Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production

Abstract

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

Comparative testing of energy yields from micro-algal biomass cultures processed via anaerobic digestion

“…Although lignin is not a component of algal cells, similar cross-linked macromolecular structures exist in the cell walls offering protection to the cell contents or simply making the cell wall carbon non-digestible [47,48]. Mussgnug et al [42] noted that Scenedesmus obliquus and Chlorella kessleri have hemicellulose-containing carbohydrate-based walls which make them tougher to digest, S. obliquus especially so as it contains a sporopollenin-like biopolymer.…”

“…A wide range of microalgal cell wall compositions and structures have been reported in the literature, some of which reflect intrinsic differences between species [47,49]. In a detailed study of the susceptibility of C. vulgaris and other micro-algae to enzymatic degradation Gerken et al [48] also noted, however, that major changes in cell wall composition might depend on very small differences in growth conditions, as well as on factors such as the culture age. To draw firm conclusions on the optimum strategies to enhance anaerobic degradation, further fundamental work is required looking both at the composition and molecular structure of micro-algal cells, and at factors affecting this.…”

“…Harvesting and storage methods may themselves act as pre-treatments [48,51], with both freezing and centrifugation potentially capable of rupturing cells and thus leading to increased methane yields or improved production kinetics. T. pseudonana has a solid silica cell wall consisting of two frustules, which can open during centrifugation: this may have contributed to the high values for BMP and for TMP conversion on a VS basis compared to those for the micro-algae with organic cell walls in the current study.…”

Cell disruption technologies

“…The layer has a high cellulose content (Rodrigues and da Silva Bon, 2011), and chitin-like glycan is a predominant amino sugar in the rigid cell wall. The rigid wall components embedded within a more plastic polymeric matrix are composed of uronic acids, rhamnose, arabinose, fucose, xylose, mannose, galactose, glucose, and pectin (Gerken et al, 2013). In addition, the outer cell wall of different species may include a trilaminar algaenan or form a thin homogeneous monolayer.…”

Effect of Soaking Pre-Treatment on Reactive Extraction/<i>in situ</i> Transesterification of <i>Nannochloropsis occulata</i> for Biodiesel Production

“…However, the cell wall's resistance adversely affects the efficiency of algal bioprocesses such as genetic transformation, fermentation, anaerobic digestion, oil extraction and biodiesel production. Indeed, it leads to significant solvent requirements and energy load during the extraction processes [5]. For instance, direct FAME production requires a significant methanol to oil molar ratio, which can be as high as 1570:1 [6].…”