Human Practices: From Waste Oil to Sustainable Squalane

Overview

Our project began with curiosity about the ingredient “Natural Plant Squalane” on a lipstick label. This simple observation prompted us to explore the hidden ecological costs behind cosmetic ingredients and inspired our mission to create sustainable alternatives. To ensure our solution is both scientifically rigorous and socially responsible, we engaged in in-depth exchanges with numerous experts from academia, industry, business, government, and civil society, as well as staff and iGEM teams. We also listened to public opinions and suggestions regarding biotechnology as a new productive force for sustainable development. Each interaction provided unique insights, clarified our project’s social value, and significantly influenced our progress. Discussions with local fishermen confirmed the unsustainability of shark-derived squalane. Dialogues with cosmetic companies like Proya and Bloomage Bio revealed the industry’s strong preference for non-animal sources and the critical importance of safety standards. Conversations with waste oil recyclers highlighted the environmental burden of used cooking oil and its untapped potential as a valuable resource. These diverse perspectives prompted us to learn from research experts and continuously refine our technical approach—from enhancing lipase secretion for efficient waste oil degradation and peroxisomal compartmentalization for high-yield squalene synthesis, to developing our software and integrated hardware system. Every piece of feedback was carefully considered and incorporated, allowing us to optimize the “Squoilene” product into a solution that achieves both scientific innovation and meets practical application needs. Throughout this process, we consistently listened and adapted, resulting not only in the creation of a microbial cell factory but also in a responsible biotechnology solution that addresses real environmental problems and meets industry demands.

Stakeholder Engagement Overview

HP Stakeholder Type	Specific Representatives	Role in Ensuring Values & Scientific Rigor
Experts	Prof. Yan Yunjun, Post-doctoral Xie Xiaoman, Prof. Tang Qiang, Prof. Ning Kang, Prof. Li Aitao, Prof. Sylvain FISSON ,Prof Shi Mang ,Doctor Wang Bohan	Validated scientific and innovative aspects of the technical route; ensured realization of scientific values. Provided key guidance on metabolic pathways, computational design, compartmentalization strategies.
Enterprise & Industry (Raw Material & End Brands)	Proya, Bloomage Bio, Wuhan Bairka Biomedical Co., Ltd., Hubei Hongfuda Biotechnology Co., Ltd., Wuhan Canuos Company, Hubei Weideli Chemical Reagent Co., Ltd., Canuos Technology. Zhejiang Zhengda Environmental Protection, Zhejiang Lanhaixing Salt Products Co.,Ltd, YposKesi	Ensured project alignment with market demands and industry standards (Social Values). Clarified safety requirements (Ethical Values), understood pain points in waste oil treatment (Environmental Values).
Government & Public Institutions	Wuhan Municipal Ecology and Environment Bureau, Ministry of Agriculture and Rural Affairs	Ensured project compliance with national environmental policies and shark protection regulations (Environmental & Ethical Values), advanced within a compliant framework.
Frontline Producers & Practitioners	Fisherman Uncle Sun from Weihai, Manager Cai from waste oil recycling company	Obtained first-hand information on shark fishing and waste oil treatment, ensuring the project addresses real social and environmental problems (Social & Environmental Values).
Engineering & Design Experts	Yang Xuezhi (National Bio-manufacturing Industry Innovation Center), Wang Jinchun (HUST Engineering Innovation Center)	Ensured hardware design feasibility for industrialization (Social Values - promoting industrialization), considered durability and contamination resistance (Scientific Values - rigor).
Public & End Consumers	200+ questionnaire respondents	Directly understood public acceptance, concerns, and preferences for the project; ensured product positioning and narrative align with social values (leading sustainable consumption) and respond to ethical values (transparency).

