

[0003] As methods for observing the activity of microorganisms (including fungi such as mycorrhizal fungi) in soil, a method of observing soil in a pot (Non-Patent Document 1) and a method using a microfluidic device (Non-Patent Document 2) have been proposed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Storer, Kate, Aisha Coggan, Phil Ineson, and Angela Hodge. 2018. “Arbuscular Mycorrhizal Fungi Reduce Nitrous Oxide Emissions from N2O Hotspots.” The New Phytologist 220 (4): 1285-95. [Non-Patent Document 2] Doktycz, and Scott T. Retterer. 2019. “Pore-Scale Hydrodynamics Influence the Spatial Evolution of Bacterial Biofilms in a Microfluidic Porous Network.” PloS One 14 (6): e0218316. Summary of the Invention [Problem to be solved by the invention]

[0005] However, the inventors have recognized that the prior art methods have a problem in that it is difficult to simultaneously control the physical structure and chemical properties of soil. For example, the device of Non-Patent Document 1 is designed to control the chemistry of a specific area of ​​soil, but it is difficult to control the fine physical properties of soil. The device of Non-Patent Document 2 uses a microfluidic platform to mimic the fine physical structure of soil, including the morphology and density of soil particles, but it is difficult to reproduce the chemistry of soil.

[0006] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a soil observation device that can control the physical structure and chemical properties of soil, and a manufacturing method thereof. [Means for solving the problem]

[0007] A soil observation device according to one aspect of the present disclosure includes a chamber portion that holds soil grains and through which liquid can flow, and a liquid introduction portion that introduces liquid into the chamber portion.

[0008] A manufacturing method for a soil observation device of one embodiment of the present disclosure includes introducing soil grains and a photocurable resin into a chamber portion of a transparent substrate having a chamber portion and being masked, exposing the chamber portion to light through the mask, and curing the photocurable resin in areas not masked by the mask to form a resin. Effect of the Invention

[0009] According to the present disclosure, it is possible to provide a soil observation device capable of controlling the physical structure and chemical properties of soil, and a method for manufacturing the same. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing the whole and a part of a soil observation device according to an embodiment. [Diagram 2] FIG. 2 shows an outline of a method for manufacturing the soil observation device according to the embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings.

[0012] <Soil observation device>

[0013] FIG. 1a is a schematic diagram showing the whole and part of an example of a soil observation apparatus according to an embodiment. The soil observation apparatus shown in the figure has a chamber section 1, a liquid inlet section 21, a liquid outlet section 22, a soil grain inlet section 31, and a soil grain outlet section 32. The left side of FIG. 1a shows an overview of this soil observation apparatus seen from above (soil grains under the cover of the chamber section 1 are not shown), and the right side of FIG. 1a shows a schematic diagram of the chamber section 1 containing soil grains 11 seen from above. Such soil observation apparatuses can be provided in different sizes depending on individual applications, and can be, for example, microfluidic device-like apparatuses in which the diameter (or maximum diameter) of the chamber section is 10 cm or less or 5 cm or less.

[0014] The chamber section 1 is a section having a space for holding soil grains 11 for observation. The chamber section 1 is configured to allow liquid to flow therethrough, and this characteristic makes it possible to introduce liquid from a liquid inlet section 21 and to discharge liquid to a liquid outlet section 22. A transparent substrate such as glass may be used as the substrate for the chamber section 1 so that it can be easily observed from the outside.

[0015] The soil grains 11 may include aggregates. Aggregates are soil grains or sand particles that have solidified, and are classified into microaggregates (diameter less than 1 mm) and macroaggregates (diameter 1 mm or more) according to size. In this disclosure, the terms "soil grains" and "sand" may be used interchangeably. In nature, microaggregates are typically formed by soil grains solidified by bacterial secretions, etc., and macroaggregates are typically formed by the entanglement of microaggregates, fungal hyphae, and plant roots, etc. In this disclosure, aggregates may be microparticles, macroparticles, or a mixture thereof.

[0016] Generally, aggregates are divided into aggregates that break down when exposed to water and aggregates that maintain their structure even when exposed to water, and the latter can be called hydrophobic aggregates. The aggregates of the present disclosure may be any of these aggregates or a mixture thereof.

[0017] In this disclosure, the term aggregate refers to an individual aggregate and the term aggregate structure refers to the soil structure formed by a group of aggregates.

