Overview #

In recent years, more and more people are becoming aware of the “white pollution” problem, and the idea of applying recycling products to large-scale of manufacture is becoming widespread in major provinces of China, like Zhejiang, Jiangsu, Shanghai, etc. To biologically break down PET for commodity reproduction and therefore enhance the economic efficiency of plastic, we at IvyMaker-China ascertained to develop a self-assembled multi-enzyme display system – the PET Buster.

The PET Buster is a two-phase project. In 2021, we developed a whole-cell biocatalyst by displaying PETase and MHETase on the surface of a yeast cell (Candida tropicalis) separately to degrade PET waste. First, we chose a robust Candida tropicalis as the host and deleted the URA3 gene of genome to block the uracil synthesis by CRISPR-Cas9. Then, we built a model to predict the anchor protein candidates that could display the protein more efficiently. Finally, we selected the most effective model and fused it with PETase/MHETase and found that both enzymes were active and had obvious degradation effects on PET film and powder.

This year, in 2022, to increase the effectiveness and convenience of the degradation of PET plastic, we plan to display MHETase and PETase at the same time, which is to develop a self-assembled multi-enzyme display system of Candida tropicalis.

We first consulted professors and doctors from related backgrounds about the problems we wanted to alleviate in the model and in the lab and received helpful advice which later shaped our final project design. Additionally, we interviewed representatives of enterprises upstream and downstream of the PET industrial chain and conducted some trips to gain a more nuanced understanding of how our solutions should fit in and work in the real world. Last but not least, we sent out questionnaires to individuals with the intention of buying products made out of bio-degraded PET to fulfill the industrial loop.

1. Lab Design Interview with Professor Xianzhong Chen #

Background #

Professor Xianzhong Chen is an experienced professor who has actively engaged in physiological and metabolic engineering research of industrial microorganisms for over twenty years at Jiangnan University. As our project is a two-phase project, we initially consulted him last year when he gave us valuable advice on the project as a whole, including the idea to use anchor proteins to express the enzymes. We believed that he would be the best candidate to advise us on the feasibility of our methodology and enlighten us on possible improvements to the current design as well as cautionary measures to combat potential problems.

Questions Prepared #

We first researched to finalize the experimental procedure and found that SnoopTag, SpyTag, peptide connections, and two separate gene promoters could be applied. Thus, we prepared several questions about the plausibility of our research as well as further large-scale application.

1. What is a better way to demonstrate both enzymes on the surface of Candida tropicalis?
2. Is there a strategy to control the proportion of enzymes on display?
3. Currently, the scheme using SnoopTag and SpyTag is experimentally proven to work. What are the limitations of this scheme?
4. After a detailed investigation, we found that peptide ligase has the potential to synthesize protein or polymer chains by enzymatic cross-linking. Is using peptide ligase to link PETase and MHETase a feasible solution?
5. Is it feasible to use two promoters to demonstrate two enzymes? If so, what would be the subsequent effect on enzyme activity?
6. Are large-scale applications feasible at the time?

Feedback #

According to Dean Chen’s answers, there are many ways to achieve our goals, but many factors should be considered carefully, such as the levels of transcription, cell metabolism, and proteins in addition to cell-cultured conditions. At the present stage, we can control the molecular weights of PETase and MHETase, referring to cellulosomes that hydrolyze cellulose. As for the Catcher-Tag system, we are still in the exploration stage. For our method, we can complete intracellular assembly by adding tags and promoters ahead of the target protein expressed. The protein could then be transported to the surface, followed by the detection of immune tags which test the enzymes’ activity. However, the Catcher-Tag system requires extra energy to form covalent bonds between enzymes, and tags and catchers can be incompatible during the experiment. Additionally, enzymes are not always biologically stable due to the impact of both internal and external environments. This interview stimulated us to do more experiments on lab design to test theoretical knowledge and provided valuable insights on how to apply that knowledge.

2. Model Design Interview with Dr. Zhongyi Cheng #

Background #

Dr. Zhongyi Cheng is an associate researcher at the School of Bioengineering, Jiangnan University. He specializes in protein modeling, molecular docking and molecular dynamics simulation to investigate the relationship between protein structure and function, and rational/semi-rational modification of industrial enzymes. We wished to gain his insights on the choice of the model suitable for our project and how we could display the results more scientifically.

Questions Prepared #

After conducting some research to investigate protein modeling technology and planning our experiment, we wanted to improve our experiment by constructing a more efficient protein model. Therefore, we prepared some questions to better understand modelling to improve our experiment’s feasibility and efficiency.

