1. Overview

Our ultimate goal is to deploy the device containing the genetically engineered E. coli we have developed into agricultural fields. Over an extended period, we aim to cultivate these E. coli on-site and produce deer odorant to protect crops from deer. However, there is currently no device that fulfills the performance requirements necessary for its realization. This is why we devised the following hardware.

Our device has the basic function of repeatedly oscillating and culturing E. coli without using any electricity or fuel. The only energy source required is the latent heat of water vaporization. With just this energy, our device not only aerates the culture via constant agitation but also continuously supplies device itself with water to continue driving. In addition, we developed a mechanism to enhance oxygen uptake and odorant release by improving the culture tank. This new hardware can be created inexpensively, is easy to handle, and can be left to function autonomously. In other words, this system fully meets the functionality required to realize our projects' goals.

However, this new device's safety must also be a concern. In our dialogues with the affected parties, we received numerous concerns about the safety of cultivating genetically modified E. coli outdoors. Taking these into consideration, we re-evaluated our roadmap to achieve our goals in two stages. For this year, we aimed to complete a device that can be operated in indoor environments such as laboratories and aim to enter testing in controlled field research grounds in the second phase.

Through both modeling and real world testing, we continued to improve our prototype devices. Feedback from potential users was pivotal in making our device sufficiently functional for use in practical environments. Ultimately, through indoor testing, we demonstrated the device's functionality and utility. The current version of our device can efficiently incubate E. coli in indoor settings, utilizing clean energy that emits no CO₂.

Although we haven't progressed to the second phase of the project, we have delineated the plan for the device's social implementation in the latter section of this document. During this planning, we conducted user interviews and tests with farmers and researchers, incorporating their feedback into our proposals. Once the necessary additional features are integrated and rigorous safety tests are performed, this device might be used for deer repulsion in outdoor cultivation.

Lastly, to enable other researchers to use and enhance this device, we've provided comprehensive information to replicate all facets of the device, including prototypes (3D print data, detailed parts, and straightforward assembly instructions). Groups interested in cultivating cultures for various purposes, not limited to producing deer-repelling molecules, with zero CO₂ emissions are welcome to use this information.

2. Requirement Definition

The ultimate goal of our project is to deploy devices containing engineered deer-repellent E. coli around fields or plant conservation areas and protect them from deer. Such a device should fulfill the following requirements:

1. An agitation mechanism (for culturing E. coli )

2. Low cost (to ensure sufficient quantity)

3. Long-term autonomous operation (to reduce user burden)

In addition, we also considered the following as necessary conditions:

4. Efficient release of odor substances

5. Operate without the use of any electricity or fossil fuels.

6. Compliance with SDGs

3. Conceptualization and Design

Fig.1 Schematic diagram of our device

Left: The water supply system set on a table
Right: The drive system with two culture tanks perpendicularly attached

Our device has three major parts:

The drive system agitates the E. coli culture using a constant bobbing motion which is driven only by the heat of vaporization of water.

The water supply system continuously provides a stable supply of water to the drive system.

The dedicated culture tank amplifies the stirring effect of the oscillation and facilitates gas exchange between the interior and exterior of the device.

3-1. Driving system

Perhaps the most challenging aspect was meeting both requirement 1. and 5. due to the necessity to avoid using electricity. Initially, we considered using the water flow of a river as a power source. However, this would limit where we could deploy our device. After researching alternative ways to utilize water as a power source, we came across a traditional toy called the Drinking Bird (DB).

DB has a simple structure of two hollow glass spheres connected by a thin glass tube and performs a continuous pendulum motion using only the heat of water [3].

Principle

When the glass sphere covered with a sponge at the head gets wet, the internal dichloromethane vapor contracts due to the release of the heat of vaporization, and the internal dichloromethane liquid phase level rises through the glass tube. The center of gravity of the tube shifts upwards, and DB rotates by several tens of degrees due to the generated moment, and “drinks water” (right photo). At this point, vapor from the lower glass sphere enters the head through the glass tube, and the liquid phase level and center of gravity are reset. At this time, a secondary pendulum motion occurs. This operation is repeated until there is no drinking water.

Using this mechanism, we designed a device that can keep agitating E. coli culture without using electricity or fuel.

Fig.2 Structre of drinking bird

Here is a video of the DB in action.

