Lynn van Boxtel

Background: Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers produced by microorganisms and represent a promising alternative to fossil‑derived plastics. Cupriavidus necator accumulates polyhydroxybutyrate (PHB), a major PHA type, under nutrient‑limited conditions. Optimizing detection and extraction methods is essential for developing sustainable microbial bioplastic production workflows.
Methods: C. necator was cultivated in White medium under nitrogen limitation. Growth was monitored via OD₆₀₀, with optimal PHB accumulation observed around OD ~0.7 (6–19.5 h post‑inoculation). PHB granules were visualized using Sudan Black and Nile Red staining; fluorescence microscopy with Nile Red provided superior contrast. Three extraction methods were compared: (1) lysis with a NaOH/SDS buffer, (2) sodium hypochlorite treatment, and (3) ethyl acetate extraction. For selected methods, the effect of freeze‑drying prior to extraction was assessed. PHB purity was evaluated using Fourier‑transform infrared (FTIR) spectroscopy and compared to a commercial PHB reference.
Results: Freeze‑drying improved PHB purity by increasing cell porosity and facilitating polymer release. Sampling time strongly influenced purity: early stationary‑phase samples (OD ~0.8) yielded purer PHB than late‑phase samples (OD ~1.9), where PHB reserves had already been metabolized. Among the tested extraction methods, sodium hypochlorite produced the highest‑purity PHB, with FTIR spectra closely matching the reference and showing minimal impurities.
Conclusion: This study confirms C. necator as an effective microbial PHB producer and demonstrates that sampling phase and freeze‑drying significantly enhance extraction efficiency. Sodium hypochlorite treatment proved the most effective and sustainable isolation method, contributing to optimized bioplastic production within circular biotechnology.