A microfluidic system for the cultivation of cyanobacteria with precise light intensity and CO2 control: enabling growth data acquisition at single-cell resolution

Abstract

Quantification of cell growth is central to any study of photoautotrophic microorganisms. However, cellular self-shading and limited CO₂ control in conventional photobioreactors lead to heterogeneous conditions that obscure distinct correlations between the environment and cellular physiology. Here we present a microfluidic cultivation platform that enables precise analysis of cyanobacterial growth with spatio-temporal resolution. Since cyanobacteria are cultivated in monolayers, cellular self-shading does not occur, allowing homogeneous illumination and precise knowledge of the photon-flux density at single-cell resolution. A single chip contains multiple channels, each connected to several hundred growth chambers. In combination with an externally applied light gradient, this setup enables high-throughput multi-parameter analysis in short time. In addition, the multilayered microfluidic design allows continuous perfusion of defined gas mixtures. Transversal CO₂ diffusion across the intermediate polydimethylsiloxane membrane results in homogeneous CO₂ supply, with a unique exchange-surface to cultivation-volume ratio. Three cyanobacterial model strains were examined under various, static and dynamic environmental conditions. Phase-contrast and chlorophyll fluorescence images were recorded by automated time-lapse microscopy. Deep-learning trained cell segmentation was used to efficiently analyse large image stacks, thereby generating statistically reliable data. Cell division was highly synchronized, and growth was robust under continuous illumination but stopped rapidly upon initiating dark phases. CO₂-Limitation, often a limiting factor in photobioreactors, was only observed when the device was operated under reduced CO₂ between 50 and 0 ppm. Here we provide comprehensive and precise data on cyanobacterial growth at single-cell resolution, accessible for further growth studies and modeling.