Fig.3 Prototype with 15 mL culture tubes as substitute culture tanks

As shown in the figure above, we redesigned the shaft and base parts that support the DB and made a small prototype.

In this model, the cylindrical culture tanks are attached to the shaft, and the culture fluid is stirred as the DB moves. The culture tank can be easily attached and detached via the connecting part protruding from the shaft. The amount of culture fluid and the ratio of the gas phase in the culture tank can be freely adjusted by adjusting the cylinder's dimensions.

We considered installing these cylinders parallel to the main glass tube to maximize the stirring effect, but this positioning may cause the contents of the culture tank to be transmitted to the DB's main body, disturbing its behavior. Thus, we decided to install the cylinder so that its central axis is perpendicular to the main glass tube of the DB.

3-2. Water supply system

A consistent water supply is necessary for the long-term operation of the DB. This is because wetting the head with water from the wet beak during the “drinking action” supports the generation of latent heat. However, if the water level in the cup decreases due to evaporation in a short period, the DB's water supply will be cut off. Inspired by automatic water dispensers used for pets, we designed a water supply device that provides a “cup” filled with water at a constant water level while suppressing evaporation.

Fig.4 3D model of the water supply system

Fig.5 Mechanism of our water supply system

The rectangular tray on the right side of the device is where the DB's beak will “drink” from.

Our water supply device is designed to be able to directly connect to a common 500 mL PET bottle. The water in the bottle flows down into the tray by gravity. When the tray is full, the gap that allows air to enter the bottle is blocked, and the negative pressure from the remaining air in the bottle stops the water from flowing out. When the water level decreases due to the movement of the DB, the air hole opens and water is replenished again. By increasing the capacity of the bottle, water can be supplied for even longer.

In this water supply device, water is lost from evaporation from the tray, and from the water that remains in the water tank below the air gap. To minimize these effects, we designed the opening area of the tray to be as small as possible without hindering the drinking action of the DB. Additionally, we designed the interior of the device to minimize the amount of stagnant water at the bottom.

3-3. Culture tank

As the DB is powered only by energy from water evaporation, but its rotational movement is still limited compared to a typical incubator.

To compensate for this reduction in efficiency, it is necessary to maximize the stirring effect. It is also needed to rapidly release the odorants produced by the culture.

We achieved this by strategically placing a stirring plate and air holes in the cylinder, as in Fig.8.

Fig.6 3D model of culture tank

Fig.7 Cross-sectional view

Fig.8 Mechanism of action of our culture tanks

As shown in Fig.8, when the culture tank tilts to the left due to the movement of the DB (center figure), the culture fluid moves from right to left through the lower side of the stirring plate. The water flow passing through the lower part of the stirring plate stirs the culture fluid.

Simultaneously, external air flows into the space where the culture fluid has flowed out and provides oxygen (right figure). This is expected to have the effect of promoting the growth of E. coli, suppressing acetic acid production by anaerobic respiration, and keeping the medium in good condition [1][2].

To prevent contamination of the medium from the incoming air, it is recommended to attach a filter to the air hole of the culture device.

We anticipate that this mechanism can efficiently culture E. coli and efficiently release volatile components into the outside air.

4. Demonstration of Device Functionality and Practicality

Here is a video of the working device.

We confirmed that the assembled device works as expected. We conducted detailed experiments and modeling on each element and demonstrated their functionality and practicality.

We demonstrated the functionality of being able to agitate culture tanks of appropriate size without any problem via a working prototype of the drive system. We also modeled the mechanisms of how it works.

(2) Water supply system
We demonstrated the functionality and practicality of being able to operate the drive system for about 30 days by using a common 500ml PET bottle as a water tank with the prototype.

(3) Culture tank
We showed by experiment that a clear amplification of the stirring effect occurs compared to a common tube. We also observed by experiment the functionality of significantly promoting the release of volatile substances inside.

Lastly, we demonstrated through actual cultivation that the oscillation effect generated by this device (which has not yet been fully maximized) is sufficient to promote the growth of E. coli. From the above results, we concluded that this device has the functionality of culturing E. coli outdoors at the necessary time and releasing odor substances.

4-1. Driving system

Demonstration of driving function

We created a prototype of the drive system that fits the size of a commercially available DB. The prototype maintains the almost same mobility as the commercial product. This prototype also has a connection part that makes it easy to exchange various types of culture tanks.

