Soil diagnosis device

Abstract

To make soil biological diagnosis easier, faster, and cheaper, and to provide a widespread technology that producers can easily use.SOLUTION: A soil diagnosis device has a biosensor composed of an oxygen electrode (A), a microbial filter (B), and a microbial fixation device (C). A tip shape of the oxygen electrode (A) is convex, and is provided with a flat part on its peripheral portion. Tip parts of the microbial filter (B) and the microbial fixation device (C) are flat, disc-shaped, and made of a flexible material that can deform to conform to the tip shape of the oxygen electrode (A). The microbial filter (B) is mounted between the oxygen electrode (A) and the microbial fixation device (C), and is shaped to allow them to fit tightly together.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a soil diagnostic device having a biosensor. [Background technology]

[0002] Soil diagnosis, both physical and chemical analysis, has become a widespread technique and is used by ordinary producers. However, soil biological diagnostic techniques have long remained a black box due to the sheer number, complexity, and diversity of the types and types of soil microorganisms. As a result, no inexpensive, simple, or rapid method has been established, and the technique has not become widespread and easily usable by ordinary producers.

[0003] A soil diagnostic biosensor was created by applying a BOD sensor that rapidly measures the pollution of river water (Patent Document 1, Non-Patent Document 1). Furthermore, Sakata Seed Corporation created a commercial model device (Non-Patent Document 2). Since then, many research and testing institutions have been studying how to use the device (Non-Patent Document 3), and active research and development has been carried out on applications to paddy field soil (Non-Patent Document 4), methods for evaluating soil disease suppression in field soil, and methods for measuring rhizosphere microbial activity (Non-Patent Document 5).

[0004] However, this device had not been improved at all since it was manufactured in 2006. The tip of the oxygen electrode manufactured at that time was flat, and the attached microbial filter and microbial fixation device were also flat to match this shape. In order to make soil biological diagnosis a widespread technology that could be used by a wider range of producers, it was necessary to improve it to something cheaper, easier, faster, and more stable for long-term use. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4528939 [Non-Patent Document 1] Microbes Environ. Vol.23, No.1, 35-39, 2008 [Non-patent document 2] Research and Development of Soil Diagnostic Biosensors | Katayanagi Research Institute, Tokyo University of Technology (teu.ac.jp) [Non-patent document 3] Quarterly Fertilizer. 109, 98-109, 2008 [Non-patent document 4] Abstracts of the 33rd Annual Meeting of the Japanese Society for Microbial Ecology, 2019 [Non-patent document 5] Crop Production and Soil Improvement. Vol. 55, No.573, 19-27, 2023 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention was made in light of these circumstances, and its purpose is to provide a device with a biosensor that enables soil diagnosis at lower cost, more simply and quickly, has high sensitivity, and can be used stably for a long period of time. To achieve this, it is necessary for the microorganisms attached to the sensor part of the electrode to be stably retained. It is easy to wash away impurities such as soil particles that adhere to the sensor part. The shape of the sensor tip makes it difficult for air bubbles to adhere. The microorganisms on the microorganism fixing device attached to the tip of the oxygen electrode must be able to survive stably for a long period of time. The sensor response must be fast. [Means for solving the problem]

[0007] The present inventors have conducted various studies to solve the above problems, specifically, the shape and diameter of the electrode, the circumferential size, the size of the microbial filter, and the prototype of a microorganism fixing tool shaped to fit these. The tip of a conventional oxygen electrode was flat, and the microbial filter and attached microbial fixation device were also flat to match this shape, but by changing the shape of the sensor part so that the tip of the oxygen electrode (A) was convex and the tip of the microbial fixation device (C) was made of a flexible material that could deform to follow the shape of the electrode, and a microbial filter (B) was attached in between, improving the device to a shape that allows the oxygen electrode (A), microbial filter (B), and microbial fixation device (C) to be in close contact, the escape of microorganisms to the surrounding area was greatly reduced and the retention rate of the fixed microorganisms was improved. However, it was found that the microbial filter (B) attached between the convex oxygen electrode (A) and the condom-shaped microbial fixation device (C) wrinkled and caused unevenness. Therefore, by providing a flat portion around the oxygen electrode (A), wrinkles are less likely to form on the attached microbial filter (B), and the above problem can be solved. [Effects of the Invention]

