The development of a multi-purpose four step-cascading biorefinery scheme for the valorization of red grape pomace (GP) was proposed. The consecutive processes were respectively dedicated to (a) the recovery of polyphenols by supercritical CO₂ extraction, (b) the production of volatile fatty acids (VFAs) by anaerobic acidogenic digestion, (c) the exploitation of produced VFAs as the precursors for the biotechnological production of polyhydroxyalkanoates (PHAs) and (d) the production of a CH₄-rich biogas by the anaerobic digestion of solid leftovers from the acidogenic process. More than 2.7 g of total polyphenols (as gallic acid equivalents) per 100 g of dry matter were extracted. A high content of valuable proanthocyanidins occurred in the recovered polyphenolic fraction. The dephenolized GP was anaerobically digested under batch acidogenic conditions, obtaining about 20 g L⁻¹ of total VFAs in the liquid effluent. The latter matrix was employed to feed a pure culture of a Cupriavidus necator strain, which was induced to produce and store PHAs under nitrogen-limiting conditions. The process was carried out in 0.5 L-shake flasks by using a two-step production approach. In particular, pre-grown biomass was fed with different concentrations of the acidic effluent (20 or 40% v/v) in two sequential batch processes. Poly(3-hydroxybutyrate) was accumulated up to 63% of the cells dry weight when pre-grown biomass was fed with 40% of the acidic effluent. No inhibitory effects due to non-VFA compounds occurring in the actual acidogenic effluent were observed. Finally, the anaerobic digestion of the exhausted solid fraction from the acidogenic process allowed obtaining 113 mL of biomethane per gram of fed volatile solids.

Two novel protocols for the chemical valorization of polyhydroxybutyrate (PHB) were developed, aiming at the production of two bio-based molecules: methyl 3-hydroxybutyrate (MHB) and methyl 3-methoxybutyrate (MMB). Optimized reaction conditions were applied to pure PHB and PHB inclusions inside bacterial cells as starting materials. MHB was synthesized through a single-step catalytic methanolysis, while MMB was synthesized through a three-step process: thermolytic distillation to give crotonic acid (CA), esterification to give methyl crotonate (MC), and oxa-Michael addition of MeOH. The obtained MHB and MMB were tested as solvents for the recovery of PHB itself both from freeze-dried single strain cultures (SSC) and mixed microbial cultures (MMC) with low to medium contents of PHB (22–57 wt %). High PHB recovery was achieved: up to 96 ± 1% through MHB and up to 98 ± 1% through MMB. Extraction from MMC slurry (with a PHB content of 39% on dry weight) was also performed, recovering 77 ± 2% using MHB and 92 ± 2% using MMB. High purities and excellent molecular weights and polydispersity indexes of extracted PHB were obtained with both MHB and MMB. Solubility in water, octanol/water partition coefficients (log K_ow), and aerobic ready biodegradability of both solvents were also evaluated.