Protein-functionalized cellulose fibrils, having various amounts of covalently bonded proteins at their surface, were successfully extracted from the tunic of Pyura chilensis tunicates using successive alkaline extractions. Pure cellulose fibrils were also obtained by further bleaching and were used as reference material. Extraction yields of protein-functionalized cellulose fibrils were within the range of 62-76% by weight based on the dry initial tunic powder. Fourier-transform infrared and Raman spectroscopy confirmed the preservation of residual protein at the surface of cellulose fibrils, which was then quantified by X-ray photoelectron spectroscopy. The protein-functionalized cellulose fibrils were found to have relatively high crystallinity and their cellulose I crystalline structure was preserved upon applying alkaline treatments. The extracted cellulosic materials were found to be constituted of fibrils having a ribbon-like morphology with widths ranging from ∼30 nm up to ∼400 nm. These protein-functionalized cellulose fibrils were found to have outstanding thermal stability with one of them having onset and peak degradation temperatures of ∼350 and 374 °C, respectively. These values were found to be 24 and 41 °C higher than for bleached cellulose.

Protein functionalized cellulose fibrils were isolated from the tunic of Pyura chilensis and subsequently used to produce protein functionalized cellulose membranes. Bleached cellulose membranes were also obtained and used as reference material. FTIR and Raman spectroscopy demonstrated that the membranes are mostly constituted of cellulose along with the presence of residual proteins and pigments. Protein functionalized cellulose membranes were found to possess ∼3.1% of protein at their surface as measured by X-ray photoelectron spectroscopy. Powder X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis were used to identify and semiquantify the amount of residual sand grains present within the structure of the membranes. The presence of residual proteins was found not to significantly affect the tensile mechanical properties of the membranes. Streaming ζ-potential was used to assess surface charges of the membranes. Below pH 4, nonbleached cellulose membranes possessed highly negative surfaces charges and also significantly less negative surface charges at physiological pH when compared to bleached cellulose membranes. No significant difference was found with respect to growth kinetics of myoblasts at the surface of the membranes for cell culturing times of 48 and 72 h. After 48 h of culture, protein functionalized cellulose-based membranes that possess ∼3.1% of proteins at their surface (H1) were, however, found to promote higher cell density, cell spreading, and more orientated shape cell morphology when compared to the other cellulose-based membranes (H3 and B) evaluated in the present study.