Establishment of a Single‐Cell Minimal Medium: A Case Study for Microfluidic Cultivation of <i>Corynebacterium glutamicum</i>

Single-cell technologies are emerging as a complementary approach to conventional laboratory-scale cultivation setups. Particularly in microfluidic cultivation and screening, precisely controlled environmental conditions and defined minimal media become important for quantitative studies. Nevertheless, conventional bulk media optimized for other scales, are often employed despite differences in nutrient requirements across varying scales and cultivation modes. This study demonstrates the limitations of using bulk media for single-cell analysis of Corynebacterium glutamicum and highlights the need for revised minimal media for microfluidic cultivations under perfusion. The commonly used CGXII medium contains protocatechuic acid (PCA), which functions both as an iron chelator and a carbon source, thereby compromising its role as a minimal medium with one limiting substrate. We systematically evaluated alternative iron chelators to replace PCA using microfluidic single-cell cultivation. We show that not all chelators are applicable with C. glutamicum at the single-cell level and identify citrate as a suitable replacement that ensures iron availability without supporting growth in the absence of glucose. Based on these findings, we established a revised minimal medium, termed 'single-cell CGXII'. This medium was validated across different scales and enables robust, reproducible investigation of cellular physiology at single-cell resolution, improving accuracy and comparability in quantitative single-cell research.