Colony sheath formation is accompanied by shell formation and release in the green alga Botryococcus braunii (race B)

Abstract

a b s t r a c tBotryococcus braunii race B is a promising candidate for renewable biofuel production. Its colony organization is unique. Cells are connected by extracellular biopolymers containing hydrocarbons at their basolateral regions, enclosed by a retaining wall, and form a sphere inside a colony sheath composed of polysaccharide fibrils. This unique colony structure is a key for the hydrocarbon production and storage, but how the colony forms remains unknown. We studied ultrastructure of colony sheath and its formation process by rapid freezing and freezesubstitution electron microscopy. Fibrils of colony sheath extend from the retaining wall and are branched, and repel India ink. The sheath formation starts in mitosis and completes during the following daughter cell maturation. In this process, the older retaining wall and thin layers at the cell apex are released as shells and new fibrils formed on the new retaining wall extend into and merge with the pre-existing colony sheath. Its formation also accompanied by the hydrocarbon secretion and accumulation at the basolateral region. The fibrils of colony sheath that extend from a cell wall are replaced by new fibrils after each cell division, while fibrils in the extracellular biopolymers accumulate to form the remaining part of sheath, without replacement.

The surface structure of Botryococcus braunii colony prevents the entry of extraction solvents into the colony interior

“…The colony organization of the three samples observed under an electron microscope is shown in Figure 1-A2, B2, and C2. In NT, a fibrillar layer of colony sheath of about less than 5 µm (indicated by double-ended arrows) covered the entire colony surface, as reported previously [21]. The fibrillar layer of CB or PH could not be clearly observed unlike than in NT under this low magnification.…”

“…The colony structure of B. braunii hinders the extraction of hydrocarbons despite their presence in the extracellular biopolymer. In the colony interior of race B, three to six thin layers containing hydrocarbons cover the basolateral side of each cell surface and connect the cells [7,21]. On the colony surface, these cells are entirely enclosed by a retaining wall covered with fibrillar structures, i.e., the colony sheath [21,22].…”

“…In the colony interior of race B, three to six thin layers containing hydrocarbons cover the basolateral side of each cell surface and connect the cells [7,21]. On the colony surface, these cells are entirely enclosed by a retaining wall covered with fibrillar structures, i.e., the colony sheath [21,22]. Largeau et al identified these fibrils on the cell apex and showed that they continuously surrounded a part of the colony by using ruthenium red treatment [13].…”

“…Subsequently, these fibrils were found to cover not only the apical region of the cells but also the entire colony surface [22]. Moreover, Uno et al used rapid-freezing and 6 freeze-substitution electron microscopy and showed that fibrils on the cell apex were generated in a different manner from those on the extracellular biopolymer [21]. Fibrils on the cell apex were replaced during every cell division, whereas those on the extracellular biopolymer were never replaced.…”

“…Fibrils on the cell apex were replaced during every cell division, whereas those on the extracellular biopolymer were never replaced. Electron microscopy analysis revealed that these fibrils showed positive signals after silver hexamine treatment [21], a kind of periodic-acid Schiff's reaction; hence, they were thought to contain saccharic components. Therefore, these fibrils might act as a hydrophilic fibrillar layer and prevent the entry of non-polar solvents such as n-hexane in the colony interior during hydrocarbon extraction.…”