Raman Spectroscopy-Based Glucose Feed Control Automates Malic Acid Fermentation

Fermentation optimization and automation are essential for advancing biotechnology's economic viability. A key aspect is maintaining optimal substrate availability; nevertheless, traditional substrate feeding often leads to imprecise control or carbon limitation due to a lack of online monitoring. This work introduces Raman spectroscopy for closed-loop glucose feed control during malic acid production, a promising platform chemical with diverse applications in food, pharmaceuticals, and biodegradable plastics. The production host Ustilago trichophora displays maximal productivity at substrate concentrations well above the substrate affinity constant; however, the feed rate is severely affected by biomass growth, cell aging, or product inhibition. Real-time online monitoring of glucose and malic acid concentrations is achieved using a Partial Least Squares regression model, with a root mean square error of prediction of 2.7 g/L and 5.3 g/L, respectively. The implemented automated feedback system uses a PID controller that dynamically adjusts the feed rate based on real-time metabolic demands to maintain a constant glucose concentration at 20 g/L during the production phase. Simultaneously, the product concentration is monitored, and the feed is stopped automatically to prevent substrate loss due to product inhibition. This work highlights the practical relevance of Raman-based fermentation control, extending its significance beyond mere technical feasibility.