Overview

Throughout the project, we consistently prioritized the goal of "making synthetic biology truly serve society." We integrated science, ethics, regulations, education, and commercial feasibility into a cohesive process using a practical, human-centric approach. We treated Human Practices (HP) not as a side project but as a priority equal to our laboratory work. Every interview, questionnaire, and scenario-based assessment informed our Design-Build-Test-Learn (DBTL) engineering cycle, directly influencing design parameters and implementation paths. It was through this "listen first, design second, and repeatedly verify" model that we transformed our green mosquito repellent solution—centered on biofermented citronellal and engineered controlled release—from a concept into a practical solution for campuses and communities.

The project began with problem identification, focusing on real-world pain points. During summer and autumn, frequent mosquito bites disrupt sleep and hinder academic concentration. Many students find the smell and irritation of traditional sprays and aerosols unpleasant. For outdoor workers, mosquito bites significantly impact productivity. Concurrently, public health concerns about mosquito-borne diseases like dengue fever periodically intensify.

During the "understanding the problem" phase, we consulted experts on three key questions: "Is it truly green and sustainable?" "Will it induce resistance?" and "Is it friendly to sensitive populations?" These discussions helped us translate the concept of "green" from a slogan into measurable and verifiable engineering indicators.

As we moved into the product design phase, insights from Human Practices informed nearly every key decision. To verify feasibility, we transformed HP into multi-dimensional scenario simulations and incorporated "incense core consumables" as a driver for repeat purchases in our financial model. Each conversation led to specific engineering adjustments, moving beyond merely recording feedback to actively iterating on our design.

At the "publicity and education" level, we implemented the other side of HP-public communication-through targeted content and tools.

Our team was founded in December 2024. Our members are high school students from across China who are passionate about synthetic biology. Prior to this, we had no formal laboratory training. Despite this, we have adhered to the iGEM principle of human practices from the beginning. Our activities focused on two key goals:

• Engage with different stakeholders and exchange ideas.
• Reflect on and strengthen our project based on lessons learned.

Looking back at the final phase, from project initiation to the Wiki freeze, integrated Human Practices (iHP) enabled our technical and social approaches to mutually reinforce each other. This allowed us to continuously calibrate our project within the framework of "sustainability, verifiability, and usability." More importantly, iHP taught us to remain humble and transparent amidst uncertainty—keeping precautions upfront, evidence nearby, and the user at the center. This embodies what we understand as the iGEM spirit of human practices: transforming a responsible idea into a truly usable product in the real world.

1. Discover the problem

1.1 We found that mosquito problems are very common and serious for different groups of people.

1.1.1 Interview log

sanitation workers

Yu Haoyang, a college student

Ms. Xu, an outdoor enthusiast

1.1.2 Interview Analysis

Before finalizing our project's direction, we conducted semi-structured interviews with three groups: university students, sanitation workers, and outdoor enthusiasts. These interviewees represented three typical risk scenarios: semi-enclosed indoor environments (dormitories/study rooms), high-exposure public environments (e.g., hot and humid mornings in urban areas), and extended outdoor activities (hiking/camping). The interviews helped us characterize the mosquito problem across four dimensions: frequency, pain points, existing countermeasures, and desired outcomes.

Firstly, the problem's frequency is higher than commonly assumed. Students reported being bitten "almost daily" in dormitories and campus green spaces from late spring through summer. The buzzing and constant swatting during nighttime study sessions cause distraction and sleep deprivation. Sanitation workers are "exposed daily" in the early morning heat and humidity, especially near garbage dumps and stagnant water after rains. Outdoor enthusiasts almost invariably encounter bites near woodlands and water bodies, particularly during early morning and evening hours, with camping near water posing the highest risk. These accounts consistently identify high humidity, proximity to water, and peak activity at night and early morning as key factors, with severity escalating in summer and autumn.

Secondly, the primary pain point extends beyond itching to the systemic impact on learning and work. Students reported that persistent bites affect sleep quality and concentration, especially during exams. Sanitation workers cited decreased efficiency, emotional exhaustion, and occasional secondary infections from numerous bites; some even require sick leave due to mosquito-borne illnesses, creating staffing pressures. Outdoor enthusiasts found their relaxation ruined by the need to frequently reapply repellents. All three groups perceived the combined burden of "disease risk, itching, and compromised efficiency/experience" as significant.

Existing products present a trilemma of "effectiveness, comfort, and safety." Plug-in heated liquids and mosquito coils are effective short-term in dormitories and rest areas but suffer from strong odors, irritant evaporation, and indoor air quality concerns. Sprays and pastes work quickly outdoors but are often short-lived, weakened by sweat and rain, feel sticky, and have a chemical odor. While DEET/picaridin are considered highly effective by outdoor users, concerns about material compatibility (damaging equipment), odor, and irritation hinder broader acceptance. Wearables like wristbands and patches offer convenience but provide inconsistent strength and coverage, proving unreliable in high-pressure environments. This explains why users often combine multiple products yet remain dissatisfied.

Users expressed a clear preference for "green" solutions but were unwilling to sacrifice effectiveness. Students preferred natural, safe options with mild odors for semi-enclosed spaces. Sanitation workers prioritized long-lasting protection (≥6–8 hours), sweat and rain resistance, affordability, and employer provision. Outdoor enthusiasts sought long-lasting protection (8-12 hours), gear-friendly performance, and low odor/irritation.

Therefore, we distilled the problem into four engineering-focused demand criteria:

Form Safety and Usability: Suitable for semi-enclosed and high-exposure scenarios; ideally non-spray, skin-friendly, and material-friendly to reduce indoor air burden and equipment damage.

Timeliness and Release Curve: Features an initial rapid barrier establishment followed by a plateau steady-state release, targeting ≥8–12 hours of protection.

Comfort and Olfactory Experience: Low irritation, low or light fragrance, non-sticky, and suitable for long-term wear during study, work, or camping.

Accessibility and Fairness: Affordable and available. For high-exposure groups like sanitation workers, employer/community supply and subsidies can create synergy between public health and personal protection.

1.2 questionnaire survey on intention to use natural insect repellent products

1.2.1 Results

Question 1: What method do you currently use to repel mosquitoes?

Question 2: Are you concerned about the side effects of chemical mosquito repellents (e.g., irritation, allergies, or unfriendliness to pets)?

Question 3: Compared to chemical sprays, which method of mosquito repellent do you prefer?

Question 4: Have you heard of "citronellal"?

Question 5: If citronellal can effectively repel mosquitoes and is harmless to humans, would you be willing to try it?

Question 6: Are you willing to spend more money on natural, safe, and environmentally friendly insect repellent products?

Question 7: Do you have children or pets, and therefore have higher requirements for the safety of insect repellent products?

Question 8: What do you think is the most important feature of an ideal insect repellent product?

Question 9: Have you ever experienced bedbugs (e.g., in a dormitory, hotel, or home)? If so, what are your current solutions?

Question 10: Regarding citronellal products for bed bugs (e.g., mattress sprays, crevice patches), what are you most concerned about?

Question 11: In what situations do you usually need insect repellent products?

Question 12: What do you think is the reasonable duration of effectiveness of an insect repellent product?

Question 13: If citronellal products are available in multiple forms (spray, sticker, sachet, solid sustained-release tablet), which one would you prefer?

1.2.2 Results Analysis

After visualizing and analyzing data from 99 valid samples, we found that the most commonly used mosquito repellent method is "natural plant-based products," accounting for 67.68% of respondents, significantly higher than chemical sprays (10.10%), mosquito repellent creams/essential oils (11.11%), and wearables (9.09%). Only 2.02% of respondents never use any method at all, indicating that "low-irritation, low-burden" natural solutions are more popular in daily life and in semi-enclosed settings. Further examining attitudes toward the side effects of chemical repellents, 27.27% expressed "great concern" and 44.44% expressed "some concern," totaling 71.71% with varying degrees of concern. This aligns with our focus on "DEET-free, low-VOC, and spray-free" design. When asked to choose between chemical sprays and natural products (such as citronellal), 44.44% clearly preferred chemical sprays, while 28.28% still chose chemical sprays, and 27.27% cited "convenience," demonstrating that effectiveness and convenience remain key trade-offs alongside safety. On the cognitive level, 21.21% said they had "heard of and used" citronellal, 46.46% said they had "heard of it but not used it," and 32.32% said they had "never heard of it." However, on the question, "If it's proven effective and harmless to the human body, would you be willing to try it?" 86.87% were very willing, 11.11% said they would "give it a try," and only 1.01% each said they were "indifferent" or "unwilling." This suggests that "gap in awareness does not equal gap in demand," providing a window into the verification of scientific knowledge and regulations. Regarding willingness to pay, 86.87% said they were "willing (at any price)," 11.11% said "it depends on the price," and only 2.02% were unwilling, suggesting that tiered pricing and refill packs can be used to address these preferences. The constraints on scenarios and people are also obvious: 73.74% "must consider safety" because they have children/pets, 10.10% "hope for gentleness" even without such constraints, and only 16.16% "don't care too much"; in actual usage scenarios, outdoor activities (61.62%) and home (54.55%) are the most prominent, followed by travel (50.51%) and office/dormitory (48.48%), pointing to the dual-line demand of "portable + indoor location". The ranking of desirable product characteristics is as follows: safe and mild (77.78%) > effective (59.60%) > reasonable price (52.53%) ≈ fresh scent (50.51%) > biodegradable (46.46%) > natural ingredients (37.37%). The majority of respondents expressed a desire for a "reasonable duration of effect" of 4–12 hours (46.46%) and 12–24 hours (22.22%), with another 14.14% hoping for more than 24 hours. This aligns with our controlled-release goal of "rapid initial barrier establishment followed by an 8–12-hour plateau phase." Regarding form factor preference, stickers/bracelets (62.63%) and solid sustained-release tablets (60.61%) were the most popular, followed by sprays (57.58%) and sachets (48.48%), providing direct support for our SKU matrix (clothing patches, replaceable fragrance bracelets, solid/gel sustained-release, and a small number of sprays for supplemental use). In addition, for special scenarios such as bed bugs, respondents are most concerned about whether the product can kill adult bugs and eggs (62.63%), how long it lasts for one use (56.57%), and whether the odor is persistent and pungent (53.54%). They are also concerned about furniture corrosion (37.37%) and price (37.37%). At the same time, 21.21% of those who have experienced bed bugs "have not found an effective solution", suggesting that we should be cautious in claiming bedding/crevice products, strengthen the long-lasting and low-odor experience, and supplement material compatibility verification and usage guidelines.