[0008] The improvements to the sensor section have been found to have the following effects: 1. Microorganisms were stably maintained and their escape to the surrounding area was reduced. 2. It has become easier to wash away impurities such as soil particles that adhere to the sensor part of the electrode. 3. Microorganisms gathered at the sensor at the tip and were able to survive stably for a long period of time. 4. It is now possible to maintain a high local water flow at the sensor area. 5. The sensor response is faster and more sensitive. 6. The localized water flow in the sensor area is faster, allowing the baseline to stabilize quickly. 7. Even if air bubbles adhere to the tip of the sensor, they move quickly and are less likely to remain at the tip. As a result, noise during measurement is reduced. 8. The problem of wrinkles on the microorganism filter has been eliminated. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 is a diagram showing the structure of a soil diagnostic device. [Figure 2] FIG. 1 shows the response, sensitivity and baseline stability of the sensor. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a soil diagnostic device with a biosensor. The device has a biosensor consisting of an oxygen electrode (A), a microbial filter (B), and a microbial immobilizing device (C), in which the tip of the oxygen electrode (A) is convex with a flat portion provided around it, and the tips of the microbial filter (B) and the microbial immobilizing device (C) are flat, disk-shaped and made of a flexible material that can deform to follow the tip shape of the oxygen electrode (A), and the microbial filter (B) is attached between the oxygen electrode (A) and the microbial immobilizing device (C) so that they can be tightly attached to each other. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0011] The biosensor may be made of any material that does not affect the dissolved oxygen and volatile substances in the soil suspension or the respiratory activity of microorganisms, and is composed of an oxygen electrode, a microbial filter, and a microbial fixation device.

[0012] The oxygen electrode (A) can be any commonly used oxygen electrode, but one with a convex tip rather than a flat one should be used. A thin oxygen-permeable membrane is attached to the tip of the electrode, and an internal electrolyte and two types of metal are placed inside. Measurements are made using the oxidation-reduction reaction that occurs between oxygen and the metal; galvanic cell-type or polarographic-type membrane electrodes are used. In addition to these, optical sensors can also be used, in which a sensor cap coated with a fluorescent or phosphorescent substance is attached to the tip, a light source and photodetector are placed inside, and measurement is made using the light emitted by the fluorescent (phosphorescent) substance.

[0013] A method for providing a flat portion around the periphery of the oxygen electrode (A) having a convex tip shape is to attach and fix a plastic, resin, or metal material that is easy to process to the outside of the electrode around the tip of the electrode, thereby attaching a flat portion of the required size.

[0014] The microbial filter (B) is a membrane with fine mesh that can hold and fix microorganisms, and is strong enough to hold the microorganisms without affecting their survival. It is made of a flexible material that can deform and conform to the shape of the electrode, and is permeable to oxygen. For example, a 0.45 μm nitrocellulose membrane, acetyl cellulose membrane, or nylon membrane can be used.

[0015] Methods for immobilizing microorganisms on a filter include collecting cultured microorganisms using a centrifuge, dropping the washed microbial suspension onto a filter made of nitrocellulose, acetyl cellulose, nylon membrane, or the like, and then aspirating or pressurizing the filter from below to remove water and water-soluble substances, thereby immobilizing the microorganisms on the filter; or immobilizing the microorganisms in a gel such as calcium alginate and then preparing thin-film sections.

[0016] The outer holding part of the microorganism fixing device (C) can be made by molding using easily processable plastic, resin, or metal. The tip that comes into close contact with the electrode has a mesh made of a flexible material that can deform and follow the shape of the electrode, and nylon netting or wire mesh is used.

[0017] The tips of the microbial filter (B) and the microbial fixing device (C) are initially flat, and when attached to the electrode, they deform to conform to the shape of the electrode. Therefore, simply by attaching the microbial fixing device (C) with the microbial filter (B) attached to the oxygen electrode (A) with a convex tip, the oxygen electrode (A), the microbial filter (B), and the microbial fixing device (C) can be brought into close contact with each other.

[0018] The oxygen electrode (A) and the fixture (B) can be fixed together by a screw-type method or by using an O-ring, a tube, or the like.

[0019] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0020] [Example 1] Fabrication of oxygen electrode and microbial fixation device It was found that when a condom-shaped fixture was created simply to fit the convex oxygen electrode, the microbial filter would wrinkle significantly, resulting in unevenness. Therefore, we purchased a convex oxygen electrode (easySense O2 21 Sensor) from Mettler Toledo, placed a flat part on the outside of the oxygen electrode, and created a microbial fixation device that could deform to follow the shape of the electrode. The oxygen electrode (A) is used with the tip of the convex part facing downward. [Comparative Example 1] Comparison electrode The standard flat oxygen electrode used in the Soil Dock (manufactured by Sakata Seed Corporation) and a flat microbial fixation device that was designed to fit the electrode were used.

