A microfluidic photobioreactor for simultaneous observation and cultivation of single microalgal cells or cell aggregates

Abstract

Microalgae are an ubiquitous and powerful driver of geochemical cycles which have formed Earth's biosphere since early in the evolution. Lately, microalgal research has been strongly stimulated by economic potential expected in biofuels, wastewater treatment, and high-value products. Similar to bacteria and other microorganisms, most work so far has been performed on the level of suspensions which typically contain millions of algal cells per millilitre. The thus obtained macroscopic parameters average cells, which may be in various phases of their cell cycle or even, in the case of microbial consortia, cells of different species. This averaging may obscure essential features which may be needed for the correct understanding and interpretation of investigated processes. In contrast to these conventional macroscopic cultivation and measuring tools, microfluidic single-cell cultivation systems represent an excellent alternative to study individual cells or a small number of mutually interacting cells in a well-defined environment. A novel microfluidic photobioreactor was developed and successfully tested by the photoautotrophic cultivation of Chlorella sorokiniana. The reported microbioreactor facilitates automated long-term cultivation of algae with controlled temperature and with an illumination adjustable over a wide range of photon flux densities. Chemical composition of the medium in the microbioreactor can be stabilised or modulated rapidly to study the response of individual cells. Furthermore, the algae are cultivated in one focal plane and separate chambers, enabling single-cell level investigation of over 100 microcolonies in parallel. The developed platform can be used for systematic growth studies, medium screening, species interaction studies, and the thorough investigation of light-dependent growth kinetics.

Fig 1. Microscopy and illumination setup.

(A) Inverted microscope with the inserted chip and connected tubing (red). (B) Objective revolver with inserted LED illumination device in illumination mode and (C) in imaging mode. (C) LED below the chip for illumination. (D) Exploded assembly drawing of the LED with the heat sink and connector. (E) Photograph of the assembled lighting unit.

Fig 2. Microfluidic single-cell cultivation platform used in this study.

(A) Photograph of the assembled microfluidic chip. (B) Overview over the four channels on each chip. (C) Detail of the cultivation chambers (green, dimensions 80 μm × 90 μm, height 4.5 μm) between two supply channels (blue, width 100 μm, height 15 μm), red arrows show cultivation medium flow. (D) SEM image of the PDMS chip showing one chamber.

Fig 3. Examples of growth for different light intensities.

Scale bar is always 5 μm. (A) For a light intensity of 19 μmol/(s m²) the average observed growth rate was 0.041/h, (B) at an intensity of 110 μmol/(s m²) it was 0.084/h, and (C) for light intensity of 1941 μmol/(s m²) it was 0.109/h. For the purpose of illustration the figure contrast was enhanced.

Fig 4

(A, B, C) Examples of cell death at high light intensity (1941 μmol/(s m²)). (D) Infection of algae with a parasite. Scale bar is always 5 μm. For the purpose of illustration the figure contrast was enhanced.