Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of noninvasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments.We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

As a result of the steadily ongoing development of microfluidic cultivation (MC) devices, a plethora of setups is used in biological laboratories for the cultivation and analysis of different organisms. Because of their biocompatibility and ease of fabrication, polydimethylsiloxane (PDMS)-glass-based devices are most prominent. Especially the successful and reproducible cultivation of cells in microfluidic systems, ranging from bacteria over algae and fungi to mammalians, is a fundamental step for further quantitative biological analysis. In combination with live-cell imaging, MC devices allow the cultivation of small cell clusters (or even single cells) under defined environmental conditions and with high spatio-temporal resolution. Yet, most setups in use are custom made and only few standardised setups are available, making trouble-free application and inter-laboratory transfer tricky. Therefore, we provide a guideline to overcome the most frequently occurring challenges during a MC experiment to allow untrained users to learn the application of continuous-flow-based MC devices. By giving a concise overview of the respective workflow, we give the reader a general understanding of the whole procedure and its most common pitfalls. Additionally, we complement the listing of challenges with solutions to overcome these hurdles. On selected case studies, covering successful and reproducible growth of cells in MC devices, we demonstrate detailed solutions to solve occurring challenges as a blueprint for further troubleshooting. Since developer and end-user of MC devices are often different persons, we believe that our guideline will help to enhance a broader applicability of MC in the field of life science and eventually promote the ongoing advancement of MC.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS, is a crucial parameter for characterizing growth within bioreactors. State‐of‐the‐art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS. In this work, a recently developed substrate‐limited microfluidic single‐cell cultivation (sl‐MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high‐frequency biomass measurement (HFB) and substrate‐limited respiratory activity monitoring (sl‐RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod‐type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl‐RA, and 2.66 ± 0.99 mg/L with sl‐MSCC was obtained. Whereas HFB and sl‐RA are suitable for a fast initial kS estimation, sl‐MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl‐MSCC lays the foundation for strain‐specific kS estimations under defined environmental conditions with additional insights into cell‐to‐cell heterogeneity.