PUB - Publikationen an der Universität Bielefeld

Microorganisms can live in their natural habitats in multi-species communities where all community members perform designated metabolic tasks and are affected by complex interactions. Cooperative interactions between cells, such as cross-feeding or division of labor, contribute to enhancing overall community fitness and increase the chance of cellular survival.
An emerging trend in biotechnological research is to exploit the aspect of division of labor and cross-feeding by using synthetic co-cultures for industrial production processes to develop more sustainable and robust bioprocesses on the one hand and to improve existing ones on the other. Hence, synthetic co-cultures must be characterized in terms of the individual performance of its community members to ensure efficient substrate conversion and optimal growth rates.
The present work solves the current limitation of quantifying individual performance based on a synthetic co-culture consisting of the sucrose-utilizing lysine-producing *Corynebacterium glutamicum Lys4ΔptsF*(pVWEX1-crimson) and a fructose-utilizing lysine auxotrophic *Escherichia coli Lys1*(pVWEX1-gfpUV) strain. Here, a growth kinetic has been developed that allows for growth behavior and performance prediction depending on initial sucrose concentration under various species-seeding ratios. To accomplish this, the substrate affinities, commonly referred to as k_s, of *C. glutamicum* wild-type regarding glucose were systematically investigated by the recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method. A k_s of 2.66 ± 0.99 mg∙L-1 has been estimated with sl-MSCC and was in agreement with reported literature values derived by conventional methods. Hence, sl-MSCC was successfully validated as a valuable tool for providing strain-specific k_s estimations under defined environmental conditions while getting more profound insights into cell-to-cell heterogeneity. Furthermore, a material balance based on single-cell data obtained through different microfluidic single-cell cultivation techniques has been mathematically derived from the specific kinetics of substrate uptake and growth stoichiometry for glucose-growing *E. coli* MG1655 K12. For the first time, microfluidic growth kinetic parameters were shown to yield comparable values to conventional methods.
This laid the foundation for determining the specific k_s values of the synthetic co-culture along with additional performance parameters to generate a kinetic growth model on a single-cell database. For *C. glutamicum Lys4ΔptsF*(pVWEX1-crimson), a k_s of 3.30 ± 1.53 mg∙L-1 regarding sucrose, and for *E. coli Lys1*(pVWEX1-gfpUV), a k_s of 2.33 ± 0.99 mg∙L-1 regarding fructose and a k_s of 0.09 ± 0.03 mg∙L-1 regarding lysine has been estimated with sl-MSCC respectively.
Based on reported growth kinetic parameters and obtained k_s values, a growth kinetic model has been compiled that allows growth and performance predictions regarding i) varying initial sucrose concentrations under ii) various species-seeding ratios while considering iii) cellular heterogeneity. In the future, model validation and revision of obtained data will increase data accuracy and generate new valuable information, which cannot be resolved with conventional cultivation techniques.
Ultimately, the present work lays the foundation for studying and quantifying growth kinetics in axenic and synthetic co-culture by utilizing microfluidic single-cell cultivation techniques and allows additional applications such as strain-specific k_s estimation of bioprocess-relevant organisms or knock-out mutants.