When plastic was invented, little did Leo Hendrik Baekeland know the problems behind its convenience. For decades, plastic products have been carelessly discarded in landfills—or worse, into the ocean. Today, plastic is so widespread and indispensable that banning it is no longer a practical solution. However, the contaminant left by it certainly needed to be resolved. To address this, the solution comes to two major concerns: recycling or degradation.
As for recycling, it is a task everyone should bear. Sadly, few have their time dedicated to it. What most people do, intentionally or inadvertently, is to dump trash without waste sorting. Consequently, we aimed our project mainly to solve the latter concern: degradation. While in HP, we also take our effort to solve the waste sorting problem by holding target fields in our campus.
We aimed at a plastic called PLA, which is biobased as well as biodegradable. By constructing and screening a set of bacterial mutants, we identified strains of Proteinase K exhibiting enhanced ability to hydrolyze PLA, demonstrating a potential method to accelerate PLA biodegradation.
Polylactic acid (PLA) is a bio based plastic, however, seldom has it been appropriately recycled in our country, Taiwan. Accordingly, McDonald's and lots of shops stopped the use of PLA from May 2020[1]. In turn, the Environmental Protection Administration (EPA), Executive Yuan, R.O.C. (Taiwan), has prohibited PLA since August 2023[2].
Although Taiwan has a relatively well-developed recycling system for conventional plastics, PLA has not been included in the framework. Recycling operators usually lack the equipment to distinguish PLA from PET, leading to contamination. Moreover, without financial incentives or clear regulations, private sectors have little motivation to invest in PLA treatment, causing the lack of compost sites.
In conclusion, the inappropriate recycling system of PLA in Taiwan could be attributed to two dominating problems: the lack of compost sites and the hardness of sorting it from other types of plastics. With these two hampers existing, it would be a low prospect of promoting the use of PLA in Taiwan. Therefore, we hoped that we could address the issue of lacking composting facilities by enhancing the degradation of PLA. In consequence, we searched for papers mentioning the degradation of various types of bacterias. Subsequently, we found a commercialized enzyme derived from the Tritirachium album. It is proven to degrade PLA, though, not aiming at PLA. As a result, we made our effort to specialize this enzyme against PLA. Apart from that, we have also thought of a method to conquer the obstacles while PLA faces the challenge of sorting from others. It is to set up a target field which only provides products made of PLA in a closed area . In this case, there might be a chance to restart PLA.
When it comes to biodegradables, PBS, PBAT, PCL, PEA, PGA,PLA, PHA (PHB), TPS, Cellulose-based, Bio-PBS, Bio-PBAT and so on would be a choice. But if we delete the ones which are petrol-based, PLA, PHA (PHB), TPS, Cellulose-based, Bio-PBS, Bio-PBAT will be left as the options. One step further, while comparing the proportion of usage, PLA and Cellulose-based, each one third of proportion, would be the dominant material of biobased products[3]. While respecting the range the materials could apply, PLA takes the throne. Thus, we choose PLA as our project because of the following reasons: biodegradable, biobased, predominant, and applicable.
PLA is a linear aliphatic polyester. It contains myriads of ester bonds. And luckily, ester bonds can be hydrolyzed by enzymes that commonly exist in nature, including esterases, proteases, and lipases. Under industrial composting conditions (58–65 °C and ~60% humidity), PLA can be fully decomposed within several months[4]. Enzymatic degradation is also feasible, as specific hydrolases such as Proteinase K, lipases, and PLA depolymerases are capable of effectively breaking down PLA. Experimental studies have shown that under standardized industrial composting environments, PLA can be completely mineralized within 180 days[5]. Compared with other biodegradable plastics such as PBAT and PBS, PLA exhibits a relatively faster degradation rate under composting conditions, while in sharp contrast to conventional petro-based plastics like PET, PE, or PP, which are almost non-degradable.
Corn, sugarcane, cassava, and sugar beet are the typical raw materials used to produce PLA. These raw materials are first processed into glucose, which is then fermented to generate lactic acid. The lactic acid is then purified and undergoes condensation to form lactide, a cyclic dimer. This lactide is subjected to ring-opening polymerization (ROP) under the action of a catalyst, producing high-molecular-weight PLA. Finally, the PLA is processed into pellets or shaped into various products such as films, fibers, and containers for practical applications. Different from plastics which are partially made of petroleum such as PBAT and PBS, PLA is fully biobased. In other words, usage of PLA plastic reduces our reliance on petroleum resources, which in turn makes it less harmful to the Earth. As a result, it has a lower carbon footprint. What’s more, thanks to its carbon source which is derived from CO₂ and sequestered by plants, PLA contributes to carbon neutrality. Life Cycle Assessment (LCA) studies further show that PLA’s CO₂ emissions are lower than those of conventional petro-based plastics, confirming its environmental advantage[6].
According to European Bioplastics, the global production capacity of biobased biodegradable plastics in 2020 confirms that PLA and cellulose-based plastics each occupied roughly 30 percent[3]. In fact, PLA slightly leads in first place and is the most industrially mature biobased biodegradable plastic. This industrial maturity is reflected in its complete supply chain, from raw material cultivation, fermentation, and polymerization, to processing and final applications. In contrast, cellulose-based plastics often rely on blending and modifications, resulting in lower technological maturity and less consistent performance.
It is a wide range of items that PLA can become: packaging, textile, medical implants, and 3D printing. These multiple realms PLA could partake truly identifies its widespread use, whereas cellulose-based is usually concentrated at disposable utensils and plastic bags, showing a limited functionality and market versatility in contrast.
