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• When we brainstormed for this year's project on rare earth recycling, Dr. Yu suggested that we use a rare earth binding protein and present it to the cell surface through the yeast surface display to capture rare earth metal ions in rare earth wastewater.
• When it comes to how cells that capture rare earth ions should be recycled, Dr. Yu Haoran proposed that yeast cells saturated with rare earth elements can be genetically engineered to settle to the bottom of the medium in a way of cell self-flocculation, which is convenient for cell recovery.

• Through brainstorming with Dr. Yu and our other three Primary PIs, our team finally determined the project framework for this year's rare earth recycling.
• As for the recovery method after the adsorption of rare earth saturation, we believe that yeast cells should fully contact the wastewater to be treated during the adsorption of lanthanide ions, rather than just adding yeast cell cultures to the rare earth wastewater. We finally selected the membrane bioreactor as the hardware carrier for wastewater treatment through in-depth investigation, and planned to achieve the yeast carrier surface adhesion by overexpressing FLO11 gene of FLO gene family related to yeast flocculation.

• For the separation of rare earth elements, the elements can be separated one by one by ion exchange or serial extraction after co-separation by biological leaching.
• Concentration detection of rare earth elements for biosorption: The protein can be modified and we can measure the metal concentration with different fluorescence wavelength and intensity. For example, uranium and terbium, one red and the other green, can be detected by fluorescence.
• As to whether microorganisms can survive in highly acidic wastewater, immobilization means can be used to enhance the tolerance of microorganisms to low pH in mine wastewater.
• At present, for the treatment of rare earth mine wastewater, the most concerned is the removal of radioactive elements such as uranium and thorium.
• In our previous literature research, we had concerned that bioadsorption proteins might not be highly selective between lanthanides and calcium. Dr. Shaw told us not to worry about this, as the two can be easily separated using conventional chemistry methods.

• We decided to adopt Dr. Xiao's suggestion that after the enriched rare earth elements are obtained by biological adsorption, chemical methods can be used for further differentiation and purification.
• We decided to further strengthen the immobilization of biofilms to achieve the purpose of adapting to the wastewater environment.
• After knowing the current status of treatment of rare earth mine wastewater, our team will further consider expanding the application of our project in the treatment of radioactive elements and extraction of industrial resources (lanthanide and actinide, lithium, etc.).

• Professor Ji thought our project was very interesting and reminded us of the specificity of the protein binding to the target metal. He suspected that the orbital similarity between metal ions was likely to cause the proteins on the yeast surface to have difficulty in highly specific binding to rare earth elements, which leads to a large amount of protein waste.
• In addition, Ji recommended that we carefully consider whether the protein was stable under the pH condition of the wastewater, which also affects the binding efficiency of the protein and the target metal.
• Ji also advised us to increase the expression of the modified protein on the yeast surface as much as possible, which is also to improve the bioadsorption efficiency.
• Our team members also consulted Professor Ji about some metal coordination issues. The chemical knowledge provided by Professor Ji is helpful for undergraduates majoring in biology to better understand the principle of binding and adsorption of proteins and metal ions.

• Our team improved the structure of the proteins presented on the surface of yeast so that the proteins have stronger specific binding ability to rare earth elements.
• A review of the literature shows that the effect of orbital similarity of metal ions on the binding efficiency of proteins to rare earth elements is limited and is acceptable from an engineering point of view.
• In subsequent experiments, it was confirmed that the proteins displayed on the yeast surface could maintain certain activity in the chemical environment of the wastewater.
• We also specially selected a more stable and efficient surface display system to improve the binding efficiency of biosorption proteins with rare earth elements.

• Membrane contamination and clogging in membrane aeration biofilm reactors is a concern for our engineered yeast. The construction of biofilm is generally suitable for microorganisms with slow growth and easy loss. However, for yeast, the growth rate is fast, and the iteration may form layers of cells on the carrier filler, resulting in membrane pollution and blockage.
• After the bioabsorption process, allowing yeast to self-flocculate into clumps may be a more economical and common option than attaching to carrier fillers.
• To apply genetically engineered laboratory strains to actual production, biosafety needs to be considered from the perspective of intensity and feasibility of horizontal gene transfer, as well as ecological competition with natural microorganisms.