[0021] [Example 2] Microorganisms escaping to the sensor area Using the sensor produced in Example 1 and the sensor produced in Comparative Example 1, a biosensor was produced by culturing natto bacteria overnight and placing it on a filter. A 100 ml beaker was filled with water and the sensor was immersed in it. Under bright lighting, the bacterial solution was observed to ooze out of the sensor, resembling a faint smoke. The results showed that the biosensor produced in Example 1 lost less microorganisms than the biosensor produced using Comparative Example 1.

[0022] [Example 3] Comparison of cleaning of sensor part The sensor manufactured in Example 1 and the sensor manufactured in Comparative Example 1 were immersed in a soil suspension and stirred for 20 minutes, and then both sensors were washed using a washing bottle filled with water. When the degree of dirt on the sensor part was checked with the naked eye, it was found that the tip of the oxygen-detecting sensor in Example 1 was convex, so dirt on the sensor part was removed more easily and quickly.

[0023] [Example 4] Adhesion rate of air bubbles to the sensor part The sensor of Example 1 and the sensor of Comparative Example 1 were immersed in a beaker, and the state of adhesion of air bubbles was observed with the naked eye while changing the stirring flow rate. As a result, in Example 1, which had a convex tip, even if air bubbles adhered to the tip, they tended to immediately move to the peripheral side and rarely remained there. On the other hand, because the tip of the sensor in Comparative Example 1 was flat, even if air bubbles adhered, they often remained there for a long time, often causing noise during measurement.

[0024] [Example 5] Continuous use time of microbial electrode Bacillus subtilis var natto, which had been cultured overnight, was immobilized on the sensor of Example 1 and the sensor of Comparative Example 1, and a yeast extract solution was added over time to check the sensor response. The results are shown in Table 1. The values ​​in Table 1 are sensor response values ​​(dissolved oxygen concentration (mgO2 / L)), Ave is the average value, and SD is standard deviation. For the sensor of Comparative Example 1, the sensor response became unstable after one week (December 5th), making measurement difficult. For the sensor of Example 1, the sensor response was stable even after two weeks (December 8th).

[0025] [Table 1]

[0026] Example 6: Sensor response, sensitivity and baseline stability A graph of the differential values ​​was drawn from the data obtained in the experiment of Example 5, and the speed and sensitivity of the sensor response were investigated from the height of the differential value and the time at which the peak appeared on the horizontal axis. The results are shown in Figure 2. As a result, it was found that the sensor of Example 1 had better sensitivity than the sensor of Comparative Example 1, had a more stable baseline, and had less data fluctuation.

[0027] [Example 7] Rapid stabilization of baseline and increase in number of analysis points When the sensor of Example 1 was used, the baseline quickly returned to its original position after 20 minutes of soil diagnosis, and the next soil analysis became possible after about 5 minutes. When the sensor of Comparative Example 1 was used, after 20 minutes of soil diagnosis, it took 15 minutes or more for the baseline to return to its original position, stabilize, and become possible to perform the next soil analysis. As a result, when continuous analysis was performed for 8 hours a day, it was predicted that a maximum of 19 points could be analyzed using the sensor of Example 1, but only a maximum of 13 points could be analyzed using the sensor of Comparative Example 1. [Industrial Applicability]

[0028] By enabling simpler, faster, and cheaper soil biological diagnosis, the present invention can be used in diagnostic techniques such as soil preparation for farmland, predicting the risk of pest outbreaks, determining soil fertility, measuring microbial activity in the rhizosphere, and determining the maturity of compost. [Explanation of symbols]

[0029] (A) An oxygen electrode with a convex tip and a flat area around it (B) A flat, disk-shaped microbial filter made of a flexible material that can deform to fit the tip shape of the oxygen electrode (A). (C) A microorganism fixing device having a flat disk-shaped tip and made of a flexible material that can deform to follow the tip shape of the oxygen electrode (A).

Claims

[Claim 1] A soil diagnostic device having a biosensor consisting of an oxygen electrode (A), a microbial filter (B), and a microbial fixing device (C), wherein the tip of the oxygen electrode (A) is convex and has a flat portion around it, the tips of the microbial filter (B) and the microbial fixing device (C) are flat, disc-shaped and made of a flexible material that can deform to follow the tip shape of the oxygen electrode (A), and the microbial filter (B) is attached between the oxygen electrode (A) and the microbial fixing device (C), and the soil diagnostic device is shaped so that they can be in close contact with each other.

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