[0018] For example, the soil grains 11 may be sieved soil or may be artificially compacted soil. The soil used for this purpose may be sterilized. The sterilization of the soil may be carried out by a method known to those skilled in the art, such as autoclaving.

[0019] By altering the size, shape and chemical composition of the soil particles and aggregates, the physical structure and chemical properties of the soil that the Soil Monitor reproduces can be controlled.

[0020] FIG. 1b is an example of a schematic cross-sectional view of the chamber section 1 seen from the side, and the lower side of the figure represents the bottom surface of the chamber section 1. As shown in FIG. 1b, the soil grains 11 are held to the base material of the chamber section through fixation by the resin 12. The type of resin 12 is not limited as long as it can fix the soil grains 11, and can be selected by a person skilled in the art based on ordinary knowledge. The resin 12 is preferably a resin obtained by hardening a photocurable resin 15. Although not shown, soil grains that are not directly fixed to the base material of the chamber section by the resin 12 can also be held in the chamber section 1 through interaction with the fixed soil grains (for example, sandwiched between two or more fixed soil grains). The amount of resin 12 only needs to be sufficient to fix the soil grains 11, and it is not necessary for the entire surface of each soil grain 11 to be covered with the resin 12. By not covering the entire surface of the soil grains 11 with the resin 12 and exposing at least a part (typically a large part) of the surface, gaps through which liquid can penetrate are maintained between the soil grains as in real soil, and the effect of the chemical properties of the soil grains 11 can be reproduced.

[0021] The chamber section 1 may have a cover 16. For example, in FIG. 1b, the cover 16 is arranged on the side opposite to the bottom surface of the chamber section 1 (see also FIG. 2e). As a result, the chamber section 1 forms a space closed by the bottom surface, side wall surface, and cover 16, and the soil grains 11 are enclosed in the space. The cover 16 may be made of a transparent material such as glass so that it can be easily observed from the outside.

[0022] The term photocurable resin in this disclosure refers to a resin or a precursor thereof that is at least partially cured by irradiation with light. Photocurable resins are also called photosensitive resins. The photocurable resin 15 may be a UV-curable resin that is cured by irradiation with ultraviolet (UV) light. Depending on the context, the term photocurable resin in this disclosure may refer to a non-solid or liquid component before being cured by irradiation with light. For example, the photocurable resin 15 in this disclosure may include a monomer or oligomer component before being polymerized by irradiation with light.

[0023] The photocurable resin 15 in the present disclosure may be any of a solvent-based, non-solvent-based, and water-based photocurable resin. The photocurable resin in the present disclosure may be a radical curable type or a cationic curable type. The photocurable resin 15 in the present disclosure may contain a polymerization inhibitor and other additives.

[0024] Examples of components of the photocurable resin 15 include, but are not limited to, acrylates such as urethane acrylate, epoxy acrylate, acrylic acrylate, polyester acrylate, and multifunctional acrylate.

[0025] Examples of resin 12 include, but are not limited to, a resin obtained by irradiating a photocurable resin containing an acrylate exemplified in this specification as a component of photocurable resin 15 with light, an organic-inorganic hybrid resin, an amide-imide resin, a polysiloxane composite resin, and a vinyl ester resin.

[0026] The chamber section 1 preferably includes a region 17 that does not include soil grains 11 and resin 12 in the depth direction (also referred to as a "soil grain free region" in this disclosure). The "depth direction" in the context of the chamber section 1 means the thickness direction of the layer of soil grains held in the chamber section 1 when the group of soil grains is viewed as a layer. More specifically, the depth direction is a direction perpendicular to the surface of a mask when masking the chamber section as described below. This specification is generally described assuming that a soil observation device is used with the layer of soil grains in the chamber section 1 horizontal as a typical embodiment, but this does not necessarily exclude embodiments in which a similar soil observation device is used with the layer vertical, for example. The depth of the chamber section 1 (or the thickness of the layer of soil grains in the chamber section 1) may vary depending on the individual application, but may be, for example, 1 cm or less, 5 mm or less, 1 mm or less, 100 μm or less, or 10 μm.

[0027] The region in the chamber 1 that does not contain the soil grains 11 and the resin 12 in the depth direction, i.e., the soil grain free region 17, may be, for example, a passage shape (like a corn maze), a well shape, or any other shape when viewed from above. There may be multiple soil grain free regions 17 in the chamber 1. Providing the soil grain free regions 17 allows further diversification of the physical structure in the chamber 1, and allows the design of specific liquid flow dynamics.