Figure 2 The types of groups involved in iHP

Part I: Problem Definition & Value Discovery

Part II: Construction of Technical Pathway & Iteration of Key Designs

Part III: Product Positioning & Engineering Implementation

Part IV:Compliance, Safety & Future Planning

Part 1: Problem Definition & Value Disco

Project Initiation

Figure 3 project inspiration-lipstick

Figure 4 Words cloud

Team members, during the summer vacation of 2024, became curious about the words “Natural Plant Squalane” on a cosmetic product at home. How could natural plants be linked to the animal attribute “squalane”? This curiosity led us to investigate, and we learned about a high-value cosmetic ingredient – Squalane/ene. Through research, we found that in today’s booming beauty and health industry, the traditional sources of the star ingredient squalene are in trouble: animal sources involve overfishing of sharks, raising ecological and ethical concerns; plant sources, due to the high environmental cost and price of olive cultivation, struggle to meet market demand.

Regarding Animal-derived Squalene

Oceana has repeatedly exposed in its reports the threat posed by the cosmetic industry’s use of shark squalene to shark populations. They pointed out that consumers, unknowingly, indirectly participate in the hunting of this endangered species by purchasing skincare products containing shark ingredients. Simultaneously, we noted that China’s Ministry of Agriculture and Rural Affairs introduced a new policy in September 2024 explicitly stating “no new construction or renovation of fishing vessels primarily targeting sharks,” further restricting the future of animal-derived squalene at the national level. To understand the specific situation, we interviewed fisherman Uncle Sun from the Nanhai New Area, Weihai City, Shandong Province, in August 2024. He mentioned that while sharks are present in the Yellow Sea, they are mostly in deep waters and less common near the shore. He emphasized that there is no specialized shark fishing industry locally, only occasional bycatch.

Figure 5 member Yu ziheng with fisherman in Shangdong,China

Regarding Plant-derived Squalene Issues

To gain a deeper understanding of the squalene market, we conducted phone interviews with Wuhan Bairka Biomedical Co., Ltd., Hubei Hongfuda Biotechnology Co., Ltd., Wuhan Canuos Company, Hubei Weideli Chemical Reagent Co., Ltd., and Canuos Technology. We learned that non-animal derived squalane is more favored by the market, and “insufficient production of non-animal squalane” is a real problem. We also contacted Engineer Jin Feng from Proya Chemical Co., Ltd. She explained that squalene and squalane appear in the high-end skincare series of companies like Proya, indicating clear demand. She also pointed out that the cosmetic industry has very high requirements for raw material safety; any new ingredient introduction requires strict entry approval and comprehensive technical index testing. This led us to define the final product as squalene and plan to introduce a complete third-party safety testing process to meet cosmetic industry needs.

Discovering the Great Potential for High-Value Transformation of Waste Oil

At the same time, we noted that China produces tens of millions of tons of waste cooking oil (“gutter oil”) annually, which is both an environmental and food safety hazard and an underutilized vast resource. Through contact with General Manager Xu Zhixin of Zhejiang Zhengda Environmental Protection Equipment Co., Ltd., we learned that even converting waste oil into biodiesel does not yield high economic profits. Manager Cai from waste oil recycling in Hong’an County, Hubei, also stated that current treatment methods offer meager profits, and there is a desire to explore new high-value conversion pathways. Common waste oil treatment produces biodiesel for energy use, with a market price of only about 8.5 RMB/kg, while every 20mg of high-purity squalene is worth 800 RMB. This huge price gap reveals a highly potential industry opportunity: in the era of resource recycling and sustainable development, can we break through traditional paths and redefine the value of waste resources? Our project thus embarked, aiming to create a green cycle “from waste oil to squalene,” not only addressing environmental and social pain points but also striving to provide a new, sustainable solution for the beauty industry.

Project Positioned in the “Fashion” Track

We released a questionnaire about our project’s product story, collecting 200+ valid responses. The results showed: over 70% of young consumers are willing to try sustainable skincare products “regenerated from waste oil,” especially when the product story and environmental concept can be combined with fashion. This indicates that consumers not only want to pay for environmental protection but also hope this behavior can become a “social currency” that demonstrates their attitude and taste. Simply emphasizing technology or efficacy fails to resonate emotionally with mainstream young consumers. This also guided us to choose the fashion track.

