Self-assembly of bio-cellulose nanofibrils through intermediate phase in a cell-free enzyme system

The current study was aimed to investigate the synthesis and self-assembly of cellulose nanofibrils in in vitro environment through intermediate phase analysis and compare with the microbial cell system. The presence and stability of cellulose synthase and other essential enzymes were qualitatively validated through liquid chromatography-mass spectrometry/mass spectrometry linear trap quadrupole (LC-MS/MS LTQ) Orbitrap analysis of the culture medium after each batch (15 days' point) of in vitro incubation. Monitoring of bio-cellulose synthesis in the in vitro environment revealed that beta-1,4-glucan chains were de novo synthesized within the culture medium which crystallized and formed the premature bio-cellulose and bio-cellulose pellicles. The pellicle moved to the surface of the medium and ultimately formed a bio-cellulose sheet at the air-medium interface. In contrast to aerobic synthesis of cellulose (bacterial cellulose) by microbial cells, bio-cellulose was synthesized anaerboically by the cell-free enzyme system and the thickness of the sheet at the air-medium interface increased from the bottom surface facing the culture medium as confirmed by the field-emission scanning electron microscopy (FE-SEM). The current study will provide a base for in situ development of bio-nanocomposites of cellulose with various bactericidal and polymeric materials for broad spectrum applications.