Biodegradation of Polylactic Acid: Enzymatic and physicochemical approach along with ecotoxicological assessment in Daphnia magna

Abstract

Introduction:
Biodegradable plastics such as polylactic acid (PLA) are increasingly used as sustainable alternatives to conventional plastics, yet their degradation in natural environments remains inefficient.
Background:
Although enzymes such as cutinases can hydrolyse ester bonds in PLA, their behaviour in the presence of PLA nanoparticles and potential inhibitory effects are not well understood. Additionally, little is known about how PLA degradation products interact with environmental nanoparticles or affect aquatic organisms.
Aim:
This study aimed to evaluate how cutinase interacts with PLA nanoparticles and iron oxide nanoparticles, how these interactions affect its structure and catalytic activity, and whether the resulting PLA particles exhibit acute toxicity toward Daphnia magna.
Methods:
Cutinase was expressed, purified, and characterized through SDS-PAGE, FPLC, and circular dichroism. PLA was mechanically degraded into nanoparticles, purified via Vivaflow filtration, and analysed using NTA and FTIR. Protein-particle interactions were assessed through catalytic activity assays, DLS and sedimentation centrifugation. Other techniques were also used in the study such as HPLC, mass spectrometry and colorimetric assays. Ecotoxicity tests were performed on Daphnia magna for two weeks using UVB treated and untreated particle suspensions.
Results:
Cutinase lost significant structural integrity and catalytic activity upon interaction with PLA nanoparticles, as shown by CD spectra and activity assays, whereas the untreated enzyme remained stable. Iron oxide nanoparticles altered protein-nanoparticle behaviour and also hindered cutinase’s activity. PLA degradation did not produce detectable lactic acid, and degradation products differed chemically from macro-PLA. Toxicity assays revealed low mortality and normal reproduction in Daphnia magna, indicating low acute toxicity at the tested concentrations.
Conclusion:
PLA nanoparticles and iron oxide diminish cutinase activity limiting the enzyme’s effectiveness in PLA degradation. Despite this, PLA nanoparticles displayed low acute toxicity in Daphnia magna.

Popular Abstract

Plastic pollution is one of today's major environmental problems. Although many biodegradable plastics have been developed to reduce this impact, they do not always break down effectively in real-world environments. One of the most widely used biodegradable plastics is PLA, which is often found in packaging, 3D printing, and disposable items. Despite being labeled as biodegradable, PLA degrades very slowly outside of industrial composting facilities.
This project investigated whether cutinase can help break down PLA more effectively. Enzymes are natural biological catalysts, and some of them are known to break down plastics by cutting specific chemical bonds. The main purpose of this project was to understand how well cutinase works on PLA, how the degradation products behave, and whether the resulting nanoparticles can have toxic effects on aquatic organisms.
PLA is mechanically broken down into nanoparticles, purified, and analyzed using several scientific methods. These included techniques for measuring particle size, chemical composition, and structure. After testing how cutinase behaved in the presence of PLA nanoparticles, it was discovered that the enzyme lost much of its activity. Its structure changed and it did not work as effectively as expected. This suggests that the interaction between the enzyme and the plastic surface may interfere with the enzyme's ability to break down PLA.
We also studied how iron oxide nanoparticles interact with cutinase. The experiments showed that iron oxide nanoparticles tend to form aggregates and can affect how the enzyme behaves, which also affects its catalytic activity.
To understand whether these nanoparticles can be harmful to living organisms, toxicity tests were performed on Daphnia magna. Daphnia is a freshwater crustacean that is often used as an indicator species in environmental studies. The results showed that even after UV exposure, which can modify nanoparticles, there was no short-termed toxicity and no impact on reproduction.
Overall, this study highlights both the potential and the current limitations of using enzymes to break down PLA. Although cutinase can interact with PLA, it quickly loses its activity, suggesting that other enzymes may be needed for practical applications. At the same time, PLA nanoparticles in the tested concentrations showed low toxicity, which is positive from an environmental perspective. The results contribute to ongoing efforts to develop better strategies for biodegradation and to understand how biodegradable plastics behave when they enter natural ecosystems.

Popular Abstract (Swedish)

Plastföroreningar är ett av dagens största miljöproblem. Även om många biologiskt nedbrytbara plaster har utvecklats för att minska denna påverkan, bryts de inte alltid ned effektivt i verkliga miljöer. En av de mest använda biologiskt nedbrytbara plasterna är PLA (polylaktid), som ofta förekommer i förpackningar, 3D-utskrifter och engångsartiklar. Trots att PLA är märkt som biologiskt nedbrytbart bryts det ned mycket långsamt utanför industriella komposteringsanläggningar.
I detta projekt har man undersökt om ett enzym som kallas kutinase kan bidra till att bryta ned PLA mer effektivt. Enzymer är naturliga biologiska katalysatorer, och vissa av dem är kända för att bryta ned plast genom att kapa specifika kemiska bindningar. Huvudsyftet med detta projekt var att förstå hur väl kutinase fungerar på PLA, hur nedbrytningsprodukterna beter sig och om de resulterande nanopartiklarna kan ha toxiska effekter på vattenlevande organismer.
PLA bryts mekaniskt ned till nanopartiklar, renas och analyseras med hjälp av flera vetenskapliga metoder. Dessa inkluderade tekniker för att mäta partikelstorlek, kemisk sammansättning och struktur. Efter att ha testat hur kutinase betedde sig i närvaro av PLA-nanopartiklar upptäcktes att enzymet förlorade mycket av sin aktivitet. Dess struktur förändrades och det fungerade inte så effektivt som förväntat. Detta tyder på att interaktionen mellan enzymet och plastytan kan störa enzymets förmåga att bryta ner PLA.
Vi studerade också hur nanopartiklar av järnoxid interagerar med kutinase. Experimenten visade att nanopartiklar av järnoxid tenderar att bilda aggregat och kan påverka hur enzymet beter sig, vilket också påverkar dess katalytiska aktivitet.
För att förstå om dessa nanopartiklar kan vara skadliga för levande organismer utfördes toxicitetstester på Daphnia magna. Daphnia är ett sötvattenskräftdjur som ofta används som indikatorart i miljöstudier. Resultaten visade att även efter UV-exponering, som kan modifiera nanopartiklar, fanns det ingen akut toxicitet och ingen påverkan på kortvarig reproduktion.
Sammantaget belyser denna studie både potentialen och de nuvarande begränsningarna med att använda enzymer för att bryta ned PLA. Även om kutinase kan interagera med PLA förlorar det snabbt sin aktivitet, vilket tyder på att andra enzymer kan behövas för praktiska tillämpningar. Samtidigt visade PLA-nanopartiklar i de testade koncentrationerna låg toxicitet, vilket är positivt ur miljösynpunkt. Resultaten bidrar till pågående insatser för att utveckla bättre strategier för biologisk nedbrytning och för att förstå hur biologiskt nedbrytbara plaster beter sig när de kommer in i naturliga ekosystem.

• author: Pérez Sánchez, Lucía
• supervisor: Tommy Cedervall; Martin Lundqvist
• organization: Department of Chemistry
• course: KEMP30 20252
• year: 2026
• type: H1 - Master's Degree (One Year)
• subject: Chemistry; Earth and Environmental Sciences
• keywords: toxicity, plastic degradation, PLA, cutinase, nanoparticles, iron oxide, biochemistry
• language: English
• id: 9218742
• date added to LUP: 2026-01-20 11:46:57
• date last changed: 2026-01-20 11:46:57