• The potential for yeast to grow faster than wild strains in the natural environment is seen as a competitive advantage, which would help get better niche competition. We decided to continue using yeast as a chassis cell, but at the same time began to consider the issue of biofilm contamination.
• In the biosafety module, we will continue to design more comprehensively and thoughtfully from the perspective of horizontal gene transfer and competition between different strains of ecological niche.

• There are three main options for the formation of biofilm in wastewater: select a suitable carrier for cells to adsorb on directly allow yeast cells to naturally suspend to form a biofilm use hardware such as membrane bioreactor (MBR) to form a biofilm.
• There are different treatment schemes for recycling biofilms under different conditions: When there is a carrier, the carrier, and the system are separated, and then the cells on the carrier are recycled; If there is no carrier, the cell and the water environment should be separated, such as making the cell settle and pumping the water in the system.
• Using proteins to adsorb rare earth elements does not have to be displayed on the surface of yeast. Functional proteins can also be adsorbed to the carrier column through protein immobilization after biofilm formation to form functional membranes similar to immobilized enzymes.

• We finally decided to adopt the scheme of membrane bioreactor (MBR) to form yeast biofilm.
• The immobilization of purified lanthanide binding proteins onto carrier columns expanded our previous approach of displaying proteins on yeast surfaces as whole-cell adsorbents. And we decided to consider this as an alternative.

• For the issue of biofilm clogging, Dr. Lai suggested that we consider the ratio of packing material to inoculum source when designing biofilm reactors. Additionally, we can also try selecting different biofilm carriers for experimental comparison to identify the most suitable carrier for extracting rare earth elements from slag wastewater. Dr. Lai also pointed out that the main function of biofilms is for substance exchange and transformation in microorganisms, and the advantage of biofilm reactors is their long sludge retention time (SRT), meaning they have a strong ability to retain microorganisms. When designing a biofilm reactor, it is necessary to consider the stress factors and tolerance ranges of microorganisms. In addition, apart from the MBBR reactor we proposed, Dr. Lai also suggested that we try a combination of various MBR reactors containing hollow fiber membranes to improve adsorption efficiency. Dr. Lai suggested that when designing a rare earth recycling scheme for slag wastewater, in addition to experimental and technical considerations, we should also incorporate economic analysis to evaluate costs and benefits, while also considering environmental and social affects. For the issue of mixed bacterial contamination, Dr. Lai Chunyu suggested that we can solve this by irradiating wastewater with ultraviolet light or sterilizing wastewater in advance. If necessary, we can also use gene editing techniques to achieve our goal.

• We have added a UV sterilization step after the bioreactor to ensure biosafety and prevent contamination by unwanted microorganisms. We ultimately designed a combined biofilm reactor of MBBR and MABR coupling to improve adsorption efficiency.

• Porous materials, such as two-dimensional elastic materials, are constructed with metal ligands (e.g., Cu, Fe) through coordination bonds. These porous materials, with various ligands and material structures, can adsorb target substances such as water, carbon dioxide, organic pollutants, and microorganisms.
• By altering the material's stacking method, such as A-A stacking or A-B stacking, the pore size of the material can be adjusted. Alternatively, modifying the concentration of solvents surrounding the material can also achieve the separation of target substances.

• We considered using advanced porous materials to adsorb target microorganisms. For instance, porous ceramics could be used as carrier materials in biofilters to immobilize microorganisms, thereby enhancing wastewater treatment efficiency. Incorporating porous organic polymer materials into wastewater treatment equipment could selectively adsorb organic pollutants, thus improving the sustainability and environmental friendliness of the project.

• Dr. Lai first affirmed the integrity of our engineering design and the richness of the content, and expressed his anticipation that we could gradually implement the experiment of each module. Since our project involves the adsorption of lanthanide metal ions, it is necessary to focus on characterizing the binding force between protein and rare earth. The part of engineering practice is mainly the formation of yeast biofilm, which needs to consider the degree of combination of strains and vectors. Professor Lai himself is also the instructor of the iGEM team of Hong Kong University of Science and Technology. Therefore, we referred to the design of their non-model strains for metabolic engineering, hoping to strengthen cooperation in gene design parts such as intestinal strains in the future, so as to realize the transformation of new achievements.

