First, we approached experts in the field of nitrogen issues to understand the issue itself.

Participation in a Symposium on Nitrogen Issues

This symposium was hosted by researchers from the Research Institute for Humanity and Nature who are involved in a project called "Sustai-N-able," aimed at sustainable nitrogen use.

Initially, our understanding of the nitrogen issues was vague, and we needed an opportunity to acquire accurate and detailed information. Therefore, two of our members, who had previously discussed this with researchers working on the "Sustai-N-able" project aimed at achieving sustainable nitrogen use, participated in the symposium in person. The goal was to gather information about the current state of the issue and its countermeasures while also engaging with nitrogen experts.

• In the symposium, the issues of current nitrogen use were highlighted, such as the low NUE (Nitrogen Use Efficiency) and the depletion of natural resources. NUE refers to the ratio of output (crop yield) to nitrogen input in agricultural fields. NUE decreases as input is increased, and its current global average is at a low level of 47%. In developing countries, the overuse of natural nitrogen through excessive farming and fishing is also a problem.
• The complexity of the nitrogen issues was also emphasized. Reactive nitrogen released into the environment flows through various conditions and ecosystems, creating complex impacts (which is called nitrogen cascade). For example, while the planetary boundary for artificial nitrogen use has been exceeded for more than 15 years, considering the damage caused to climate change, air pollution, ecosystems, and other factors, the problem becomes even more serious.
• It was also pointed out that Japan has a unique issue: through food imports, Japan forces nitrogen emissions at the production stage onto other countries. This amount is the highest in the world, at 3.6 million tons annually.

From the discussions on NUE and resource depletion, we reaffirmed the need to optimize nitrogen use. The concept of the nitrogen cascade highlighted the necessity of considering multiple stakeholders in our project. After the symposium, we approached the speaker, Dr. Kentaro Hayashi, and later visited him for a more detailed discussion.

Dr. Kentaro Hayashi (1)

The speaker at the nitrogen symposium. He is also the leader of the Sustai-N-able project, which is for future planning towards the realization of sustainable nitrogen use.

To build our project by considering the current situation regarding nitrogen use, we contacted Dr. Hayashi. We also learned about his approaches in the Sustai-N-able project.

• Nitrogen runoff from farmland can be categorized as either horizontal runoff into rivers or vertical leach-out into groundwater. While runoff can be addressed with filters, preventing leach-out is considered difficult.
• Rice paddies use less nitrogen since lower protein content results in tastier rice. However, for crops like leafy greens, wheat, and corn grown in fields, a large amount of nitrogen fertilizer is applied.
• In Japan, nitrogen runoff due to Japanese consumer behavior is not visible, and awareness among farmers and consumers is low. In response, the Sustai-N-able project has been engaging in activities that allow consumers to understand the connection between food and the environment, such as providing experiences to calculate their nitrogen footprint.

Based on the current fertilization trend, we decided to shift the focus of our sensing target from rice paddies to fields. Given that physical prevention of groundwater leach-out is difficult, we recognized the demand for sensors that could minimize the cause of runoff itself. We also decided to engage in Human Practices targeting consumers to raise their awareness.

In iGEM, it is necessary to conduct Human Practices regarding existing approaches to avoid "reinventing the wheel". Early in the project, we interviewed companies using existing sensors to explore the potential for ShowgNs to impact society.

Mr. Seiji Matsumoto (1)

A staff member at the Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Centre, who conducts research and consultations on agriculture.

To understand the current state of fertilizer management.

• Mr. Matsumoto introduced us to slow-release coated fertilizers and localized fertilization as measures to save fertilizer. The slow-release coated fertilizer, which provides nutrients gradually through holes in a plastic membrane, can reduce nitrogen-based fertilizers by 30%. However, this method contributes to environmental pollution as the plastic eventually becomes microplastic. Localized fertilization, which applies fertilizer only near the crop roots, can reduce fertilizer use by 20%.
• The frequency of fertilizer application is left to the discretion of the farmers, with some using electrical conductivity (EC) values as a reference. However, Mr. Matsumoto pointed out that while EC is correlated with nitrate concentration, it only provides a rough estimate based on empirical correspondences and is not very precise. He also pointed out that the NPK ratio of base fertilizers is determined based on soil analysis results, but soil analysis takes a day, and it would be desirable to shorten this time.

Given the current state of fertilizer management, desirable characteristics of a sensor would include the ability to measure specific fertilizer components, higher accuracy than EC values, and shorter measurement times. Mr. Matsumoto also introduced us toMr. Funakoshi, a Marine Centre expert, and Mr. Morita, a farmer knowledgeable about fertilizers.

PLANTX (1)

A company developing technology for closed-type artificial light plant factories. They develop the hardware and software needed for environmental control in plant factories. We interviewed Mr. Masahito Takeyama, an employee at PLANTX.

We initially planned to build a conversion system that could achieve the optimal NO3-/NH4+ ratio based on the nitrogen concentration detected by sensors. However, as we progressed through literature research, we encountered difficulties in realizing this conversion system in a cell-free manner, and thus we aimed to check if there was sufficient demand for such a system. We also explored the possibility of integrating our system into the software already used at PLANTX to save labour.

• Mr. Takeyama suggested that it would be more distinctive to focus on sensing devices, and that creating a recovery and reuse system would better align with our goals than a conversion system.
• He also pointed out that compared to water, soil is not uniform, making it difficult to measure accurately. The amount of fertilizer and its effect on plants varies depending on where the measurement is taken, so careful consideration of measurement points is necessary.
• SAIBAIX (system for closed-type artificial light plant factories invented by PLANTX) controls single-fertilizer ions by mixing concentrated solutions in arbitrary ratios. Since there are no sensors that can continuously measure the concentration of single fertilizer ions, there is a demand for such a device, and it would be desirable for the system to be applicable not only to ammonium and nitrate ions but also to other ions like potassium.

We decided to abandon the development of the conversion system and focus on developing the sensing method. Although it was extremely difficult to achieve the conversion system in a cell-free manner, by switching to sensing, we were able to aim for a fully cell-free system. We also added versatility, continuous measurement, and specificity as necessary elements for the sensing system. We planned to discuss measurement point considerations with a soil expert and arranged to meet with Horiba, Ltd., a company that manufactures existing nitrogen sensors.

PLANTX (2) (Factory Tour)

After the discussion with Mr. Takeyama, one of our members participated in a factory tour and a roundtable discussion organized by PLANTX. The representative Mr. Kosuke Yamada, Managing Executive Officer Nobu Nishimura, and Ms. Tabasa Nagai from the Management Department, attended the discussion.

Given the learning outcomes from the discussion with Mr. Takeyama, we participated in the factory tour to gain more insights by observing the site.

