Engineering

Overview

BAID - China is dedicated to addressing the increasingly severe issue of microplastic contamination in bottled water and beverages. We have developed an eco-friendly and efficient biobased filtration membrane using synthetic biology methods, which can achieve over 90% microplastic removal. This groundbreaking filtration device is specially designed to fit onto environmentally friendly and renewable materials used in bottle caps. It is expected to be available for mass production at a price not exceeding $0.25 per unit, with an estimated reuse capacity of 20-30 times.

In addition, we have introduced a calculator program to help consumers estimate their daily intake of microplastics. This tool aims to raise awareness about microplastic ingestion, encouraging individuals to reduce their reliance on plastic products in their daily lives.

For the biobased filtration membrane, we employ bacterial cellulose as the cellulose matrix. To enhance adsorption efficiency, we first engineer oat proteins into the cellulose matrix using cellulose-binding modules (CBMs). We have created a triblock structure component called "CBM3-Globulin-CBM3" to strengthen the binding between cellulose and oat proteins. This combination forms the foundational membrane for filtering microplastics, along with CBM3-OP-CBM3 composite proteins. Furthermore, we tested the addition of substances like APT, tannic acid, and catechins to increase microplastic adsorption sites on the membrane. In microplastic removal efficiency tests, we utilize a microscopic Raman spectrometer to characterize the composition (types) of microplastics in water. We have pioneered an AI-based algorithm for high-precision microplastic counting in drinking water.

For the bottle cap structure, we experimented with using a common agricultural waste, sugarcane bagasse. We combined cellulose microfibers and lignin extracted from sugarcane bagasse to create an alternative material for bottle caps, replacing traditional plastic materials. Surface treatment with sodium alginate and calcium lactate made the bottle caps waterproof and resistant to deformation. We manufactured the mentioned bottle cap structures using 3D printing technology and mold methods, reducing individual bottle cap production costs to only 0.2% of traditional machining methods.

We hope our product will become an effective alternative to plastic bottle caps in the bottled water market, potentially reducing the production and usage of billions of plastic bottle caps each year and lowering consumer microplastic ingestion by 40%-60%.

Target 1: Production of Oat Protein

• Design

• Oat protein is a globulin extracted from byproducts of processing oat β-glucans. In recent years, oat β-glucans have gained prominence due to their health benefits in reducing blood cholesterol and regulating blood sugar levels. Consequently, they have been incorporated into 76 different health-related components in food and cosmetics. Oat protein is favored for its low cost and environmental sustainability.

  The decision of taking oat protein as an additive is grounded in the research conducted by Wang and colleagues in 2021. Their study reported that oat protein sponges exhibited a removal efficiency of 75% to 82% for PS particles (a form of microplastic). Their model indicated that the binding sites for these microplastics on the sponges are likely hydrophobic interactions between protein side chains and the phenyl rings on PS. Considering that they primarily produced oat protein hydrogels through chemical cross-linking and subsequently created oat protein sponges through liquid nitrogen freezing and freeze-drying, a process that minimally affects the protein's side chain groups, we theorize that oat protein possesses the necessary characteristics for adsorbing microplastics. Therefore, we have chosen to incorporate it into our biomaterials.

• Build
• Using the method proposed by Wu et al. in 1977, oat flour, which had been defatted using n-hexane and sifted to remove bran, was subjected to multiple centrifugation cycles and freeze-dried to obtain oat protein powder (as elaborated in the notebook).
• Test