We attached 15 mL tubes simulating the culture tanks to this machine and observed the drinking water movement while changing the weight loaded. We filmed a video, observed the approximate total rotation degree in 1 minute, and evaluated its mobility.

Fig.9 Mobility testing of the device

Water evaporation was also measured at the same time. This experiment was conducted at a temperature of around 25~27°C and a humidity of around 40~60%.

Based on the results above, we concluded that the DB-based drive unit has practical-level functionality to oscillate cultures of sufficient size.

These data, along with some additional data observed and measured from the DB, were used as support for creating a Model for how the behavior of the DB changes with the payload weight (See Model Part 6 for details).

This modeling allowed us to derive the maximum culture size that DB can load and how the behavior changes when carrying cultures of any size. Using this modeling, we can effectively design and optimize the size of the device in the future.

Temperature sensitivity of device operation

All the experiments discussed thus far have the DB deployed at room temperature. However, the temperature outdoors is less controlled than indoors. Because vaporization of water is less likely to occur at lower temperatures, we were interested in knowing if our device could still effectively function at lower temperatures. The video below shows footage of our device continuously operating overnight outdoors at a temperature of around 17°C.

Here is a video of the experiment of running DB overnight.

The DB continued to operate throughout the night. In this video, water-filled 15 mL tubes were used instead of E. coli culture tanks.

Through this, we can say that our device is not significantly affected by temperature, and can at least operate during spring to autumn.

4-2. Water supply system

Measurement of DB's water consumption

Before measuring the water consumption of the water supply system, we measured the amount of water used by the DB for its motion.

During the DB's drive tests, we measured the weight of water lost from both the drinking cup (where the DB consumed water) and the control cup. We calculated the DB's 24-hour water usage for shaking cargoes of different weights.

The water usage of the DB was found to be approximately 12 g / 24 hours, regardless of the loaded item's weight. This suggests that the evaporation rate of water is the limiting step for water consumption.

Functionality and practicality demonstration

We produced a water supply system and confirmed its functionality. The drinking spout was filled to the expected water level, and when the water level in the drinking spout decreased, the tank supplied water to restore it to the original level.

Fig.10 Water-supplying device

Fig.11 Drinking spout

The two holes in the tray are for air intake (top) and water refills (bottom).

Here is a video of the confirmation that the water server is functioning properly.

We also measured the rate of water evaporation, which was approximately 2.26 mL per 24 hours at a temperature of 30°C and a humidity of about 50%. Additionally, from the structure of the device, 6 mL of stagnant water remained inside the tank. This means 500ml water can operate the DB for up to one month, successfully demonstrating our device's low level of necessary maintenance.

4-3. Culture tank

Demonstration of volatility effect based on design of culture tank

We created a culture chamber and control without a stirring plate. Both had a volume of approximately 180 mL.

The ethanol evaporation rate was compared by placing about 70 g of 70% ethanol in the center of the seesaw at 3.75 rpm and a maximum incline angle of 10° in a temperature of around 25~27°C and a humidity of around 40~60%.

The weight loss was measured to compare the evaporation rate.

Fig.12 The designs of the experimental culture chambers without the stirring plate (left) and with the stirring plate (right)

Fig.13 EtOH level before assay

Fig.14 Assaying

Fig.15 After assay

This experiment revealed that our culture tanks can efficiently release volatile material and that the stirring plate facilitated efficient oxygen supply to the culture medium due to the rapid gas exchange between the interior and exterior.

Demonstration of stirring effect based on culture tank shape

We assessed the stirring effect of the culture chamber. Because of biosafety concerns, this was not conducted by measuring the growth of E. coli but only visualized using colored water.

We dropped colored water into a DB with the newly designed culture tank and another into one with a simple cylindrical control tank. These tanks were then shaken in the center of the seesaw to visualize and observe the stirring effect. The following video shows the results of that experiment.

Here is a video of the effect of dividers on diffusion of culture medium.

Our observation showed that the culture tanks, with their limited oscillation, provided a significantly stronger stirring effect compared to the simple cylindrical control chamber.

4-4. Actual cultivation of E. coli

From prior research, it is verified that oscillation of the culture plays a significant role in the cultivation of bacteria, and the ample supply of oxygen is also suggested to contribute significantly [6].

Our device possesses both of these functionalities. Among them, we conducted verification to demonstrate the practicality of the oscillating function in our device.

