Demand-driven dual carbon delivery strategy for maximizing CO2 utilization in poly(3-hydroxybutyrate) biosynthesis by Cupriavidus necator H16

Chemoautotrophic production of poly(3-hydroxybutyrate) (PHB) from CO 2 by Cupriavidus necator H16 offers a sustainable carbon upcycling route, but conventional continuous gas supply modes suffer from excessive unreacted gas discharge and poor utilization efficiency. Here, we demonstrate a novel demand-driven dual carbon delivery strategy that significantly reduces this gas wastage. Exploiting the acidic nature of CO 2 , a primary CO 2 -pulsed pH-stat loop was integrated to supply the gaseous substrate matching the cellular metabolic consumption. However, this single-loop system encounters an operational “deadlock” phenomenon, a state where carbon limitation halts the pH feedback loop. To overcome this limitation, a secondary fed-batch control was implemented. By utilizing a nominal NaHCO 3 dose as a primer and monitoring the real-time CO 2 inactive duration, the system dynamically replenished the dissolved inorganic carbon pool while aligning the restricted CO 2 pulse flow with the actual metabolic uptake. This automated dual-loop control effectively synchronized substrate injection with cellular demand, preventing deceptive pH surges. Consequently, the system achieved a reactor-level apparent carbon recovery efficiency of 76.1%, representing a 3.05-fold improvement over the continuous mode (24.9%). Notably, this enhancement in carbon recovery was accomplished without compromising fermentation performance, yielding a final dry cell weight of 14.0 g/L and a PHB content of 77.9%. Relying on readily monitorable hardware signals rather than expensive sensors, this approach provides a smart biomanufacturing platform to significantly improve the economic feasibility of biological carbon utilization processes.