• Through precise environmental control, crop yields are increased fivefold compared to typical plant factories. Moreover, it is possible to increase the nutritional and medicinal content of crops through growth condition adjustments.
• The SAIBAIX system receives the quantity of fertilizer as an input from sensors, and it seems likely that this system can be applied to open-field cultivation and profitably scaled. However, since PLANTX focuses on plant factories, they have not yet ventured into open-field applications.

If SAMURAI enables more precise environmental control, there is potential to maintain and improve crop yields. We realized that there is technical potential for integrating our sensor device with nitrogen management software like SAIBAIX. On the other hand, it became evident that developing independent crop management software would be extremely labor-intensive and time-consuming. Since SAIBAIX already possesses sufficient capabilities, we decided to halt our crop management software development.

Horiba, Ltd. (1)

Horiba, Ltd. is a company that develops and manufactures analytical and measurement systems, headquartered in Kyoto with global offices. We spoke with three representatives: Mr. Tatsuoki Muroga, Ms. Ayumi Hamada, and Ms. Saki Tachidokoro.

(From left to right: Mr. Tatsuoki Muroga, Ms. Ayumi Hamada, Ms. Saki Tachidokoro)

To understand the mechanisms and features of existing NO3- and NH4+ sensors, as well as the current demand from farmers. Our goal is to develop a user-friendly device by leveraging the strengths of biosensors while compensating for their weaknesses.

• Horiba's NO3- sensor uses the electrode method to measure concentration. Its strengths include a wide measurement range and the ability to measure a variety of sample types, such as solids, powders, and sheets. However, one downside of the electrode method is that interference from halide ions can cause discrepancies between actual and measured values.
• NO3- sensors are primarily used for improving yield by adjusting fertilizer levels and preventing nitrate poisoning in livestock, with very few purchases motivated by awareness of nitrogen issues. The frequency of use ranges from daily to twice a month. Currently, EC meters are more widely used due to their lower cost. Users have expressed a desire for an expanded measurement range, simplification of the calibration process, and a reduction in the required sample size.
• NH4+ sensors are mainly used in sewage treatment plants, with little demand from farmers. This lack of demand is attributed to low awareness of the nitrification process. As a result, the development of portable devices has not been pursued.

To increase adoption, affordability is key. The main advantage of biosensors is their specificity, and there is a need for sensors that do not react to halide ions. Information on the nitrification process and the properties of different nitrogen forms should also be included in educational materials.

Agricultural DX

We participated in a symposium where we spoke with experts about the data-driven agriculture in Kagawa Prefecture and the Nile Bank platform, which integrates various data management systems for agriculture.

To understand the current state of smart agriculture and data management in farming, as well as the demand for sensors.

• Smart agriculture is broadly divided into two categories: data-driven and robotics-based. In Kagawa Prefecture, efforts are being made to advance the former, with plans to also focus on plant growth diagnostics using drones. The main focus is on strawberry production. Due to the current reliance on experience and intuition, there is a yield gap among strawberry producers, so the goal is to standardize and visualize techniques while nurturing data-driven farmers.
• One challenge is that the data currently relies heavily on human input, making the analysis dependent on the skill of the individual. Missed opportunities for timely fertilization lead to increased costs and greater environmental impact. Nile Bank uses drones to collect, analyze, and apply data, but some farmers have expressed dissatisfaction, stating that smart agriculture "does not match the reality and does not produce the expected results." This mismatch seems to stem from a disconnect between the desired outcomes and the chosen methods.

To advance smart agriculture, it is essential to visualize data and reduce the yield gap between experienced and less experienced farmers. The need for user-friendly devices catering to a diverse range of users, across generations and levels of demand, was highlighted.

When planning stakeholder mapping and human practices based on it, concerns arose that creating the map from only our perspective might lead to oversights. Therefore, we sought expert opinions for creating the stakeholder map and planning our human practices.

Dr. Makoto Usami

Dr. Usami is a legal philosopher at Kyoto University's Graduate School of Global Environmental Studies, researching justice in intergenerational and international contexts related to environmental issues.

To ask for guidance on methodologies for effectively integrating a particular technology within existing social systems, and to discuss approaches for considering stakeholders in environmental issues.

• Dr. Usami pointed out that not all stakeholders are involved in decision-making, and many people are unaware of or do not understand the nitrogen issues. Given the low awareness of nitrogen issues despite its wide-reaching impact, he suggested that it is better to first convey the mechanisms of nitrogen issues before conducting interviews with stakeholders.
• He also emphasized the importance of determining how far into the future we consider when including future generations as stakeholders. In the case of nitrogen issues, considering a timeframe of several decades is sufficient, which allows the present generation to represent the interests of future generations.

We decided to create educational materials about nitrogen issues to share with stakeholders before conducting human practices. You can view the materials we prepared here.

Dr. Kentaro Hayashi (2)

To seek advice from Dr. Hayashi, an expert on nitrogen issues, on additional stakeholders to consider.

• Dr. Hayashi suggested considering additional stakeholders, including agricultural cooperatives, the food supply chain, and agencies responsible for agricultural and environmental regulations.

Based on Dr. Hayashi's advice, we revised the stakeholder map and used it to plan and execute further human practices. Additionally, he introduced us to the LEACHM research team, who study nitrogen dynamics in agricultural land.

Mr. Takahiro Okamoto

A farmer cultivating tomatoes in greenhouses in Kumamoto Prefecture.

To clarify the issues to be addressed through the project by hearing about the realities of fertilization from farmers.

• After applying basal fertilizer once before planting, he performs top-dressing at a rate of 0.5 to 3 times per day, depending on the season. An automatic machine measures the EC value to apply the optimal concentration of liquid fertilizer. While basal fertilizer (solid) remains in the soil, the liquid fertilizer, introduced five years ago, is well absorbed by the plants and doesn't remain in the soil, which is beneficial. In the land where farming has been done for a long time before the introduction of liquid fertilizer, residual fertilizer components accumulate in the soil, causing concentration damage to the crops, reducing their water absorption and ultimately lowering yield. Even attempts to correct this accumulation have been challenging.
• Compared to 30 years ago, fertilizer prices have increased by about 50%. Recent price hikes have been significant, increasing the burden on farmers, especially with the costly liquid fertilizers.

We need to devise ways to minimize fertilizer usage to alleviate the economic burden on farmers. This is also important to prevent fertilizer residues in new agricultural lands.

Miyama no Sato

A direct sales shop of agricultural products located in the mountainous area of Ibaraki City, Osaka Prefecture. They sell vegetables, rice, and processed products made by local farmers. We interviewed four farmers (Mr. Ogami, Mr. Inoue, Mr. Ogami, Mr. Harada) who provide their products there.

To understand the realities of farming and the needs of farmers, as well as their awareness of nitrogen issues, to design a user-friendly sensor.

