Direct imaging of the alternating disordered and crystalline structure of cellulose fibrils via super-resolution fluorescence microscopy



Cellulose, the primary component of the plant cell wall, has fueled the wood, textile, pulp and paper industries for centuries, and has recently been used for the production of renewable nanomaterials. The tight crystalline packing of glucan chains within cellulose fibrils is responsible for its superior mechanical properties but renders this material recalcitrant to biochemical and chemical breakdown and limits its use as a green resource. The presence of nanoscale dislocations within cellulose fibrils has been postulated for decades and is thought to be responsible for the production and size of cellulose nanocrystals (CNCs) following acid hydrolysis. However, dislocations have never been directly visualized and their prevalence and size have remained elusive. In this study, we have used super-resolution (SR) fluorescence microscopy to directly visualize and measure alternating crystalline and disordered regions within individual fluorescently labelled bacterial cellulose fibrils. The measured size of the crystalline regions ranges from 40 – 400 nm and shows striking overlap with the length distribution of bacterial CNCs produced through sulfuric acid hydrolysis, supporting the fringed micellar model for the supramolecular structure of cellulose fibrils. The disordered regions were found to be 20 – 120 nm in length and show heterogeneous accessibility, which directs fibril cleavage during the initial stages of cellulose acid hydrolysis. Two-colour SR imaging of cellulose fibrils and bound exoglucanases (Cel7A), in combination with degree of crystallinity measurements suggest that these dislocations are nanoscale in size, and do not result in amorphous cellulose pockets large enough to accommodate enhanced enzyme binding. Through characterization of disordered regions in cellulose fibrils, we have gained insight into the role of cellulose nanostructure in its breakdown by chemical and enzymatic means.


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Supplementary material

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Six supplementary figures and one table
Figure S1. Labeling BC with different reaction schemes and with spectrally distinct fluorophores. Figure S2. Continuous localization of fluorophores along Cy5-BC fibrils and identification of bright regions using a local median threshold method. Figure S3. Different thresholding methods used for quantitatively characterizing the labeling pattern of DTAF-BC and Cy5-BC fibrils. Figure S4. AFM images of BC and CNCs produced following various durations of the acid hydrolysis treatment. Figure S5. Lengths of CNCs produced from bacterial cellulose with different durations of the acid hydrolysis treatment compared to Cy55μM-BC spacing lengths measured with different peak-picking thresholds. Figure S6. Proposed mechanism for the acid hydrolysis of bacterial cellulose fibrils in the production of cellulose nanocrystals. Table S1. Characterization of purified BC and bacterial CNCs resulting from time-lapsed acid hydrolysis.