1. What kind of problem does modelling solve?
2. What is the process for linking the sequences of two different proteins in modeling?
3. Does the order of protein sequences affect the modeling results? If so, how can we reduce the influence?
4. Will the same sequence modelled on different platforms yield different results?
5. How can we objectively assess the credibility of the model?
6. What is the best method to optimize the protein models?
7. What are the specific areas being optimized?

Feedback #

According to Professor Cheng, it is fairly simple to obtain the first-level structure of the sequence with modern developments in nucleic acid sequencing technology. However, structural data is harder to access than sequence data, so modeling is needed to detect the protein structure without costly wet lab experiment.

As we are linking the two proteins artificially with a designed linker (small peptide segment) that forces the two segments together and acts as a transition, the function of the protein could undergo changes.

Thus, before we type in the sequence of the two proteins, it is essential that we consider their order. The order of proteins can influence their conformational morphology and function, especially for the protein placed in the back.

According to Professor Cheng, results might also vary if we enter the same sequence on different platforms, as platforms have different algorithms of their own, leading to significant differences in the structure of the same sequence of proteins.

Once we have obtained different models, we can evaluate them using several tools, and we derive their scores through calculations with the aforementioned tools. The models that have a score within the range will be considered credible. Among these models, we need to select the most stable structure and test its performance using a wet experiment. If the protein structure we selected does not work, we need to then optimize its structure.

For optimization, there are several commercially available forms of software (discovery studio/Schrödinger). When we submit the protein structure, these platforms will minimize the energy that the protein carries, simulate its molecular dynamics at the nanosecond level, and optimize each amino acid chain. Therefore, such optimization makes the model more advantageous than the original structure. This interview enabled us to understand modeling more thoroughly, which granted us more opportunities to improve both our enzymes’ functions and structures.

3. Implementation Interview at Haili Environmental Protection Technology Co., Ltd. #

Background #

Haocheng Teng and Lichang Wang visited Zhejiang Haili Environmental Protection Technology Co., Ltd. on August 12, which is a recycled polyester resource comprehensive utilization enterprise. Haili factory has two channels to retrieve plastic bottles: one collects bottles scattered outdoors from waste recycling stations and transports them to the factory through household suppliers, and the other collects bottles from residential households and other specialized locations via recycling individuals together to platform recyclers, who will then sort and pack them uniformly to the factory. The interviewees were Mr. Fang, the vice general manager of HEPT Co., Ltd., and Ms. Lin, the sales manager. The main purpose of this interview was to understand the whole process of recycling plastic bottles in a professional factory and the current supply situation. Also, we hoped to obtain suggestions from professionals to improve our project in the future.

Q&A #

In advance, we prepared several questions, which were answered patiently by Fang and Lin, respectively. Before the interview, we also introduced our project.

1. What kind of process does the factory use to accomplish the complete green industrial chain of recycled polyester resources? And how long would the whole process take?

   “The entire industry chain is very complete, and the factory will begin with collecting the plastic bottles and end with manufacturing the final products. Also, the whole process accords with national standards and is quite sustainable.” - Lin

   “There are two aspects concerning time. For example, if there is only one plastic bottle, it will only take one day to manufacture the final product. Conversely, if a large number of bottles are mixed, it will probably take 15 to 30 days for a complete assembly line.”

2. What are the main sources of plastic that the factory is using to recycle? What is the standard applied to measure recyclable plastic?

   “We collect our bottles in three categories. The first category is bottles found outdoors, the second category is bottles found in public establishments, and the third category is bottles thrown from individual households.”

   “The standard we have is that plastic from the agricultural, medical, and automobile industries are forbidden to recycle since it has detrimental effects on health.”

3. What are the aspects that your factory mainly pays for? If possible, what would you say is the approximate cost?

   “Mainly, the cost includes buying raw materials, about 5000 RMB/ton, and transportation, nearly 150 RMB/ton. Also, we need to wash and clean the bottles, which usually costs us 2000 RMB/ton, including a loss of 20%, approximately. In addition, the wage is another significant expense due to skill training.”

4. We find that the factory uses a physical degradation method which is different from our biological method. What are the advantages of this method? How would physical energy consumption differ from others?

   “In our opinion, the advantages of the physical method involve low operation cost as well as high efficiency. This method does not damage the plastic’s original physical properties.”

5. Have you heard about our method? What is your general opinion on it?

   “The general manager received word about this biology procedure from French workers who already started this process. For our method, it’s not necessary to consider colors since enzymes on yeast cell surfaces can thoroughly degrade the plastic to small monomers. However, there are disadvantages to our method. Enzymes require optimum circumstances to work efficiently and can only produce in small amounts with high costs.”