• 

  We conducted SDS-PAGE experiments on the extracted proteins, with results shown in the figure above. The result revealed prominent bands at 22kDa, 32kDa, and 55kDa, which is consistent with the research conducted by Shotwell et al. in 1900. Their research identified that oat protein 12S comprises two subunits of 22kDa and 32kDa, which would be separated during electrophoresis. The presence of a band near 55kDa suggests the existence of 7S proteins, while other bands correspond to other proteins. Consequently, it can be inferred that the extracted oat protein exhibits a purity ranging from 60% to 70%, which is within the normal and slightly higher range.
• Learn
• Through this engineering cycle, a convenient method for oat protein extraction has been successfully proposed and validated using readily available materials. Additionally, the corresponding part, BBa_K4714002, have been created, allowing future teams to easily choose different methods for oat protein extraction as per their experimental requirements. Notably, oat protein has remained unexplored in previous iGEM teams. Owing to its pertinent attributes and straightforward extraction, oat protein holds great potential as a research focus for future teams in the fields of nutrition, agriculture, and related disciplines.

Target 2: Oat Protein as an Additive to the Base

Target 2.1 Maillard reaction

• Design
• To assess the impact of oat protein(OP) on the filtration efficiency and determine the optimal ratios, the following experimental groups were established. This section will focus on the first five groups.

Index	Base	Additive
1	BC Only
2		OP only
3	BC	OP concentration 1
4	BC	OP concentration 2
5	BC	OP concentration 3
6	BC	CBM3-CBM3
7	BC	CBM3-OP-CBM3

The first and second groups serve as control groups. BC membranes were prepared by vacuum filtering BC suspensions in the first group. The second group, on the other hand, involved the production of a hydrogel using oat protein powder and then freeze-dried, creating a structure akin to the oat protein sponge mentioned prior in the work of Wang et al, 2021.

The Maillard reaction occurs when proteins and carbohydrates are mixed at elevated temperatures (Kim & Lee, 2008). For instance, Lii, Chen, Lu, and Tomasik (2003) reported the formation of covalently bonded CMC-casein complexes via electrosynthesis. These complexes exhibit exceptional stability in response to changes in pH and ionic strength, alongside excellent emulsification properties and thermal stability. Hence, the approach taken here involves the incorporation of oat protein into BC through the Maillard reaction, using a simple blending process. Blending is an effective method for polymer modification, with strong economic incentives, as it imparts enhanced properties to polymers through conventional processing techniques. After pouring the blended solution onto stainless-steel plates coated with an anti-sticking agent, then drying at 50 degrees Celsius for 5 hours in a drying oven, the membrane can be readily separated from the stainless-steel plate.

• Build
• To determine the optimal dosage of oat protein, a ratio gradient was set while preparing the oat protein solution: 5 wt%, 10 wt%, and 15 wt%. The first five groups were prepared based on the experimental process detailed in the notebook.
• Test
• The experimental groups with varying concentrations of oat protein were tested for their microplastic filtration ability using the method described on our measurement page (link). A microplastic suspension for standardizing the filtration efficiency test was prepared by diluting PS microspheres with diameters of 100nm, 1μm, 5μm, and 10μm. Following measurements, it was determined that the optimal ratio for oat protein was 15wt%.
• Learn

• [Technical Iteration]
  Maillard Reaction Round 1
  Problem 1: There’re visible cavities on the protein membrane surface, suspected to be a result of bubbles introduced during the stirring process, becoming apparent during pouring. This phenomenon undoubtedly compromises the filtration effectiveness of the membrane.

  Problem 2: The mechanical properties of the protein membrane are suboptimal. While it can withstand the pressure exerted by water flow, it is relatively fragile, posing challenges in industrial operational settings.

  Maillard Reaction Round 2
  Problem 1 Resolved: Vacuum degassing was performed before pouring the solution onto stainless steel plates. In cases where this process proved inefficient, brief ultrasonication (10-20 minutes) was introduced. As a result, no longer were holes observed on the second batch of membranes due to bubble formation.

  Problem 2 Resolved: By incorporating some AR (analytical reagent) level glycerol into the membrane, substantial enhancements were achieved in the mechanical properties of the membrane, particularly in terms of increased tensile strength.

