The Hazards of Polyethylene terephthalate

PET (polyethylene terephthalate), one of the most widely used plastics worldwide, has caused multi-dimensional hazards to the environment, ecosystems, and even public health due to its poor degradability and improper disposal methods—and this serves as the core driver for launching relevant projects. From an environmental perspective, PET has a natural degradation cycle of hundreds of years. Discarded PET bottles, packaging films, and medical supplies tend to scatter in soil, water bodies, and ecologically sensitive areas. For instance, in Hunan Badagongshan National Nature Reserve, PET accounts for over 70% of total waste: it not only seeps into soil to damage soil fertility but also pollutes drinking water sources. This forms long-term "white pollution" that is hard to eliminate, continuously eroding the environmental foundation

The harm of PET to wildlife is equally direct and fatal. Birds, mammals, and aquatic organisms often mistake fragmented PET for food. After ingestion, PET can block their digestive tracts, leading to death by starvation. Staff at the Badagongshan Reserve have discovered multiple cases of wildlife dying from plastic ingestion. A more hidden risk lies in microplastics: as PET degrades over the long term, it produces microplastics that enter the food chain and accumulate layer by layer via the bioaccumulation effect. This may eventually threaten human health, forming a cross-species ecological risk chain.

Current Situation and Challenges

Traditional PET disposal methods—landfilling and incineration—fail to eliminate pollution and even trigger secondary hazards. Landfilling occupies large amounts of land resources; moreover, during PET’s slow degradation, microplastics in landfill leachate can seep into groundwater, and their environmental risks have not yet been fully evaluated. While incineration can reduce volume and recover energy, mixing PET with chlorine-containing plastics (e.g., PVC) during combustion may increase HCl emissions and release toxic pollutants like dioxins. Even with advanced emission control technologies, completely eliminating these pollutants remains difficult. More importantly, this method treats recyclable PET as "waste," fully wasting its potential for high-value utilization.

Conventional physical recycling of PET also has significant limitations and falls into a "downcycling loop." After 3–5 cycles of melting and granulation, PET’s intrinsic viscosity (IV value) drops from an initial 0.8–0.9 to 0.4–0.5. As a result, it can only be used to produce low-value products (e.g., low-end fibers, plastic buckets) and cannot re-enter the production chain of high-grade PET products. This inefficient recycling not only fails to unlock PET’s resource value but also drives up processing costs. Take Sangzhi County (Zhangjiajie City, Hunan Province,China) as an example: the cost of domestic waste treatment there is approximately 200 yuan per ton, yet PET’s resource potential is severely wasted due to inefficient recycling.

Even in biodegradation, PET’s hazards have not completely disappeared: if ethylene glycol (EG) and terephthalic acid (TPA)—the products of PET degradation—are not treated promptly, new problems will arise. EG exerts competitive inhibition on PET hydrolases, reducing degradation efficiency; meanwhile, accumulated TPA interferes with microbial metabolism, undermining the sustainability of the biodegradation process. This means simple "degradation" is insufficient to solve the PET problem—supporting product conversion technologies are also required.

It is precisely these multi-dimensional hazards—difficulty in degradation, high pollution from disposal, low recycling value, and superimposed ecological risks—that have driven the development of "PET biodegradation + high-value conversion" technologies. The goal is to fundamentally resolve the dual dilemma of PET pollution and resource waste. To address these issues, the UESTC-China team has proposed a microplastic identification and upcycling solution: Repeat.

Our Solutions

Repeat is a specialized, precise, and efficient approach to address PET. Our work mainly consists of the following two parts:

1. Degrading

To tackle the problems of poor recoverability and insufficient stability of free PET-degrading enzymes, our project developed an immobilized dual-enzyme (TfCa and Tfh) system based on functionalized magnetic nanoparticles. Using K5C—a fusion protein of ELP and SpyCatcher—as a template, we prepared the magnetic carrier K5C@Fe₃O₄ via biomineralization. We then used two covalent self-assembly systems (SnoopTag/SnoopCatcher and SpyTag/SpyCatcher) to achieve ordered immobilization of the dual enzymes.

We successfully constructed three plasmids (piGEM25_01, piGEM25_02, piGEM25_03) for expressing related proteins and verified the soluble expression and purification of these proteins. After optimization (with 3.0 M NaOH as the optimal mineralization concentration), the sequential immobilization strategy (first immobilizing TfCa, then Tfh) achieved the best PET degradation efficiency, with the TPA concentration reaching 1.2 mM. After 10 cycles of recovery and reuse, the system retained 61% of its initial activity, demonstrating good operational stability and reusability. Our project realizes efficient PET degradation; future work will further improve system performance by optimizing the dual-enzyme ratio and adjusting the linker peptide structure.