2. Understanding the Problem

2.1 Understand the necessity and urgency of biological control

2.1.1 Interview Records

Interview with Dr. Lü from National University of Defense Technology

2.1.2 Interview Analysis

At its core, green pest control is necessary and urgent because it achieves long-term pest and disease vector management with minimal ecological externalities: it reduces reliance on chemical agents, avoids persistent water and soil pollution and harm to non-target organisms, and enhances system resilience by restoring or leveraging ecological processes (food webs and natural enemy regulation). This approach aligns with public health and agricultural production stability in the context of climate change, and aligns with broader goals such as the UN Sustainable Development Goals. This ensures that green pest control is not a "nice-to-have" supplement but rather a key path forward. For schools and communities, this means integrating "personal protection + breeding ground management + behavioral repellent" measures, prioritizing environmentally friendly and verifiable solutions.

At the promotion level, the government and businesses need to divide the work, collaborate, and work in the same direction. The government's role is to set incentives and boundaries: by restricting high-risk chemicals, encouraging registration and R&D funding for biocontrol products, and conducting public education and cross-regional collaboration, the barriers to entry for green solutions can be lowered and the public value amplified. Businesses, on the other hand, play a role in "transforming scientific research into usable products": investing in the process development and scale-up of technologies such as microbial/natural enemy/plant-based repellents to ensure accessibility and affordability, and building trust through compliant production and third-party verification. A standard combination of "third-party efficacy and safety reports + campus/community demonstration sites" can be used for entry into public settings (schools, properties, camps), combined with policy-friendly labeling and popular science materials to form a closed loop of "evidence + education + supply."

Education and community mobilization are key levers for implementing green pest control. Incorporating ecological pest control and IPM (Integrated Pest Management) concepts into middle school and university curricula and community activities can directly impact the cognitive-decision-making process among young people and their parents. Community-wide workshops, guides, and practical activities (such as clearing stagnant water, habitat management, and the use of insect-repellent plants and physical barriers) can transform "knowledge" into "action." Products can serve as practical vehicles in classrooms and community activities: By clearly explaining "what citronellal is, why controlled release is necessary, and how to use it correctly," coupled with environmental management recommendations and risk communication, they can reduce misuse and improve compliance.

The real obstacles stem from five main areas: public and institutional dependence on chemical repellents and the psychological expectation of "instant and powerful effects"; the stereotype of natural products' "unstable effectiveness"; initial production cost and price pressures; insufficient risk perception and environmental health awareness; and the complexities of green product approval and compliance in different regions. The solution is to prioritize evidence, validate scenarios, and implement a cost-effective roadmap. Standardized efficacy, safety, and stability data are used to align procurement standards with those of schools and communities. Controlled-release engineering is used to improve consistency in efficacy, while fermentation-based scale-up and domestic material production mitigate costs. Clear labeling and educational materials are also used to manage expectations and proper use (e.g., when combined with physical protection and space equipment).

Looking at the spectrum of urban and rural issues, mosquitoes, cockroaches, rodents, and bedbugs are the primary pests in cities, where population density and stagnant water/garbage areas determine exposure to vectors and sanitary pests. In rural areas, agricultural pests, livestock ectoparasites, and wildlife damage are more prominent. Common challenges on both ends of the spectrum are the spatiotemporal reshaping of insecticide resistance and climate change-driven changes in pest distribution. This requires a layered approach based on IPM as the framework, green measures as the first priority, and chemical agents as a "last resort." This approach can be directly integrated into urban personal protection and school/community settings, while in rural and camp settings, it can be coordinated with breeding ground management and physical measures.

Compared with traditional chemical methods, biological control offers unique mechanisms for delaying resistance: stronger targeting, targeting key stages in the pest lifecycle, lower single-target selection pressure, and a diverse combination of cultural, physical, and behavioral interventions. This can transform an "arms race" into a "multi-channel diversion." Adopting a "green, low-selective pressure" approach with non-lethal, behavioral repellents is inherently beneficial for resistance management; the key lies in establishing a stable and verifiable time-concentration curve for the "initial barrier + plateau steady-state release" strategy.

Looking ahead 5-10 years, research and innovation must focus on "stability and consistency," "resistance ecology," "scalable formulation and delivery," and "regulatory science and standardized evaluation." On one end of the spectrum lies the foundations and engineering: maintaining consistent efficacy under varying temperature, humidity, UV, and wind conditions; understanding the co-evolution of pest and control agents; developing more robust microbial and plant-based products; and materials-driven controlled-release and carrier systems. On the other end, the application and governance aspects involve developing comparable and reproducible scenario-based evaluation and labeling standards, and establishing policy-friendly access channels and risk communication paradigms. On the fermentation side, efforts should be focused on cost and purity stability; on the formulation side, on photothermal and antioxidant homeostasis; and on the evaluation side, third-party verification of efficacy and safety in classrooms, dormitories, and campsites should be conducted. Reproducible technology and evidence should be used to truly implement green control measures in society.

2.2 Understanding diseases caused by mosquitoes, flies, and other vectors

2.2.1 Interview Records

Interview with Postdoctoral Fellow Huang from Johns Hopkins University (Biomedical Expert)

2.2.2 Interview Analysis

This interview seamlessly connects the dots between the need, implementation, and verification. Starting with the risk landscape, Dr. Huang emphasized mosquito-borne diseases, represented by dengue fever, Zika, malaria, West Nile, and Japanese encephalitis. These diseases primarily come from urban and semi-urban Aedes mosquitoes, Culex mosquitoes, which remain active during the hot season, and Anopheles mosquitoes, which carry the malaria parasite in specific areas. Flies, primarily houseflies, spread intestinal and eye diseases like dysentery, cholera, and trachoma through mechanical transport and short-term survival in the stomach. In real-life campuses and communities, summer and early autumn, the return to school and military training periods, and garbage and stagnant water around cafeterias and dormitories create high-risk zones in space and time, overlapping with these pathogens. This reality ensures that personal protective equipment is not just a "nice-to-have" measure, but rather a primary line of defense alongside environmental governance.

At the mechanistic level, Dr. Huang reminds us to focus on the critical link of "exposure-infection-pathogenesis." Mosquitoes transmit the virus through a closed loop of "intestinal replication-salivary gland enrichment-saliva injection during the bite." The anticoagulant and immunomodulatory molecules in saliva not only facilitate pathogen entry but also trigger a local inflammatory response. Flies, on the other hand, typically follow a chain of "mouthpart and foot contamination-recontamination by regurgitation or excretion." This means that the correct positioning of over-the-counter personal protective equipment should be to "reduce the probability and dose of bite and contamination exposure," rather than replacing medical treatment. Supporting health education should emphasize early, nonspecific, but critical warning signs: low-grade fever, rash, headache, muscle aches, red eyes, or a cyclical "chills-fever-sweating" pattern that occurs several days after a bite, as well as gastrointestinal discomfort and conjunctival symptoms. If combined with travel history or local epidemics, medical attention should be sought promptly rather than delaying.

Differences in scenarios and seasons necessitate scenario-based product and user guide design. Summer and early autumn are peak seasons for mosquitoes and flies. Waterlogged areas after rain and on sweltering days, air conditioning drain pans, and garbage collection points are the most common breeding grounds. Bites are particularly frequent in the early morning and evening, significantly increasing the risk of outdoor activities near woodlands and water bodies. For campuses and communities, the most important aspect is the continuous chain from "breaks - evening self-study - dormitories - club outings," as well as coordinated environmental management efforts at cafeterias and garbage collection points. It was in this context that Postdoctoral Fellow Huang affirmed our "non-spray, low-irritation, and material-friendly" form factor and suggested developing a portable and shareable solution as a kit: primarily for daily wear, combined with screens, fan turbulence, and electric heating devices as needed to enhance overall protection stability.