Verification of cultivation enhancement by oscillation

First, we investigated whether gentle oscillation provided by the DB is sufficient to promote the growth of E. coli. In place of the actual DB, a seesaw set to a similar rate of oscillation to the DB was used (60 vibrations / min(3.75rpm), 37°C, no fixation) and measured against a negative control (37°C, no agitation).

We placed 3 mL of culture medium into 15 mL tubes and then measured the OD600 of the cultures over four hours. We then recorded the average of three tubes. The following table shows the results of this experiment.

From this, we can conclude that even relatively gentle oscillation was sufficient to significantly promote the growth of E. coli.

Cultivation experiment with our driving system

We conducted a cultivation experiment using the actual prototype of the device to verify whether the device's oscillation can support E. coli growth.

For this purpose, we prepared two 15 mL tubes with and without stirring plates and installed them into two devices. We also prepared two stationary 15 mL tubes as controls. All tubes contained LB medium with kanamycin, and the OD600 was measured after 6 hours. We recorded the average of two tubes.

Here is a video of E. coli culture test with DB device.

From above, we can see that both the oscillated samples exhibited better growth compared to those that were not oscillated, confirming that the device's motion promotes E. coli growth effectively. (The dimensions of the agitation plate did not precisely match the 15 mL tubes, and the tubes were too small (which increases the influence of the viscosity and surface tension of the culture fluid), so it is likely that only a portion of the expected amplification of the stirring effect was obtained.)

Previous studies have demonstrated that E. coli can grow within the average outdoor temperature range during the temperate spring to autumn periods [5].

From these experiments, we have demonstrated that our device can cultivate E. coli outdoors during the crop-growing season and produce and release repellent substances.

5. Roadmap to Social Implementation and Engagement with Users

In this section, we briefly describe the discussions we had with stakeholders, including potential users, and how they greatly improved our plan and device design. The details of the discussions with stakeholders are described on Human Practices page.

5-1. Initial plan

Our initial plan was to implement this device in a real agricultural field setting this year. However, after receiving feedback as below and the consideration of safety, we found that many challenges were difficult to solve in a short period. Therefore, we revisited our project roadmap and divided it into two phases.

In the first phase to be implemented this year, we aimed to create and implement a device that can be operated in the laboratory. In the second phase, the goal would be to create and implement a device that can be deployed in the fields.

Interview 1

We introduced our initial plan at Kyoto University Academic Day, an event organized by Kyoto University aimed to connect the general public with researchers, and received feedback from various listeners. These listeners were not only the targeted end users but also ordinary citizens who would be greatly affected by the device as consumers of agricultural products.

The opinions we received included the following:

• Concerns about leakage of genetically modified E. coli due to mischief by children, etc

• Risks to food safety due to deploying genetically modified E. coli in agricultural production sites

• Concerns about contamination of culture tanks

In response to these, we revised our roadmap. We also devised a solution strategy for these challenges and summarized them as below.

5-2. Implementation in the laboratory

We made an implementation plan in the laboratory and conducted user interviews and user tests using the prototype. We made improvements based on the feedback. We also received suggestions for other uses besides culturing E. coli.

Implementation plan in the laboratory

Install the current device in the laboratory and use it as an oscillating culture device. (Scale up the device as needed.)

This device provides an excellent oscillating culture method from the perspective of SDGs (see below).

User test & interview 2

We conducted a user test & interview using the prototype with Assistant Professor Makoto Kitabatake of the Institute for Life and Medical Science, Kyoto University. The opinions we obtained were as follows:

• There is an advantage in being able to continue culturing even during power outages

• Aside from culturing, this device could also be used in other methods such as Rotator, nickel bead binding, Western blotting antibody binding, etc

• It is considered to have great significance for experimental equipment manufacturers who consider SDGs

• It would be better to have variation in what containers can be attached to the device

In response to this, attachments that allow various tubes to be attached to the device were additionally designed.

Fig.16-1 Designed connectors for various lab containers

From left to right, eppendorf tubes x 6, a 50 mL tube, and a 15 mL tube.

Fig.16-2 A DB with a 15 mL tube attached on one side and six eppendorf tubes on the other

Fig.16-3 How to use the attachment

Downloadable for 3D printing from 8. Materials for Replication.

5-3. Toward implementation in agricultural fields

We have outlined implementation plans for agricultural fields and a societal implementation roadmap. Furthermore, we conducted user interviews and tests using prototype devices.