Target 2.2 Cellulose Binding Module (CBM)

• Design
• Wet Lab Design

  The cellulose-binding module (CBM) is a specific region on certain cellulase enzymes comprised of amino acids with aromatic residues (tyrosine or tryptophan). These residues establish firm bonds with cellulose chains through van der Waals interactions (Perez & Samain, 2010). CBMs have found extensive and enduring application in the design and modification of protein-polysaccharide composites (Lapidot et al., 2012). In our experiment, we employed CBM3 from Ruminiclostridium thermocellum (Protein Data Bank (PDB) accession: 1NBC). This component was initially proposed by LinksChina 2021. Previously, the Imperial College 2014 team used the cellulose-binding domain, BBa_K1321356, individually as an additive to enhance the adsorption of heavy metal pollutants by bacterial cellulose membranes. Moreover, research by Mohammadi et al. in 2019 indicated that CBM3-CBM3 dimers, when binding to cellulose, exhibited greater mechanical strength and pressure tolerance compared to CBM3 monomers combined with cellulose. Therefore, CBM3-CBM3 was designed and expressed for testing CBM3's adsorption capabilities.

  Furthermore, due to the special structure of CBM3 - featuring regions with affinity for both cellulose and proteins, it can emulate the function of natural cellulose cross-linkers. This ability ensures them to secure various proteins onto cellulose to produce cellulose-CBM and fusion protein composites. The "triblock protein architecture" model proposed by Mohammadi et al., involving CBM, functional proteins, and bacterial cellulose, provides a pathway for manufacturing highly adjustable composite materials. In our project, we adapted this binding pattern, substituting spider silk proteins with oat proteins. This approach retains the adsorption sites on oat protein side chains while introducing the CBM3 component, thereby maximizing the efficiency of microplastic adsorption.

  In the study, we designed the amino acid sequences of CBM3-CBM3 and CBM3-Globulin-CBM3 fusion proteins, and reverse-translated and codon-optimized their expression sequences based on the Escherichia coli codon usage. Prior to assembling the expression sequences, we conducted transmembrane region prediction, hydrophobicity analysis, and disorder analysis on the designed sequences. After confirming that the designed sequences met the desired criteria, the sequences were synthesized. The synthetic sequences were then introduced into the pET28a vector using BamHI-HindIII restriction ss sites for plasmid construction. The constructed plasmid was subsequently introduced into Escherichia coli strains for induction expression.

  Following the completion of plasmid assembly, we performed protein purification using a His tag and conducted preliminary analysis of expression results through SDS-PAGE electrophoresis. Furthermore, although CBM3 has been confirmed for soluble expression in Escherichia coli on multiple occasions, the solubility expression of oat 11S globulin, which has been less studied, remains unknown. Even if the oat 11S globulin itself exhibits good solubility expression, it is uncertain whether the fusion protein will alter this desirable characteristic. Therefore, we induced expression in modified bacterial strains and conducted soluble expression analysis on their protein products, obtaining a solubility result of 0.8504. This further confirms the utility of the designed fusion protein.

  

• Build
• Tsingke Biotech used Escherichia coli TOP10 to synthesize the designed plasmid, and helped us with protein expression and purification. The obtained proteins were used for further experimental analysis.
• Test
• The experimental group that only contains CBM3-CBM3 and BC achieved a removal efficiency of 79.77% for the microplastic mixture. In the group containing OP (CBM3-OP-CBM3), the removal efficiency increased to 91.70%. This result aligns with the outcomes of computer simulations, suggesting that specific functional groups on the target protein's side chains, as well as its isoelectric characteristics, may facilitate electrostatic and hydrophobic interactions with microplastics, enhancing the adsorption of composites to microplastics. The increase in adsorption efficiency proves the effectiveness of our system.
• Learn

• 

  To evaluate the interaction between proteins and membranes, an Energy Dispersive Spectrometer (EDS) analysis was conducted on samples containing BC+Oat membrane, BC membrane, and BC+CBM-Globulin-CBM+APT membrane. The obtained results demonstrated the occurrence of nitrogen in both BC+Oat membrane and BC+CBM-Globulin-CBM+APT membrane, with a measured nitrogen content of 10.8%wt. In contrast, the absence of nitrogen in the BC membrane confirmed the successful binding of proteins to the membrane.

