Study and monitoring of the multiple fission cell cycle of Chromochloris zofingiensis: a high-throughput approach

Microalgae are photosynthetic microorganisms that typically live in freshwater, soil or marine habitats, but also more extreme habitats, such as salt, sulfur-rich lakes or on snow surfaces. They are becoming increasingly important in the bioeconomy and biotechnology sectors as an attractive, sustainable source of value-added products, owing to their enormous potential for the production of industrially relevant, high-value products, e.g., pigments with antioxidant and antibacterial activity such as carotenoids (astaxanthin, canthaxanthin, β-carotene and lutein), polysaccharides, polyunsaturated and omega-3 fatty acids. Some microalgae species grow and divide through binary fission, in which one cell divides into two cells, while others divide through multiple fissions, in which a cell can produce up to several daughter cells; in some cases, both binary and multiple fission can be executed in response to changes in ambient conditions or life cycle phase.

Chromochloris zofingiensis is a unicellular freshwater microalga, considered as a promising natural producer of high-value carotenoids such as astaxanthin. It can reproduce itself via asexual multiple fission cell cycle, under which DNA replication and nuclear division are executed multiple times prior to cell division. Thus, allowing the observation of polynuclear cells (PNC) (Figure 1), in which the number of daughter cells within a cell is determined by the number of DNA replications and nuclear division events before cell division. Though, as C. zofingiensis cells progress through their cell cycle, they grow, commit to divide, execute DNA replications and nuclear divisions, therefore creating the PNC sub-populations in which 2n daughter cells are produced, with the exponent “n” indicating the number of DNA replications that occurred and representing a degree of heterogeneity within the same population (Figure 2).

Figure 1. Microscopic observation showing the evolution of the cleavage process of C. zofingiensis mother cell about to release its daughters.

Figure 2. Microscopic observation showing high cell population heterogeneity of C. zofingiensis cultivated in non-limited photoautotrophic conditions.

Precise and quick monitoring of key microalgae population features such as cell count, cell size, cell morphology, and DNA content is crucial for applications in cell culture research to develop feasible algae-based bioprocesses. Thus, analysis and examination of these different physiological state parameters must be precise, accurate, and characterized by quickly measurements. In this perspective, one of the main objectives of our project is to develop new single-cell analysis techniques and methods to study the population heterogeneity of the non-conventional microalgae C. zofingiensis. Based on Flow Cytometry (FCM), a method that allows high-throughput analysis and acquisition of key data on cell populations at a single-cell level.

Therefore, a sensitive routine is currently being developed to allow us to detect and recognize microalgal population heterogeneity as well as different phases of the cell cycle, by combining DNA staining dyes with flow cytometry and image cytometry analysis. The latter offers visual inspection of the cell population and exact quantification of sub-populations proportions since it provides different images for each analyzed cell (Figure 3).

Figure 3. High throughput Image Cytometry analysis of C. zofingiensis population heterogeneity at the single-cell level.

by Yob Ihadjadene