• In addition to rice, they grow various vegetables (tomatoes, eggplants, pumpkins, potatoes, soybeans, perilla, etc.) on a scale of about 2 to 5 hectares each. Recent concerns include rising fertilizer prices and an aging workforce.
• They apply both basal and top-dressing fertilizers by hand using solid fertilizers. They also use green manure by plowing in clover fields to utilize its nitrogen content, but since clover growth is unstable, they supplement with chemical fertilizers. The amount and timing of top-dressing vary depending on the vegetable, and decisions are made based on experience. For leaf and root vegetables, top-dressing is done about once every two months, while for fruit vegetables, it is applied after fruiting, and no fertilizer is applied for rice cultivation. For outdoor cultivation, no watering is done after planting except at the time of planting.
• They do not currently use soil sensors due to two reasons: (1) Previous soil analysis results varied greatly depending on the sampling location, making them distrustful, and (2) The effort required was too much compared to the benefits. However, given the current reality that organic farming alone is not feasible, they would appreciate the presence of a sensor for proper fertilization. On the other hand, for outdoor cultivation, small-scale, part-time farmers find it difficult to manage soil meticulously, and it was suggested that we gather opinions from full-time farmers. They also advised consulting with JA for expertise on fertilizers and crop issues.
• The farmers expressed a need for sensors that can identify the cause of individual crop growth failures (e.g., nitrogen deficiency or excess). The sensor's price, size, and shape should be comparable to a thermometer, and the output should be visualized, such as through colours or lights, rather than numerical data.
• While awareness of nitrogen issues is high among farmers due to the promotion of eco-friendly farming, few farmers are taking specific measures due to the effort involved.

Currently, the amount and timing of fertilization are based on experience, and considering the high fertilizer prices and increased awareness of nitrogen issues, there is a demand for nitrogen sensing. For outdoor cultivation, it is undesirable to trigger sensing based on watering. The sensor should be a simple, portable model that minimizes effort. We decided to engage in Human Practices with JA and full-time farmers as well.

Farmer Survey

To gather opinions from more farmers, we conducted an online survey using Google Forms from June to September 2024. We contacted nearly 60 farmers and received 6 responses.

• Fertilization: Both liquid and solid fertilizers are used equally. Some fertilize by hand, while others use machines. Some farmers do not apply top-dressing at all.
• Watering: Frequency and timing vary among farmers. The most frequent cases involve watering multiple times a day. Some farmers consider weather and crop conditions, and some use solar radiation sensors and soil moisture meters.
• Soil Sensors: Two out of six respondents used soil sensors. They measured soil moisture, pH, temperature, and EC.
• Awareness of Nitrogen Issues: Four out of six farmers were somewhat aware or conscious of nitrogen issues. Some attributed the primary cause of nitrogen issuess to chemical fertilizers. On the other hand, one respondent had heard of nitrogen issues but was not conscious of them, and one had no knowledge of them at all.

Since the existence and methods of top-dressing and watering vary widely, it may be difficult to design hardware that suits all cases. Therefore, it is necessary to investigate the demand for soil diagnosis before applying basal fertilizer. While some farmers use soil sensors, many do not, so it is important to create a user-friendly form. There are significant differences in awareness of nitrogen issues among farmers. The path forward involves continued discussions with farmers who are aware of the issues and developing a design that can be easily used by all farmers.

Mr. Yoshihiko Morita (1)

A full-time farmer in Kamigamo, Kyoto City, who focuses on fertilizer management. We interviewed him as a farmer highly aware of nitrogen issues.

To determine the demand for SAMURAI among full-time farmers who have used soil sensors, and to understand the issues with existing soil sensors for designing user-friendly hardware.

• Although organic farming is currently booming, it is almost impossible in Japan due to its drastically changing seasons. Mr. Morita currently uses a combination of chemical and organic fertilizers.
• Before fertilization, he outsources soil analysis to a company, but it is costly. The benefit of soil analysis is that proper fertilization maximizes crop growth and shortens the time to harvest. However, few farmers around him conduct soil analysis.
• He also mentioned the challenges of labor shortages, aging, and corporatization in agriculture. He believes that the primary reason for the lack of new farmers in Japan is that it is not profitable enough.

There is a significant demand for biosensors that do not require outsourcing for farmers who focus on fertilizer management. If the cost issue can be addressed and agricultural efficiency improved, it may lead to the promotion of new farmers.

Consumer Survey

One of the reasons for the slow adoption of existing nitrogen sensors is the lack of visible cost-effectiveness. Therefore, we conducted a survey on purchasing desire for crops grown using SAMURAI and asked about factors that might influence this desire. Method, Questions, and Results can be found here → Download

• The factors "Age," "Awareness of Environment," "Awareness of Nitrogen Issues," "Knowledge about Nitrogen Issues," and "Knowledge about Biosensors" had little impact on the desire to purchase crops produced using biosensors.
• Sixty percent of respondents had never heard of nitrogen issues, indicating a clear lack of awareness. This needs to be improved through education and other means.
• The average purchasing desire for agricultural products grown using biosensors was 4.87 out of 7, but a certain number of respondents expressed no desire to purchase them. This could be due to an unconscious negative perception of biosensors, as about 80% of respondents had never heard of biosensors. When moving toward practical implementation, it will be necessary to document the benefits of being cell-free and the biosafety measures taken.
• At this stage, it is unclear whether the widespread use of biosensors would have a positive effect on consumers. For those with negative perceptions, it could potentially reduce purchasing power.

To raise awareness of nitrogen issues, we have publicly shared previously created materials on official SNS platforms.

JA Ibaraki City Farmers' Market Mishima Kan, Kyoto City

Our activities are related to agriculture, but we have come to realize that we do not have a sufficient understanding of actual farming practices. In order to develop truly useful products, we need to have a clear grasp of the current situation. Therefore, we conducted surveys with farmers, as mentioned above. However, we believe that in addition to understanding individual farmers' perspectives, it is also important to gain a broader view of agriculture from those who are closer to the field but can still provide an overall perspective. With that in mind, we decided to speak with the Japan Agricultural Cooperatives (hereinafter referred to as JA). First, we visited Mishima Kan, a farmers' market operated by JA located in Ibaraki City, Osaka Prefecture, and spoke with one of the staff members.

To gain a deeper understanding of the current state of agriculture in Japan by hearing opinions from individuals who have a broad perspective on the agricultural landscape, especially regarding the gap between ideal and reality in terms of sustainability. Additionally, to learn about the initiatives that JA is undertaking to address these challenges.

Although Osaka Prefecture ranks 46th out of 47 prefectures in terms of cultivated land area, it ranks 19th in agricultural output per hectare, utilizing the advantages of its proximity to urban areas. In Osaka, approximately 64% of farmers are classified as self-sufficient, with a farm size of less than 30 ares and an annual sales amount of less than 500,000 yen. However, around 3,000 individuals have been recognized as certified farmers or Osaka-certified farmers. JA operates about 2,200 farmers' markets nationwide, where consumers can purchase a wide variety of seasonal agricultural products, with clear visibility of the producers. Products that meet the criteria—such as not exceeding the number of pesticide applications set by Osaka Prefecture for each crop, not exceeding the prescribed amount of chemical fertilizers, and not being genetically modified—are branded as "Osaka Eco Agricultural Products." JA Group Osaka is involved in a variety of initiatives, such as providing farming guidance and labor management training to foster diverse new farmers. It also promotes the use of community gardens and advocates for policies to preserve farmland.

