A microfluidic platform for high-precision growth quantification of photosynthetic microorganisms

RWTH Publications (RWTH Aachen)

Abstract

Conventional methods of quantifying microbial growth, such as optical density or backscatter measurements, cannot distinguish between cell division, growth and changes in morphology. This issue is particularly pertinent for phototrophic microorganisms, given that circadian regulation is known to affect these variables with a 24-hour period. Furthermore, in conventional photobioreactors, self-shading of cells inevitably creates light-intensity gradients, which makes it difficult to determine the amount of light impinging on a cell. This thesis addresses these issues by outlining the development of a novel experimental platform for phototrophic microorganisms. Microfluidic cultivation technology is used to restrict cell growth to monolayers, ensuring constant and accurate illumination. Combined with timelapse imaging at single-cell resolution, this technology enables the precise quantification of the relationship between light intensity and growth rate. In addition, cell division and cell growth can be distinguished. Furthermore, the experimental platform can test multiple light intensities and CO2 concentrations simultaneously, thus accelerating the acquisition of growth data. The first part focuses on setting up the platform and conducting proof-of-concept studies. First, a ringlight connected to an LED was integrated into the microscope’s periphery to provide precisely regulated illumination for photosynthesis. The design enables the ringlight to illuminate the cultivation chip either homogeneously or with a spatial light intensity gradient, allowing multiple intensities to be tested simultaneously. Stable long-term cultivation for up to twelve days was demonstrated for cyanobacteria as well as extremophile microalgae. No CO2 limitations were observed during microfluidic cultivation in ambient air. Moreover, the CO2 concentration in the monolayer growth chambers can be controlled by flushing defined gas mixtures through a CO2 control layer. This allows growth under CO2 limiting conditions to be studied. The method, in combination with Python-based workflows for cell segmentation, allows multiple cyanobacteria strains to be characterized in parallel in just four days. In the second part, the device is employed in two biological case studies to investigate fundamental properties of cyanobacteria. (i) The characterization of Synechocystis mutants lacking the ability to synthesize intracellular glycogen as carbon storage (ΔglgC mutant) and (ii) the development of a method for reporter-free observation of the circadian rhythm in individual Synechocystis cells. In both studies employing single-cell resolution time-lapse imaging revealed effects occurring within individual cells otherwise masked in bulk cultivation, thereby demonstrating the advantages of the device. Finally, the potential applications of the device in future research studies regarding phototrophic microorganisms are discussed.

Authors 1

1. Lennart Witting corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen