Structure elucidation of uniformly 13C-labeled bacterial celluloses from different Gluconacetobacter xylinus strains

Abstract

The morphological and supramolecular structures of native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769) were investigated. Samples had been statically cultivated in Hestrin-Schramm medium containing fully 13 C-labeled b-D-glucose-U-13 C 6 as the sole source of carbon. The results are compared with structure data of bacterial celluloses with a natural 13 C abundance of 1.1%. Non-enriched and 13 C-labeled cellulose pellicles formed crystalline structures as revealed by cross-polarized/magic-angle spinning (CP/MAS) 13 C{ 1 H}-NMR and near infrared (NIR) FT-Raman spectroscopic measurements as well as wide-angle X-ray diffraction (WAXD) investigations. Atomic force microscopy (AFM) was applied for analyzing fiber morphologies and surface properties. For the first time, details about the manipulation of fiber widths and pellicle formation were shown for different bacterial strains of G. xylinus depending on the use of b-D-glucose-U-13 C 6 for the biosynthesis.

Spectral assignments and anisotropy data of cellulose Iα: 13C‐NMR chemical shift data of cellulose Iα determined by INADEQUATE and RAI techniques applied to uniformly 13C‐labeled bacterial celluloses of different Gluconacetobacter xylinus strains

“…As for the nonenriched samples, the quantitative analysis of the C4 resonances of the crystalline (∼90 ppm) and amorphous (∼84 ppm) components indicated that only about one fourth of the 13 C-labeled material occurs in noncrystalline regions. [21,27] The data also show that the 13 Cenrichment of samples 1 and 2 was successful with about 94% yield, virtually uniformly at all carbon positions. In view of their rather well-resolved 13 C-NMR lines compared to BC DSM 13 368, [10] with slight modifications (Avance 500, 4 mm, ν R = 12.5 kHz, ns = 256, t w = 1 s, t CP = 5 ms, TPPM: ±20 • , 10 µs; echo time for J-couplings = 3.04 ms) of uniformly 13 C-labeled bacterial cellulose from Gluconacetobacter xylinus NQ-5 (1).…”

“…[20,21] The optimized RAI sequence thus proves to be the most appropriate method for de-termining the full information on 13 C chemical shift anisotropies of cellulose in moderate experimental time.…”

“…These insights on the distinctive character of cellulose samples from different strains of G. xylinus have also been confirmed by analytical methods other than solid-state NMR and will be published elsewhere. [21] Using 2D refocused INADEQUATE experiments on uniformly 13 Clabeled samples, accurate 13 C chemical shifts δ iso were obtained for all carbons of the cellulose allomorph I α in BC NQ-5 (1) and BC AY-201 (2). These values were taken into account when determining the principal chemical shift tensor components with the aid of an optimized 2D RAI experiment.…”

“…Further details about crystallinity and the morphological structure of these 13 C-labeled bacterial cellulose pellicles will be published elsewhere. [21] NMR spectroscopy 1D CP/MAS 13 C{ 1 H}-NMR spectra were recorded on a Bruker AMX 400 MHz (AMX 400) spectrometer operating at 100.58 MHz for 13 C, with a 4-mm MAS double resonance probe and ZrO 2 rotors. The measurements were carried out at 6.5 kHz MAS.…”

“…Furthermore, differences in the morphological structures of BC pellicles produced by the two different G. xylinus strains occur. [21] To investigate the supramolecular structures of these bacterial celluloses, NMR experiments are indispensable. The corresponding 13 C isotropic chemical shift values of cellulose I α could be assigned using 2D refocused INADEQUATE experiments.…”