Source: CORNELL UNIVERSITY submitted to NRP
ENZYMATIC REMEDIATION OF MICROPLASTICS IN FOOD, WATER, AND AGRICULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032575
Grant No.
2024-67011-42996
Cumulative Award Amt.
$180,000.00
Proposal No.
2023-11553
Multistate No.
(N/A)
Project Start Date
Aug 15, 2024
Project End Date
Aug 14, 2027
Grant Year
2024
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
Over the past 50 years, the global production of polyethylene terephthalate (PET) plastic has surged to 80 million metric tons annually due to its durability and cost-effectiveness. However, less than 20% of PET is recycled, with the rest ending up in landfills or incinerated, contributing significantly to plastic pollution. PET breaks down into microplastics, posing environmental risks, including contamination and toxicity that affect plant and soil health. A promising solution involves the enzyme PETase (PETase), which can break down PET, producing water-soluble byproducts with minimal environmental impact. While research has explored PETase's mechanisms and enhancements, practical applications remain limited.This research focuses on immobilizing PETase onto solid supports like activated charcoal (AC), commonly used in water purification, to improve its stability and reusability in extreme conditions. This approach aims to enhance microplastic degradation in water treatment systems, addressing a significant research gap in the application of PET hydrolase enzymes for bioremediation. Key objectives include optimizing the immobilization conditions and demonstrating the effectiveness of immobilized PETase in degrading microplastics in simulated water systems. This research aims to provide practical solutions for integrating PETase into industrial water treatment, ultimately improving water quality and reducing environmental plastic pollution.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330210104050%
1335350104025%
1330199100025%
Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
5350 - Domestic and community water supply facilities and systems; 0210 - Water resources; 0199 - Soil and land, general;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The goal of this project is to conduct research on biological methods to remove polyethylene terephthalate (PET) and develop an activated charcoal (AC) immobilized PET degrading enzyme (PETase) for use in municipal water treatment plants.Objective 1: Preliminary Research. Determine optimal binding and immobilization conditions for PETase. Identify the reusability and recovery of immobilized PETase.Objective 2: Application Research. Create a simulated water treatment filtration system to demonstrate the use of immobilized PETase. Evaluate the enzymatic activity within this simulated filtration system and on polyester-based consumer plastics that may be found in the environment.
Project Methods
Objective 1: PETase target sequence and AC binding protein will be put into a commercial vector. Polymerase chain reaction and High Fidelity Gibson Assembly Method will amplify and join DNA fragments for transformation into E. coli DH5α. The DNA sequence will be sequenced after purification using QIAprep Spin Miniprep Kit. Purified DNA will be transformed into an expression strain of E. coli, BL21(DE3). Protein expression will be carried out in Luria Bertani supplemented with Ampicillin (LB-Amp) broth and then inoculated at into auto-induction media and expressed overnight. Protein purification will take place at 4°C and will be purified by His-Pur Cobalt Resin following manufacturer's instructions. Eluted proteins will be applied to desalting columns to remove imidazole. Protein content of concentrated proteins will be measured by Bicinchoninic Acid Assay (BCA) and put in varying conditions to test AC immobilization capacity. A range of binding time, temperature, and pH will be tested. Enzyme activity is determined by High-Performance Liquid Chromatography (HPLC) of degradation products. pH and temperature stability of free and immobilized enzymes will be compared using Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Once binding conditions have been optimized, reusability of PETase will be tested evaluating the residual activity in the washes. Recovery of immobilized enzymes will be evaluated to quantify recovery yields using BCA and SDS-PAGE. Objective 1 will result in (1) Verification of genetic modifications via Sanger Sequencing. (2) Confirmation of purified enzyme and affinity towards AC. (3) Quantification of free and immobilized PETase activity through HPLC analysis of PET degradation products. (4) Demonstration of immobilized PETase reusability and recovery.Objective 2: Building from Objective 1, purified enzyme will be used for application on AC filters. Using the optimal binding conditions determined in Objective 1, PETase will be immobilize onto the AC filters. Binding efficiency onto AC filters, in simulated environments will be tested. Flowthrough will be collected and tested for protein content using BCA to determine immobilized enzyme content. Contact time required by PETase for PET degradation will also be tested on a bench scale continuous flow system with manually incorporated PET powder and flowthrough evaluated via HPLC analysis. Lastly, I will collect a variety of postconsumer polyester-based plastics to test the hydrolytic capacity of PETase. Post-consumer plastic degradation will carried out for at least 10 days. Objective 2 will result in (1) Confirmation of enzyme immobilization onto AC filters. (2) Collection and analysis of flowthrough for enzyme binding capacity. (3) Application of continuous flow system to test PET hydrolytic capacity through a filter. (4) Inspection of degraded postconsumer plastics.