Based on the information obtained, we believe that demand will increase for products that can be easily adopted by self-sufficient and small-scale farmers, so we have decided to prioritize "affordability" in product development. We were also introduced to the JA Ibaraki City Farming and Economic Center, where we could learn more about fertilizers, and plan to visit there as well.

JA Ibaraki Einou Centre

We visited the JA Ibaraki City Farming and Economic Center, which was introduced to us at Mishima Kan, and spoke with Mr. Mukai, the head of the JA Ibaraki City Farming and Economic Department.

To gain a deeper understanding of Japan’s current agricultural situation regarding fertilizers by hearing opinions from someone with a broad perspective on agriculture, specifically about the gap between the ideal and reality in terms of sustainability. Additionally, to learn about the initiatives JA is undertaking to bridge this gap and understand the distribution channels through which JA sells fertilizers. Outcomes:

• There are relatively few farmers using liquid fertilizers, with most using solid or coated fertilizers. In Ibaraki City, many farmers are small-scale and part-time, making it difficult for them to allocate enough time to farming. As a result, many farmers opt to grow rice, which, although it incurs machinery costs, requires comparatively less daily attention. This explains the higher prevalence of coated fertilizers.
• In an effort to reduce the amount of microplastics used in fertilizer coatings, the packaging of fertilizers previously sold in 20 kg bags has been changed to 15 kg bags, while still containing the same amount of active ingredients.
• JA provides guidance to farmers on appropriate fertilizer application based on crop conditions and soil diagnosis, ensuring that fertilizer is not applied indiscriminately.
• As the national "Green Food System Strategy" advocates for balancing productivity and sustainability, focusing solely on sustainability risks neglecting the need to ensure current food production. It is important to maintain a balance between the two.
• Fertilizers must be applied before symptoms like leaf discoloration appear, as there is a delay before they take effect. In this regard, chemical fertilizers are preferred for their relatively quick efficacy, second only to liquid fertilizers.
• JA Ibaraki City offers free soil testing (measuring nitrogen, phosphorus, potassium, calcium, magnesium, EC value, pH, etc.) and promotes the use of green manure farming with legumes. However, only about 5% of all farmers utilize the free soil testing service. Aside from this, the main soil improvement tool recommended by JA, considering cost, is typically just a pH meter.
• Regarding fertilizers, most of the products sold by JA are bulk purchases made by Zennoh, which handles economic operations within the JA Group, from companies. These fertilizers are sold at certain JA-affiliated centers. In the past, each JA branch used to sell fertilizers, but due to the current sluggish economy, only the Ibaraki City Farming and Economic Center continues to sell fertilizers, as a cost-cutting measure.
• The JA Farming and Economic Department prioritizes the interests of farmers, so the potential reduction in total fertilizer sales due to our project’s goal of reducing fertilizer usage is not seen as a major issue for them.

We concluded that in order to ensure farmers actually use the product, we need to emphasize not only its affordability but also its potential to improve productivity. Additionally, the soil analysis methods currently in use require time and cost, such as drying the soil, but our project has the potential to serve as an alternative. Given the current situation, our product could also help reduce the use of chemical fertilizers, which are valued for their immediate effectiveness when determining fertilization amounts. Therefore, we decided that "immediacy" should be another key focus in the development of our product.

Shiga Prefecture's Lake Biwa Conservation and Restoration Division

We spoke with Mr. Shunji Terauchi, who is responsible for the conservation of Lake Biwa in Shiga Prefecture, and Mr. Takayuki Takayama, who is in charge of the agricultural sector in Shiga Prefecture. Lake Biwa, the largest freshwater lake in Japan, has a history of dealing with freshwater red tides. Shiga Prefecture is also known for its flourishing agriculture.

(From left to right: Mr. Terauchi, Mr. Takayama)

To understand the role played by citizens and the government in reducing nitrogen inflows into Lake Biwa, and to identify our own responsibilities. Additionally, we aimed to grasp the current fertilizer application status in Shiga Prefecture and dicover the most effective use case for SAMURAI.

• Lake Biwa undergoes monthly water quality surveys, which show a decreasing trend in total nitrogen levels. However, they highlighted the lack of good methods for investigating the fertilizer application phase. While soil monitoring is carried out by analysis companies and agricultural cooperatives, it has significant cost and time drawbacks, making SAMURAI a potentially suitable tool for this stage.
• The primary causes of algal blooms are suitable water temperatures, water stagnation, and water quality deterioration. Cyanobacteria, which cause blue-green algae, can proliferate even at a water temperature of 30°C, which is unsuitable for other organisms. This raises concerns that the worsening of blue-green algae blooms may occur with global warming.
• In terms of technical methods, slow-release fertilizers and banded fertilizer applications are being used. Microplastics from plasters used in slow-release fertilizers are becoming a concern, so alternatives like sulfur are being employed. However, due to the higher efficiency, precision, and ease of production of plastic-based fertilizers, they continue to be used. There is a roadmap to phase out plastic-based coatings by 2030.
• In the northern part of Lake Biwa, over-fertilization occurs in some areas because organic matter is less likely to decompose due to winter snow.
• The primary cause of the red tide in Lake Biwa in 1977 was phosphorus from synthetic detergents. As a result of citizen movements advocating for the use of soap instead of synthetic detergents, Shiga Prefecture enacted regulations in 1980 to ban the use of synthetic detergents, regulate factory wastewater, and promote the development of sewage systems. This was a pioneering effort ahead of national legislation. Additionally, July 1st, the day the ordinance was enforced, was designated as "Lake Biwa Day," with activities like the removal of invasive fish and cleaning efforts. To further raise awareness, the Mother Lake Goals (MLGs), modeled after the SDGs, were established.

As Dr. Yagi also mentioned, the most suitable use case for SAMURAI would be soil diagnostics before fertilization. We have decided to design the hardware with this use case in mind. Specifically, in addition to the usual automatic measurements during the farming season, we are considering implementing a mode that allows for measuring a desired amount of soil by inserting it into the machine. As it is likely that restrictions on plastic-coated slow-release fertilizers will be introduced in the future, SAMURAI's effectiveness in fertilizer management was demonstrated. The importance of improving citizen awareness was also shown to potentially lead to broader improvements in regulations at the municipal and national levels. To enhance awareness of nitrogen issues, the idea of creating a "Nitrogen Issues Day" or nitrogen-related SDGs emerged.You can view the NCGs (Nitrogen Cycle Goals), which we created as the nitrogen issue version of the SDGs, here.