We have gained assurance that our device is indeed user-friendly and affordable as per our intended design. We received feedback and made adjustments to our implementation plans and societal implementation roadmap.

With the help of our targeted end users, we have already initiated the initial stages of the societal implementation roadmap.

Implementation in agricultural fields

Our device features a user interface tailored for implementation in agricultural fields with the following benefits

• It operates without the need for an electricity source or fuel

• The culture tanks are easily attachable and detachable with simple connectors, and they function until our "long-lasting" E. coli bacteria deplete the nutrients in the culture medium

• Refilling the water in the water supply system and replacing the culture tank is required only approximately once a month

• The device is cost-effective.

The current version uses expensive commercial DB and 3D printed components, but the material cost for the water supply system and drive system is $7.7, and the culture chamber (180 mL) costs $1.1. Mass productions are expected to further decrease these costs.

Before this device can be deemed safe to use outdoors, "Physical Issues" and "Biological Safety Concerns," (See 6. Discussion).

We will conduct improvements and safety tests carefully. We designed a roadmap for future implementation with the finalized device.

Roadmap

Before implementing the device in agricultural fields, we plan to establish an NPO with the support of national and local governments to address deer damage in agricultural fields. This device will serve as the cornerstone of this initiative.

We will begin trials in neighboring plant protection areas such as the Mizoro Lake Plant Protection Zone, and collaborate with local researchers to gather feedback and performance data.

Based on our results, we will establish partnerships with Japanese Agricultural Cooperatives in Kyoto. We will raise awareness among consumers regarding the safety and effectiveness of the device while conducting pilot trials on some farms.

Upon successful results, we will expand the implementation, create project manuals, and promote the device's adoption in regions beyond Kyoto.

The NPO will manufacture the device and culture tanks with E. coli, providing them affordably to farmers. With common tools and a facility for E. coli subculture, the NPO will be able to operate.

User test & interview 3

We conducted a user test and interview with Mr. Tsutsumi, a farmer and end-user, via Zoom. The following feedback was received:

• An initial cost of around $15 is considered to be cost-effective( Our estimate is even more budget-friendly)

• Maintaining the device about once a month will not be troublesome

• Concerns about the water supply system getting clogged by algae or pollen and other debris were raised

• In addition to government support, there are also research and development grants available through industry-academia partnerships. Utilizing these grants during the development phase would be a beneficial starting point.

Taking this feedback into consideration, we plan to add protective covers and cages to the water supply system. We will pursue the acquisition of grants and have already made inquiries with the Kyoto Prefecture department through Mr. Tsutsumi. In other words, we have already initiated the roadmap for implementation!

Moving forward, we plan to continue to collaborate with Mr. Tsutsumi and other users to work toward the implementation of the roadmap.

6. Discussion

In this section, we will discuss the current limitations of our hardware and possible solutions. We will focus on the eco-friendly design of our hardware, and we propose several applications for it in a context beyond our intended use.

6-1. Scaling up the device

From Model Part 6 and experiments, the maximum weight of culture that can be agitated with commercial DBs is approximately 650g. This is because the friction between the drive shaft and the base increases with increasing weight.

However, to achieve a sufficient oscillation effect that promotes the growth of E. coli, it is possible that a smaller culture may be more suitable.

Also, in implementation, there may be a need to oscillate larger cultures to produce a sufficient amount of odorant. (See Model Part 6)

To address this, scaling up the drive section is a potential solution. This would increase the moment during operation and allow it to overcome friction. Considerations regarding scaling up the device are as follows.

(Additional experiments confirmed the weight-carrying capacity of the culture tank using a commercial DB. While a 143g culture could be agitated, a 245g one could not, likely due to distortions in components like the driving unit's shaft and imbalances in weight distribution. These results showcase the ability of commercial DBs to stir substantial cultures. However, scaling up is crucial for practical use and meeting the required agitation weight (See Model Part 6), emphasizing the need to reinforce component rigidity for scaling up.)

Does behavior change through scaling up?

• As indicated in Model Part 6, when simple geometric scaling is performed, the mobility of the DB tends to decrease

• During geometric scaling, the overall volume and weight increase by a factor of three cubed, while the surface area increases by a factor of two squared. Consequently, the ratio of surface area to volume for the head decreases, leading to a reduction in cooling efficiency, which results in slower operation.

