Integration of online pH monitoring and control in orbitally shaken bioreactors for microbial cultivations

Erlenmeyer shake flasks are widely used in bioprocess development but lack standardized and user-friendly online monitoring techniques. This thesis presents 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. 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 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. The high-frequent and sensitive pH-monitoring was particularly advantageous for registering subtle and transient metabolic phenomena. The non-invasive pH sensor spots were further utilized for the development of a new Continuous parallel shaken pH-auxostat (CPA). The CPA combines the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting adaptive laboratory evolution (ALE) experiments. The CPA consists of six parallel shaken cylindrical reactors with real-time pH control, achieved using pH sensor spots and a programmable pump module to adjust the fresh medium dilution rate. Two different strains of the methylotrophic yeast O. polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, strains with up to 4.8-fold faster growth rates were selected. The increased maximum specific growth rates of the selected strains were confirmed in subsequent batch cultivations. This thesis demonstrates the successful integration of non-invasive online pH monitoring and control technologies in milliliter-scale orbitally shaken bioreactors used for microbial cultivations.