Sequential Yeast Fermentation Platform for Recycling and Separating PLA and PHA Bioplastic Mixtures

Overview

Mixed bioplastic waste streams containing polylactic acid (PLA) and polyhydroxyalkanoates (PHA) are difficult to recycle using conventional methods because the polymers degrade under overlapping conditions. This project addresses that challenge with a sequential yeast fermentation platform that selectively breaks down each polymer in a mixture, producing recoverable monomers such as lactic acid and PHB hydroxyalkanoate monomers.

The approach uses Pichia pastoris, a yeast known for high-yield extracellular enzyme production, low cost, and industrial scalability. By housing engineered P. pastoris strains expressing PLA hydrolase in a first reactor and PHB depolymerase in a second, the system enables step-wise, substrate-specific hydrolysis of PLA–PHB blends. This delivers simultaneous recycling and separation in a single process flow, offering a sustainable route for managing mixed bioplastic waste.

Technical specifications

Key features:

• Engineered P. pastoris hosts expressing PLA hydrolase and PHB depolymerase under the methanol-induced AOX1 promoter using pPICZ/pPICZA vectors
• Optimized secretion signal identified from prior work that outperforms the commonly used α-MAT signal for extracellular enzyme release
• Strain selection validated: X33 identified as the highest-yielding P. pastoris strain, surpassing KM71H and GS115 for extracellular enzyme production
• Induction condition optimization: 1.0% methanol induction yields maximum laccase production without notable cell toxicity
• Sequential reactor design: first reactor degrades PLA to lactic acid; residual solids transferred to a second reactor where PHB depolymerase converts PHB to monomers and oligomers
• Monitoring framework: LA, PHB monomer, and oligomer concentrations tracked over time to evaluate selectivity and yield
• Analytical validation: extracellular enzyme production confirmed by Western blot and activity assays

Technology readiness level

The platform is at an early-to-mid stage of development (TRL 3–4). Prior work has demonstrated successful production and secretion of multiple enzymes (manganese peroxidase, lignin peroxidase, laccase) in P. pastoris and identified optimal strains, induction conditions, and secretion signals. The current project aims to deliver proof-of-concept validation by cloning PLA hydrolase and PHB depolymerase genes, confirming extracellular expression, and demonstrating sequential degradation of a PLA–PHB blend in a two-reactor system. Next steps toward commercialization would include scaling reactor volumes, expanding to additional PHA variants, and integrating monomer recovery and purification.