Novel one-pot recovery and in-situ crystallization of polyhydroxybutyrate and hydroxyapatite/tricalcium phosphate biocomposite microparticles with comparative life cycle assessment

Abstract

Formation of polyhydroxybutyrate (PHB) biopolymer composites with bioceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) is essential in achieving mechanical properties needed for novel bone tissue engineering using PHB. However, composite microparticle synthesis typically requires multiple steps, including 1) PHB recovery and purification, 2) dispersion of HA and TCP particles in the melt or solvent-dissolved polymer liquid, and 3) micro-droplet drying. In this study, PHB/HA/TCP composite microparticles were successfully produced by one-pot biosynthesis. This was achieved during acid-based PHB recovery by utilizing the crystallization of native-amorphous granule PHB within Cupriavidus necator. In-situ PHB crystallization was successfully monitored by real-time attenuated total reflection-Fourier transform infrared (ATR-FTIR). Additionally, the in-situ crystallization behavior was elucidated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The most suitable conditions for synthesis of the PHB/HA/TCP composite were pH 2, and 20 min of reaction time, which capitalizes on the amorphous nature of the in-situ PHB during recovery. The HA and TCP did not function as nucleating agents, thereby not impacting accumulation and homogeneity. This allows HA/TCP bioceramics to be inserted into the polymer during the PHB recovery period, and after the crystallization step is completed, the composite microparticles could facilely form. The crystallization mechanism was found to be sporadic, and the morphology was a disc with two dimensions. Additionally, the life cycle assessment (LCA) revealed that the one-pot method reduced global warming potential (GWP) emissions by 50% and non-renewable energy use (NREU) by a comparable margin, compared to the conventional multi-step method for HA/TCP (20:80) production. These findings emphasize the environmental advantages of the one-pot approach alongside its cost and process efficiency. The demonstrated one-pot synthesis method would allow for more streamlined and cost-effective production of PHB/HA/TCP biocomposites. The materials produced and insights gained will be beneficial for future development of biopolymer composite processing and biomedical applications.