Target 3: APT as an Additive to the Base

• Design

• Attapulgite (APT), a low-cost and abundant clay mineral, exhibits a porous, rod-shaped crystalline structure, a large surface area, and high cation adsorption capacity. It is frequently employed in anti-diarrheal medications (Haden et al., 1967). Research has demonstrated that BC-APT membranes (BAM) produced by combining APT with bacterial cellulose possess excellent microplastic adsorption capabilities (Zhang et al., 2023). The irregular, randomly distributed clusters formed by BC nanofibers crosslinked with APT bundles increase water flux, generating an abundant and uniform pore structure ideal for microplastic filtration (see figure). Furthermore, microplastics in water experience a repulsive effect with APT as they pass through the membrane due to their weak negative charge, preventing PS particles from entering the membrane pores. This allows the solvent in the filtrate to pass through, maintaining a high flux for BAM during water filtration.

  Therefore, we aimed to add APT as an additive to the base membrane for its superior effect. Since a relatively toxic crosslinking agent was used during the production process of BAM, 3-aminopropyl triethoxysilane, the actual application of APT as a necessary component is still under consideration. However, the wide-spread application of 3-aminopropyl triethoxysilane in tissue engineering and other areas that requires no toxicity and the toxicity test performed in both literature and our wet lab proves the little effect of APT under the BAM circumstance.

• Build

• BAM membranes were fabricated using the method described by Jiang et al., 2023. In essence, they were prepared through vacuum filtration of a mixture of BC and APT, followed by in-situ crosslinking.

  Firstly, a pre-treatment of APT was conducted. APT was placed in ultrapure water, stirred, and allowed to settle for 8 hours, resulting in the collection of fine particles in the intermediate layer. The obtained APT was then acidified in HCl, and the mixture was subjected to ultrasonication for 30 minutes to ensure the removal of any internal organic matter. Subsequently, the APT was ultrasonically washed in ultrapure water until the wash solution reached a neutral pH. Finally, it was dried and sieved.

  The BC-APT membrane was prepared by vacuum-assisted filtration of a BC/APT suspension onto a PVDF membrane. Typically, a specific quantity of APT powder and 3-aminopropyltriethoxysilane were added to the BC suspension, and the mixture was sonicated for 30 minutes. The suspension was then vacuum-filtered, and the resulting composite membrane was dried at ambient temperature with an APT weight ratio of 5% to the BC.

• Test
• Characterization

  Characterization of BAM was conducted using scanning electron microscopy (SEM). Fiber structures highly resembling those reported by Jiang et al. were observed, providing partial confirmation of the successful production of BAM.

  Toxicity Test

  Due to the addition of 3-aminopropyltriethoxysilane in the production of BAM, a commonly used chemical cross-linker in tissue engineering and materials science, it can immobilize functional molecules on various materials but carries some toxicity and environmental impact. In order to investigate the potential toxicity of MPs/NPs on water quality after filtration, acute toxicity tests were conducted using D. magna Straus as a model organism. D. magna Straus was cultured for 1 to 3 generations, and individuals aged between 6 hours and 24 hours were selected for the experiment, with 10 water fleas in each group. Filtrate from BAM filtration was used for the toxicity tests, and water was used as a control. Each group was exposed in triplicate, and the water fleas were maintained under starvation conditions during the experiment. Monitoring was carried out at 24 and 48 hours to determine the number of water fleas exhibiting activity inhibition (including mortality) as an indicator of the toxicity of the experimental solution.

  The data from the four experimental groups were analyzed, and the results are presented in the table and graph.

