<p>Mixed microbial culture (MMC) fermentation holds promising potential for polyhydroxyalkanoates (PHA) production due to its operational simplicity. However, maintaining process stability and ensuring continuous PHA production remains critical for its industrial application. In this study, the cyclic and long-term fermentation performance of MMC was evaluated. The maximum concentration of PHA was maintained at 3200–3571 mg·L^− 1 within a six-cycle butyric acid fermentation system. Subsequently, continuous co-polyester synthesis with mixed butyric-valeric acids substrate achieved the highest PHA content of 2301.1 mg·L^− 1 among five cycles, with a PHA composition comprising 35.5% 3-hydroxyvalerate. In light of the stable accumulation of PHA, a rapid gradient expansion of volume was conducted. The corresponding PHA outputs were recorded at 694.4 mg·L^− 1 (20&#xa0;L system) and 358.2 mg·L^− 1 (50&#xa0;L system). The dynamic detection of microbial populations revealed that the predominant functional species in continuous fermentation and scale-up culture systems included <i>Achromobacter</i>, <i>Pseudomonas</i>, <i>Paracoccus</i>, and <i>Aquamicrobium</i>. This study explores the stability of PHA synthesized by mixed consortia from a different perspective, highlighting the potentials of large-scale industrial PHA biomanufacturing.</p> Graphical Abstract <p></p>