The direct implications for product design and validation can be summarized as "stable, comfortable, and verifiable." Regarding effectiveness, the natural monoterpenoid volatilization profile, typically high at the beginning and low at the end, needs to be engineered into a profile with a rapid initial barrier buildup followed by a steady-state plateau release over an 8-12-hour period to meet mainstream expectations for efficacy over a half-day to full-day duration. Furthermore, the product should be low-odor, low-irritant, and fabric- and plastic-friendly, making it suitable for long-term use in semi-enclosed spaces like classrooms and dormitories. Regarding evidence, third-party scenario-based efficacy testing should be mandatory for product introduction into campuses and communities, complemented by data on skin compatibility, inhalation exposure, photothermal and UV-accelerated stability, and material compatibility, ensuring that its "green and effective" claims can be externally verified. Postdoctoral researcher Huang emphasized the importance of risk communication: labeling and education should avoid medicalized language, clearly define the intended purpose of "reducing the chance of bites and lowering the risk of exposure," and clearly outline proper use, additional physical protection measures, and precautions to both prevent misuse and manage expectations.

Regarding social implementation, it is recommended to initiate large-scale promotion through a "small-scale, strong-evidence" pilot program. At the school level, two or three model schools with different climates and management conditions can be selected for simultaneous trials in classrooms, dormitories, and club activities, establishing a closed-loop data system encompassing "supply distribution - compliance tracking - bite complaint rates - adverse event monitoring." At the community level, collaboration should be conducted with neighborhood committees, property management companies, and grassroots health service agencies to include patches and wristband scent cores in summer and autumn emergency supplies, concurrently with environmental governance measures such as "clearing stagnant water, sealing garbage, and strengthening coverage." After each round of pilots, these will be solidified into a reusable "scenario-based usage guide," with publicly available summarized data. This transparent and verifiable evidence will lower the adoption barrier for parents and school authorities.

Looking ahead, climate warming and reshaped rainfall patterns will extend the mosquito season and expand the distribution of mosquito species. This, combined with high temperatures and garbage exposure, will increase the risk of fly breeding and foodborne illness. This compels us to upgrade our strategy from a "seasonal response" to a "semester-based, regularized management": risk warnings, material replenishment, classroom education, and environmental remediation are incorporated into the school calendar and updated on a rolling basis; third-party testing and scenario-based efficacy evaluations are incorporated into annual reviews; and on the supply chain side, high-temperature, dark-proof transportation and peak-season inventory plans are prepared to ensure continuous supply. Only by simultaneously implementing the three pillars of "scientific evidence, product experience, and social governance" can our green mosquito repellent solutions be both responsible and truly effective in the real world.

2.3 Professor Fang suggested that we use citronellal to repel mosquitoes

2.3.1 Interview Records

Interview with Professor Fang from Georgia Institute of Technology

2.3.2 Interview Analysis

To demonstrate the scientific feasibility of a "citronellal-based green mosquito repellent" approach, we systematically organized our interviews with Professor Fang around four key themes: mechanism of action, adaptation/resistance risk, differences from synthetic agents, and key R&D and application considerations. The overall conclusion is that citronellal reduces bite probability by interfering with mosquitoes' olfactory perception. This behavioral repellent approach is highly consistent with the safety and sustainability demands of campuses and communities. However, to ensure reliable effectiveness in real-world scenarios, it must be supplemented with controlled-release formulations, scenario-based validation, and engineered risk communication.

1. Mechanism: From "Smelling You" to "Not Smelling You." Professor Fang pointed out that citronellal can directly affect the olfactory receptor neurons on mosquito antennae and maxillary palps, potentially blocking the binding of host-attracting odorants (such as lactic acid and octenol) to receptors. It can also trigger negative feedback, desensitizing the olfactory circuit, and ultimately weakening the "host-seeking-location-biting" behavioral chain. In other words, it achieves its repellent effect by interrupting key cues in the perception phase, rather than increasing pressure on the killing phase, which is also a key source of its green properties.

2. Adaptation and Expression: Why Controlled Release and Rotation? The interview emphasized two types of "time-dependent" adaptation that require attention: First, olfactory desensitization/sensory adaptation—after sustained or repeated exposure, the response amplitude of related olfactory neurons decreases, and the repellency strength decreases accordingly; second, slow adjustments on the expression side—receptor genes associated with host cues can be downregulated or their response thresholds altered, further reducing sensitivity to human odors.

3. Difference from Synthetic Repellents: Not Utilizing the GABA Pathway. Unlike some synthetic repellents that may cause broad-spectrum neuroinhibition by interfering with insect GABAergic signaling, citronellal's core effect is not "neural inhibition-induced disruption" but rather "distortion/absence of olfactory cues." This explains its relatively favorable safety profile and suggests appropriate management expectations: it reduces the probability and frequency of bites, rather than providing a "drug-like" immediate sedative or killing effect. Labeling and educational materials should clearly state that "perceptual intervention does not equal treatment," and should be coordinated with physical protection and environmental management.

4. Resistance and Metabolism: Minimizing Selection Pressure. Interviews suggest that long-term or single use of any repellent can induce adaptation pathways such as receptor site mutation, upregulation of detoxification enzymes (such as P450), or behavioral avoidance. Therefore, product strategies should reduce "single-channel pressure" at the source: employing a multi-channel combination of behavioral repellency and scenario-based physical approaches, and reducing over-reliance on a single molecule through formulation and release profile design.

5. From Science to Engineering: Transforming "feasible" into "usable and verifiable." For real-world applications, Professor Fang identified key constraints stemming from environmental instability and rapid volatilization: UV rays, high temperatures, and wind accelerate the dissipation of citronellal, resulting in a decrease in sustained efficacy. Scenario-specific efficacy verification (for classrooms/dormitories/commuting/camping locations, etc.) coupled with third-party reports on skin/inhalation safety, photothermal and UV-accelerated stability, and material compatibility will form an "evidence package" for use in schools and communities. Rotating and overlaying usage guidelines will be implemented, combining them with screens, deflector fans, or space equipment during periods of high mosquito pressure and after rain to manage expectations and enhance the user experience. These recommendations are highly consistent with our established path and will serve as milestones for subsequent iHP evaluation and productization.

6. Project Alignment: Green, Steady-State, and Reproducible. Aligning interview key points with the team's roadmap, our three-tiered closed loop of technology, product, and implementation is as follows: On the technical level, we adhere to a bio-fermented citronellal × controlled-release formula to reduce batch-to-batch variability and establish a steady-state release. On the product level, we prioritize clothing patches and replaceable fragrance bracelets, supplemented by low-irritation ointments/solid blocks for use in dormitories and travel locations. On the implementation level, we conduct small-scale, evidence-based pilots on campuses and in the community, focusing on a closed loop of "supply distribution - compliance tracking - bite complaint rate - adverse events." Clear labeling and risk communication (perception intervention, proper use, and integration with physical measures) lower the barrier to adoption. These conclusions and evidence, all derived from Professor Fang's mechanistic explanations and application recommendations, provide solid support for advancing "green mosquito repellent" from concept to a practical social solution .

2.4 Determining the anthelmintic efficacy of citronellal

2.4.1 Interview Records

Interview with Assistant Researcher Pang, an expert in the field of biopesticides

2.4.2 Interview Analysis

Based on the key points of Assistant Researcher Pang’s interview, we have provided a coherent chain of evidence on “whether citronellal is reliable in repelling insects and how to make it into a product that is stable and effective in real scenarios”: First, citronellal has a clear broad-spectrum repellent activity on major mosquito vectors such as Aedes, Anopheles, and Culex, and can also be effective against houseflies, blowflies, fruit flies, and some whiteflies and ticks. Its core mechanism is olfactory intervention - by masking or disrupting host cues such as lactic acid and octenol and modulating the response of olfactory receptor neurons, it weakens the behavioral chain of “seeking the host-locating-biting”, so it reduces the probability and frequency of bites rather than poisoning; secondly, the citronellal content in natural sources (such as citronella, palmarosa, etc.) can reach 30-75%, indicating that “natural "It can be effective immediately", but due to its fast volatility and great influence by ultraviolet light/temperature/wind field, it is difficult to provide long-lasting and reproducible protection in an open environment by relying solely on plant tissues or simple essential oils. The engineering answer must fall on controlled release (microcapsules/gel networks/solid sustained release) and stabilization (antioxidant + anti-UV), and reshape the volatility curve of natural monoterpenes from "high at the beginning and low at the end" to "initial rapid barrier building + 8-12 hours plateau steady state"; again, from the perspective of resistance and safety, because it acts on the behavioral perception link, the risk of resistance at the genetic level is relatively low, but sensory desensitization/behavioral tolerance and metabolic adaptation (such as P450 upregulation) may occur in long-term single use, so compounding and rotation are required (such as with citronellol, geraniol, PMD and other compounds) and superimposed on physical barriers/space equipment in high mosquito pressure windows to reduce the pressure of single-channel selection; for humans and the environment, it is generally well tolerated according to the recommended exposure concentration, and only a few sensitive people may be mildly irritated or sensitized. It is easily degraded by microorganisms in the environment, and has low ecological accumulation and secondary toxicity, which is suitable for the ethical and compliance boundaries of campuses and communities; at the same time, although natural compounds have synergy, the release is unstable, and the pure product has high strength but is more susceptible to oxidation/photolysis. The best solution is to take into account both synergy and spectrum coverage in the compound, and to unify "strength and durability" with a controlled-release carrier and a stabilization system; finally, it is necessary Differentiated usage guidelines were developed: clothing patches and solid/gel slow-release tablets were prioritized in semi-enclosed spaces such as classrooms and dormitories to maintain effective vapor concentrations; replaceable fragrance wristbands were primarily used during school commutes and light outdoor activities; physical protection or space equipment were added after rain, at dusk, and during periods of high mosquito pressure. Third-party reports on scenario-specific efficacy (classroom/dormitory/commuting/camping), skin compatibility and inhalation safety, photothermal/UV-accelerated stability, and material compatibility were provided as a "evidence package" for use in schools and communities. Labeling and educational materials clarified the "behavioral repellent ≠ treatment" boundary and proper usage instructions. Thus, Assistant Researcher Pang's proposal crystallized our three-tier closed loop of technology, product, and implementation: We adhered to bio-fermentation citronellal and controlled-release engineering to ensure the stability of the raw material and release profile; employed compounding and rotation to delay adaptation; anchored our performance around an 8-12 hour plateau period; and utilized scenario-based validation and risk communication to translate the "green and effective" concept into a real-world, replicable solution.