Figure 7 Respondents finishing questionaire

Part 2: Construction of Technical Pathway & Key Iterations

Ideal Chassis Strain Selection:

In the early stages of the project, we contacted Prof. Tang Qiang from the University of Science and Technology of China. We explained our project goal: to find a microbial chassis that can survive in waste oil and efficiently utilize waste oils. After hearing our needs, Prof. Tang clearly stated: “Yarrowia lipolytica is the ideal choice for your project. It is not only recognized as an ‘oleaginous yeast,’ naturally possessing strong lipid accumulation and metabolism capabilities, but more importantly, it itself has a complete MVA pathway, which is the foundation for squalene synthesis.” We adopted Prof. Tang’s advice, formally selected Yarrowia lipolytica as the project chassis, and began designing strategies to strengthen its endogenous MVA pathway.（Detailed design specifics can be found in the Design section.）

Figure 8 member Zou wanting with professor Wang qiang

Lipase Introduction:

However, in subsequent experiments, we encountered a bottleneck: even when using relatively simple edible oil for fermentation, the oil degradation efficiency of our engineered strain did not meet expectations. With this new problem, we consulted Prof. Yan Yunjun from HUST, who has profound expertise in enzyme engineering and multi-enzyme catalysis. After analyzing our data, Prof. Yan pointed out incisively: “The intrinsic lipase activity of Yarrowia lipolytica might indeed be insufficient to support high yield requirements. I suggest you introduce an exogenous, efficient, and stable lipase as an ‘enhancement module.’ Consider introducing the TLL enzyme from Aspergillus oryzae. This enzyme has good thermal stability and broad substrate specificity, with high hydrolytic activity against various triglycerides, effectively decomposing waste oil into free fatty acids.” Ultimately, we chose the TLL enzyme to enhance waste oil degradation.

Figure 9 member Zou wanting and Yuan tianxu with professor Yan yunjun

Rate-Limiting Enzyme Optimization:

To optimize the key rate-limiting step, we contacted Prof. Ning Kang from HUST. He suggested introducing ProteinMPNN and Rosetta. Integrating summer research progress and the team’s existing technical stack, we finalized the optimization path: using ProteinMPNN for scaffold sequence redesign under structural constraints, supplemented by Frustratometer2 for quantitative assessment and iterative screening of local and global conformational frustration, and utilizing Rosetta for fastrelax. The goal is to reduce local frustration, smooth the energy landscape, and overall enhance the thermodynamic stability and folding robustness of tHMGR.

Figure 10 Professor Ning kang

Figure 11 members with Ning kang

Mining Novel HMGR Homologs Based on Structural Biology:

As the project progressed, we committed to optimizing key enzymes in the squalene synthesis pathway through protein engineering. We wanted to first mine all proteins in nature with catalytic activity similar to HMGR using a certain method. Therefore, how to accurately and quickly screen the enzymes we want at the three-dimensional structural level and guide rational design? For this, we contacted Prof. Shi Mang from Sun Yat-sen University. The expert pointed out that the core of our problem lies in functional annotation from the perspective of structural homology. He explained that traditional tools like MMseqs2 primarily rely on amino acid sequence similarity, but their predictive power drops significantly when sequence identity is low. He recommended a powerful new tool – Foldseek. This tool can directly compare the 3D structures of proteins, effectively identifying structurally homologous and potentially functionally related proteins even when sequence similarity is low, greatly improving the accuracy of functional annotation. The expert also shared his team’s successful experience, setting the structural consistency (TM-score) threshold at 90% as a reliable standard for functional inference. This consultation opened a new door for us. We immediately integrated Foldseek into our protein engineering workflow. The introduction of this tool moved our enzyme optimization work from “guessing” to “structure-based rational design,” significantly improving the success rate and efficiency of our engineered strain design.(Detailed design specifics can be found in the Model section.)

Compartmentalization:

While strengthening the MVA pathway and knocking out downstream squalene pathways, we found that MVA pathway optimization did not significantly increase squalene yield, and knocking out downstream genes severely affected normal life activities. To solve this problem, we contacted Post-doctoral Xie Xiaoman from HUST. She stated: “The cytoplasmic environment is complex; squalene accumulation might produce toxicity, and negative feedback regulation exists. She suggested adopting a ‘compartmentalization’ strategy, transferring the synthesis pathway to independent organelles.” We subsequently made a major technical route iteration, deciding to localize the entire MVA pathway to the peroxisome, creating a more optimized micro-reaction environment. The dry lab team immediately began systematically screening efficient signal peptides.

