High‐sensitivity real‐time monitoring of <scp>pH</scp> and respiration activity unveils metabolic dynamics in shake flask cultures

Erlenmeyer shake flasks are widely used during the first steps of bioprocess development. Despite their broad application in academia and industry, shake flasks usually lack standardized and user-friendly online monitoring techniques. In this work, the pH and Respiratory Activity MOnitoring System (pH-RAMOS) for the non-invasive online measurement of the oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and pH in up to eight parallel shake flasks under sterile conditions is presented. The OTR and CTR are quasi-continuously measured in the headspace of the shake flasks using dedicated oxygen and carbon dioxide sensors, enabling precise respiratory quotient (RQ) evaluation. Self-adhesive pH sensor spots are used for the high-frequent real-time pH monitoring of the culture. These prototype pH sensor spots stand out due to their simple sterilizability and subsequent one-point calibration in the cultivation medium. The long-term stability of the pH sensor spots was assessed in a 28-day long abiotic experiment. The novel pH-RAMOS was validated with different eukaryotic and prokaryotic microorganisms, such as Ogataea polymorpha, Ustilago trichophora, and Vibrio natriegens. The combination of online OTR, CTR, RQ, and pH signals allowed for identifying various metabolic phenomena, such as oxygen limitations, substrate limitations, diauxies, and the production or consumption of specific compounds, based on their degree of reduction or change of pH. The high-frequent and sensitive pH-monitoring was particularly advantageous for registering subtle and transient metabolic phenomena.