3. Project design , mechanism verification , path optimization and bottleneck breakthrough

3.1 Comparison of different synthesis and production methods of citronellal, and determination of bio-fermentation production

3.1.1 Interview Records

Interview with Dr. Wang from Tsinghua University

3.1.2 Interview Analysis

To design a replicable and scalable route within the engineering framework of project design-mechanism verification-pathway optimization-bottleneck breakthrough, we conducted a systematic review of our interview with Dr. Wang from Tsinghua University, comparing three mainstream methods of citronellal production: chemical synthesis, plant extraction, and microbial fermentation.

The overall conclusion is clear: when weighed across four dimensions-sustainability, scalability, quality consistency, and long-term cost trajectory-microbial fermentation holds the greatest industrial potential and should serve as the baseline technology for this project, with continuous improvement from synthetic biology and process systems engineering.

From a process standpoint:

• Chemical synthesis begins with petrochemical-derived feedstocks, offering high conversion efficiency and instantaneous capacity. However, its high carbon footprint, reliance on hazardous chemicals, and the burden of byproduct disposal create compliance and ESG pressures, and clash with the "green insect repellent" product narrative.
• Plant extraction aligns with the "natural" label but suffers from yield variability (climate/region/season), heavy land and water resource use, and high purification costs due to the complexity of essential oil mixtures, making it unsuitable for long-term stable supply and consistent quality.
• Microbial fermentation, in contrast, uses renewable carbon sources and a controlled reaction environment, enabling stable supply, high-purity output, and replicable scale-up. This approach better fits schools' and communities' expectations of "green, safe, and traceable" public health solutions.

From an industry and regulatory perspective, demand for "bio-based/natural equivalents" is rising in personal care, fragrance, and biopesticide sectors. Meanwhile, regulations are tightening worldwide on carbon emissions, VOCs, and hazardous chemicals. These trends make "fermentation replacing parts of the petrochemical chain" an increasingly predictable direction. Fermentation can also integrate with the circular economy (using molasses, agricultural residues, or food waste as feedstock), offering resilience in supply and better cost stability compared to single-source petrochemical or agricultural chains. Thus, fermentation not only meets compliance but also strengthens market narrative and acceptance.

In terms of engineering implementation, Dr. Wang recommended coordinating across four layers: strain-flux-process-materials.

• Strain level: use synthetic biology to enhance the IPP/DMAPP → GPP → geraniol → citronellal pathway while suppressing side routes and byproduct formation.
• Process level: target a mesophilic aerobic window (pH 5.5-7.0, 25-35 ^oC, DO ≥ 2-30% saturation), prioritizing fed-batch or continuous steady-state operations.
• Materials level: apply dynamic regulation (inducible promoters/biological sensors) to allocate carbon flux across growth and production phases, preventing "premature production" or "overloading."
• Integration strategies: explore co-culture systems (dividing precursor generation and terminal oxidation), and adopt online monitoring/feedback control to minimize drift during scale-up.

For fermentation-specific bottlenecks, the interview provided clear countermeasures:

1. Cytotoxicity and volatility of products and intermediates → use in situ product recovery (two-phase extraction, gas stripping, adsorption) and tolerance engineering.
2. Carbon source costs → replace part of glucose with agricultural residues or second-generation hydrolysates, and use chassis strains tolerant to inhibitors, combined with pre-treatment optimization.
3. Oxygen transfer and heat management at scale → employ efficient gas-liquid distribution, impeller optimization, and digital twin models for predictive scale-up.
4. Stability and consistency → build quality systems with online biosensors, closed-loop control, and statistical process control (SPC) across batches and seasons.

Together, these strategies ensure replicability from laboratory to pilot scale and ultimately to industrial production. Compared with the cost trajectories of chemical synthesis and plant extraction, fermentation may require higher upfront investment in strain construction, process development, and online monitoring. However, unit costs decrease significantly with flux improvement, continuous operations, and feedstock substitution. More importantly, fermentation’s batch-to-batch consistency and verifiable “bio-based” attributes directly translate into brand value and pricing power, creating barriers in procurement for public-sector buyers such as schools and communities.

As such, fermentation emerges as the optimal solution across the technology-market-regulation triad.

Route Decision for This Project:

• Establish microbial fermentation as the baseline process for citronellal production.
• Build the scale-up path through synthetic biology (flux enhancement and dynamic regulation) + process systems engineering (fed-batch/continuous operation, online monitoring, digital twins).
• Define bottleneck breakthroughs in three directions: feasibility of co-culture, low-cost feedstock substitution, and ISPR to increase spatiotemporal productivity.

Milestones include:

1. Lab-scale identification of optimal parameter ranges and dynamic regulation strategies.
2. Pilot-scale validation of > g·L^-1 steady-state productivity and batch-to-batch consistency.
3. Third-party completion of quality/safety/environmental compliance packages.
4. Scenario-based validation in schools/communities, providing real-world evidence of efficacy and stability.

This route not only responds to social expectations of "green, safe, and trustworthy" products, but also provides a clear engineering roadmap for subsequent mechanism verification, pathway optimization, and bottleneck breakthroughs.

3.2 Determining the Optimal Chassis Cell

3.2.1 Interview Records

Interview with Dr. Jin from Tsinghua University

3.2.2 Interview Analysis

Focusing on the question of "Which is more suitable as the chassis for citronellal biosynthesis?" Dr. Jin systematically compared yeast and E. coli based on their biological properties and engineering feasibility, providing a clear rationale for selecting the right approach. First, in terms of development speed and cost, E. coli offers significant advantages, with its faster doubling time (20–30 minutes) and simpler, less expensive culture medium requirements. This allows for a shorter cycle time for the DBTL closed loop of strain construction, phenotypic assessment, and scale-up validation. This is crucial for high-throughput screening of enzyme mutants and rapid validation of throughput optimization. Second, in terms of the maturity and programmability of genetic tools, E. coli possesses the most comprehensive library of standardized components and workflows (inducible expression vectors, CRISPR/Cas editing, leader peptides and secretion pathways, periplasmic folding chaperones, etc.), enabling superior "design-build-test-learn" turnaround efficiency, throughput, and reproducibility compared to yeast. This means that more combinations can be tested in parallel in a shorter time and effective constructs can be rapidly transferred to high-density fermentation conditions. Thirdly, in terms of productivity and scalability, E. coli is more readily capable of reaching extremely high cell densities in fed-batch systems, achieving higher total protein expression and metabolic yields per unit volume. While high cell densities can be prone to issues such as acetate overflow, plasmid burden, and stress responses, these are readily manageable through well-established mitigation pathways (carbon source feeding, coupled DO and pH control) and host-plasmid engineering. Yeast, by contrast, primarily offers advantages in its tolerance to eukaryotic post-translational modifications (such as disulfide bonds and glycosylation) and long-term processes. However, our target pathway (IPP/DMAPP → GPP → geraniol → citronellal) does not rely on complex eukaryotic modifications and presents no inherent barriers to its application in E. coli. Furthermore, E. coli effectively addresses the need for secretory/periplasmic folding through its signal peptides (such as PelB ) and Dsb systems. In terms of compliance and safety, both products operate at the BSL-1 research safety level. Despite public concerns about Gram-negative endotoxins, our products are small molecule metabolites that can be trapped outside the cell, not parenteral biologics, and do not involve the release of live bacteria into the environment. These risks can be controlled within acceptable limits through routine downstream removal and finished product standards. After comprehensively weighing the four dimensions of development speed/cost, tool maturity/programmability, high-density amplification/unit yield, and regulatory compliance, choosing E. coli as the base was the decision that best aligned with engineering rationality and the project schedule .

3.3 Problems encountered in the experiment

3.3.1 Interview Records

Interview with Dr. Wang from Tsinghua University

3.3.2 Interview Analysis

Focusing on the key pain points we encountered in advancing the experiment, Dr. Wang put forward systematic engineering suggestions from six aspects: "consistency of experimental exposure, formulation stability, reliability of molecular construction, complementarity of expression-transcription characterization, yield optimization path, and dry experiment empowerment". These suggestions helped us transform scattered problems into verifiable and replicable solutions, and clearly embed them into the subsequent DBTL cycle.

First, to address the fundamental issue of uneven citronellal odor diffusion affecting behavioral and efficacy testing, it is recommended to establish a "reproducible odor field" through exposure control. On the physical level, this can be achieved by using small wind tunnels/directional airflow, micro fans, or air pumps to create a stable flow field, and by overlaying permeable isolation materials (mesh covers/porous media) to eliminate "hot-spot-cold-spot" concentration gradients. On the supply side, sustained-release pads, diffusion chambers, or solid-phase reservoirs can be used instead of single-shot additions to create a smoother concentration-time curve. If necessary, online/offline sampling (sensor arrays or GC) can be performed at multiple spatial locations to calibrate the exposure distribution, transforming "uniform exposure" into quantifiable and traceable experimental conditions. This modification directly improves the signal-to-noise ratio of the "test" and reduces the sample volume required for replicates and controls.