Mr. Yuki Funakoshi

Mr. Yuki Funakoshi is an employee at the Marine Centre of the Kyoto Prefectural Agriculture, Forestry and Fisheries Research Centre, where he conducts research and consultations related to the fishing industry.

We met with Mr. Funakoshi, who was introduced to us by Mr. Matsumoto, to understand the current situation of nitrogen issues in the fishing industry and the demand for sensors.

• Red tides are no longer a major issue nationwide, including in Kyoto Prefecture. On the contrary, seaweed and bivalve shellfish farmers are suffering from reduced quality and yields due to nitrogen deficiency. In coastal regions along the Pacific Ocean and the Seto Inland Sea, some wastewater treatment plants deliberately reduce their treatment rates to release nutrient-rich water into estuaries.
• There is demand for inexpensive, on-site nitrogen sensors at seaweed and bivalve farms. Currently, outsourcing one test (300ml) costs about 70 dollar, and purchasing an Auto Analyzer for self-testing costs about 70,000 dollar. Nitrogen detection paper used in tropical fish tanks can only provide qualitative measurements, so if affordable quantitative measurements become possible, the demand would be high.

We decided to develop sensor concepts based on three key points: affordability, on-site usability, and visibility.

Dr. Hideaki Yoshimura

An assistant professor at the Graduate School of Science, the University of Tokyo, specializing in bioimaging.

Our team aimed to create a biosensor focusing on visibility based on the concentration of RNA purified by T7RNAP transcription. During literature research, we found papers discussing the reconstitution of split GFP according to RNA concentration[3] and the reconstitution of split luciferase[4]. We interviewed the authors of these papers to explore potential applications for ShowgNs and to deepen our understanding of fluorescence and luminescence.

• Dr. Yoshimura confirmed that it is possible to generate fluorescence and luminescence in response to RNA concentration using the beta-actin gene sequence and the DNA-binding protein mPUM.
• We received detailed explanations of the papers, and Dr. Yoshimura agreed to send us the sequence information and plasmids for mPUM.
• Both fluorescence and luminescence have drawbacks: fluorescence is challenging to handle due to the irreversibility of split protein reconstitution, and luminescence is unsuitable for observing changes over time as the substrate diminishes.
• Outdoor sensing using drones to excite light may be difficult, and detecting either fluorescence or luminescence would be challenging unless in a dark environment.

We concluded that RNA-binding proteins and split proteins provide a viable sensing mechanism. Hence, we decided to experiment with mPUM and split luciferase. Given the shortcomings of split fluorescent proteins, we began considering alternative methods, such as RNA aptamers. Additionally, while drone-based detection remains under consideration, we recognized the need to explore alternative approaches due to the difficulty of detecting fluorescence or luminescence in outdoor settings.

Dr. Makoto Yoshimoto

A researcher at the Faculty of Engineering, Yamaguchi University, studying the properties of artificial lipid membrane liposomes.

To explore whether liposome properties and examples of their use could be applied to cell-free sensors.

• Factors destabilizing liposome structures include temperature, pH, proteins, and air bubbles. By adjusting lipid design (e.g., changing carbon chain length or introducing double bonds), one can manipulate liposome properties. High molecular weight polymers on the surface can also affect stability by encouraging protein adsorption.
• We explored the possibility of using liposomes in open-field cultivation, with findings suggesting that embedding them in alginate beads could enhance stability. While choosing oxidation-resistant lipids can ensure physical-chemical stability, too few double bonds may compromise flexibility (a trade-off between stability and flexibility).
• Liposome-based sensing technology exists, such as enhancing reactivity in microspaces or measuring membrane destabilization or interaction.
• The biggest challenge in implementing liposomes as sensors is cost, with 100 mg costing several tens of thousands of yen. Large-scale production would require addressing the issue of air bubbles.

While liposomes are suitable for creating targeted capsules, freeze-drying the entire system may be more effective when considering costs.

Dr. Hideki Nakamura(1)

An associate professor specializing in synthetic biology at the Graduate School of Engineering, Kyoto University.

We sought feedback from Dr. Nakamura, a synthetic biology researcher, on potential issues and improvements for ShowgNs.

• Dr. Nakamura pointed out that mPUM has low specificity and suggested considering highly specific RNA-binding proteins like MS2.
• For outdoor drone-based sensing, both luminescence (due to measurement difficulties) and fluorescence (due to the excitation light issue) pose challenges. He advised considering a system where colour changes according to RNA concentration.
• Protein modification might be feasible using tools like AlphaFold.
• He warned that temperature and pH changes in the field could significantly impact protein activity, necessitating calibration. Buffer conditions may need to be optimized.
• The concept has broad applicability and could be adapted for other purposes.

We decided to consider other RNA-binding proteins besides mPUM, ultimately selecting MS2 and PP7. We also began exploring systems where dye changes according to RNA concentration. Based on his recommendation, we interviewed Dr. Mahiro Noji about AlphaFold. Calibration for field conditions was incorporated into the project plan, and we aimed to develop a platform adaptable to various sensing applications.

Dr. Mahiro Noji(1)

A research fellow at Kyoto University's Institute for Advanced Study, specializing in protein design using AlphaFold.

As we needed to create fusion proteins for our project, we sought advice from Dr. Noji on protein design using AlphaFold.

• AlphaFold allows unlimited in silico trials, and evaluations can be made by observing results visually or using parameters like pLDDT and PAE. However, AlphaFold should be used in balance with wet experiments.
• For linkers, flexible ones like GGGGS and rigid ones like EAAAK should be used depending on the purpose. For our objective, GGGGS linkers would likely improve mobility.
• To assess whether the original folding of proteins is maintained, RMSD values between proteins can be checked.
• For systems involving protein interactions, individual 3D structures can be examined in PyMOL to discuss alignment.
• For predicting multimers, residue limitations might necessitate discussions based on monomers.

We brought the fundamental knowledge gained from this session back to the Dry Lab and began protein design ourselves.

Dr. Mahiro Noji(2)

Following the first discussion, we attempted protein design using AlphaFold. We then sought further advice on the linker length for GlnR-Leucine zipper and the feasibility of attaching a Leucine zipper to GlnA.

• Long linkers are generally acceptable as long as they are not excessively long. Ensuring sufficient length for interaction is crucial.
• Attaching a Leucine zipper to the N-terminus is valid, given prior research showing the functionality of proteins with N-terminal His-tags.
• For 14-mer GlnA, care must be taken to prevent homotypic interactions of Leucine zippers. Given the residue limitations and reliability concerns in predicting 14-mers, conclusions should be drawn from smaller predictions.

We used slightly longer linkers for GlnR in wet experiments and created versions with Leucine zippers attached to the N-terminus. We also referred to the relevant literature to ensure heterotypic interactions for GlnA-Leucine zipper proteins and proceeded with the wet experiments.

