Formation of extracellular β-chitin nanofibers during batch cultivation of marine diatom Cyclotella sp. at silicon limitation

Diatoms are unicellular algae that make nanostructured biosilica shells called frustules possessing ordered pore arrays. Dissolved silicon (Si) in the form of Si (OH)(4) is the required substrate for cell wall biosynthesis and cell division. The extracellular formation of beta-chitin nanofibers by the centric diatom Cyclotella sp. was followed during batch cultivation of the cell suspension within a bubble-column photobioreactor at a low initial Si concentration (0.25 mM) leading to 1-2 cell divisions, and high Si concentration (1.7 mM) leading to 4-5 cell divisions. Fibers were excreted from 20 specialized pores (fultoportulae) lining the rim of each frustule valve face. In Si-limited batch cultivation, 80% of the fibers formed during the last cell division after dissolved Si was depleted from the medium. Within 24 h after the final cell division, fibers were nominally 56 nm diameter and 60 mu m length, with 85% of the fibers between 20 and 80 mu m, and one fiber per fultoportula was formed. After the final cell number density was maintained for 72 h, a second fiber per fultoportula was formed, presumably after breakage or scission of the first fiber from the cell. X-ray diffraction (XRD) analysis confirmed that isolated fibers were composed of pure beta-chitin. The unprecedented length and purity of the beta-chitin nanofibers suggest future advanced material applications.