• It was considered that increasing the size ratio of the head and enlarging the surface area through modifications to the head's shape could serve as a solution. In the pursuit of productivity, an example of a head shape with a significant surface area was devised, ensuring that the surface area ratio does not decrease during scaling up, as illustrated in Model Part 6. This significantly improved the mobility of the DB. (Note: Due to restrictions on the use of dichloromethane, physical tests could not be conducted. Furthermore, the actual wetted area and changes in evaporation efficiency due to changes in head shape have not been taken into account in this study.)

Moreover, the following improvements are also conceivable.

• Reducing the thickness ratio of the glass tube could allow dichloromethane to efficiently reach the head, resulting in an expected increase in the moment per unit vaporization heat that can be extracted

• Glass has a low thermal conductivity. Changing the material of the head to aluminum or similar material is expected to enhance the cooling efficiency even with the same heat of vaporization.

• Although limited to daylight hours, painting the DB's body black to increase its warmth and facilitate the upward movement of dichloromethane from below is also a possible approach

Based on the discussions above and the outcomes derived from the Modeling based on the actual behavior of the device, it is suggested that scaling up is achievable through simple changes in design and materials while maintaining mobility.

Can it be produced? Can the price be maintained?

Scaling up plastic components is straightforward. The price can be further reduced by using an injection molding or blow molding instead of 3D printing.

The current toy DB's chambers are made of glass, but DBs made to agitate E. coli, do not need to be made of glass. Instead, a sturdy material with efficient heat conduction such as aluminum can be used.

Of course, developing the manufacturing process in large-scale facilities will also contribute to cost-effective production.

6-2. Obstacles to implementing the device in agricultural fields and solutions

Physical issues

• Damage or function lost due to strong winds (resulting in functional failure and the release of bacteria and dichloromethane)

• Blockage of the water supply system and drive components malfunction due to debris or external factors

• Contamination/leaching from damaged parts(Release of dichloromethane, plastic particles, and so on)

• Risk of breakage from the rain

• Drying out of the cultivation medium

Biological safety issues

• Accidental leakage of E. coli from the culture tanks

• Health risks and public perception issues associated with genetically modified E. coli

• Contamination of the cultivation medium

Solutions to physical issues

• Reevaluate the device materials (use high-strength materials such as FRP or biodegradable materials like polylactic acid as needed)

• Design and implement covers and cages to protect the device from debris and rain

• Optimize the volatilization rate through air hole dimensions and filters

• Conduct careful durability testing

Solutions to biological safety issues

• Incorporate a kill switch in the E. coli to ensure they cannot survive outside the cultivation chamber

• Filters in the air holes of the cultivation tank

• Add antibiotics to the cultivation medium

• Establish non-harmful E. coli strains

• Conduct thorough safety testing

• Use this information to educate consumers

The former set of solutions is considered to have low technological novelty and can be achieved relatively early. The latter, dealing with E. coli, is a long-term endeavor and requires patience. Since these processes are common for cultivating genetically modified E. coli outdoors, we will collaborate with groups developing various E. coli devices.

6-3. Is it possible to add a heating function?

In cold regions or during the winter, having a heating function would undeniably be effective for cultivating E. coli. Is it feasible to incorporate a heating function into this device without compromising its strengths? Dr. Uechi, whom we interviewed, has been researching a mechanism that uses DB movement to generate electricity.

Covering the cultivation chamber with expanded polystyrene foam to trap the heat inside is expected to be easily feasible. In the future, it is believed that this device could evolve into a "complete" incubator with a heating function without the need for electricity or fuel, all while preserving its existing capabilities.

6-4. On-site production of odorants: pros and cons

If the goal is to repel deer using odorants, it might seem logical to just place odorant products produced at facilities near the fields. However, after comparing the advantages and disadvantages, we believe that on-site odorant production with E. coli is a superior approach for resolving harm by deer.

• Risk of E. coli or dichloromethane leakage

• Risk of public perception issues related to the use of genetically modified organisms

• Increased complexity of the device

• No need for large-scale factories to produce and refine odorants. Simple production facilities can provide a stable supply of devices. This allows for wide adoption of the device.

• In the future, by utilizing the biological clock of E. coli, odorant production can be concentrated during specific time intervals, or the odors can be cyclically altered for more effective deterrence.

• With potential improvements to the "long-lasting" system and co-cultivation with algae, it is possible to develop a system that uses nutrients more efficiently over a longer period.