LEACHM Researchers（Dr. Sadao Eguchi, Dr. Kei Asada, Dr. Tetsuo Yagi）

These researchers improved the nitrogen dynamics model LEACHM for application to Andosol, a major soil type in Japanese agricultural fields. They were introduced to us by Dr. Hayashi.

Source:NARO No.32

To understand the mechanism and characteristics of the nitrogen dynamics model LEACHM and evaluate its applicability to our modeling. Additionally, to inquire about the impact of SAMURAI on nitrogen runoff from the perspective of soil experts.

• Data sets necessary for modeling can be obtained from the Japanese Soil Inventory, but ideally, they should be collected from actual fields.
• A simple device for collecting soil leachate is the monolith lysimeter. When it rains, water passes through a cylindrical tube and collects in a plastic tank, where it is then drawn into a vacuum-sealed gallon bottle. - A more straightforward method involves using a ceramic rod-based soil solution extraction pipe.
• Due to the widespread use of KCl in fertilizers, agricultural fields tend to contain large amounts of chloride ions.
• Placing devices in fixed positions, like for long-term observations, might interfere with tractors. It would be more practical to use them for soil analysis before planting (base fertilization). A diagonal arrangement of the devices across the field may balance simplicity and accuracy in this case.
• A variety of depths for measurement is preferable. The root zone, 20-30 cm, could be one important target.

We explored whether part of the monolith lysimeter's mechanism could be replicated for extracting soil solution. The high concentration of chloride ions in the soil, being a monovalent anion, could lower the accuracy of electrode-based sensors. We need to confirm that the protein used in our biosensor does not have a binding site for chloride ions. We also discovered that there is a demand for pre-fertilization soil diagnosis not only among farmers but also researchers.

Dr. Akihiko Ito

A professor at the University of Tokyo researching forest resources and the global environment. We reached out to him after reading a paper on the process-based terrestrial ecosystem model VISIT.

To understand nitrogen cycle simulations using the ecosystem model VISIT and assess its potential application in our modeling.

• He introduced us to Dr. Kazuya Nishina, who is conducting more nitrogen cycle-focused research as a co-author.

We conducted a Human Practice with Dr. Nishina.

Dr. Kazuya Nishina

A researcher at the National Institute for Environmental Studies, working on nitrogen issues and involved in the development of the nitrogen dynamics model VISIT.

To understand nitrogen cycle simulations using the ecosystem model VISIT and evaluate its application in our modeling. Also, to discuss the current status and outlook on nitrogen issues and countermeasures.

• VISIT uses a 50° × 50° grid, which is much larger than the scale of the model we are aiming for in the Kyoto project. It would be preferable to use simpler and smaller-scale models like LEACHM for our modeling. If time allows, building a model ourselves is an option, but for now, running an existing model with default values is a good approach.
• A resolution on sustainable nitrogen use was adopted at the 2019 UNEA (UN Environment Assembly). While the goal is to reduce nitrogen emissions, the issue receives little attention in many countries, including Japan.
• There is no open data on fertilization practices across Japan, but globally, Japan has very high fertilization rates.

Given the significant difference in prediction scale, we concluded that VISIT would be difficult to apply to our modeling. In the early stages of developing our own nitrogen model, we attempted to simulate ammonium and nitrate dynamics in the soil using LEACHN, a component of LEACHM, as recommended. Although we eventually simplified the equations and customized many of the parameters, the concepts from LEACHN and VISIT informed the design of our equations in the modeling process.

Mr. Seiji Matsumoto (2)

During the prototyping stage of our device, we needed to collect soil samples. We contacted Mr. Matsumoto, whom we spoke with in "2 Understand Problems," for advice on the best sampling methods.

• Compared to soil leachate, soil itself is a more suitable sampling target for measuring nitrogen levels in agricultural fields. This is because the nutrient concentration in leachate varies depending on the amount of water input from rainfall and the volumetric water content of the soil.
• Nutrient concentrations in soil vary greatly depending on whether it is in the rhizosphere or not, whether fertilizers are applied throughout or locally, precipitation, irrigation amount and location, and root growth patterns. Therefore, the optimal sampling location must be determined through repeated testing.

We chose to sample soil itself. We planned multiple tests in actual fields to optimize sampling locations, and consulted soil science researchers for further advice.

Dr. Shinya Funakawa

A professor at the Faculty of Agriculture, Kyoto University, researching soil science.

To determine the optimal placement for hardware and seek advice on protocols for transporting soil samples and ion extraction.

• To monitor changes in soil ion concentration, if the goal is to observe nutrient conditions, it is best to collect soil water at the root zone depth; to monitor leaching, it should be collected below the root zone. On the other hand, when sampling soil itself to assess the amount of nitrogen available to plants, the root zone depth is ideal.
• However, due to the heterogeneity of resource distribution and dynamics in soil, and unpredictable weather conditions, it is difficult to precisely determine the placement of collection devices.
• Since reactions like nitrification proceed at room temperature, collected samples must be transported in a cooler to minimize microbial activity. It is also preferable to store them in a refrigerator in the lab.
• A 1 mol KCl solution should be used to extract nitrate and ammonium ions from soil.

We decided to place the device at the root zone depth for now, but given the many dynamic environmental factors, further refinement is necessary. Collected samples will be refrigerated and extracted with a 1 mol KCl solution.

Ms. Sayuri Ishida

A master's student in Dr. Funakawa's lab researching nitrogen cycling in tropical soils. She was introduced to us by Dr. Funakawa.

To hear opinions from someone researching soil nitrogen and optimize the design and measurement methods for the biosensor.

• When transporting soil samples back to the lab, it is common practice to place them in a Styrofoam container and cool them to 4°C to minimize microbial impact.
• We are considering two methods for hardware design: one where rainwater passing through the soil is collected in a funnel-like part, and another where soil is drilled and mixed with liquid inside the device. For the latter, she pointed out the possibility of needing complex and large hardware.
• If the concentration ratio of the measurement target to the protein in the system is too high, the output level could saturate. In such a state, the device would not be usable, so it is necessary to mix the soil water and each protein at an appropriate concentration.
• She shared the need for real-time measurement in her own research. She is currently conducting culture experiments to collect data on the nitrification process, but real-time measurement could allow them to monitor net nitrate production in the field and visualize it. It would be great if the device could measure organic nitrogen in the future as well.

To maintain appropriate concentrations, we designed a mechanism to standardize the amount of soil collected and the amount of reagents mixed.

Symbiobe(1)

A venture company from Kyoto University working on the development of nitrogen fertilizers using photosynthetic bacteria. We spoke with Mr. Kato, who was previously involved in agricultural automation projects in the U.S.

To understand the current state of the fertilizer industry, investigate the efforts made by fertilizer companies toward fertilizer optimization, and learn about organic fertilizers being developed by Symbiobe. Additionally, to assess the demand for nitrogen sensing from the perspective of a fertilizer company.

