Uncovering the complex cell wall of Neochloris oleoabundans, a promising green microalga

An ever-increasing world population together with industrial development at an accelerated pace have push scientists to identify promising bio-resources to produce food, feed and fuel in the framework of carbon-neutral bio-economy. Amongst the bio-resources available, there has been a growing interest in the exploitation of microalgae biomass as an alternative green feedstock. However, at this moment, the microalgae-derived products are not economically feasible on account of a low return in investment. One way to enhance the economic viability of microalgae production and utilization is to design effective and sustainable methods for the extraction of its components. To design such methods we need to understand the biology of the microalgae cell. N. oleoabundans, the studied green microalgae in this thesis, contains valuable components enclosed within a morphologically complex cell wall. Thus, before accessing the intracellular components the cell wall barrier needs to be deconstructed. The overall aim of this thesis was to generate in-depth insights into cell wall building blocks of N. oleoabundans. Fundamental knowledge on the physicochemical structure and the underlying molecular determinants of cell wall biosynthesis and modification are the main focus. In Chapter 1 of this thesis, we presented an introduction about the importance of microalgae as a promising renewable feedstock. Further, we described the enormous biodiversity among the cell walls of different life kingdoms and in particular Chlorophyta green microalgae. In Chapter 2, we have made a comprehensive characterization of the N. oleoabundans cell walls. The results showed that N. oleoabundans cell walls are composed of about 2.4.3% carbohydrates, 31.5% proteins, 22.2% lipids and 7.8% inorganic compounds. A rather scarce amount of glucose in the N. oleoabundans suggested that non-cellulosic polysaccharides may underpin the cell wall rigidity. We concluded in this chapter that N. oleoabundans cell walls are very different from (higher) plant cell walls, and that similar functionalities can be achieved by (combinations of) different polymers. In Chapter 3, we evaluated the wall composition and properties of N. oleoabundans cells grown under freshwater nitrogen-replete (optimum culture) and -depleted conditions, and seawater nitrogen-replete and -depleted conditions. Results from this chapter substantiated that variation in cell wall composition was notably dependant on the growing conditions, supporting the importance of the cell wall as a dynamic structure to adapt to different environments. In Chapter 4, microscopic approaches were applied to describe the morphologic characteristics of cell wall development throughout the cell cycle. We observed that throughout the cell cycle N. oleoabundans cell walls are mostly doubled. Electron microscopy images enabled us to propose a model for the daughter cell wall regeneration in N. oleoabundans. In this model, daughter cell wall deposition occurs at the beginning of the growth phase. In Chapter 5, we performed a comparative transcriptomics study followed by biochemical characterisation to identify the molecular mechanisms underlying the cell wall development throughout the cell cycle. We disclosed that glucosamine, glucose, galactose and rhamnose were the main carbohydrates of N. oleoabundans cell wall that undergo controlled variations through the cell cycle. An important factor identified in cell wall dynamic process throughout the cell cycle was an increase in the bulk of uridine diphosphate glucose (UDP-D-Glu) during the growth phase, which was mainly related to chitin and galactose degradation. In Chapter 6, we integrated the entire knowledge and new insights generated within this research. We discussed the uniqueness of N. oleoabundans cell wall, considering its constitutive components, its morphological properties, and changes in response to the environment and developmental phase. Furthermore, we proposed a new approach, based on the fundamental knowledge generated on cell wall-degrading enzymes, that could facilitate a valorisation of the whole microalgae biomass.