To ensure formula stability and outdoor usability, the approach is "anti-degradation + controlled release + compatibility." For chemical protection, antioxidant and UV protection systems are prioritized to mitigate degradation and inactivation caused by light, heat, and oxygen. For delivery, microcapsules/gel networks, nanoemulsions, lipid nanocarriers, or mesoporous silica are used to achieve plateau-phase steady-state release. Process optimization utilizes solvent systems and nonionic surfactants to optimize dispersion and spreading, ensuring compatibility with different carrier materials (non-woven fabrics, silicone, TPE, and fabrics). These "Build" improvements at the formula level are ultimately validated in a closed-loop "Test" phase using third-party data on long-lasting efficacy and scenario-specific efficacy (classroom/dormitory/outdoor).

Regarding molecular construction reliability, Dr. Wang noted that common failure points in seamless cloning focus on PCR fragment quality, homology arm length, and vector self-ligation. High-fidelity polymerases and optimized annealing-extension protocols can significantly improve fragment quality; homology arms of 20-40 bp help increase recombination efficiency; and vector dephosphorylation and a high-fidelity recombination system effectively reduce empty vector background. To avoid orientation errors and missing gaps, colony PCR/restriction enzyme digestion rapid screening followed by final sequencing is an essential "test-and-learn" closed-loop approach. Plasmid and primer design emphasizes a holistic approach encompassing "backbone-regulatory-compatibility": host compatibility between the replicon and selection marker, matching the expression strength of the inducible promoter with the RBS, and, if necessary, the addition of a tag or signal peptide to improve detection and folding. Tools such as Primer3 and Benchling are used to pre-check secondary structure, primer dimers, and Tm compatibility to ensure high success rates and reusability in subsequent constructions.

Dr. Wang clarified the complementary relationship between qPCR and Western blotting in characterization systems: Western blotting demonstrates "whether a protein is present/in place," while qPCR explains "why it is present/in place." When mRNA is upregulated but protein expression is weak, this suggests bottlenecks in translation efficiency, folding stability, or degradation pathways; when both are consistently upregulated or consistently low, this points to problems in transcription or further upstream, respectively. This "dual-dimensional readout" allows pinpointing the "phenomenon" to the "link." Furthermore, improving Western blotting success rates requires a comprehensive approach to sample/transfer/antibody integration: sufficient protease inhibition, quantitative sample loading, and transfer verification ( Ponceau S staining). According to the manufacturer's instructions and preliminary experiments, determine the primary antibody incubation time range of 4^oC overnight or room temperature for 1-2 hours, and the secondary antibody incubation time range of 30–60 minutes, and optimize the dilution. For the sole purpose of "proving presence," running an internal control may be omitted, provided strict equal loading and good transfer are maintained. However, when comparing expression across multiple conditions, an internal control becomes essential.

For yield optimization, the core approach is response surface methodology (RSM), which constructs a statistical model based on a "small number of experiments and multiple factor interactions." This prioritizes the quadratic and interaction terms of carbon/nitrogen source, pH, temperature, DO, inoculum size, and induction program. Central composite or Box-Behnken designs are used to generate regression equations and contour plots, directly providing an optimal window and process robustness assessment. The advantage of RSM lies in transforming the "Design-Build-Test-Learn" process into a computable equation, significantly reducing trial-and-error costs and providing a quantitative basis for the "process drift absorption capacity" during scale-up.

To rationally design enzymes to empower dry experiments, we recommend a triple "sequence-structure-dynamics" approach to screening enzyme mutants: Sequence-wise, PSSM/conservation analysis should be used to identify tolerable mutation sites; structurally, homology modeling and energy scoring should be used to assess the effects of mutations on folding stability and the active cavity; and kinetically, docking/molecular dynamics should be used to observe coordinated changes in substrate entry, retention, and product release, prioritizing combinations that simultaneously improve stability and catalytic efficiency. Computational results should be validated on a small scale in parallel, quickly incorporating discrepancies between "prediction and measurement" into the model, accelerating the "learn to next round of design" process.

Ultimately, Dr. Wang translated these suggestions into an executable DBTL timeline: On the "Design" side, exposure control, controlled-release formulations, RSM factors, and dry-test screening parameters were solidified as design inputs; on the "Build" side, standardized cloning-plasmid-primer processes and risk mitigation measures were used to improve first-pass success rates; on the "Test" side, standardized data packages were established based on "odor field consistency → qPCR/WB dual-channel → scenario-based efficacy and stability"; and on the "Learn" side, both failed samples and successful conditions were incorporated into statistical models and rule libraries to form the next round of parameter updates and construction priorities. This framework translated "problems" into "engineering tools" and provided a quantifiable and reproducible roadmap for subsequent mechanism verification, path optimization, and bottleneck breakthroughs.

4. User feedback and product iteration details

4.1 Conduct product trials, use them on campus, collect feedback, and continue to optimize the product

4.1.1 Results

Question 1: Your grade:

Question 2: Frequency of being bitten by mosquitoes or insects on campus:

Question 3: Wearing time of the wristband:

Question 4: Comfort level of wearing the bracelet:

Question 5: Mosquito repellent effect after wearing the bracelet (compared to the unworn phase):

Question 6: Wristband odor perception:

Question 7: What do you think of the bracelet's appearance design:

Question 8: What improvements or additional features would you like the wristband to have?

Question 9: Are you willing to recommend this wristband to classmates or friends?

Question 10: What do you think is the appropriate price range:

4.1.2 Analysis

Drawing on 100 valid responses to the "Feedback on Citronella-Based Insect Repellent Products" questionnaire administered in a school environment, the sample is concentrated in Grades 10-12 (29%, 33%, and 38%, respectively), underscoring that our primary users are adolescents navigating classrooms, corridors, sports fields, and after-school activities; this age distribution, together with the recorded 100 valid entries, anchors our interpretation in a consistent campus context. Exposure to mosquitos is substantively high: 48% report being bitten "very frequently," 42% "occasionally," and only 10% "hardly any," which both validates the problem salience and explains the universal appetite for practical, low-effort protection during school hours. Against this need, the observed wearing pattern clusters around mid-length sessions, with 46% wearing 1-3 hours and 26% wearing 3-6 hours, while 15% wear under 1 hour and 13% exceed 6 hours; these windows suggest that formulation, loading, and diffusion design should emphasize a reliable, perceivable efficacy arc across a 1-6-hour interval, with secondary attention to brief bursts (under 1 hour) and extended activities (over 6 hours). Experience indicators are uniformly and strikingly positive: comfort is rated "very comfortable" by 100% of respondents, scent is unanimously "fresh and natural,"" and aesthetics are universally judged "very nice,"" while perceived efficacy is 100% "significantly reduce mosquito bites," establishing strong face validity for both the material/fit and the functional user experience in this cohort. Even with such high baseline satisfaction, the improvement wishlist points directly to productization levers: 67% want more color/size options, 63% request an adjustable fit, 62% prioritize greater durability (water- and sweat-proofing), and 52% would prefer a more delicate fragrance. Translating these signals into engineering and design action, our next iteration should expand SKU variety and adopt a sizing system that accommodates smaller wrists and growth-related changes, implement adjustable geometries (e.g., micro-ratchet or multi-hole systems) for comfort and stability, and harden the device against sweat, rain, and abrasion through elastomer selection, seal integrity, and coating choices; parallel refinement of scent-either via tuned top notes or controlled-release matrices-can sustain "fresh" perceptions over the dominant 1-6-hour wear block without elevating intensity beyond school-appropriate thresholds.

Commercial viability indicators align with a two-tier pricing architecture anchored in the stated willingness-to-pay: 80% select 10–20 RMB and 20% select 20–30 RMB, with zero responses below 1 RMB or above 30 RMB, which supports a standard model positioned around 19.9 RMB and an enhanced, durability-oriented model priced in the 24.9–29.9 RMB band; bundling (two- or three-packs) could lift average order value and facilitate rotation across activities or terms. Word-of-mouth potential is exceptional, as 100% declare they would “definitely recommend” to peers, implying that if we preserve perceived efficacy and comfort at the target price points, diffusion through class cohorts, clubs, and sports teams should be rapid and self-reinforcing. Methodologically, such unanimous positives may reflect presentation effects, social desirability bias, or the controlled conditions under which the product was demonstrated and evaluated; accordingly, we will expand validation beyond a single school setting to multiple seasons and venues (dormitories, athletic fields, lakeside greenbelts), introduce blinded or controlled comparisons, and pair subjective ratings with objective endpoints (standardized bite counts, third-party observations, repeated-measures tracking across different wear durations). In engineering, we will focus on heat and sweat robustness, release-rate stability within the 1–6-hour window, and packaging that avoids skin occlusion or irritation under movement; in product development, we will prioritize adjustable architectures, water/sweat resistance, and colorway updates mapped to school uniforms and sports attire; and in Human Practices, we will design clear guidance on correct wear, safe use, and disposal, and we will run a structured feedback loop through campus activities to capture longitudinal satisfaction, durability outcomes, and real-world efficacy. Together, these measures convert the survey’s strong demand and satisfaction signals into a concrete, evidence-backed plan for iteration, scale-up, and community education, while preserving the user-centered logic that guided the original concept.

