Solvent extraction and analytical characterization of polyhydroxyalkanoate inclusions produced by the microeukaryote...

The increasing global burden of petroleum-derived plastic pollution has intensified the search for sustainable and biodegradable alternatives to conventional plastics. Polyhydroxyalkanoates (PHAs) are biodegradable microbial polyesters with considerable potential to replace petroleum-based plastics; however, research has predominantly focused on bacterial production systems, while microeukaryotic microorganisms remain largely unexplored. In this study, the ciliated protozoan Paramecium jenningsi, isolated from stagnant freshwater, was investigated as a microeukaryotic platform for PHA biosynthesis. Growth conditions were optimized using Bold Basal Medium (BBM), and intracellular PHA accumulation was initially screened using Sudan Black B and Nile Blue A staining. PHA production was induced under glucose-enriched and HgSO₄-stressed conditions, and polymers were extracted after 24, 48, and 72 h of exposure. The highest polymer yield (0.36 g/L) was obtained from glucose-treated cultures after 24 h, significantly exceeding that of HgSO₄-treated and control cultures. The recovered polymers were characterized using Fourier-transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). FTIR analysis revealed characteristic functional groups associated with PHAs, while GC-MS detected prominent 2-butenoic acid ester derivatives indicative of polyhydroxybutyrate (PHB)-related polymers. Notably, GC-MS also revealed medium- and long-chain-length hydroxyalkanoate monomers (C4-C19) alongside the short-chain-length PHB-associated derivatives, indicating that P. jenningsi produces a structurally heterogeneous PHA copolymer rather than PHB alone. These findings demonstrate the ability of P. jenningsi to accumulate PHB-like biopolymers under both carbon-rich and metal-stressed conditions and provide the first evidence supporting this species as a potential microeukaryotic host for PHA production. The study expands the diversity of microbial systems available for biopolymer research and highlights the potential of protozoan platforms for sustainable bioplastic development.