To ensure our project is both effective and socially responsible, we engaged with various stakeholders through questionnaires, interviews, and site visits. At the outset, we conducted a survey to gauge public awareness of ethanol and synthetic biology. We also held two interviews with experts in synthetic biology and environmental science to deepen our understanding of the project's feasibility, ensuring that our technology is suitable for laboratory experiments and aligned with social development goals, particularly in addressing current environmental challenges and promoting the SDGs. As we progressed with our business plan, we consulted experts in biology and entrepreneurship to assess the feasibility of our product. These interactions significantly enhanced our understanding, allowing us to refine our approach based on the valuable feedback and insights we received.

In the initial phase of our project, we wanted to find out the general public’s knowledge and attitude on second-generation biofuel ethanol to provide insight for publication and education activities later on. Thereby, we designed an anonymous online questionnaire.

We sent out the questionnaire through WeChat Moments and group chats. So far, we have collected a total of 256 samples. More than half of our respondents are students under 18 years of age and many are middle age people between 31 and 50 who were most likely parents of the students.

Fig. 1. Age distribution of all respondents

Fig. 2. Occupation of all respondents

67% of all respondents to our questionnaire said that they have heard of biofuel ethanol. The main ways they learned about biofuel ethanol include public platforms and institutional and personal connections.

Fig. 3. Answers to the question “Have you heard of ethanol biofuel?”

Fig. 4. Answers to the question “Where did you get to know about ethanol biofuel?”

In terms of deeper knowledge of biofuel ethanol, we asked the respondents about their understanding of biofuel ethanol. A large proportion of the respondents considered ethanol biofuel as a fossil fuel substitute and a renewable resource. However, 15% of the respondents thought that biofuel ethanol was a kind of fossil fuel. Misunderstanding of this new type of energy source existed among the public and it is our responsibility to raise awareness in people.

Fig. 5. Answers to the question “In your mind, what is ethanol biofuel?”

Then, we move on to find out the public’s attitude towards using biofuel ethanol in their lives. Nearly half of the respondents said they were willing to choose gasoline that contains biofuel ethanol and almost another half said they were not sure. It is difficult for the general public to make informed decisions about the choice of gasoline given that there is asymmetrical information between the producers and consumers. Therefore, we could inform the public about this new energy source, including both the advantages and disadvantages to help them make a decision best fit for them.

Fig. 6. Answers to the question “Would you choose gasoline that contains ethanol biofuel over conventional gasoline?”

According to our sample, the biggest potential problem of ethanol gasoline that people are worried about are the reduction in the dynamic performance of vehicles and the reduction in the life span of vehicles. When publicizing biofuel ethanol, we need to find out whether ethanol gasoline affects these aspects and address the issues to the public.

Fig. 7. Answers to the question “What do you think is the biggest potential problem if ethanol gasoline replaces conventional gasoline?”

Through our findings, it can be concluded that we still need to raise the public’s awareness of biofuel ethanol and the second generation of biofuel ethanol, and to inform them fully of the new technology, addressing the impacts on the performance of vehicles, so that the demand for ethanol gasoline could potentially increase, creating more business opportunities for the production of ethanol utilizing lignocellulose.

In the early stages of our project, we sought to gain a comprehensive understanding of lignocellulosic conversion technology and its current advancements. To this end, we interviewed Professor Wang Xin, a leading expert in synthetic biology and industrial biotechnology. Professor Wang, currently a lecturer at Henan University of Technology, focuses his research on microbial cell factory construction, particularly in converting agricultural by-products into valuable chemicals.

During the interview, Professor Wang provided an overview of the ethanol production process, emphasizing the high costs and challenges associated with traditional fermentation methods. He highlighted that using synthetic biology to engineer yeast strains could significantly reduce the need for external enzymes, thereby lowering production costs. He also suggested focusing on optimizing the pretreatment of lignocellulosic biomass to maximize the yield of fermentable sugars. Regarding the use of agricultural and forestry waste for ethanol production, Professor Wang noted that while these raw materials are cost-effective, their efficient conversion remains a challenge. He recommended conducting small-scale pilot studies to identify the most effective pretreatment methods before scaling up to industrial production. This approach would help mitigate risks and ensure the feasibility of using such materials in a larger production context.