2. Upgrading and Recycling

The upgrading and recycling of EG and TPA (PET’s degradation products) are the core of the project. Among them, EG is the focus: we achieve its high-value conversion through "bioinformatics analysis for target identification + wet experiments for practical conversion." Meanwhile, TPA is synchronously converted into high-value intermediates, forming a complete recycling system.

(1) Bioinformatics Recommendations

First, we conducted bioinformatics analysis to clarify the direction of research: Using E. coli MG1655 (a strain with proven EG metabolic capacity) as a reference, we performed comparative genomics analysis on BL21 (DE3). The results showed that the proportion of core orthogroups shared by the two strains exceeds 99%, and EG metabolism-related genes (e.g., gcl, fucO) show structural collinearity—confirming that BL21 (DE3) has potential EG metabolic capacity. Subsequently, we reconstructed its genome-scale metabolic model (supplementing the complete "EG→PEAs" pathway) and identified key regulatory genes (including fucO, aldA, gcl) via flux analysis (FBA, FVA, etc.), providing targets for subsequent strain engineering.

(2) Upgrading and Reconstruction

At the wet experiment level: We first constructed the plasmid piGEM25_04 (containing the key genes) and transformed it into BL21 (DE3) to generate the EG-BL21 engineered strain. This strain can grow in a medium with EG as the sole carbon source (reaching an OD600 of approximately 0.8 after 72 hours and consuming around 22 mM EG). Then, via co-transformation of piGEM25_07 (for synthesizing PEA monomers) and piGEM25_08 (for monomer activation and polymerization), we constructed the PEA02 engineered strain. Nile Red staining and fluorescence detection (with a strong emission peak at 610 nm) confirmed that EG was successfully upcycled into PEAs, a biodegradable plastic.

Highlight: Close Integration of Wet Experiments and Dry Experiments——In the high-value conversion of EG, we first used bioinformatics analysis (dry experiments) to clarify the metabolic direction, reconstruct the metabolic model, and identify key genes. We then relied on wet experiments (plasmid construction, strain modification, functional verification) to execute the conversion pathway. This forms a closed loop of "theory guiding practice and practice validating theory," upholding the scientific rigor and feasibility of the technical route.

(3) Identification of EG

EG identification faces two practical challenges: first, it is prone to interference in complex matrices, making accurate quantification difficult; second, traditional detection equipment is expensive and poorly portable, incapable of adapting to grassroots-level processing scenarios. However, accurately obtaining EG concentration is crucial for determining PET degradation efficiency, regulating the EG metabolic process of engineered bacteria, and ensuring the quality of PEA synthesis—it is a core link in the "PET degradation→product conversion" technical chain. Therefore, we adopted a combined scheme of "colloidal gold for standard curve construction + self-developed spectrometer for quantitative detection," which precisely meets the dual needs of experimental research and grassroots application.

1. The colloidal gold method serves as the foundational support for EG quantification: First, prepare wine-red gold nanoparticles; then prepare a series of EG-LB mixed solutions with concentrations ranging from 0 to 150 mM; take 0.8 mL of the gold nanoparticle solution, mix it with the aforementioned EG solution, and determine the absorbance at 525 nm with an ultraviolet-visible spectrophotometer. Finally, a linear equation (y=0.0025x+0.7085, correlation coefficient R=0.988) is fitted, laying an accurate standard foundation for subsequent quantitative calculation of EG concentration in samples.

2. The self-developed spectrometer enables low-cost, portable EG detection: It adopts Arduino UNO R3 as its core controller, equipped with 6 narrow-band LEDs (with a full width at half maximum, FWHM, of ≤20 nm) and an AS7341 visible light spectral sensor. The LEDs are driven by pulses to light up sequentially; after penetrating the sample, the transmitted light intensity is collected by the sensor. According to the Beer-Lambert law, the light intensity signal is converted into discrete absorbance readings, and a smooth 380–780 nm spectral curve is generated via cubic spline interpolation. The cost of this device is two orders of magnitude lower than that of conventional spectrophotometers, with a wavelength accuracy of ≤±3 nm. It can directly call the standard curve constructed by the colloidal gold method to quickly calculate EG concentration, meeting both the needs of accurate laboratory detection and low-cost grassroots application.

(4) Upgrading and Recycling of TPA

The focus of TPA upgrading and recycling is high-value conversion: We constructed the plasmid piGEM25_09 (harboring the TphA1, TphA2, TphA3, and TphB genes) and realized the soluble expression of the corresponding enzymes in BL21 (DE3). When the bacterial OD600 was optimized to 60, HPLC detection showed that the yield of protocatechuic acid (PCA), a pharmaceutical intermediate derived from TPA, reached 1250 μM. This not only solves the problem of microbial metabolic inhibition caused by TPA accumulation but also upgrades TPA’s resource value.