4.2 Provide consultation on product safety to ensure product biosafety

4.2.1 Interview Record

Interview with Assistant Researcher Pang, an expert in the field of biopesticides

4.2.2 Interview Analysis

To ensure that our green mosquito repellent products are both usable and safer on campus and in the community, we conducted in-depth interviews with Assistant Researcher Pang on six key areas: product safety definition, potential risks, key monitoring indicators, use limits for sensitive populations, pre-market safety testing, and safety thresholds and labeling. This led to the iteration of this round of formulation and implementation strategies. The consensus reached during the interviews first clarified the criteria for biopesticide safety: this includes both acute and chronic toxicological assessments for humans and companion animals (skin/eye irritation, sensitization, inhalation exposure, etc.), as well as systematic assessments of non-target organisms (pollinators, beneficial insects, aquatic organisms) and the environment (persistence, bioaccumulation, and degradation pathways), all of which must be corroborated with efficacy. This means that safety is not a single point of data, but a comprehensive package of evidence spanning humans, animals, and the environment.

Regarding the intrinsic properties of citronellal, experts point out that its overall toxicity is lower than that of most traditional chemical pesticides. However, at high concentrations or when used improperly, it may still cause skin/eye mucosal irritation, individual sensitization, and respiratory irritation under poorly ventilated conditions. Accidental ingestion or high-dose exposure by pets may also cause gastrointestinal reactions. In addition, on the environmental side, attention should be paid to acute toxicity to aquatic organisms and potential disturbances to soil microbial communities. Although citronellal is generally easily degradable and not prone to ecological accumulation, local high-concentration emissions or improper disposal still pose risks. Therefore, we have incorporated "reducing peak exposure, controlling release rates, and avoiding entry into water bodies" into the core objectives of engineering and usage guidance.

Regarding key monitoring indicators, interviews suggested quantifying three areas in parallel: first, human and companion animal safety (acute/subchronic toxicology, skin/eye irritation and sensitization, and inhalation exposure); second, environmental and non-target effects (soil/water degradation, bioaccumulation, and toxicity to bees, beneficial insects, and aquatic organisms); and third, product stability (light/heat/humidity stability and shelf life, and the presence of harmful degradation products). This framework is closely coupled with our controlled-release objectives: a stable plateau release not only correlates with efficacy but also directly impacts the safety margin for inhalation and skin contact.

Experts have adopted more cautious guidelines for sensitive populations (infants, pregnant women, and those with respiratory diseases): Any form of direct skin contact is not recommended for infants (especially those under 6 months old); pregnant women and those with asthma or COPD should avoid high-concentration vapor exposure and diffusion in enclosed spaces. If use is necessary, ensure good ventilation and choose a controlled-release form that does not require direct application (such as clothing patches or replaceable fragrance bracelets), strictly adhering to the principle of the lowest effective dose. For this reason, we have clearly included the following requirements on labeling and campus usage guidelines: "Do not apply to bare skin, keep away from the face, nose, and mouth, and maintain ventilation."

Regarding pre-market safety testing and compliance, experts recommend a four-pronged approach for school and community procurement: toxicology, environmental testing, stability testing, and non-target testing. These include completing acute and sub-chronic toxicity, skin and eye irritation and sensitization, and inhalation exposure assessments; conducting soil and water degradation and bioaccumulation testing; verifying accelerated stability and shelf life in light, heat, and humidity; and supplementing with ecotoxicology testing for bees, beneficial insects, and aquatic organisms. Complementary labeling compliance should include: ingredients and content, intended and contraindicated populations, proper use, risk warnings (e.g., avoid contact with water, keep pets and children away from undried product), storage and disposal methods, and instructions for handling irritants. Based on this, we refined our third-party testing plan and labeling language to ensure the scope of the "reduced risk of bites" claim is clear and not overly medical.

Regarding safety thresholds and formulation decisions, interviews suggested referencing NOAEL/LOAEL and acceptable exposure limits (AELs) to determine the upper limit of product internal control. Daily chemical products intended for direct skin contact often limit citronellal to ≤10-15% to reduce the likelihood of irritation and sensitization. Airborne exposure is recommended to be well below the common irritation threshold (≈5 mg/m³ is used in the literature as a reference level for unlikely significant respiratory irritation). In line with our "non-sprayable, non-epidermal" approach, we prioritize release rate per unit time as a core control metric. We utilize a microencapsulation/gel network to flatten the peak release, achieving equivalent safety margin by reducing exposure dose. Furthermore, we employ antioxidant/UV stabilization systems to prevent the formation of irritating degradation products under high-temperature exposure. These thresholds and engineering measures have been incorporated into batch release standards and scenario-based SOPs.

4.3 Legislative Advice and Legal Consultation

4.3.1 Interview records

Interview with Lawyer Wang

4.3.2 Interview Analysis

To truly implement the green mosquito repellent solution of "bio-fermented citronellal x engineered controlled release" on campuses and in the community, we must first clarify the regulatory framework: how the product is classified, who oversees it, what evidence is required before it can be marketed, and how to maintain compliance after it is released. Attorney Wang's professional interpretation provided us with an actionable roadmap for our compliance path.

First, regarding product attributes and regulatory pathways, mosquito repellent/mosquito-killing products containing citronellal are generally categorized as pesticides (biopesticides) in China, requiring pesticide registration and efficacy, safety, and environmental evaluations. However, if the product's form and claims are positioned as personal care/cosmetics (such as skin lotions/sprays), the NMPA will review it under cosmetic regulations, with the data focus on skin compatibility and consumer health. This principle of "use/claim determines regulatory pathway" directly influences the data package and labeling language we will subsequently prepare.

Secondly, the division of responsibilities among regulatory authorities needs to be clarified: the Ministry of Agriculture and Rural Affairs (MARA) leads the registration and standards for pesticides (including biopesticides); the National Medical Products Administration (NMPA) oversees cosmetics and personal care products; the National Health Commission (NHC) focuses on health risks and residue limits; the Ministry of Ecology and Environment (MEE) is responsible for environmental and non-target biosafety; the State Administration for Market Regulation (SAMR) ensures labeling and advertising compliance; and customs/inspection and quarantine are responsible for import and export compliance. Provincial and municipal authorities are responsible for local implementation. Our evidence and processes must align with these regulatory roles to form a closed loop.

Regarding market access and licensing, if commercialization is carried out according to the pesticide route, it is necessary to apply for a pesticide registration certificate from MARA (submitting a full set of information such as active ingredients, toxicology and ecotoxicology, environmental fate, efficacy field/scenario testing, etc.), and the production end must comply with GMP and quality systems; if the cosmetics/personal care route is followed, it is necessary to complete NMPA registration/filing (focusing on skin irritation/sensitization, inhalation exposure, ingredient compliance and labeling), and ensure quality consistency in the production and distribution links; if it involves import and export, the corresponding customs clearance and quarantine permits must also be obtained.

Regarding pre-market biosafety assessments, Attorney Wang recommends packaging the "toxicology-environmental-ecological-efficacy-labeling" evidence package into a standard set of criteria: acute/subchronic toxicity, skin/eye irritation and sensitization, inhalation exposure; ecotoxicity to non-target organisms such as bees, beneficial insects, and aquatic organisms; degradation and bioaccumulation in soil/water; and efficacy verification consistent with the intended use scenario and exposure control methods in wind tunnels/semi-confined spaces. Labels should also include first aid information, dosage/frequency, precautions , contraindications, and disposal methods to ensure consistency across the "evidence-labeling-claim" process.

Regulations specifically address the specific requirements for residential and public use, emphasizing indoor air exposure safety, ventilation conditions, warnings for vulnerable populations, and risk control for non-target organisms and the environment. Aerosol/airborne formulations also have additional air quality and packaging safety specifications. This means our campus-specific formulations must adhere to the principles of non-aerosol, near-body controlled release, no application to bare skin, and away from the face, nose, and mouth. Exposure must be managed using internal control indicators for release rate and total loading.

In terms of mandatory labeling, whether it is "pesticide" or "personal care" route, the active ingredients and content, usage, dosage and frequency, safety warnings and first aid, expiration date and batch number, storage and disposal must be clearly defined; in view of the characteristics of citronellal, precautions and environmental impact tips for children/pregnant women/people with respiratory diseases should also be added (avoid entering water bodies, avoid periods when pollinating insects are active, etc.), and promotional language should be strictly restricted to avoid medicalization or exaggeration.

As for the strategy to promote the inclusion of biopesticides in sustainable control legislation, the path is "evidence + alliance + public": using high-quality toxicological/ecological/cost-benefit data to form policy evidence; establishing alliances with industry associations, environmental protection and agricultural organizations to advocate for the simplification and clarification of biopesticide registration guidelines and use incentives; and transforming "green control" into social consensus through popular science education on campus and in the community, thereby reversely promoting policy improvement.

