Abstract
<title>Abstract</title> <p>Graphical abstract Highlight Culture conditions for Heterocapsa pygmaea were optimized using RSM. The modified C5 medium significantly enhanced biomass and PCP performance. Flow cytometry enabled rapid in vivo tracking of PCP physiological status. FCM offers a scalable strategy for real-time PCP monitoring during extraction. Authentic PCP complexes with molecular weights of 10–15 kDa were confirmed. ABSTRACT Dinoflagellates are promising sources of high-value light-harvesting proteins; however, their commercial utilization remains limited by insufficient optimization of cultivation conditions and the lack of efficient physiological monitoring tools. This study developed a cultivation and monitoring framework to enhance biomass productivity and peridinin–chlorophyll a–protein (PCP) performance in Heterocapsa pygmaea. PCP was characterized using SDS-PAGE and HPLC, confirming a molecular weight of 10–15 kDa and a pigment composition dominated by peridinin and chlorophyll a. Systematic evaluation of environmental (temperature and light intensity) and nutritional (phosphorus, nitrogen, and iron) factors identified optimal cultivation conditions as 24 °C, 100 μmol photons m⁻² s⁻¹, 60 µM phosphorus supplied as mixed sources, nitrate or mixed nitrogen conditions, and 17.55 µM iron. Flow cytometry enabled the simultaneous assessment of cell density and intracellular fluorescence intensity, providing a rapid in vivo indicator of PCP-associated physiological performance across cultivation treatments. Incorporation of the optimized parameters into a modified C5 medium significantly improved biomass productivity and fluorescence responses compared with conventional f/2 and K media (p < 0.05). These findings demonstrate that combining cultivation optimization with flow cytometry-guided physiological monitoring is an effective strategy for improving PCP production in H. pygmaea. This study establishes a scalable framework for the cultivation of PCP-producing dinoflagellates and highlights the broader potential of flow cytometry-assisted monitoring for microalgal bioprocess development and industrial biotechnology applications.</p>