• 3D printing
• biodegradable plastics
• extrusion
• microbial incorporation into plastic
• PLA
• plastics
• sustainability

In recent decades, the use of plastics and its production has increased considerably due to their relative low cost, chemical stability and physical characteristics. However, due to poor waste management, unsuitable recycling activity and inappropriate disposal, the success and dominance of plastics have also led to environmental risks. The same attributes that make plastics such an excellent material also pose a threat to terrestrial, aquatic and marine ecosystems. Indeed, most plastics when disposed are durable and degrade very slowly, in the order of centuries. The chemical make-up is resistant to natural degradation processes. When discarded, these products enter land, waterways and thus reach the seas and oceans, directly or indirectly. There, they persist many years and thus have a significant negative impact on ecology and wildlife.
To decrease the impact of plastic pollution, some actions from political, social and scientific points of view are being taken. The latter includes extensive research to find solutions in terms of recycling methods and “biodegradable” plastics. Although these plastics represent a promising and more sustainable alternative than traditional plastics, their biodegradability is limited only to industrial settings where favourable pH, moisture content and temperatures can be set. Therefore, “biodegradable” plastics are not really biodegradable in the environment, as their name would suggest. For example, studies report that poly(lactic acid) (PLA), among the most famous “biodegradable” plastics, shows no evidence of microbial degradation or hydrolysis within a range of 3-12 months in marine environments.
The present thesis is part of a wider project aiming at developing an alternative interdisciplinary approach to have a broader PLA biodegradation spectrum and an increased PLA biodegradation rate in natural environments. Moreover, it also aims at preventing the formation of microplastics.
The project is focused on the development of a “Two-cycle-system” concept based on the production of plastic objects using 3D-printing. This Two-Cycle-System involves two different streams: a technical cycle and a connected biological cycle. Thus, following this method, 3D-printed plastic objects could be either technically recycled or biodegraded once discarded into the environment. Biological degradation is based on exploring the possibility of making PLA biodegradable by incorporating bacteria in plastics during the filament generation (by then called ‘poly-biotic plastics’-PB-PLA) for 3D printing applications. If this step can be successfully carried out, the biological degradation could occur when the product enters a natural environment. PB-PLA would indeed be an “alive” material, with bacteria degrading the plastic and producing non-toxic compounds ready to be used in ecological carbon cycle, while also limiting the release of microplastics.
The thesis project was then aimed at investigating biotechnological, chemical and engineering opportunities to incorporate spore-producing microbes into PLA product lifecycle and to evaluate to what extent this new approach can be suitable for future applications.

In the first part of the study, we evaluated 3D-printing & 3-Devo extrusion settings and conditions that could allow and enhance endospore survival during incorporation into plastic and printing. Also, a protocol for mixing bacteria and medium with PLA to create poly-biotic PLA was developed. Thus, the presence of the incorporated bacteria into the plastic was verified. Preliminary results confirmed the generation of PB-PLA filaments containing bacteria.
The second part of the project focused on the study of mechanical and physical properties of poly-biotic PLA such as Young’s modulus (E), tensile strength, glass transition state, melting temperature (Tm) and polymer chain length.
Finally, ecotoxicology tests on Daphnia Magna and Chironomid Riparius assesed no toxicity of PB-PLA films within 48 hours. Moreover, bacteria are being modified to optimally express a PLA degrading enzyme, with the aim to increase the biodegradation rate of PB-PLA compared to normal PLA.
Therefore, this interdisciplinary work lays the foundation for a new type of material. It also paves the way for future biodegradation tests and further studies on the feasibility of PB-PLA as a sustainable solution to deal with plastic pollution.
The present work was carried out at the Fair Fusion laboratory (EMMTEC services – Eerste Bokslootweg 17, 7821 AT Emmen, The Netherlands) under the supervision of Dr. Nicolò Mossink. Prof. Arianna Tavanti was the internal supervisor from the Department of Biology, University of Pisa.