6. Would this process pollute the environment, like producing waste gas or water? If the answer is yes, how would you deal with it?

   “Certainly, our process will not pollute the environment. It does produce waste gas and water, but it will be collected into a sewage disposal system where 80% of the treated water can be reused and the rest will be sent to the government. Additionally, there are solar panels on every workshop’s roof, which can produce 10-20% energy for the whole factory,” said Fang proudly.

7. What fields is the recycled plastic used in currently?

   “For the most part, final plastic spinning would be used in textiles, clothing, and container. The product can be used for garment accessories like zippers. It can make tents, dresses, and even Coca-Cola bottles!”

8. Does IKEA have the requirement for the product? If the answer is yes, how would the factory adjust?

   “Like many clients of ours, IKEA holds very high standards on product quality and safety. It asks us to provide track records of every product to ensure safety, and it also requires us to post the danger sign in the workplace and to offer employees enough relaxation time.”

Tour of the Factory #

After the interview in the office, we were guided by Fang and Lin to the factories. There, we closely observed the machines and workers toiling away. Surprisingly, almost the whole process was led by robots with mechanical arms, which automatically maintain the factory flow line. During the visit, we took a lot of pictures.

Feedback #

After visiting the factory, we understood the whole plastic recycling chain and also got the opportunity to observe the interior of a professional factory closely. During the interview, we know that our method still has some degree of limitation which needs to be perfected in the future, like the external environment control of yeast growth. When we finished the interview, we started to become curious about the products remade from plastics. Through the Haili Factory interview, we learned about our project more comprehensively and decided to further interview the downstream market-- IKEA in the future.

4. Implementation Interview at IKEA Baoshan #

Background #

We have learned from Mr. Fang, the vice general manager of Haili, that the recycled polyester has been used in most of the textile products in IKEA, such as in curtains, carpets, and other pieces of furniture. So on Aug. 16th, Lichang Wang, He Zhang, and Xuan Li visited Baoshan IKEA, Shanghai, to see the downstream market of recycling products. Also, we interviewed both the sales assistant and sales director as well as a few customers to discuss in detail the relationship between the materials and sales and to observe consumers’ reactions.

Questions Prepared #

We spoke to a sales assistant and asked about the most frequently asked questions by customers and if they would notice or care about the materials used. She told us that customers cared most about the comfort level and ease of cleaning the products while most of them did not even notice the materials used.

We then randomly interviewed some customers, showed them the product tag, and asked if they had ever noticed that the products they bought were made by recycling material. While all said no, the majority were quite impressed at the idea.

Afterwards, we interviewed the purchasing manager of IKEA Shanghai, Mr. Wang. According to him, IKEA has been devoted to protecting the environment for years, and they wish to realize the goal of all materials and products sold becoming recyclable or reproducible by 2030 and is purchased in a responsible manner. Currently, most of the textiles in IKEA are made from recycled plastic waste, and IKEA’s quality and standards in addition to the traceability of their products will remain unchanged as for new materials. It is without doubts that the cost was higher than before, but IKEA is working towards regulating the price so that everyone could afford the green product.

Feedback #

During this offline event at IKEA, we learned how companies further down the plastics recycling chain operate. During the tour, we analyzed the labels of textile products and found that many contained recyclable materials, and there were plastic recycling signs all over the mall. This shows the increase in action that major companies are now taking to recycle plastic. In addition, during the interviews with passers-by, we realized that the concept of plastic recycling is not yet popular in mainstream Chinese society and there is still a long way to go to promote it. In the future, we hope that our project can spread the concept of environmental protection to a much larger audience, and this offline visit helped because it provided us with a deeper insight into the market of recycled products and information about their sales as well as customers’ tendencies towards them. Our next step in this process is to conduct a public questionnaire to accommodate a larger sample size.

5. Implementation Design Public Questionnaire #

Background #

To further learn about the level of people’s attention to the products’ materials, we carried out an online questionnaire on the Wenjuanxing platform and distributed it amongst individual members’ WeChat Moments to get as much feedback as possible. We tried our best to ensure the formality of the questionnaire by informing people of our project’s idea and the questionnaire’s purpose.

Results #

In total, we obtained 2324 answers from all corners of society. According to the results, gender and age were relatively balanced among the respondents.

In the first place, 1424 respondents which are around 61.27% in proportion will be concerned about ingredients and sources of a product in their daily lives, among whom 934 people, approximately 65.59%, will pay attention to whether the materials are recyclable and renewable.

Furthermore, we focused on respondents who once bought textile goods in IKEA, our focused downstream market, to learn about their awareness of the sign “recyclable” on the labels. It turned out that 775, around 62.8%, of 1234 IKEA textile product buyers noticed before.