• The Japanese fertilizer market is flat, but there is a growing movement to reduce the overall amount by shifting from chemical fertilizers to organic ones and optimizing fertilization.
• He wasn't sure about the exact demand for sensors, but it seemed low. Even if nitrogen levels were known in real time, the data would be meaningless if it couldn't be used to manage fertilization.
• Three key elements needed for a sensor are "automatic data acquisition," "the ability to take action based on the data from the sensor," and "asset management (particularly important for U.S.-style farming)."
• For business purposes, it is necessary to increase customer resolution.

We decided to design a fully automated device capable of wireless transmission.

This diagram illustrates how ShowgNs has evolved with IHP. How each part of the flowchart was integrated into the project is shown in five steps: Project assembly, Wet experiments, Nitrogen modeling, Hardware design, and Implementation. Please refer to "Integration into the Detailed Design of the Project" for specific integration methods at each step.

OverviewProject-assemblyWet-experimentsNitogen-modelingHardware-designImplementation

Integration into Project Assembly

• Through Human Practices with nitrogen experts and reviewing prior research on nitrogen issues, we learned that agriculture contributes significantly to the nitrogen issues. We concluded that optimizing the amount and timing of fertilizer application is essential to solving this issue.
• From our Human Practices with plant factories, agricultural research institutes, and sensor manufacturing companies, we learned the following, which led to the goal of creating a biosensor that leverages the specificity of biological recognition systems.
  • Soil sensors are used as one technique to optimize fertilizer application.
  • Existing nitrogen sensors have challenges in terms of specificity, labor, and cost.
• During Human Practices to examine the specific style of the biosensor, we learned that fertilizer application is not done in rice paddies, so we shifted the measurement target from rice paddies to fields. Additionally, we found that there was not enough demand for the initially conceived conversion system, so we decided to focus solely on the sensing system and aim to achieve it as a cell-free system.
• By incorporating insights from philosophy of law and environmental science, we created a comprehensive stakeholder map for ShowgNs. This map served as a foundation for advancing the subsequent IHP.

Integration into Wet Experiments

• To explore whether liposomes, artificial lipid membranes, could be used as a technology to realize a cell-free system, we consulted with experts in chemical engineering. As a result, we found that stability and cost would be issues, so we decided to pursue an alternative approach.
• When assembling a biosensing system using a cell-free system, we needed to find RNA binding proteins with high sequence recognition specificity that could fuse with split proteins. Through consultation with multiple synthetic biology experts and reviewing prior research, we selected MS2 and PP7.
• As potential outputs for outdoor use, we considered fluorescence, luminescence, and pigments. After discussions with several experts, we developed the concept of a modular sensing system called MITSUNARI, which encompasses all these outputs.
• We learned the know-how for protein design using AlphaFold from experts and successfully designed fusion proteins. We also consulted experts to resolve questions that arose during the design process.

Integration into Nitrogen Modeling

• To estimate the impact of ShowgNs on society, we decided to create a predictive model for nitrogen leach out/runoff using SAMURAI. Referring to previous research, we focused on two models—LEACHN and VISIT—and consulted with experts on whether these models would be suitable for our purpose. As a result, we decided to build a simplified, customized model based on LEACHN, which was better suited to the scale of our predictions.

Integration into Hardware Design

• Drawing on the needs we heard from farmers, agricultural and fisheries research institutions, and government bodies, we identified the following essential features for the sensor: affordability, on-site usability, visualization, higher accuracy than EC (electrical conductivity) values, short measurement times, versatility, continuous measurement, specificity, automatic measurement, and wireless transmission.
• During the hardware design process, we learned that refrigerating samples during transport is the optimal method for sample collection. Using this method, we successfully conducted several test runs of sample collection.
• We discovered that the best format is to collect soil and mix it with reagents, and we learned the proper handling of soil within this mechanism.

Integration into Efforts Toward Implementation

• As a result of surveys conducted with consumers, we found that the project's limitations include uncertainty about whether the widespread use of biosensors would have a positive effect on consumers. For people who hold a negative image of biosensors, it could even decrease their purchasing power.
• For details beyond this point, see sections "6 Implement" and "7 Re-evaluate."

Wet Experiments

Regarding the transcription sensor (ammonia) using the Two-Hybrid system, protein purification and the proof of concept are not perfect, but have been achieved to some extent. On the other hand, the nitrate sensor using Split T7 RNAP has been challenging, as culturing and purification have not gone smoothly. Additionally, while we have been able to collect data on luminescence and fluorescence for the output system, we have not yet obtained any data on dye changes. See the Results page for more information.

Modeling

Based on advice from Professor Noji, we provided information on the designed protein to the Wet Lab team, and they proceeded with protein synthesis. For nitrogen modeling, we are developing our own modeling system by referencing LEACHEM and VISIT. See the Modeling page for more information.

Hardware

Based on the needs obtained from surveys of farmers and insights gained from discussions with nitrogen experts, we developed hardware that automatically samples soil. We also showed a model to actual farmers, gathered their feedback, and improved the hardware accordingly. See the Hardware page for more information.

We created a business plan from the project's current concept for future social implementation. This business plan was developed through Human Practices involving key stakeholders essential for advancing the solution, such as venture capitalists, entrepreneurs in the fertilizer sector, and end-users like farmers. Particularly, we received significant support in areas that are difficult to develop without input from those actively involved, such as milestones for social implementation.

Miyako Capital Co., Ltd.

As venture capitalists who actively support startup businesses, Mr. Tamiya and Mr. Otani provided us with advice on creating our business plan.

To receive ongoing advice on business plan creation from a mentor-like position.

Outcomes:

• They taught us the importance of demonstrating impact through numbers to add credibility to our business plan.
• We were advised to utilize existing frameworks like SWOT analysis and TAM, SAM, SOM for analyzing business strengths and competition.

We decided to utilize various frameworks to quantitatively demonstrate the project's impact and analyze the project.

Norinchukin Capital Co., Ltd.

Mr. Taiki Hino, introduced by Mr. Otani of Miyako Capital, is an investor who has been involved in a wide range of investment projects, from startups to large corporations.

To receive advice on our business plan.

• He pointed out that we lacked a narrative and that it is essential to focus on storytelling when creating a business plan.
• He also advised us that our project would be more persuasive if we could express in monetary terms how it will impact the lives of farmers.

We created a new "Real Use Case" section under the Entrepreneurship section to improve storytelling and make real-life usage scenarios easier to visualize.

Organic nico Co., Ltd.

Ms. Fukuda, introduced by Mr. Otani of Miyako Capital, is a farmer and researcher in the cultivation methods division of Organic nico, an agricultural corporation that focuses on high-value-added organic farming and agricultural consulting.

To hear from a farmer about real-life use cases.

• We learned when and on what aspects soil analysis is currently performed, especially the need to monitor concentration levels during topdressing.
• She also shared insights on the most helpful media and formats for providing measured information from the perspective of those in the field.