One of the critical challenges discussed was the conversion of xylose to ethanol. Professor Wang pointed out that the efficiency of this process is crucial for the economic viability of lignocellulosic ethanol production. He advised focusing on the development of yeast strains that can tolerate higher concentrations of xylose and convert it more efficiently. Additionally, he recommended conducting comparative studies on different genetic engineering approaches to identify the most promising strategies for improving strain performance.

Throughout the interview, Professor Wang shared valuable insights into using synthetic biology to optimize yeast metabolism, which will significantly advance our project. Key insights include:

• Strain Optimization: Develop yeast strains that efficiently convert xylose to ethanol to improve economic and environmental viability.
• Cost Reduction: Use synthetic biology to lower enzyme costs and enhance cost-effectiveness.
• Comparative Analysis: Compare genetic engineering strategies to identify the best approaches for improving yeast performance.
• SDGs: Align research with SDGs to maximize positive environmental and social impacts.

In conclusion, the project supports Responsible Consumption and Production (SDG 12) by converting waste into ethanol, thus reducing land use and preserving biodiversity. It contributes to Climate Action (SDG 13) by offering a renewable energy source that cuts greenhouse gas emissions. Innovations in synthetic biology promote Decent Work and Economic Growth (SDG 8) by potentially creating jobs and boosting the industry. Additionally, it helps with Life on Land (SDG 15) by mitigating land use pressures and supporting conservation. These findings will guide us in refining our approach for more sustainable and cost-effective bioethanol production.

Our project aims to replace the use of certain fossil fuels with ethanol from an environmental perspective and policy trends. Still, we lack a comprehensive understanding of the usage, environmental impact, and related policies of fossil fuels, first and second generation ethanol. Therefore, we interviewed Professor Bao Cunkuan, an expert in environmental science.

We have learned that the position of fossil fuels in the energy structure will continue for a period of time and can only be relegated to supplementary energy rather than eliminated. However, in the future, biofuels are still expected to become the main energy source in the order of "standing first and then breaking down." In addition, he pointed out the limitations of ethanol in specific application scenarios, such as photovoltaic power generation, wind power generation, etc. He pointed out that any energy source has its particular limitations. In addition, biomass conversion is not only about energy utilization but also includes the conversion of raw materials.

Regarding environmental impact, agricultural waste and residue can be used as raw materials for lignocellulosic ethanol, achieving sustainable development, reducing waste generation and emissions, and improving efficiency. But compared to the straw we once imagined (because it has many uses), the waste from the production of corn, cotton, and fruits, as well as the pruned branches of trees and shrubs, have better utilization prospects.

Regarding policy incentives, the political environment is favorable for the development of cellulose ethanol, not only because of the dual carbon target but also because of China's current energy cooperation with Middle Eastern countries. Secondly, policy incentives vary by region. For example, the establishment of hydrogen refueling stations may provide greater incentives for the use of hydrogen energy, competing with fuel ethanol.

The interview with Professor Bao Cunkuan has provided us with a new understanding of fuel ethanol's positioning and new insights into product commercialization strategies, such as adapting our product commercialization to local conditions.

As we began to develop our business plan, we encountered some challenges. Fortunately, we consulted with an expert who has a background in both biology and finance.  We discussed this with Dr Wang Qingzhuo, a Ph.D. in synthetic biology from the Chinese Academy of Sciences and the co-founder of Shiqi Biochemicals. He had been within the biochemical industry for years now.

During our discussion, Dr Wang helps point out a lot of the problems within our Business Plan. We are selling the right to use our yeast strains rather than selling strains directly. The companies that are going to buy our strains are fermentation companies themselves, so they would not need to buy our yeast strains repeatedly. He was also able to give a better idea, with us selling the right for use first, and then receiving a commission based on how helpful our yeast strain is. Another core problem is electrically powered cars such as TESLA taking over the car industry, leaving less of the market for alcohol-based fuel, like ethanol, methanol, and butanol. Dr Wang agreed to this insight on the future, and suggested we not only look at the fuel market. There are more uses for ethanol than the fuel industry, and those are the future market for these alcohol-based chemicals. In conclusion, our discussion with Dr Wang made us realize a lot of our mistakes and the way to fix these problems.

For more details about the three interviews mentioned, you can refer to the provided transcript document.

