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Insights into cell wall disintegration of Chlorella vulgaris.

ABSTRACT: With their ability of CO2 fixation using sunlight as an energy source, algae and especially microalgae are moving into the focus for the production of proteins and other valuable compounds. However, the valorization of algal biomass depends on the effective disruption of the recalcitrant microalgal cell wall. Especially cell walls of Chlorella species proved to be very robust. The wall structures that are responsible for this robustness have been studied less so far. Here, we evaluate different common methods to break up the algal cell wall effectively and measure the success by protein and carbohydrate release. Subsequently, we investigate algal cell wall features playing a role in the wall's recalcitrance towards disruption. Using different mechanical and chemical technologies, alkali catalyzed hydrolysis of the Chlorella vulgaris cells proved to be especially effective in solubilizing up to 56 wt% protein and 14 wt% carbohydrates of the total biomass. The stepwise degradation of C. vulgaris cell walls using a series of chemicals with increasingly strong conditions revealed that each fraction released different ratios of proteins and carbohydrates. A detailed analysis of the monosaccharide composition of the cell wall extracted in each step identified possible factors for the robustness of the cell wall. In particular, the presence of chitin or chitin-like polymers was indicated by glucosamine found in strong alkali extracts. The presence of highly ordered starch or cellulose was indicated by glucose detected in strong acidic extracts. Our results might help to tailor more specific efforts to disrupt Chlorella cell walls and help to valorize microalgae biomass.

SUBMITTER: Weber S

PROVIDER: S-EPMC8759652 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Over the last decades, microalgae have gained popularity due to demand for novel environmental green solutions and development of innovative mass-production sources for multiple processes, including animal feed and human diet, turning microalgae into an exquisite candidate for several ecofriendly technologies. Notwithstanding, there is a catch. Most species of microalgae, as the case of common Chlorella vulgaris (C. vulgaris) display a recalcitrant cell wall, characterized by a complex matrix of polysaccharides and glycoproteins, which constitutes a major barrier for monogastric species digestibility and extraction of inner valuable 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 in monogastric diets, namely at high levels of incorporation. In order to disclosure novel combination of feed enzymes to disrupt C. vulgaris cell wall, a lab protocol was implemented by our research team containing the following key steps: after microalgae cultivation and having available a repertoire of two hundred 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, defined as the microalgae suspension incubated with PBS) and the determination of reducing sugars released by the 3,5-dinitrosalicylic acid (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) along with characterization of each enzyme thermostability and resistance to proteolytic attack, to which feed enzymes are subjected in the animal gastrointestinal tract; step 3 is the assessment of C. vulgaris cell wall degradation degree by measuring the amount of reducing sugars released by the DNSA method, fatty acid analysis by gas chromatography (GC) with flame ionization detector (FID), oligosaccharides quantification by high performance liquid chromatography (HPLC) equipped with an electrochemical detector (ECD), protein content by the Kjeldahl method, and various pigments (chlorophylls a and b, and total carotenoids) in the supernatant. In the correspondent residue, we also assessed cellular counting using a Neubauer chamber by direct observation on a bright-field microscope and fluorescence intensity, after staining with Calcofluor White for both control and CAZymes cocktail treatments, on a fluorescence microscope. Beyond 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.

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Insights into cell wall disintegration of Chlorella vulgaris.

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