[0028] As will be described in more detail below, the photocurable resin 15 can be used to form any desired pattern of the resin 12 (and thus the reproduced soil) within the chamber 1. Such pattern formation makes it possible to create an environment in which the dynamics of microorganisms or substances such as nutrients or toxic substances can be analyzed while still allowing greater freedom in controlling the physical structure of the soil.

[0029] A liquid can be introduced into the chamber 1 through the liquid introduction part 21. The liquid introduced into the chamber 1 by the liquid introduction part 21 may be a microbial suspension. Additionally or alternatively, the liquid introduced into the chamber 1 by the liquid introduction part 21 may be a buffer solution, a culture medium, a chemical solution, or any other solution capable of controlling the chemical properties of the soil grains 11. A pump or a syringe for introducing the liquid may be connected to the liquid introduction part 21.

[0030] The microorganisms contained in the microbial suspension introduced into the chamber 1 by the liquid introduction part 21 are not particularly limited and can be appropriately selected by a person skilled in the art depending on the purpose of use of the device. The microorganisms may be, for example, bacteria, archaea, fungi, animals, plants, or algae. The microorganisms may be soil microorganisms or soil bacteria.

[0031] The liquid in the chamber section 1 can be discharged through the liquid discharge section 22. A pump or syringe for discharging the liquid may be connected to the liquid discharge section 22. In the example shown in FIG. 1a, the liquid introduction section 21 and the liquid discharge section 22 each have a flow path that branches into a U-shape in multiple stages, but this is merely an example, and the shapes of the liquid introduction section 21 and the liquid discharge section 22 are not limited to such shapes. For example, in a simpler embodiment, the liquid introduction section 21 and the liquid discharge section 22 each may be only one passage that connects to the internal space of the chamber section 1. The points at which the liquid introduction section 21 and the liquid discharge section 22 are connected to each other in the chamber section 1 can be appropriately selected by a person skilled in the art according to the purpose of each embodiment. For example, the liquid introduction section 21 and the liquid discharge section 22 may be connected to the chamber section 1 from opposite sides as shown in FIG. 1a, or may be connected to the chamber section 1 from the same side. Also contemplated are embodiments in which one or both of the liquid inlet 21 and liquid outlet 22 are connected to the chamber portion 1 from the top or bottom side, rather than from a side of the chamber portion 1 (as shown in FIG. 1a).

[0032] The soil observation apparatus may optionally have a soil particle introduction section 31. Soil particles can be introduced into the chamber section 1 through the soil particle introduction section 31. The soil particles can be introduced into the chamber section 1 as, for example, a suspension in a liquid. The introduced soil particles may be aggregates. The introduction of the soil particles into the chamber section 1 through the soil particle introduction section 31 may be performed, for example, during the manufacturing process of the soil observation apparatus. Alternatively, the soil particle introduction section 31 may be used to additionally introduce soil particles other than the soil particles 11 held in the chamber section 1 before the soil observation apparatus is started to be used, into the chamber section 1 during the use of the soil observation apparatus. For example, additional soil particles may be introduced into the chamber section 1 from the soil particle introduction section 31 through the soil particle free area 17 having a passage shape. Alternatively, fine soil particles that can pass through the gaps formed by the large soil particles 11 may be introduced into the chamber section 1.

[0033] The soil observation device may optionally have a soil particle discharge section 32. Through the soil particle discharge section 32, for example, excess soil particles introduced during the manufacturing process of the soil observation device, unadhered flowing soil particles during the use of the soil observation device, or unadhered soil particles and liquid after the use of the soil observation device can be discharged from the chamber section 1. By discharging the soil particles from the soil observation device after use in this manner, it is also possible to use the same soil observation device repeatedly.

[0034] <Manufacturing method>

[0035] 2 shows an example of a manufacturing method for a soil observation device according to an embodiment. The manufacturing method shown in the figure includes (a) a chamber forming step, (b) a masking step, (c) a soil grain and light-curing resin filling step, (d) a light-curing step, and (e) an encapsulation step. The description of each element provided with respect to the embodiment of the soil observation device above can also be applied to this embodiment of the manufacturing method, and vice versa.