This field trip and interview aimed to enhance our business plan and gain a comprehensive understanding of the synthetic biology industry. We had the opportunity to visit Bluepha, an iGEM-incubated startup that has grown into a leading company in the field of synthetic biology. Bluepha is recognized for its innovative approaches to biomanufacturing and its commitment to sustainable development. During our visit, we engaged in a detailed discussion with one of Bluepha’s R&D experts, Doctor Zong.

During our visit, we first toured Bluepha’s exhibition hall, where we delved into the company's history and core products. This provided us with a comprehensive understanding of Bluepha’s journey, significant milestones, and its integration of sustainability into product development and production processes. We learned about the innovative measures and technologies the company employs to reduce its environmental footprint.

We then moved on to a laboratory tour, where we observed the fermentation processes across various scales, from small pilot projects to large-scale industrial production. This gave us insight into the scale-up process and the different fermentation equipment and control systems used. We also visited the genetic engineering lab, where we explored key technologies and the strain screening platform that supports the production of second-generation biofuel ethanol using Saccharomyces cerevisiae. Finally, we examined how Bluepha leverages automation technologies to enhance efficiency and precision in their R&D processes.

In our discussion with Doctor Zong, we explored the prospects of second-generation biofuel ethanol, focusing on its cost structure, potential applications, and market outlook. This provided us with a deeper understanding of the economic and commercial factors influencing the viability of these biofuels. Additionally, Doctor Zong, drawing on their experience as pioneers in iGEM entrepreneurship, offered valuable advice on refining our business plan, optimizing experiment design, and preparing for iGEM, including insights on model and wiki development.

Also, we explored the feasibility of building a factory for scaling up ethanol production. While our initial research and interviews indicated that implementing this idea in China would be challenging due to government regulations—where permits are typically reserved for large state-owned enterprises—Doctor Zong suggested considering other countries as potential locations for such a venture. This led to further discussion, and we are now contemplating the viability of pursuing this idea internationally.

With our efforts and the sincere responses from the stakeholders to our project, we have fortunately obtained some valuable findings that will help us advance our project process and understanding. From the combined insights of the questionnaire, expert interviews, and company visits, several key conclusions emerge, and our measures would focus on public education, technological refinement, and market diversification.

• Public Awareness and Educational Needs: Public Awareness and Educational Needs: We will prioritize educational campaigns to address knowledge gaps and concerns about ethanol use, focusing on vehicle performance and environmental benefits. (Questionnaire survey) For more information, please refer to our education page.
• Technological Innovation and Production Challenges: The primary effort should be on improving yeast strains for xylose conversion and reducing production costs through synthetic biology. Small-scale trials are crucial for refining these methods before scaling up. (Interview with Wang Xin, Bluepha visit)
• Environmental Benefits: Utilizing agricultural waste for ethanol production supports sustainability, aligning with global goals to reduce emissions and waste. This attitude has been reflected throughout the entire process of our project’s design and implementation and has deepened over time. (Interview with Wang Xin & Bao Cunkuan)
• Business Model and Strategy Adjustment: Shift from direct strain sales to a licensing model, earning commissions based on customer success. Additionally, explore expanding ethanol applications beyond fuel, especially in the chemical industry. (Interview with Wang Qingzhuo) For more information, please refer to our entrepreneurship page.

Who are your proposed end users?

Our proposed end users are ethanol manufacturing plants and research institutions, particularly those in the biofuel sector. These firms and laboratories are seeking innovative, cost-effective solutions to improve the efficiency of second-generation biofuel production. By selling our engineered yeast strains, we aim to meet the needs of companies looking to optimize ethanol production from xylose and other renewable sources.

How do you envision others using your project?

We envision ethanol producers using our engineered yeast to enhance the efficiency of their production processes, particularly in the utilization of xylose from lignocellulosic biomass. By incorporating our yeast strains into their fermentation processes, manufacturers will be able to improve the economic viability of biofuel production. Academic and research labs will also benefit from our yeast strains as a tool for further study and experimentation in biofuel development, advancing the field of synthetic biology.

How would you implement your project in the real world?

To implement our project, we would focus on direct partnerships with ethanol manufacturing plants and research institutions. Our business model includes both a prepayment and a dividend system, where we receive an initial payment for the use of our yeast strains, followed by dividends based on the success and continued usage of our product. Additionally, we will promote our product through industry publications and academic conferences, ensuring that we reach our target audience effectively. We are also considering expanding internationally to overcome regulatory challenges, allowing us to scale production and reach a broader market.