Part 3: Product Positioning & Engineering Implementation

Establishing Integrated Hardware Design Principles

During our research and experimental process, we found that when conducting micro-fermentation and substance extraction in the laboratory, the micro-fermentation equipment is very expensive and still cannot monitor fermentation conditions and related parameters in real-time. Our members once spent 36 consecutive hours sampling and measuring data. This process is not only cumbersome and time-consuming but also carries a high risk of contamination, greatly reducing experimental efficiency and accuracy. We contacted Yang Xuezhi from the National Bio-manufacturing Industry Innovation Center, who suggested we design an integrated hardware device; Prof. Li Aitao from Hubei University suggested using grinding methods to break cells. This led us to design the iFPS.

Achieving Hardware Material and Automation Upgrades

After the initial hardware design was completed, we contacted Wang Jinchun from the HUST Engineering Innovation Center to trial our device. Taking his advice, he recommended using photosensitive resin to improve device density, temperature resistance, and contamination resistance, and using PCB boards instead of breadboards and DuPont wires to improve circuit stability. With his help, we are integrating motors, sensors, and custom PCB control boards to improve device stability. We also contacted Dr. Chen Yulong from HUST, who has long researched microbial fermentation. He suggested we design a baffled fermentation device to increase dissolved oxygen; he also proposed new application scenarios, suggesting the device could be used for the collection and extraction of extracellular secreted fermentation products.(Detailed Hardware design specifics can be found in the Hardware section.)

Figure 15 hardware member communicate with teacher Wang jinchun

Figure 16 Communication with Ph.D Chen yulong

Part 4: Compliance, Safety & Future Planning

Strengthening Emphasis on Raw Material Safety and Product Compliance

Bloomage Bio is a famous synthetic biology company. We contacted Kan Hongling, Chief Formulator at Bloomage Bio, to ensure the technical solution can handle the complexity of real waste oil and meet the industry’s highest safety standards. Experts reminded us that waste oils from domestic sources have complex compositions and may contain components like preservatives that inhibit microbial growth. They strongly emphasized that any raw material derived from waste oils must undergo extremely strict purification and safety verification before being accepted by the industry. Therefore, we will add a waste oil pretreatment module to our plan, and we established “completing a full set of safety assessments in cooperation with authoritative third-party testing institutions” as a mandatory threshold that must be met before product launch.

Figure 17 member Zhao shijing visit Bloomage Biotech

Figure 18 Formulator Kan hong

Recognizing Downstream Industrialization Engineering Bottlenecks

To understand the practical problems that might be encountered when transitioning from a lab product to an industrial commodity, we contacted Ms. Wei Qin, General Manager of Zhejiang Lanhaixing Products Co., Ltd., and Engineer Chen Wei. They took us to visit the Zhejiang Lanhaixing filling line. Downstream packaging stages might become efficiency bottlenecks due to product physical properties (like viscosity), visually demonstrating the engineering and design bottlenecks that might be encountered during the transition from laboratory technology to industrial production. They suggested testing under different pH, temperature, and viscosity conditions, and considering transportation and shelf-life requirements. Therefore, in hardware design and future planning, we will pay more attention to compatibility and adaptability with downstream industrial equipment.

Figure 19 member Ma yichuang visit Zhejiang blue starfish salt product CO.,Ltd

Project Prospects

We contacted Prof. Sylvain FISSON, Professor of Immunology (University of Evry Paris-Saclay). We detailed our project to him. Prof. FISSON saw great potential, stating that our project has significant practical meaning and scientific prospects, providing a comprehensive solution for squalene synthesis and waste oil recycling. We will continue to promote the project’s development. Prof. FISSON also invited us to the University of Evry Paris-Saclay for discussions on the project’s further development.

Figure 20 member Zhao haixi communicate with Professor Sylvain