• Because our project design is more about protein engineering and industrial design instead of the design of gene circuits and metabolic engineering, we considered innovative modification and design of TFD-EE protein, so as to make the whole project close to the core of iGEM competition and reflect the professional application of synthetic biology.

• The largest practical application of synthetic biology products is still in metabolic engineering, that is, the construction of artificial cell factories to mass-produce target metabolites in closed fermenters. It can be sterilized directly after fermentation without the risk of microbial leakage into the environment. Our rare earth adsorption strain can also be used in a semi-enclosed water treatment unit, which can avoid biological leakage to a certain extent. Compared with biosensing products, more policy restrictions can be avoided. If it is for his biosensing products, Professor Wang prefers to develop sensors based on cell-free sensor platforms.

• We decided to confine our biosorption strains to a fixed water treatment bioreactor to avoid direct exchange between open installations and the environment. After the biosorption process of rare earth elements was completed, we decided to add ultraviolet sterilization units downstream of the water treatment bioreactor to strictly ensure biosafety.

After interviewing Professor Baojun Wang and learning that the promotion of synthetic biology products to practical applications may encounter tremendous obstacles in policy approval, we decided to ask government agencies for further information.

On July 29, we came to Zhejiang Ecological Environment Scientific Design and Research Institute to learn the current situation of using microorganisms to control environmental problems. We have learned that the application of synthetic biology in the field of bioremediating is mainly through the redesign and modification of existing contaminant-degrading strains so that they can degrade multiple pollutants at the same time. Or build a microbial community with collaborative degradation ability, and use the interaction between different microorganisms to improve the overall degradation efficiency. Retrofitting existing engineered strains would be less of a biosafety concern.

Considering the importance of introducing artificially designed lanthanide binding protein TFD, we finally chose to present the TFD protein on the surface of saccharomyces cerevisiae through cell surface display. Saccharomyces cerevisiae is generally regarded as a safe (GRAS) chassis cell, and we believe that it will face less resistance in future applications.

We visited the Zhejiang Provincial Environmental Monitoring Center with students from the College of Environmental and Resource Sciences at Zhejiang University, gaining an in-depth understanding of the advanced technologies used by the center in areas such as nuclide analysis, environmental monitoring data management, and automatic radiation environmental monitoring. These technologies ensure effective oversight of environmental safety through precise monitoring methods, enabling accurate control of radioactive substances. The center utilizes modern equipment and scientific data analysis to monitor environmental changes in real time, providing strong support for government decision-making and public health

Following this, we visited the Department of Ecology and Environment of Zhejiang Province, where we focused on learning about the operation of the "Ecological Environment Brain" real-time monitoring system. This system integrates multiple ecological datasets to achieve dynamic monitoring of the province's environmental conditions, covering various aspects such as air quality, water, and soil. The high-precision carbon emission monitoring equipment we observed in the carbon monitoring laboratory provides a solid technical foundation for achieving the "30-60" carbon targets.

This visit offered us several key insights:

• Precision Monitoring: Inspired by the multi-layered monitoring approach of the Environmental Monitoring Center, we plan to incorporate high-precision monitoring methods into our project to ensure comprehensive tracking and optimization of the engineered strain's performance.
• Collaboration Opportunities: Strengthening partnerships with environmental departments will help us develop more efficient and cost-effective environmental management technologies, ensuring the sustainable development of our project.

On July 30, 2024, members of our team went to Shanghai, China, and visited Anshengda Bioengineering Company. We interviewed the company's researcher, Dr. Huang, who graduated from the Massachusetts Institute of Technology. We introduced our project to Dr. Huang. He thought that our team's project ideas were relatively simple, and could be further improved to make it more innovative. He also emphasized the importance of biosafety and acknowledged it after learning about our copper-ion induction suicide mechanism. Dr. Huang believes that our team needs to pay special attention to the efficiency of the bioabsorption of metals. He believes that it is necessary to maximize the expression of target proteins on the surface of yeast cells in terms of plasmid copy number, promoter strength, and mRNA stability. As a researcher at a bioengineering company, Dr. Huang reminds us to pay attention to the sustainability of the hardware devices we design from a production perspective. He also reminds us that if this idea is actually put into large-scale production, there is also a cost to consider. For example, how often does the biofilm in the MBBR device need to be replaced, and would it be too costly to provide the nutrients needed for the growth of the engineered bacteria?