Taken together, PLA's large production capacity, industrial maturity, and wide application range establish its predominant position among biobased biodegradable plastics. These combined advantages make PLA the material of choice for further research and development, underscoring its environmental and commercial significance.
Against the backdrop of widespread restrictions on single-use conventional plastics in Western Europe[7], starch–polyester blends and PLA have been increasingly adopted in certain applications. These materials are commonly used to produce biodegradable plastic bags, such as shopping bags and agricultural bags. However, these alternative plastics have introduced three main problems.
As mentioned earlier, conventional plastic sorting systems are designed for petrochemical plastics such as PET, PS, and PP. Newly introduced plastics such as starch-based and polyester-based (e.g., PLA) materials can cause errors in the recycling system, which in turn affects the quality and efficiency of recycled petrochemical plastics, potentially leading to undesirable discoloration or molding defects. Consequently, these materials, including PLA, are rarely, if ever, recycled into secondary plastics, even when properly disposed of in recycling bins. Ironically, these materials often end up being incinerated or landfilled as waste[8].
PLA degrades very slowly under natural conditions. To improve efficiency, it often needs to be processed in designated industrial composting plants. However, most industrial composting facilities in Western Europe are primarily designed to handle food waste or agricultural residues, with relatively few dedicated to PLA. As a result, although PLA is technically biodegradable, the lack of appropriate processing channels prevents it from degrading effectively, posing potential environmental threats.
Collection, sorting, and treatment of PLA require significant effort and resources. In some cases, handling small amounts of PLA can even cost more than its production. Under such financial pressure, private companies are generally unwilling to take on these high costs. Worse, some local governments in Western Europe have ceased providing financial support, making the promotion of PLA even more challenging[9].
To address these issues, Western European countries have implemented two main strategies. First, to solve the problem of recycling channels, they have introduced separate collection systems, established regulatory certifications for biodegradable plastics such as EN 13432 or OK Compost, and encouraged cooperation among food companies, supermarkets, and composting plants to form an integrated supply chain[10]. Second, to promote PLA, governments provide substantial subsidies or tax reductions, lowering the costs borne by recycling facilities and consumers. This approach effectively addresses both the lack of composting facilities and the high recycling costs.
As the general problems and solutions have been outlined, we now turn our focus to the methods, challenges, and solutions in the PLA degradation process. PLA is known to degrade slowly in natural environments, and its high crystallinity makes it difficult for microorganisms in both terrestrial and marine settings to break it down. Current research and applications identify several degradation pathways for PLA:
However, each of these approaches faces practical limitations. Hydrolysis requires high temperature and humidity, making it mostly effective only in industrial composting environments. Enzymatic and microbial degradation are milder but slow, limited by PLA’s crystallinity and hydrophobicity. Photodegradation and thermal degradation require specific environmental stimuli. Consequently, although PLA is classified as a “biodegradable plastic,” it can persist for long periods in natural environments—an issue that must be acknowledged and addressed.
Among these approaches, enzymatic degradation presented specific challenges. By integrating the concept of using microorganisms from iGEM, we ultimately developed our own solution to address this problem.
This year, our goal was to promote the replacement of petroleum-based plastics with PLA in Taiwan by developing enzymatic solutions to overcome its slow degradation. While reviewing the literature, we found that although several enzymes have already been commercialized for industrial use, none of them were specifically designed for PLA degradation. This gap suggested that there remains considerable potential to optimize PLA-degrading activity. Therefore, we set out to identify existing enzymes with PLA-degrading capacity, evaluate their efficiency, and further enhance their activity through mutagenesis and screening.
In the first stage, we selected four enzymes as our wild-type candidates: Proteinase K from Tritirachium album (PK), Alcalase from Bacillus licheniformis (AC), Lipase B from Candida antarctica (CA), and a PLA depolymerase (DP) identified from the literature with relatively high substrate specificity toward PLA. These genes were synthesized and cloned into the pET-28a vector for IPTG-inducible expression in E. coli. Following verification, the expressed proteins were subjected to activity assays. Enzyme activity was first evaluated using the pNPB assay, a quick and reliable method to measure ester bond hydrolysis, followed by testing PLA degradation through lactate production assays. Our results confirmed that while all four enzymes exhibited activity toward PLA, their efficiencies varied, and even the PLA-specific DP showed room for improvement. These findings highlighted that the current enzymes were not yet optimized for PLA degradation and called for further engineering efforts.
Building upon these insights, we entered the second stage by generating large numbers of mutants and employing auto-induction for high-throughput screening. Using the same cloning and expression system, we produced mutant libraries and subjected them to the same pNPB and lactate production assays to rapidly identify promising variants. Through this iterative process, we successfully obtained enzyme variants with significantly improved hydrolytic activity on PLA compared to their wild-type counterparts.
From these two experimental cycles, we learned that although PLA-degrading enzymes already exist, they are not specialized enough to serve as an efficient biodegradation tool. By applying mutagenesis and auto-induction screening, we demonstrated that enzyme activity can be substantially enhanced, pointing toward a feasible pathway for engineering PLA-specific hydrolases. Looking ahead, we plan to continue optimizing these enzymes, exploring combinations of directed evolution and rational design, to ultimately develop PLA-degrading biocatalysts suitable for real-world applications.
Our project provides substantial value by addressing degrading limitations inherent in existing methods. Traditional approaches often face challenges such as limited operational efficiency and constrained adaptability, which can restrict their broader applicability. In contrast, our project is designed to enhance efficiency while maintaining flexibility across different scenarios. By overcoming these limitations, the project offers a more effective and sustainable solution, demonstrating clear advantages over conventional methods in terms of performance, applicability, and long-term utility.