In addition, we researched the preferences of respondents for products made from recyclable materials and their favored qualities. By grading the preferences from 0 to 10, where 0 represents the total inclination for new-ingredient products and 10 represents the total inclination for recyclable products, we obtained the respondent proportion for each level. From the graph, we can conclude that a relatively major proportion of people have gained awareness of environmental protection.

Also, the respondents were required to list three indices of normal products, among them security, practicability, durability, material, stain resistance, price, environmental protection, and origin. Then, we calculated the average composite score of each index by multiplying the frequency by weight and then dividing it by the number of respondents.

The graph demonstrates that people still tend to focus on the utility of products. However, only a relatively small proportion of the respondents seemed to prioritize the product’s sustainability.

This questionnaire has given us much information on the current situation of customers’ preferences when making purchases. In the future, we need to more widely promote the idea of environmental protection with our project.

6. Implementation Interview with Jiangyin Zhongda Soft Plastic New Material Co., Ltd Representative #

Background #

To achieve our goal of collaborating with other plastic recycling factories to further apply our biological degradation methodologies on a larger scale, we decided to interview Mr. Wang in search of possible investment opportunities and cooperation. Mr. Wang is the head of the factory, which focuses on producing plastic film for customers using recycled plastics.

Questions Prepared #

Given this background, we generated six questions:

1. You recently asserted that "in recent years, the group has grasped the development direction of new soft plastic materials, and continuously advanced to the high-tech, multi-functional energy conservation, and environmental protection”. May I ask what progress your factory has made in energy saving and the environmental protection of plastics?
2. What is the company’s current involvement in the sale of PET plastics?
3. Does the factory purchase raw materials or recycle and reprocess products? Could you give us the approximate ratio of the two?
4. Does your factory have a material R&D center? How much time does it take for the materials to undergo development and then production?
5. What is your opinion on the biological degradation method? Do you think there is an economic value from the perspective of policy and market prospects?
6. At present, we have imagined some possible application scenarios. Based on your experience, could you give us some suggestions? Is the idea feasible?

Feedback #

Mr. Wang first talked about the three basic principles of his company and the future of plastic recycling, namely:

1. Complete bio-degradation: The company currently uses PET raw materials to produce packaging films, using a proportional mix of new and recycled raw materials to design product formulations. However, as society develops and technology advances, various types of plastics such as PBAT, PBS, PLA, etc. have emerged that are fully biodegradable. The company, in the last two years, has paid more attention to the development and use of PET and other degradable plastics among its customers.
2. Easily recyclable: The company is currently working on using different grades of the same plastic to meet the different functional requirements of a product, which can facilitate recycling and reusing.
3. Energy-efficient and environmental-friendly: The company is gradually shifting from relying only on industrial electricity, which is made from coal and other unsustainable energy sources, to solar energy and additional environmentally sustainable energy sources. For example, the roof of his company has already been installed with an array of solar panels.

Then, Mr. Wang moved on to talk about the company's R&D model. Specifically, his company follows the process of "receiving the request from customer --> research and development based on the request --> trial production of a sample --> customer assessment, adjustment for reproduction --> final mass production." This entire process may take 1-3 years, depending on the complexity of the customers' requirements.

Lastly, Mr. Wang commented on the potential of our plan. He admitted that we are highly innovative in using enzymes to degrade plastic and have established a complete ecosystem of PET sorting, recycling, decomposition, and reuse, but cautioned that we would face some challenges as well.

1. Assuming the project works experimentally, we would need to consider how to do it on a mass scale -- doing it in the case of large-scale industrialization will have higher requirements. When degrading plastics at a company level, a significant amount of yeast and enzymes must be used. However, the selection of efficient or functioning enzymes easily done in experimental conditions would face great difficulty during mass production due to production conditions.
2. The sorting and recycling process is also challenging in terms of ensuring the required recyclables and the purity of the enzymatic degradation products.
3. China's safety and environmental protection policies for the transportation or reuse of organic chemical products are becoming increasingly strict, and the treatment of post-industrial wastewater or other materials needs to be carefully considered to ensure that there will be no discharge pollution, and the design of this link is very important.
4. At present, the capacity of PET bio enzyme degradation experimental stage is still low, and there is still considerable room for progress. The company needs to consider the project's short-term or long-term economic benefits when making investment decisions. If the project capacity continues to rise in the long run, we believe that it can improve the possibility of industrialization and strengthen the company's investment intention.

Mr. Wang's comments and suggestions on the project were significant for us and gave us a positive view of our research. Additionally, we were also provided with a follow-up on the direction and focus of the subsequent industrialized research.