SiMBal 2.0: Quantifying co-culture performance as a function of condition-dependent inter-cell mass exchange

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Synthetic microbial co-cultures perform complex catalytic tasks and benefit from cooperative interactions. The rational design of co-cultures facilitating beneficial interactions for biotechnological production could bring catalytic modularity into bioprocesses and hold the potential to catalyse a cascade of reactions more efficiently than axenic cultures. Rational metabolic and process engineering towards their efficient biotechnological application, however, requires quantitative and spatiotemporally resolved data on microbial performance. Relevant data typically includes specific growth, uptake and production rates, yield coefficients, and stoichiometry as a function of the co-culture interaction kinetics. Such knowledge cannot be generated from averaged readouts, based on population experiments in shake flasks or bioreactors. SiMBal 2.0 will analyse the performance-determining kinetic and stoichiometric indicators in co-cultures with single-cell resolution to apply fundamental biochemical engineering principles, such as material balancing. As interactions kinetics and co-culture stoichiometry are strongly governed by extracellular conditions, we will extend the established material-balancing framework towards condition-dependent co-culture physiology analyses. To this end, we will exploit perfusion cultivations, microfluidic batches in chambers and droplets, combined with time-lapse microscopy, mass imaging, and mass spectrometry for substrate and product analysis. The data will be used for material balancing and feed a segregated dynamic co-culture model for unravelling tuning opportunities. Dynamic modelling based on single-cell data will be used to predict the co-culture performance in terms of growth, productivity, and species ratios and also take phenotypic heterogeneity into account. The obtained model will then be tested for validity with population-based co-culture experiments.We will not only advance our technological concepts but also progress towards the analyses of eusymbiotic co-cultures. As a model for synthetic co-cultures, we will study cell-cell interactions between Corynebacterium glutamicum strains each auxotrophic for one essential amino acid, but capable of mutually complementing these auxotrophies. We will focus on relevant parameters such culture pH, substrate availability, amino acids, spatial culture arrangement, and the initial seeding ratios as the main determinants for co-culture physiology. We will push forward the understanding of co-culture interactions for rational strain and process development. Our interdisciplinary project spans the disciplines of biochemical engineering, microfluidic single-cell analysis, and culture modelling. The overarching goal of SiMBal 2.0 summarizes as the quantification of intercellular interactions of synthetic microbial co-cultures. This knowledge obtained is invaluable for future co-culture-based biotechnology.