Upcycling agricultural residues into polyhydroxyalkanoates through pilot-scale mixed microbial culture processes

Polyhydroxyalkanoates (PHAs) produced by mixed microbial cultures (MMCs) represent a promising alternative to conventional plastics, enabling the valorisation of agricultural residues within circular biorefineries. However, despite extensive laboratory-scale research, integrated pilot-scale studies encompassing the complete production chain, from feedstock fermentation to downstream polymer recovery, remain scarce. In this study, poly (hydroxybutyrate-hydroxyvalerate) (PHBV) production from agricultural residues was investigated in a pilot plant integrated within a full-scale agricultural anaerobic digestion facility. The process included acidogenic fermentation, microbial selection, PHBV accumulation, biomass recovery, while downstream extraction and purification were evaluated at laboratory scale on pilot-produced biomass. A total of 15 pilot-scale campaigns were carried out, allowing progressive optimisation. Two representative campaigns were selected: Batch 12, fed with a synthetic carboxylic acid (CA) mixture, and Batch 14, which used a real agricultural feedstock derived from cattle slurry and maize silage. Acidogenic fermentation of the agricultural substrate produced up to 16.7 gCOD/L of CAs, with valeric acid accumulation, providing suitable precursors for PHBV synthesis. Microbial community analysis confirmed the successful enrichment of recognised PHA-storing genera, including Corynebacterium, Lampropedia, Paracoccus and Thauera. Pilot-scale accumulation produced PHBV with a 3-hydroxyvalerate (HV) content of 14%–26% (w/w), while intracellular PHA contents reached 26% and 30% (w/w) for Batch 12 and Batch 14, respectively. More than 1 kg of PHBV-rich biomass was recovered in both campaigns, while 10.2 kg throughout the 15 campaigns. Among the downstream treatments investigated, a NaOH-HAc protocol provided the best compromise between polymer purity, molecular weight preservation, operational simplicity and process economics. For the synthetic-feedstock biomass, PHA content increased from 26% to a purity of 58% after NaOH-HAc treatment, whereas purification of the real-feedstock biomass reached 37%, highlighting the greater complexity of processing heterogeneous waste-derived substrates. Although polymer purities remained lower than those typically achieved with pure cultures, the PHBV obtained exhibited HV contents that may favour flexibility and support its potential use in agricultural applications requiring flexible compounds. Overall, this work provides one of the few integrated pilot-scale demonstrations of MMC-based PHBV production from agricultural residues, identifies downstream processing as a key bottleneck and contributes to advancing the industrial implementation of sustainable waste-based PHA biorefineries.