• The project can be applied to various outdoor E. coli cultivation projects beyond odorant production, such as bioremediation and environmental sensing.

6-5. Comparison to conventional commercial products?

When comparing the cost and other aspects of protecting a one-hectare square field, several outstanding advantages were identified in comparison to conventional methods. This serves as evidence that our solution can be considered new and groundbreaking.

Fences are used to physically block the deer's entry into a field. The initial cost for electric fences is around $1,000, while wire mesh fences cost around $4,000. Considering labor, electricity costs, maintenance, and durability, both options require approximately $670 annually. Electric fences come with high electricity costs and the labor of grass cutting. Wire mesh fences are extremely labor-intensive to install. Due to the high cost and labor involved, many farmers cannot afford to adopt these measures. As a substitute, nylon mesh net fences are often used, but the farmers we interviewed have reported that deer often just jump over or destroy them, rendering them ineffective.

Despite the many downsides, this method has a certain level of reliability in terms of its defensive effect, and is the the most commonly used.

Wolfpee is a brand of chemical repellent. Dispensers for wolfpee deployment are inexpensive, but the chemical itself is expensive, with a yearly stock costing approximately $2,500 to $4,200.(This is the cost when it is used throughout the year.) Since it is made from wolf urine, it has a strong deterrent effect, but its mass production is challenging. The liquid needs to be replenished monthly.

Our device Our device, as shown above, is inexpensive, with replacement culture tanks and LB medium being affordable. If the cultured E. coli is capable of exhibiting the intended deterrent effect as assumed in Modeling-6, and if the device can maintain the current prototype's price, the initial cost would be only at $20.5. In addition unlike the other methods, our device needs minimal maintenance to continue working. There is room for future improvements, and it aligns with the SDGs (as discussed below).

6-6. Relevance to the Sustainable Development Goals (SDGs)

Fig.17 TARGET 2・1

Fig.18 TARGET 2・3

Fig.19 TARGET 2・A

The implementation of this device can help reduce the damage caused by deer to crops in Kyoto and around the world. Furthermore, the ability to implement this project without requiring special facilities, electricity, or substantial funding benefits not only affluent farmers but all agricultural practitioners equally.

Fig.20 TARGET 7・2

Fig.21 TARGET 7・3

Fig.22 TARGET 7・A

Fig.23 TARGET 14・3

One notable aspect is that this device operates using the latent heat of water, a clean and untapped energy source. This ensures operation in any location and at any time with consistent temperatures. It also doesn't emit CO₂ or harmful substances throughout its operation phases. Water consumption is minimal, and water quality is not necessary. So it won't compete with potable water supply.

Fig.24 TARGET 8・4

Fig.25 TARGET 12・1

Fig.26 TARGET 12・5

Fig.27 TARGET 15・2

Our device and genetically modified E. coli promote the efficient and environmentally considerate use of resources. Our E. coli can convert a higher percentage of nutrients in the medium into effective odor components through density control and nutrient recycling. The device can potentially be manufactured using reused materials and eco-friendly plastics. In the current version, a reused PET bottle was used as the water tank, and all the 3D-printed parts were manufactured using bio-based plastic resin. The spool resin made from recycled materials, too. The use of this resin also contributes to afforestation efforts by its manufacturer.

7. Conclusion

Our device was designed to address the challenges in realizing our project and fulfill all the requirements. It has the potential for broader applications beyond this project and aligns with SDGs. (2. Requirement Definition, 3. Inspiration & Design, 5. Roadmap for Social Implementation and Engagement with Users, 6. Discussion)

All fundamental functions have worked well. Through experiments and modeling, the practicality and functionality of this device have been demonstrated. (4. Demonstration of Functionality and Practicality)

We have conducted multiple rounds of user tests and interviews. Valuable feedback from users has been incorporated into the device and the roadmap for social implementation. (5. Roadmap for Social Implementation and Engagement with Users)

3D print data, parts lists, and assembly diagrams for all prototypes and parts created in this project have been attached. Other teams can easily replicate and improve upon this device. (8. Materials for Replication)

With the above, we propose this Hardware as a new oscillation device and a promising application for solving the deer problem!

8. Materials for Replication

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All stl. date can be found here.

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References

[1] Luli, G.W., & Strohl, W.R. (1990). Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Applied and Environmental Microbiology, 56(4), 1004-1011. https://doi.org/10.1128/aem.56.4.1004-1011.1990

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