Testimony on a Successful Lab Protocol to Disrupt Chlorella vulgaris Microalga Cell Wall

OBJECTIVES: Microalgae have recently gained popularity due to demand for novel environmental green solutions. Notwithstanding, there is a catch. Most species of microalgae, as Chlorella vulgaris (C. vulgaris) display a recalcitrant cell wall characterized by a complex matrix of polysaccharides, a major barrier for monogastrics digestibility and extraction of nutritional compounds. To overcome this limitation, the development of feed enzymes, in particular Carbohydrate-Active enZymes (CAZymes) with capacity to disrupt C. vulgaris cell wall may contribute to improve the bioavailability of these microalgae compounds. METHODS: In order to disclosure novel combination of feed enzymes to disrupt C. vulgaris cell wall, a lab protocol was implemented containing the following steps: after microalgae cultivation and having available a repertoire of pre-selected CAZymes produced by high-throughput technology, the step 1 is the individual screening of the most functional enzymes on disrupting C. vulgaris cell wall (versus a control with PBS) and the determination of reducing sugars released by DNSA method; step 2 concerns on finding the best CAZymes cocktail, testing the synergistic effect of enzymes, to disrupt C. vulgaris cell wall (in parallel with running the control); step 3 is the assessment of the degree on Chlorella vulgaris cell wall degradation by measuring reducing sugars released by DNSA method, fatty acids by GC-FID, oligosaccharides by HPLC, protein content by Kjeldahl method, and various pigments (chlorophylls a and b, and carotenoids) in the supernatant. In the residue, we assessed cell counting using a Neubauer chamber on a bright-field microscope and fluorescence intensity, after staining with Calcofluor White for both control and CAZymes cocktail treatments. RESULTS: This is a study protocol. CONCLUSIONS: In addition to animal feed industry with impact on human nutrition, our lab protocol may increase the yield in obtaining valued constituents from C. vulgaris microalga for other biotechnological industries, namely those associated with biofuel, food, cosmetics and pharmaceutical applications. FUNDING SOURCES: Fundação para a Ciência e a Tecnologia (FCT, Lisbon, Portugal) through project UIDB/00,276/2020 to CIISA, FCT Stimulus of Scientific Employment Program to PAL (DL57/2016/CP1438/CT0007) and a PhD fellowship to DC (SFRH/BD/126,198/2016).