On July 3, 2024, we visited Hangzhou Lijia Environmental Services Co., Ltd., which is the largest comprehensive hazardous waste disposal center in East China. It not only handles hazardous waste through multiple incineration lines, recovering some of the heat generated during the incineration process for local heating systems or electricity generation, but also has a large hazardous waste landfill that uses a strict double-layer anti-seepage structure to minimize the risk of leakage during the waste treatment process, effectively protecting the surrounding environment.

The next day, we visited Guangda Environmental Protection Energy Hangzhou Co., Ltd., where we gained insights into the complex processes and environmental protection measures of the Jiufeng waste incineration power generation project. Notably, Guangda Environmental successfully addressed the NIMBY (Not In My Back Yard) effect by fostering understanding and support from surrounding communities through proactive public communication and transparent information sharing.

This visit provided us with several important insights:

• Resource Recovery: The slag from incineration is not only used for building materials but also the residual heat generated during the incineration process is used for electricity generation, further enhancing resource utilization efficiency. This inspires us in recovering rare earth elements from industrial wastewater, reminding us to explore multiple pathways for resource recovery to enhance the overall benefits of our projects.
• Environmental Safety: The design of the double-layer anti-seepage structure in the landfill reminds us to prioritize environmental safety when modifying biological strains, ensuring harmless treatment and avoiding secondary pollution of soil and water.
• Public Communication: Guangda Environmental's success in mitigating the NIMBY effect underscores the importance of public communication, providing valuable references for social recognition and public participation in our future project promotions.

To gain a deeper understanding of the practices in water resource management and wastewater treatment, we visited the Qige Wastewater Treatment Plant of Hangzhou Water Group Co., Ltd. The Qige Wastewater Treatment Plant adopts a semi-underground design, with some facilities located below ground to minimize environmental impact. The plant is integrated with Qiantang Ecological Park, reflecting a compact and eco-friendly design concept. The core of the plant is its biological treatment zone, which includes six A2/O reactors that combine anaerobic, anoxic, and aerobic phases for efficient wastewater treatment.

During our visit, we toured the wastewater treatment units and observed the system’s operational flow and the adjacent ecological park, gaining a clearer understanding of the current wastewater treatment processes.

In China, biological treatment processes such as AAO, SBR, and MBR are widely used, with well-established workflows and operational procedures. The primary advantage of biological treatment is its efficiency and environmental friendliness, effectively purifying wastewater without causing secondary pollution. However, its efficiency is often influenced by factors such as temperature, pH levels, and organic load, leading to variable stability.

Most wastewater treatment plants today focus on pollutant removal, with limited efforts toward resource recovery. Biological treatment processes typically rely on natural microbial communities for pollutant degradation, with minimal artificial control. We aim to apply synthetic biology techniques to engineer microorganisms capable of efficiently adsorbing and recovering rare earth elements, promoting resource recycling.

Drawing on the principles of the AAO biological treatment process used in wastewater plants, we can further enhance our system. By alternating anaerobic, anoxic, and aerobic conditions, we aim to optimize the metabolic pathways of the engineered strains and improve their efficiency in adsorbing rare earth elements.

During our exchange with Hangzhou Water Group, we presented our modular equipment design. Company representatives appreciated the flexibility of our design, noting that such an adaptable system could be adjusted based on different wastewater compositions, enhancing the equipment’s overall versatility.

With the opportunity provided by Zhejiang University's international exchange program, we were honored to visit the NEWater Visitor Centre in Bedok, Singapore, where we learned about the innovative water treatment technologies and automated systems that enable efficient resource recycling. The plant's deep tunnel sewage system and multi-stage purification processes ensure the efficient recovery and reuse of wastewater, with the final product—NEWater—meeting drinking standards.

This visit inspired us to apply the concept of multi-stage treatment mechanisms to ensure the stability and safety of microbial strains, as well as to improve the recovery rate of rare earth elements. This will help promote the environmental and economic sustainability of our project.