  

  A two-way ANOVA analysis was conducted on the data related to D. magna Straus activity inhibition. The analysis revealed a p-value of 0.896 for the interaction between D. magna Straus activity and BAM filtrate treatment. This p-value does not reach statistical significance at the alpha level of 0.05. Therefore, there is no statistically significant difference in the activity of D. magna Straus between the two groups, confirming that BAM filtrate is not toxic or exhibits minimal toxicity.

  Removal Efficiency

  The measured removal efficiency of BAM for the MP mixture was 96.17%, while Jiang et al. reported an efficiency of 99.81%. The discrepancy between these results may stem from slight human errors, or it could also be attributed to the use of a broader range of PS microspheres in our MP suspension (Jiang et al. employed 100nm, 0.5μm, and 1μm PS microspheres, whereas we used microspheres with diameters of 100nm, 1μm, 5μm, and 10μm). However, overall, the observed filtration effectiveness of BAM for microplastics is excellent. This suggests that APT, as the sole additive on the BC membrane, plays a primary role in enhancing filtration performance.

• Learn

• Toxicity Testing Round 1
  Problem: D. magna Straus, when cultured under normal conditions, did not survive for unknown reasons.
  Toxicity Testing Round 2
  Problem Resolved: By introducing a culture medium for green microalgae and regularly changing the culture medium, there was a significant increase in the reproduction rate of D. magna Straus. From 3 newborn D. magna Straus individual per day for every 10 individual group, to up to 15 newborn D. magna Straus individual per day for every 10 individual group.
  Toxicity Testing Round 3
  Reflection: D. magna Straus, as a simple planktonic crustacean, have limited representativeness in toxicity tests. In the future, it will be necessary to conduct toxicity testing using human tissue cells or other models that better mimic real-life scenarios. The reason for not using these cells is the lack of suitable laboratory conditions and animal testing certification.

Target 4: Production of the Support structure

4.1 Composition of the Support Structure-- Cellulose-Lignin Reinforced Composites

1. Design

2. Inspired by the principle of cellulose and lignin reinforcement found in natural wood, we developed all-natural, biodegradable, strong, and water-stable cellulose-lignin reinforced composites by integrating lignin into micro-nanocellulose fibers as a replacement for plastic. Following the method described in [Reference], the cellulose-lignin composite consists of approximately 74 wt% cellulose fibers (a blend of cellulose microfibers and nanofibrillated cellulose (NFC) for cost-efficiency and mechanical performance balance) and approximately 26 wt% uniformly mixed lignin powder, forming a wet film. The lignin content closely resembles that of natural wood. The untreated fragments of natural wood still maintain the intrinsic bonding between lignin and cellulose, which is somewhat unfavorable for achieving high mechanical performance and water resistance. Therefore, the use of commercially available cellulose microfibers, nanofibers, and lignin components allows us to chemically process and rebuild the cellulose-lignin reinforced composite structure, thus achieving high mechanical performance and water stability in the resulting support structure.

   

   To demonstrate the superior mechanical strength and water stability of cellulose-lignin reinforced composite materials, we further fabricated drinking straws from this wet film, followed by baking in an oven at 150°C. Due to the abundant hydroxyl groups in cellulose molecular chains and lignin, hydrogen bonds are readily formed between cellulose fibers and lignin during the drying process. Consequently, the newly formed hydrogen bonds between cellulose fibers and lignin effectively adhere to the edges of the film without the need for additional additives. Baking at temperatures higher than the glass transition temperature of lignin allows molten lignin to penetrate the pores of cellulose fibers, forming a permeable and dense structure.

   Cellulose-lignin reinforced composite straws exhibit excellent properties, including:

   1. Outstanding mechanical performance due to crosslinking between cellulose and lignin.
   2. High water stability resulting from the hydrophobic nature of lignin.
   3. Cost-effectiveness.
   4. Biodegradability, offering potential as an environmentally friendly alternative to petroleum-derived plastic straws.