Finally, Attorney Wang offered practical advice on a compliant launch strategy: determine the regulatory pathway and classification during the initial phase, and reverse engineer the testing and inspection plan; conduct GMP production system and labeling/advertising compliance reviews concurrently with R&D; secure product liability insurance, proactively manage recalls, and manage adverse events; and conduct ongoing post-launch monitoring and annual compliance reviews, dynamically tracking regulatory updates. For us, the most reliable approach is a "dual-track preparation, unified evidence" approach: for school and community B-end procurement, prepare a comprehensive set of efficacy, safety, and environmental reports and school enrollment materials based on the biopesticide pathway; for individual consumer scenarios, simultaneously explore the compliance lines for personal care/non-spray near-body controlled-release cosmetics, ensuring that labeling is consistent with the claimed "risk reduction" strategy. This approach translates the commitment to "green, safe, and effective" into auditable, traceable, and replicable compliance practices.

5. Project implementation, publicity and education

5.1 Confirmation of target market and product formt

5.1.1 Results

Question 1:What is your age group?

Question 2:What is your living environment?

Question 3:When do you need to repel mosquitoes most?

Question 4:How long do you hope the effect of mosquito repellent products will last?

Question 5:What experience do you care most about when using mosquito repellent products?

Question 6:What smell do you prefer for mosquito repellent products?

Question 7:What do you think is the most attractive feature of citronellal products?

Question 8:What potential drawbacks do you worry about with citronellal products?

Question 9:In what form do you most want citronellal mosquito repellent products to appear?

Question 10:Which way of using the product do you prefer?

Question 11:In terms of appearance design, you prefer the product to be:

Question 12:If the packaging of a citronellal product states "safe for pregnant women and infants," would you be more willing to buy it?

Question 13:When purchasing mosquito repellent products, which channel do you prefer?

5.1.2 Analysis

This questionnaire, targeting people with a high insect repellent need, collected 160 valid samples, with a relatively balanced age distribution (16.88% each for those aged 13-18 and over 46, 21.25% for those aged 18-25 and 36-45, and 23.75% for those aged 26-35). Respondents lived in urban apartments/commercial housing (32.5%), suburban/rural areas (25.63%), and school dormitories (25.63%), ensuring the results' explanatory power across the core scenarios of campus and home. In terms of mosquito repellent usage, indoors at home (31.25%) and outdoors (29.38%) accounted for the highest percentages, followed by semi-enclosed spaces such as offices and dormitories (22.5%), suggesting that products need to address both "portable" and "point-of-use" applications.

Regarding effectiveness and core appeal, users' primary expectations for duration of effect are 3-4 hours (57.5%) and more than half a day (27.5%), with 15% still seeking all-day protection. In terms of user experience, "gentle and harmless to the human body" ranked first (36.88%), followed by "strong mosquito repellent" (32.5%) and "easy to use" (18.75%). While "fresh, non-pungent scent," while important, ranked lower (11.88%). This suggests that safety and effectiveness are the primary decision-making factors, while duration and convenience determine repurchase. In terms of scent preference, users prefer floral (31.88%) and lemon (27.5%) scents, while 17.5% clearly prefer unscented options. This supports our dual-line fragrance strategy of "fresh herbal/citrus notes + fragrance-free options."

In terms of perceived value, the most attractive features of citronellal solutions are "safe for humans and pets" (34.38%), "natural plant-based ingredients" (28.13%), and "environmentally friendly and biodegradable" (25.63%). However, the main concerns centered around "lack of safety knowledge" (31.88%) and "high price" (28.75%), with another 21.88% questioning whether the product's effectiveness would be inferior to chemical sprays. This means that before products enter schools and homes, they must address the three key questions of "safety, effectiveness, and affordability" with third-party data and clear labeling. Standardized efficacy and stability testing should be used to align expectations for "strength and duration," while skin/inhalation safety and material compatibility reports should mitigate concerns about vulnerable populations and indoor air quality. Furthermore, refills and multi-tiered pricing should mitigate price sensitivity. Crucially, 100% of respondents expressed a greater willingness to purchase products when the packaging states "safe for pregnant women and infants." However, such claims must be supported by rigorous toxicology and population specificity to avoid overly medical claims.

In terms of form and usage, mosquito repellent bracelets/patches received the highest preference (56.88%), followed by sprays (48.13%) and scented candles/diffusers (47.5%). Furthermore, users preferred refillable/reusable products (53.13%), while instant sprays (31.88%) and disposable patches (15%) served as supplementary options. Considering the aforementioned timeliness and safety requirements, the most suitable SKU matrix is: a wearable bracelet with a replaceable refill as the primary product, clothing patches for both school and study time, and solid/gel slow-release tablets for bedrooms and corners. Sprays are reserved for emergency or outdoor use. In terms of appearance, cute cartoon designs (31.88%) and high-fashion designs (24.38%) each have their own audiences, suggesting the need to differentiate packaging and display strategies for "school and parent-child" products and "gift/travel" products.

In terms of channels and distribution, e-commerce platforms (53.13%) and supermarkets/convenience stores (50.63%) are the preferred purchase channels, followed closely by social commerce/recommendations from acquaintances (43.75%) and cosmetic stores (39.38%). This requires us to adopt a dual-pronged approach of "online conversion + offline engagement": online, we leverage "efficacy/safety evidence + scenario-based selling points" to drive conversions, while offline, we offer user experiences and refill displays in campus supermarkets and outdoor stores. We also focus on educational content during the summer and back-to-school season, tailored to demographic demographics, emphasizing the product's differentiation points of "non-aerosol spray, near-body controlled release, and greater child and pet friendliness." Standardized instructions and precautions are also used to enhance compliance and word-of-mouth. Overall, the survey data clearly anchored our product decision-making in the principles of "safety first, equal emphasis on effectiveness and timeliness, priority for wearables and refills, dual-line fragrance and unscented options, and modular presentation of evidence and compliance." This has helped us translate our SKU, labeling, and channel strategies into actionable plans.

5.2 Business Model Determination

5.2.1 Interview Records

Interview with Mr. Huo, the owner of a company in the field of biology

5.2.2 Interview Analysis

To determine the optimal sales model and channels and build a feasible financial and cost framework, we conducted a systematic interview with Mr. Huo of Dalian Zhiyuan Biotechnology, focusing on topics such as "how to sell, who to sell to first, what evidence to use to drive conversion, and how to maintain clear accounting and mitigate volatility." The core conclusion was "starting with e-commerce, driven by evidence, layered penetration, and dual-track growth": First, leverage content e-commerce and flagship stores to directly engage with consumers and build word-of-mouth, generating authentic repeat purchase and review data. Then, selectively penetrate chain pharmacies and maternity and baby product stores to build offline trust. Simultaneously, during high-incidence mosquito season, pilot B2B initiatives for government, enterprise, and property management companies to secure large-scale orders and stabilize cash flow. This approach not only facilitates A/B testing and demographic matching, but also meets the demand for "safe and trustworthy" channels in campus and community settings.

Channel prioritization is based on the integration of "content ecosystem x professional retail." The outdoor, camping, self-driving, and maternity verticals are highly sensitive to "green, safe, and long-lasting" concepts. Leveraging video presentations of expert reviews and comparative experiments can significantly increase conversions and repeat purchases. Offline, the company connects chain pharmacies with the maternity and infant product ecosystem, addressing the core concerns of "safety and applicable populations" through centralized display and employee education. If a product possesses community public health application value, it can be integrated with property management companies for "Community Mosquito-Free Week" programs, significantly reducing bite complaints and amplifying word-of-mouth during the three-week window. This combination of "building buzz and data online, building trust and penetration offline, and achieving scale through B2B" forms the channel path from 0 to 1, and then to N.

Differentiating strategies based on region and demographic is also crucial. Due to the risk of dengue fever and high humidity in the southern coastal and southwestern regions, the need for family and community-based governance is stronger, making government-owned enterprises and pharmacies more effective. In the north, with its short summer months and light outdoor activities, e-commerce and convenience stores offer greater flexibility. For mothers and infants, emphasis is placed on ingredients and third-party testing; for outdoor users, emphasis is placed on long-lasting efficacy, portability, and waterproofing; and for students, emphasis is placed on cost-effectiveness and group purchasing. Accordingly, small steps of differentiation are being made in formula, specifications, and materials. For example, a "bracelet + replaceable fragrance core" is the core SKU, with patches covering both school and study time, solid/gel sustained-release tablets as a solution for bedroom corners, and sprays reserved for emergency use.

Cost and financial controllability relies on a clear unit economics model. Before launching a new product, it's necessary to break down the following items: raw material and formula BOM, packaging and consumables, manufacturing conversion and yield, quality control and sample retention, registration compliance and third-party testing, channel marginal costs (rebates/platform commissions/display fees), logistics and warehousing, influencer and advertising, after-sales service and return rates, product liability insurance and recall reserves. This requires distinguishing between fixed and variable costs, creating a calculation chain from tax-inclusive gross profit to contribution gross profit, providing a boundary for pricing and investment intensity. Entering the distribution system requires factoring in channel gross profit and entry costs; e-commerce channels use platform commissions, payment processing fees, and advertising expenses as alternatives. National fulfillment requires consideration of initial weight/recurring weight, remote location surcharges, and breakage rates.

