PROBING THE MESOSCALE STRUCTURE OF CRYSTALLINE CELLULOSE IN PLANT BIOMASS USING SUM-FREQUENCY-GENERATION (SFG) VIBRATIONAL SPECTROSCOPY

Open Access
Author:
Lee, Christopher Morgan
Graduate Program:
Chemical Engineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
May 03, 2016
Committee Members:
  • Seong Han Kim, Dissertation Advisor
  • Phillip E Savage, Committee Chair
  • Enrique Daniel Gomez, Committee Member
  • Wayne Roger Curtis, Committee Member
  • Daniel J Cosgrove, Outside Member
Keywords:
  • cellulose
  • sum-frequency-generation
  • nonlinear spectroscopy
  • plant cell walls
Abstract:
However, the crystalline cellulose assembly inside plant cell walls is extremely difficult to analyze in their intact states. This study utilizes sum frequency generation (SFG) vibration spectroscopy which can selectively detect crystalline cellulose within intact plant biomass, due to the unique requirement of non-centrosymmetric ordering. In this work we found that SFG is sensitive to the molecular structure, crystal structure and mesoscale structure of cellulose inside plant biomass. Obtaining such structural information spanning multiple length scales is not achievable by conventional analytical methods. Therefore, SFG presents a unique opportunity to study the hierarchical structures of cellulose in plant cell walls which are designed for specific biological functions. At the nanoscale, the molecular and crystal structures of cellulose are determined by the covalent bonds as well as hydrogen bonding networks between glucan chains. The peak positions in the SFG spectra of native cellulose Iα and Iβ were identified through comparison to the vibration frequencies calculated with density functional theory with dispersion corrections (DFT-D2). The frequencies from DFT-D2 calculations allowed the full assignments of the dominant SFG peaks. These are the localized CH2 symmetric (2850, 2886 cm-1) and asymmetric (2920, 2944, 2968 cm-1) stretching vibrations, and the coupled 2O-H (3270 cm-1), 2,3,6O-H (3300‒3330 cm-1) and 3O-H (3370 cm-1) stretching vibrations. The observation that O-H stretching vibration modes are highly coupled through hydrogen bonds lead to new assignments for the O-H peaks which replaced the incorrect assignments propagating in the literature for more than two decades. These advances in spectral interpretation of cellulose vibrational spectra are critical to utilize SFG analysis for the identification of cellulose crystal structures. SFG was employed to distinguish between the native crystal structures (Iα and Iβ) from the artificially-induced polymorphs (cellulose II, IIII and IIIII). This study showed that SFG is sensitive to cellulose crystal structure; due to the non-centrosymmetric ordering of polar cellulose chains within the crystallographic unit cell at the nanometer length scale. The SFG intensity in the O-H stretching region was strong for crystals containing parallel chains (Iβ and IIII) and quite weak for antiparallel chains (II and IIIII) due to the symmetry cancellation of the OH stretching vibrations. The SFG intensity is also dependent on the non-centrosymmetric ordering of functional groups or crystallites over the SFG coherence length which is approximately hundreds of nanometers to microns. Two reference samples of purified cellulose Iβ were used: uniaxially-aligned crystallites and randomly-packed or aggregated crystallites. It was found that the overall SFG intensities and peak shapes of the alkyl (C-H) and hydroxyl (O-H) stretch modes correlate with the lateral packing and net directionality of cellulose microfibrils at the mesoscale. Cellulose SFG spectra of cell walls from various plant species could be identified into two groups based on the (C-H/O-H) intensity ratios. The first group included antiparallel-packed cellulose microfibrils as found in textile fibers and secondary cell walls of dicots and monocots. The second group included non-antiparallel packed cellulose as found in primary cell walls, algal cell walls, bacterial biofilms and tunicate mantles. This study showed that SFG can distinguish between cellulose within the (primary) walls of actively growing cells and cellulose within (secondary) walls of matured and lignified cells. The (C-H/O-H) intensity ratio was utilized to study the development of cotton fibers from two species, G. hirsutum and G. barbadense, which had different lateral packing at the mesoscale depending on the natural drying process that takes place during the maturation stage. The (C-H/O-H) intensity ratio was also used to determine the mesoscale packing of bacterial cellulose pellicles produced from two strains of G. xylinus. The packing pattern of crystalline cellulose was found to be different on the upper and lower portions of the pellicle and culture time of the bacteria. A broadband sum frequency generation spectrometer combined with a microscope (BB-SFG-M) was constructed in order to visualize the heterogeneous nature of cellulose across plant tissues with different cell types. We demonstrated the applicability of SFG microscopy to secondary cell walls in cotton fibers and the primary cell walls from onion epidermal cells.