Tenebrio molitor-Derived Enzyme Systems Enable Solvent-Reduced Recovery of Intracellular Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are intracellular microbial polyesters whose commercial deployment is strongly influenced by downstream processing costs and environmental burden. Here, we demonstrate a nearly solvent-free, mild aqueous strategy for the recovery of PHAs directly from wet bacterial biomass using crude enzyme systems derived from Tenebrio molitor. Under optimized conditions (0.6 wt% crude protein, pH 7.6, 40°C), near-quantitative (≥ 95%) recovery of PHB and PHB/HV and up to 60% recovery of mcl-PHA were achieved without prior biomass drying. Proteomic analysis identified abundant digestive hydrolases, including α-amylase and cathepsin and recombinant validation confirmed their contribution to polymer release, demonstrating that targeted enzyme combinations can substantially enhance mcl-PHA recovery (up to 94%-98%). Gel permeation chromatography, NMR and thermal analysis demonstrated preservation of polymer molecular integrity and crystallinity comparable to chloroform extraction. Life cycle assessment revealed a three- to seven-fold reduction in carbon footprint relative to conventional solvent and classical enzymatic methods, primarily due to elimination of drying and solvent use. These findings establish biologically driven biomass hydrolysis as a scalable downstream strategy and highlight insect-derived enzyme systems as promising tools for integrated microbial biopolymer processing.