We incorporated the topdressing timing as a use case in the business plan and decided to adopt service formats that match the actual needs when providing the service.

Symbiobe (2)

To report the progress of our hardware development to Mr. Ito, who we spoke with during a previous Human Practices session, and to seek advice on social implementation from the perspective of an entrepreneur in the agricultural field.

• He pointed out that the "value proposition"—the value we provide to whom—was unclear when considering social implementation.
• He provided entrepreneurial advice, suggesting we consider monetization methods such as introducing carbon credits.

We decided to clearly state who our project benefits and what value we provide, and we also incorporated alternative monetization methods beyond direct cost burden on beneficiaries into the business plan.

Dr. Hideki Nakamura (2)

We reported the current progress of the Wet Lab to Dr. Nakamura, a synthetic biology researcher with whom we had previously engaged in Human Practices. We sought his advice on how to improve the areas of the experiments that did not succeed, while also discussing potential applications beyond the project for the successful aspects.

• Regarding the Two-hybrid system using the leucine zipper, while our experimental results show some differentiation, the absence of non-linear feedback loops, like those found in biological systems, leads to a high background signal. This makes the system too weak to compete with intracellular systems. Therefore, it is believed that amplification can be achieved by introducing a mechanism for non-linear amplification after transcription.
• Our system’s strength, which lies in its platform based on in vitro transcription, offers advantages over more complex, costly, and unstable mechanisms like translation systems. If we can make it competitive with biological switches, we believe it will have potential applications in various fields. Additionally, we see a niche for our system in environments where the use of living organisms is not desirable, such as in outdoor settings.
• The inability to purify the Split-T7 RNAP can be attributed to common issues associated with split proteins, such as reduced solubility, difficulty in folding, increased degradation, and changes in isoelectric point upon cleavage. On the other hand, protein expression and purification still require a high level of expertise, so it is not surprising that we could not purify all proteins. In the future, the process may become easier if programs that determine protein purification methods through machine learning are developed.
• To improve protein solubility, it was suggested to attach tags like MBP or SUMO to enhance solubility and later remove the tag.

We incorporated Dr. Nakamura's insights on the potential causes of protein purification failures into our considerations. For the post-iGEM plan, we included the use of Split-T7 RNAP with solubilizing tags.

Mr. Seiji Matsumoto (3)

To report the progress of our hardware development to Mr. Matsumoto, an employee of the Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Center, who we spoke with during a previous Human Practices session, and to discuss use cases for the sensor we are developing.

• He advised us that when discussing use cases or potential damages, it is easier to relate by starting with specific cases rather than large-scale scenarios.

• He pointed out that when using buried sensors, the desired measurement depth varies depending on the crop, and the values obtained can change based on drainage conditions.

We decided to incorporate specific cases into sections where understanding and relatability are critical, such as actual use cases.

Mr. Yoshihiko Morita (2)

Objective:

To explain the tasks performed by the end user, Mr. Morita, one of the end users we interviewed during the Human Practice, using a life-size model through installation tests in an actual field.

Outcomes:

Regarding the replacement of reagents, Mr. Morita suggested that instead of refilling a fixed tank with reagents, it would be better to replace the entire tank filled with reagents. For the cartridge unit, he advised that rather than refilling the cartridge into the cartridge case, it would be preferable to replace the entire cartridge case containing the cartridge unit. As for the soil collection mechanism using a drill nozzle, concerns were raised about its operation in clayey soil. The concern was that when the drill nozzle rotates, the resistance from the clayey soil might increase, making it difficult to collect soil properly.

Instead of fixing the reagent tank and cartridge case in place using adhesives or other methods, we implemented the use of magnets for easier attachment and detachment, allowing for the whole units to be replaced. We devised a new mechanism for collecting soil in clayey soils. Detailed explanations of this mechanism can be found in the Proof of Concept section on this page. Additionally, we developed a drill nozzle design that functions effectively in clayey soil.

Horiba, Ltd. (2)

To report the detailed specifications, mechanisms, and functions of our hardware in an online user test format to Horiba Ltd., from whom we previously received insights on the current technological and commercial state of nitrate and ammonium electrode sensors. We aimed to seek advice on technical aspects and the development plan for implementation, from the standpoint of a nitrogen sensor/optical measurement equipment manufacturer.

• Several concerns were raised regarding measurement accuracy, the risk of contamination during continuous operation, and component placement. We received suggestions, including adjustments in parts arrangement and fluorescence lifetime measurement. It was also advised that some of these issues can only be verified through actual development, so completing the hardware at least once would be ideal.
• Upon mentioning that we are considering interference ions, both those already reported in literature and those specific to the target application, they recommended referring to the relevant JIS standards for testing methods.
• For development, we were advised to test durability and resistance to transportation based on JIS standards, and to design with user-friendliness in mind through discussions with end-users regarding usage and maintenance. If durability, cost-efficiency, and measurement accuracy can be achieved, the product may have great potential.
• Additional functionalities, such as integrating features to replace existing farmland sensors and measuring nitrification rates, were suggested. There is also high demand but a lack of technological solutions for phosphorus measurement, which could be a significant future offering.
• The practical lifespan of ion electrode sensors varies depending on their form, application, and usage conditions, with EC sensors typically lasting about five years, and others ranging from six months to a few years.

We incorporated some of the technical advice on hardware into our development, while integrating others into the roadmap for future implementation. The specific advice on hardware development served as a reference for creating our roadmap toward implementation. Information on the lifespan of electrode sensors was integrated into our business plan for future reference.

PLANTX (3)

To report the final project vision and hardware specifications to PLANTX, from whom we previously learned about plant cultivation management technologies, their effects, and the use and demand for nitrogen sensors in hydroponics. We sought feedback on the project and hardware.

PLANTX acknowledged that we had correctly identified the issue, noting that nitrogen problems in open-field cultivation are well recognized. Although plant factories aim to address such issues, eliminating open-field cultivation is unrealistic, and sensors are necessary to solve this problem. Regarding hardware specifications, they suggested miniaturization to enhance convenience and increase the number of measurement points. For automatic operation, they recommended referencing laboratory autosamplers. They advised against focusing too much on cost reduction, as losing the price competition could be detrimental. Instead, pursuing technical superiority is advisable. Current detection methods for ammonia and nitrate include simple detection reagents and electrode sensors for on-site use, and ion chromatography for precise analysis. A simple yet accurate sensor that bridges this gap could find demand. Looking ahead, there is potential to leverage biosensor characteristics to continuously measure difficult individual ions or extend measurements to plant-derived waste products and bioactive substances that are challenging to measure with physical or chemical sensors.

We incorporated their suggestions into the concept and mechanical improvements of our design in the roadmap for hardware implementation. Their input on market demands, especially from plant factories, served as a reference when exploring the commercial development and applications of our SAMURAI project.