[0036] (a) In the chamber forming step, a chamber 1' (a depression in the center in FIG. 2a) is formed in the transparent substrate 13. The chamber here is a depression that has a capacity to hold soil particles, and is the part that will eventually become the chamber portion 1. That is, in the chamber forming step, a new chamber 1' is formed in the transparent substrate 13 using a means known to those skilled in the art. Alternatively, if a transparent substrate having a chamber 1' from the beginning is available, the chamber forming step can be omitted. The type of transparent substrate is not limited and can be appropriately selected by those skilled in the art, but it can be glass such as borosilicate glass. Although the method of the embodiment generally relates to etching a pattern of a soil-containing region / soil-free region in the chamber portion, the chamber 1' can also be formed in a substrate 13 such as glass by a known etching method. Thus, apart from the soil-free region, the chamber portion 1 of the present disclosure itself can be provided in various patterns, such as a flow path that branches, meanders, connects, etc. For example, when the transparent substrate 13 is a glass plate, the chamber 1' can be formed by a known etching method such as a reactive ion etching (RIE) method. The size of the transparent substrate 13 is not limited, but may be, for example, within 10 cm or 5 cm in length and width, and may be 0.4 to 10 mm in thickness. In a specific example, a transparent substrate of 30 mm x 40 mm x 0.5 mm is used. The etching depth of the transparent substrate may be, for example, within a range of 5 μm to 1000 μm. After the chamber formation step, the transparent substrate 13 may be optionally subjected to a cleaning process such as ultrasonic cleaning.

[0037] In the (b) masking step, the mask 14 is aligned (mask alignment) with the transparent substrate 13. The terms masking and mask alignment have the usual meanings understood by those skilled in the art who know the principles of general etching technology. Mask alignment is typically performed on the bottom surface of the transparent substrate 13, that is, the surface opposite to the opening surface of the chamber 1'. The mask 14 is used in a pattern corresponding to the shape of the soil grain free region 17 designed in advance, that is, a pattern complementary to the pattern defined by the resin 12 in the chamber part 1. The mask 14 is not limited as long as it can block the light used in the (d) photocuring step described later according to the desired pattern, and a photomask manufactured by a known method can be used. Examples of photomasks include photomasks manufactured by forming a light-shielding film portion on glass, quartz, etc. with a thin film of chromium or blackened metallic silver.

[0038] Alternatively, if a transparent substrate having a masking is available from the beginning, the masking step can be omitted. Thus, the method for manufacturing a soil observation device in one embodiment includes introducing soil grains and a photocurable resin into a chamber portion of a transparent substrate having a chamber portion and having been mask-aligned. The method may further include forming a chamber 1' in the transparent substrate 13 and / or aligning a mask 14 with the transparent substrate 13 (mask alignment). In the example of FIG. 2, step (b) is shown after step (a), but forming the chamber and aligning the mask may be performed in either order, or may be performed simultaneously.

[0039] (c) In the soil grain and photocurable resin filling step, the soil grains 11 and photocurable resin 15 are introduced into the chamber 1. The introduced soil grains 11 and photocurable resin 15 may be the soil grains and photocurable resin described in the section on the soil observation device. The introduced soil grains 11 may form an aggregate structure. The soil grains or aggregates may be sieved before being introduced into the chamber 1' so that the diameter of the soil grains or aggregates is equal to or smaller than the depth of the chamber 1'. The introduction of the soil grains and the introduction of the photocurable resin into the chamber 1' may be performed in any order, or may be performed simultaneously. For example, the photocurable resin may be added to the soil grains, and then the mixture of the two may be introduced into the chamber 1'.

