Production and Characterisation of Polyhydroxyalkanoates from Cocoa Mucilage Using a Wild-Type Priestia aryabhattai Strain

Abstract

The accumulation of petroleum-based plastics demands sustainable alternatives such as polyhydroxyalkanoates (PHAs), biodegradable polyesters synthesised by numerous prokaryotes. However, high feedstock costs limit their commercialisation. This study evaluated cocoa mucilage, an underutilised by-product of the Ecuadorian cacao sector, as a low-cost carbon source for PHA production by a wild-type strain isolated from cocoa fruit residues. Bacteria were recovered from cocoa mucilage and pod shell fractions and screened for PHA accumulation by Sudan Black B staining with UV–Vis spectrophotometric confirmation. A single PHA-positive isolate, designated Priestia aryabhattai strain NBP01-UTN (GenBank accession OR567321.1; 99.88% 16S rRNA gene sequence identity to the type strain B8W22T), was recovered from the cocoa shell surface—representing, to the best of our knowledge, the first report of a PHA-producing P. aryabhattai from cacao fruit residues. Fermentation conditions were optimised using the response surface methodology with a central composite design evaluating temperature, pH, and ammonium sulphate concentration. The fitted quadratic model was highly significant (R2 = 0.978, p < 0.0001), indicating that temperature and nitrogen limitation were the dominant factors. Optimal conditions (40 °C, pH 7.30, 0 g·L−1 (NH4)2SO4) yielded 0.496 g·L−1 PHA at 24 h (productivity ≈ 20.7 mg·L−1·h−1). Notably, no external nitrogen supplementation was required, as the endogenous nitrogen in cocoa mucilage sufficed to sustain growth whilst triggering the nutrient imbalance needed for PHA biosynthesis. FTIR and DSC analyses provided spectroscopic and thermal evidence consistent with poly(3-hydroxybutyrate) (PHB), although definitive monomer-level confirmation requires GC–MS or NMR spectroscopy. These results demonstrate the feasibility of coupling a locally isolated wild-type strain with cocoa mucilage to produce bioplastic within a circular bioeconomy framework.