Progress 08/15/24 to 08/14/25

Outputs
Target Audience:During this reporting period, I successfully engaged a diverse range of audiences through media (for the general public), scientific conference posters and presentations (for researchers and industry partner/representatives) focused on the development and application of immobilized PET degrading enzymes that will address plastic pollution through improved wastewater treatment. The first major engagement occurred at the Institute of Food Technologists Annual Meeting. I delivered both an oral presentation and a poster presentation highlighting the benefits of immobilized enzyme-based technology to break down PET. The audience included food and agricultural industry professionals, university faculty and researchers, and undergraduate and graduate students. The next major engagement was multiple poster sessions and an oral presentation at the Gordon Research Symposium and Conference on Protein Engineering. Gordon Research Conferences are a highly regarded scientific forum on in-depth discussions of cutting-edge research. Specifically, this conference audience included biotechnology industry representatives, academic researchers, faculty, and graduate students. These outreach activities provided platforms to share scientific findings and fostered discussions with stakeholders who could influence the adoption and scaling of these immobilization technologies. Members of associated university groups, namely the advisory council, were also reached through posters and presentations at their group meetings. These efforts helped in bridging the gap between laboratory research and real-world implementation, an essential step in addressing environmental challenges. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has created valuable opportunities for science communication and technical skill development. Practice in communicating scientific concepts in both formal and informal settings, while also learning how to tailor the message to diverse audiences has been invaluable training from this project. This ability to adapt communication styles is a critical component for professional development in technical fields. This project has a clear emphasis on hands-on training for core scientific competencies, particularly in enzyme engineering and advanced laboratory techniques. These skills are directly applicable to careers in biotechnology and other related industries. Through this project I can contribute to novel research and presenting these findings at scientific conferences, where I can also gain exposure and expand my professional networks in food, agriculture, and biotechnology, enhancing my career development and understanding of real-world applications. How have the results been disseminated to communities of interest?Currently the results that have been achieved have been disseminated to communities of interest through research presentations (3 poster and 2 oral) at conferences. When ready, the results will be disseminated as research papers referred to journals. There has also been coverage of this work in media. What do you plan to do during the next reporting period to accomplish the goals?I plan to continue working diligently on the project to complete the next objectives and achieve the final goal. In the next reporting period, I intend to work towards completing Objective 1(b) to determine the binding capacity and activity of my modified enzyme in reusability studies. I also intend to develop my simulated environment to test my enzymes in Objective 2. In the next reporting period I also plan to present my research at at least one national conference. Additionally I expect to publish at least one additional paper in the coming year.

Impacts
What was accomplished under these goals? Within the major goals, I am on track with the completion of Objective 1(a) by engineering and determining immobilization conditions for the engineered PETase constructs. I have verified the genetic modifications through whole genome sequencing. Binding conditions have been determined but activity measurements are to be conducted. I am beginning work on Objective 1(b) and determining the activity, binding, and reusability of the PETase constructs that have been engineered. This significant progress was made by having preliminary research done through silica binding PETase work where linker choice was informed for this plasmid construction. Methods for protein expression, purification, and activity quantification have been developed and optimized for the parent enzyme. A thorough review of the literature has informed decisions in the research by finding material analogues to ensure that there is directed immobilization.

Publications

  • Type: Other Journal Articles Status: Submitted Year Published: 2025 Citation: Su, S., Pagar, A.D., Goddard, J.M., Silica immobilized PETase for microplastic bioremediation: Influence of linker peptides on activity.
  • Type: Other Journal Articles Status: Submitted Year Published: 2025 Citation: Su, S., Goddard, J., Synthetic Biology Approaches to Enzymology in Food and Agriculture Systems.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Su, S., Goddard, J.M., Immobilization of PETase for microplastic bioremediation (poster), Gordon Research Conference, Protein Engineering 2025 Conference and Symposium, Smithfield, RI, 30 July 2025.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Su, S., Goddard, J.M., Immobilization of PETase for microplastic bioremediation (poster), Gordon Research Symposium, Protein Engineering 2025 Conference and Symposium, Smithfield, RI, 27 July 2025.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Su, S., Goddard, J.M., Immobilization of PETase for microplastic bioremediation (oral presentation), Gordon Research Symposium, Protein Engineering 2025 Conference and Symposium, Smithfield, RI, 26 July 2025.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Su, S., Goddard, J.M., Enzymes for Bioremediation of Microplastics in Food (poster), Western New York Institute of Food Technologists, Western New York Institute of Food Technologists Poster Session, Ithaca, NY, 25 October 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Su, S., Goddard, J.M., Silica immobilized PETase for microplastic bioremediation (oral presentation), Institute of Food Technologists, Institute of Food Technologists Food Improved by Research, Science, and Technology Annual Meeting, Chicago, IL, 15 July 2025.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Su, S., Goddard, J.M., Silica immobilized PETase for microplastic bioremediation (poster), Institute of Food Technologists, Institute of Food Technologists Food Improved by Research, Science, and Technology Annual Meeting, Chicago, IL, 14 July 2025.