[0040] In the photocuring step (d), light is irradiated to the chamber 1 through the mask 14, and the photocurable resin 15 is cured in the area not masked by the mask 14 to form the resin 12. It is understood that the formation of the cured resin 12 causes the soil grains 11 to be fixed inside the chamber of the transparent substrate 13. As the photocurable resin 15 is cured in the unmasked area, a soil grain free area 17, i.e., an area not including the soil grains 11 and the resin 12 throughout the depth direction in the chamber, is generated at a position and shape corresponding to the masking pattern. The wavelength of the irradiated light is not limited as long as it can cure the photocurable resin 15, and can be appropriately determined by a person skilled in the art according to the type of the specific photocurable resin 15 used. As the wavelength of the irradiated light, for example, UV (10 to 400 nm) can be used, but it is preferable to use 320 to 400 nm, and it is more preferable to use 360 ​​to 370 nm. The intensity of the irradiated light can be adjusted so that the photocurable resin 15 is appropriately cured throughout the depth direction of the unmasked area. An embodiment is also contemplated in which selective curing is achieved at a relatively lower position (closer to the irradiation source) by deliberately setting the irradiation intensity to a moderate level. After the photocurable resin 15 is cured by light irradiation, the uncured photocurable resin 15 is washed away. When solidifying soil in a device, the problem is how to wash away the parts that are not to be solidified. In the manufacturing method of the embodiment, this problem is solved by solidifying the soil via pattern etching before covering the device, washing away the unsolidified photocurable resin 15, and then bonding the cover. Alternatively, after bonding the cover 16, this washing can be performed, for example, through the above-mentioned soil particle introduction section 31 and soil particle discharge section 32. Through this series of operations, a soil observation device in which a pattern of resin 12 and soil particles 11 is formed in the chamber section 1 can be produced.

[0041] In the sealing step (e), the chamber section 1 is sealed by attaching the cover 16. The method of attaching the cover 16 is not limited, and it may be a permanent attachment or a temporary attachment that allows the cover 16 to be removed. For example, the cover 16 can be attached by dripping diluted neutral detergent onto the edge of the chamber on the transparent base material 13, which is a glass plate, and then placing a glass plate (cover 16) large enough to completely cover the opening of the chamber while still leaving a margin, and bringing it into close contact with the transparent base material 13, and firmly fastening the margin with clips and leaving it for about 10 hours.

[0042] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0043] <Additional Notes> (Appendix 1) A chamber portion that holds soil particles and allows liquid to flow through; A liquid introduction part for introducing liquid into the chamber part is provided. Soil observation device. (Appendix 2) The soil observation device of claim 1, wherein the soil particles form an aggregate structure. (Appendix 3) 3. The soil observation device according to claim 1 or 2, wherein the soil grains are held in the chamber portion by being fixed with resin. (Appendix 4) 4. The soil observation device according to claim 1, wherein the chamber portion includes a region that does not contain the soil grains and the resin across the depth direction. (Appendix 5) 5. The soil observation apparatus according to any one of claims 1 to 4, wherein the liquid is a microbial suspension. (Appendix 6) A method for manufacturing a soil observation device, comprising: introducing soil particles and a photocurable resin into a chamber portion of a transparent substrate having a chamber portion and being mask-aligned; exposing the chamber portion to light through the mask to harden the photocurable resin in areas not masked by the mask to form a resin. A method for manufacturing a soil observation device. (Appendix 7) The manufacturing method described in Appendix 6, wherein hardening the photocurable resin in areas not masked by the mask forms an area in the chamber portion that does not contain the soil grains and the resin throughout the depth direction. (Appendix 8) The method of claim 6 or 7, wherein the soil particles form an aggregate structure. [Explanation of symbols]

[0044] 1. Chamber section 11 soil grains 12 Resin 13 Transparent base material 14 Mask 15 Photocuring resin 16 Cover 17 Soil-free area 21 Liquid introduction section 22 Liquid discharge part 31 Soil particle introduction part 32 Soil particle discharge section

Claims

1. A chamber portion that holds soil particles and allows liquid to flow through; A liquid introduction part for introducing liquid into the chamber part is provided. Soil observation device.

2. The soil observation device according to claim 1 , wherein the soil particles form an aggregate structure.

3. The soil observation device according to claim 1 , wherein the soil grains are held in the chamber portion by being fixed with a resin.

4. The soil observation device according to claim 3 , wherein the chamber portion includes a region that does not contain the soil grains and the resin across the depth direction.

5. The soil observation device according to any one of claims 1 to 4, wherein the liquid is a microbial suspension.

6. A method for manufacturing a soil observation device, comprising: introducing soil particles and a photocurable resin into a chamber portion of a transparent substrate having a chamber portion and being mask-aligned; exposing the chamber portion to light through the mask to harden the photocurable resin in areas not masked by the mask to form a resin. A method for manufacturing a soil observation device.

7. The manufacturing method according to claim 6 , wherein curing the photocurable resin in the area not masked by the mask forms an area in the chamber portion that does not contain the soil grains and the resin across the depth direction.

8. The method according to claim 6 or 7, wherein the soil particles form an aggregate structure.

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