Profit and cash flow forecasts utilize a combination of "small-sample real-world data calibration + scenario and sensitivity analysis." Small-batch trial sales are used to obtain real conversion, average order value, and repeat purchase metrics. Mid-term, sensitivity analysis and Monte Carlo simulations are used to assess the impact of fluctuations in advertising costs, raw material prices, and conversion rates on gross profit and cash flow. Long-term, rolling forecasts are developed based on seasonal factors and category baselines. Metrics such as gross profit margin, contribution margin, customer acquisition cost, repeat purchase rate, ROAS, single-store output, inventory turnover and cash turnover days, return and customer complaint rates, and out-of-stock rates are consolidated into the operating dashboard. Regarding risk mitigation, a minimum ROAS and a stop-loss threshold are set, and a flexible budget is maintained. Key raw material pricing ranges are locked in through dual supplier and framework agreements, maintaining a safety stock and alternative packaging materials. Unforeseen expenses and after-sales funds are established, and product liability insurance is purchased to ensure operating expenses cover at least one peak season.

In terms of market acceptance, the perception of "plant-based, green, and safe" continues to strengthen among families and mother-and-baby customers. However, consumers are sensitive to the boundary between "lasting effect" and "repellency vs. killing." Third-party efficacy and stability reports, skin/inhalation safety assessments, and volatility curve data are essential to answer the questions of "is it truly effective, how long does it last, and for whom is it safer?" A tiered pricing strategy can be adopted: high-value, refillable products for schools and families, and co-branded and high-end products for outdoor and gift-giving scenarios. However, this requires demonstrable evidence and clear differentiation. Past successful practices have shown that community projects with property groups and co-branded gift packs with outdoor brands can significantly increase repeat purchases and word-of-mouth, making them worth replicating during peak season.

The proposed implementation schedule is a two-phase approach, with regulatory compliance in place. The first phase involves small-scale e-commerce trial sales to establish product-target-content alignment and build positive buzz. This is followed by offline and B-side pilots to scale up and validate channel sales. The second phase, centered around the two key selling points of "long-term effectiveness and safety," involves preparing a standardized "evidence package" (including scenario-based efficacy, skin irritation/sensitization, inhalation exposure, volatility profile, and stability), which will be incorporated into product page materials and in-store merchandise. The financial model incorporates seasonality, channel rates, and return rates into a three-dimensional sensitivity surface, setting two regulatory compliance lines: gross profit margin and ROAS minimums to ensure expansion without sacrificing unit economics. This approach of "e-commerce validation - offline penetration - government and enterprise amplification - evidence-driven - regulatory compliance" provides an actionable, replicable, and quantifiable commercial implementation plan for our green mosquito repellent products.

5.3 Determine product compliance path and key milestones

5.3.1 Interview Records

Interview with Mr. Huang, a business economics expert

5.3.2 Interview Analysis

The interview centers our Human Practices on one controlling insight: before building a commercialization story, we must precisely classify what our product legally is and align all downstream activities to that category. The mentor emphasizes that every claim, channel, and usage scenario must match the chosen classification; misalignment is a hard stop for both regulators and investors. Practically, this means selecting the correct national product class early, mapping allowed indications and sales venues, and designing our R&D and documentation package to that rule set from day one.

On sequencing, the interview recommends putting regulatory engagement at the very front of our roadmap. Investors may "talk," but real commitments are unlikely without visible progress on approvals; after that, the order of market testing versus investor outreach depends on the investor's profile-some require concrete market tests before proceeding, while others with stronger sector understanding may help enable those tests once they see credible potential. Our HP plan should therefore bookend early regulatory steps with adaptive paths for either "test-first" or "investor-first" partners.

Because uncertainty in oversight can spook stakeholders if mishandled, the mentor urges transparent risk disclosure coupled with a policy-alignment narrative. We should explicitly articulate the current direction of government policy and show that our product advances those priorities; doing so reframes regulatory risk as a managed pathway rather than a binary threat, and increases investor confidence that issues are solvable rather than existential. This policy fit must be stated clearly in our materials and investor conversations.

For our business plan, the guidance is to quantify rigorously and communicate accessibly. Before approaching non-technical investors, we need a proof-backed commercial thesis-market size, why that market is accessible to us, how we mitigate competitive threats, and the evidence behind each claim. Scientific explanations should be accurate but plain-language; the business sections must be notably rigorous, with complete data logic and defensible assumptions. This dual register-"scientifically clear, commercially exacting"-is described as essential for credibility.

Financing strategy is framed around two pivotal milestones we should plan for and communicate explicitly: (1) surviving to first deployable sales or first realized value, and (2) traversing the gap from first sales to true profitability. We need to specify the time required for each phase, how those timelines are funded (equity vs. debt, number of investors), and how we will expand production and capture value in the interim. This "two-bridge" view helps align investor expectations with operational reality.

On customers and revenue logic, the mentor highlights public-health use cases and suggests that, in some regions, government could be the anchor buyer. Profitability will vary sharply by geography due to differences in vector burden and public budgets; therefore, our HP fieldwork should include region-by-region diligence on disease risk and procurement capacity, with our value proposition expressed in metrics that public buyers recognize (e.g., cost per protected person-hour, operational simplicity, risk reduction).

For compliance pathways relevant to biocontrol/biopesticide-like products, the interview outlines a two-track requirement: (i) R&Dexperimental evidence packages that meet the expectations of agricultural and (in some contexts) drug-related agencies, and (ii) commercial category management once the product is sold-each with its own documentation and procedural standards. Our HP deliverables should therefore split evidence into "research qualification" and "market authorization," making it clear which claims and channels are permitted at each stage.

Translating these interview insights into our HP plan, we will: state our intended product classification and the exact claims/channels it permits; schedule an early regulatory workstream with defined artifacts (protocols, exposure assessments, labeled claims) to unlock investor confidence; present our market thesis in quantified, evidence-backed terms; frame our financing around the two milestones of “to first sales” and “to profitability,” including runway and funding structure; and tailor outreach to regional public-health stakeholders with procurement-relevant KPIs. Throughout, we will keep scientific explanations readable while making the commercial case unusually rigorous, as advised.

5.4 Feasibility Study of Citronellal Bioproduction

5.4.1 Interview Records

On-site interviews and research at Bluepha Labs

5.4.2 Interview Analysis

Focusing on the feasibility of industrializing citronellal bioproduction, the Bluepha microbiology expert emphasized a core conclusion: to move from laboratory results to industrial application, both technology-market alignment and process reproducibility must be achieved simultaneously.

First, on the commercial side, it is necessary to build a complementary relationship between technology and brand: biotech companies need to partner with FMCG/personal care companies to match strengths, letting real-world needs drive R&D, and turning "efficacy, safety, and stable supply" into an entry pass. For To-B customers, the top priorities are batch-to-batch stability and traceable quality systems, while To-C customers rely more on brand perception and user habits. The shared underlying logic is "efficacy evidence + stable supply." Lessons learned are also clear: "having the technology" does not equal "market adoption." Early-stage small-batch trial sales and pilot partnerships should be used to refine product definition and supporting evidence.

On the engineering and scale-up side, the expert stressed phased scaling rather than "one step to full production." Key parameters from lab trials (e.g., dissolved oxygen, pH, temperature, feeding rhythm, induction program) should first be standardized, followed by stepwise pilot verification at 50 L → 500 L. Operational SOPs must be written in a way that frontline staff can apply. At the same time, assess hardware and regulatory compatibility (e.g., use of flammable solvents, safety and environmental qualifications) to avoid the mismatch of "technically feasible but not factory-feasible."

After scale-up, packaging, storage, and transport standards (e.g., product specifications, light/temperature/moisture protection, shelf-life validation) must be designed in parallel, ensuring "product-logistics-end-user consistency is incorporated into the quality system.

Continuity and risk control must be pre-set:

• Raw materials: dual suppliers and price-band frameworks.
• Equipment: redundancy for critical processes and on-call maintenance (spare units/24-hour repair response).
• Quality: apply a "minimum set of Critical Quality Attributes (CQA)" to balance cost and release efficiency. The more metrics, the stronger the consistency, but also the higher the testing cost and cycle time. Focus on the few key indicators most relevant to efficacy and user perception to build a system that is "fast to release, auditable."

For competitive disruption and IP risks, the expert recommended establishing continuous patent/intelligence monitoring. Core technologies and industrial strains should be protected through a dual-track approach of patents plus trade secrets, while technical barriers and traceable markers in processes/cultivation systems should prevent misuse.

On finance and market launch, the expert underlined cash flow security as the top red line: hedge raw material fluctuations via strain engineering and low-cost substrate substitution, and diversify revenue through multi-SKU and multi-channel strategies. For customer acquisition and trust, start with e-commerce/content channels for sampling and evidence communication, then expand to pharmacy, maternal/childcare, and outdoor retail. Gain first core clients via expos, joint launches, and grassroots promotions. Before full launch, publish third-party testing and compliance certificates to establish brand credibility and presale momentum, built around "traceable raw materials - stable processes - efficacy & safety evidence."

As for the timing of transitioning from lab to pilot and first mass production, the expert proposed a dual-threshold: (1) lab trials achieve expected yields with clearly defined parameter boundaries; (2) market channels show clear demand signals and validation scenarios. Based on past projects, after years of lab/pilot work, the practical cycle from trial production to first full-scale launch is ~8-9 months. However, this requires high-frequency iterations, intensive work pace (including holidays), and systematic documentation of each problem and solution into dynamic SOPs-ensuring the shift from "can produce" to "stable production, auditable, reproducible."

This interview provides us with an actionable roadmap for the industrialization of citronellal fermentation:

• Use demand to define product and evidence packages.
• Ensure engineering reproducibility via phased scale-up and SOP standardization.
• Safeguard quality and cash flow with multi-source supply and a minimum CQA set.
• Protect the technology moat through IP and intelligence systems.