   Cellulose fibers have a high degree of hydrophilicity due to the presence of numerous active hydroxyl groups on their surfaces. As a result, water molecules penetrate the dense hydrogen bond network, causing it to expand and subsequently reducing mechanical strength, resulting in insufficient wet strength. To improve the user experience, we chose to introduce a thin layer of sodium alginate to the surface of the support structure, which was then converted into calcium alginate. As an edible natural polysaccharide polymer, sodium alginate can infiltrate the pores of the 3D nanoscale cellulose network. Both cellulose and alginates are polysaccharides with many hydroxyl groups on their surfaces, which implies that a considerable number of hydrogen bonds can be formed at the interface, resulting in a strong connection. The addition of alginates, along with the penetration of lignin, constitutes two steps that increase the adhesion at the support structure joints, thus avoiding the use of chemical adhesives commonly found in other biomaterials like paper straws.

   Compared to paper and plastic, cellulose-lignin reinforced composite materials can simultaneously achieve high water stability and biodegradability.

3. Build

4. According to the designed methods and plans, two components were produced:

   1. Composite material thin rectangular prisms used for performance testing.
   2. Real bottle cap structures prepared using molds and baking.
5. Test

6. Due to a lack of the required equipment for mechanical performance testing, we were unable to conduct three-point bending tests or other mechanical tests on the support structures. However, literature mentions that the tensile strength of cellulose-lignin reinforced composite material (75.2 MPa) is 1.88 times higher than that of plastic straw materials (40.0 MPa), replacing the original plastic components with the support structures. Therefore, it is reasonable to assume that the increased waterproofing, due to the surface treatment, should result in superior performance compared to the literature.

7. Learn
8. Surface Treatment Round 1 Issue: During the first immersion test (without surface treatment, only baking), we observed noticeable darkening and slight expansion in the portions of contact with water. After an internal team discussion, it was unanimously agreed that this may reduce the user's experience and trust in the product. Surface Treatment Round 2 Issue Resolved: By conducting further literature research, we discovered a surface treatment technique originally used for producing BC-based straws. We applied this technique to our composite materials and achieved positive results. Surface Treatment Round 3 Reflection: Due to equipment and time constraints, detailed performance testing has not been conducted yet. This aspect will need to be addressed in the future.

The production of the Mold

• Design

• 

  The original plan was to use stainless steel molds for the most precise control of microplastic contamination. However, due to cost constraints (creating a stainless steel mold from scratch costs nearly 10,000 RMB or approximately 1,370 dollars), we decided to use 3D printed heat-resistant flexible nylon molds to produce the support structures. As shown in the figure above, the envisioned support structures were modeled using Blender and SolidWorks, and the initial drawings were used for test printing.
• Construction and Testing

• Initially, common 3D printing material PLA (in pink) was used to print plastic support structures to test the size compatibility between different components. After three rounds of test printing and adjustments, the dimensions at the bottle neck thread were modified to ensure a perfect fit with the threads commonly found on bottled water in the Chinese market, and to prevent water leakage when inverted, ensuring a good seal.

  Once the final dimensions for the support structures were confirmed, SolidWorks was used for the mold design. Elastic molds made of nylon were then printed to facilitate demolding of threaded structures.

• Learning
• Mold Design Reflection:

  Given the constraints of time, cost, and technical capabilities, the mold design currently in use is relatively simple and rough. To explore the industrial application of Plasticlear, it will be essential to investigate methods for demolding molds with internal threads using stainless steel molds, which are rigid and lack elasticity.

Summary

Through the engineering cycles described above, we successfully produced all the components of Plasticlear, assembled them, and ultimately, the experimental results confirmed the feasibility of our theoretical design and the outstanding filtration performance of Plasticlear.

It is evident that as a high school student team, there are still many areas in the industrial application phase that require improvement. However, for each part involved, we conducted a complete engineering cycle to test the component's usability and continuously enhance its overall performance.