Daisuke Sawada

Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment

BackgroundObtaining a better understanding of the complex mechanisms occurring during lignocellulosic deconstruction is critical to the continued growth of renewable biofuel production. A key step in bioethanol production is thermochemical pretreatment to reduce plant cell wall recalcitrance for downstream processes. Previous studies of dilute acid pretreatment (DAP) have shown significant changes in cellulose ultrastructure that occur during pretreatment, but there is still a substantial knowledge gap with respect to the influence of lignin on these cellulose ultrastructural changes. This study was designed to assess how the presence of lignin influences DAP-induced changes in cellulose ultrastructure, which might ultimately have large implications with respect to enzymatic deconstruction efforts.ResultsNative, untreated hybrid poplar (Populus trichocarpa x Populus deltoids) samples and a partially delignified poplar sample (facilitated by acidic sodium chlorite pulping) were separately pretreated with dilute sulfuric acid (0.10 M) at 160°C for 15 minutes and 35 minutes, respectively . Following extensive characterization, the partially delignified biomass displayed more significant changes in cellulose ultrastructure following DAP than the native untreated biomass. With respect to the native untreated poplar, delignified poplar after DAP (in which approximately 40% lignin removal occurred) experienced: increased cellulose accessibility indicated by increased Simons’ stain (orange dye) adsorption from 21.8 to 72.5 mg/g, decreased cellulose weight-average degree of polymerization (DPw) from 3087 to 294 units, and increased cellulose crystallite size from 2.9 to 4.2 nm. These changes following DAP ultimately increased enzymatic sugar yield from 10 to 80%.ConclusionsOverall, the results indicate a strong influence of lignin content on cellulose ultrastructural changes occurring during DAP. With the reduction of lignin content during DAP, the enlargement of cellulose microfibril dimensions and crystallite size becomes more apparent. Further, this enlargement of cellulose microfibril dimensions is attributed to specific processes, including the co-crystallization of crystalline cellulose driven by irreversible inter-chain hydrogen bonding (similar to hornification) and/or cellulose annealing that converts amorphous cellulose to paracrystalline and crystalline cellulose. Essentially, lignin acts as a barrier to prevent cellulose crystallinity increase and cellulose fibril coalescence during DAP.

Comparison of changes in cellulose ultrastructure during different pretreatments of poplar

Abstract One commonly cited factor that contributes to the recalcitrance of biomass is cellulose crystallinity. The present study aims to establish the effect of several pretreatment technologies on cellulose crystallinity, crystalline allomorph distribution, and cellulose ultrastructure. The observed changes in the cellulose ultrastructure of poplar were also related to changes in enzymatic hydrolysis, a measure of biomass recalcitrance. Hot-water, organo-solv, lime, lime-oxidant, dilute acid, and dilute acid-oxidant pretreatments were compared in terms of changes in enzymatic sugar release and then changes in cellulose ultrastructure measured by 13C cross polarization magic angle spinning nuclear magnetic resonance and wide-angle X-ray diffraction. Pretreatment severity and relative chemical depolymerization/degradation were assessed through compositional analysis and high-performance anion-exchange chromatography with pulsed amperometric detection. Results showed minimal cellulose ultrastructural changes occurred due to lime and lime-oxidant pretreatments, which at short residence time displayed relatively high enzymatic glucose yield. Hot water pretreatment moderately changed cellulose crystallinity and crystalline allomorph distribution, yet produced the lowest enzymatic glucose yield. Dilute acid and dilute acid-oxidant pretreatments resulted in the largest increase in cellulose crystallinity, para-crystalline, and cellulose-Iβ allomorph content as well as the largest increase in cellulose microfibril or crystallite size. Perhaps related, compositional analysis and Klason lignin contents for samples that underwent dilute acid and dilute acid-oxidant pretreatments indicated the most significant polysaccharide depolymerization/degradation also ensued. Organo-solv pretreatment generated the highest glucose yield, which was accompanied by the most significant increase in cellulose microfibril or crystallite size and decrease in relatively lignin contents. Hot-water, dilute acid, dilute acid-oxidant, and organo-solv pretreatments all showed evidence of cellulose microfibril coalescence.

X-ray crystal structure of anhydrous chitosan at atomic resolution.

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [au2009=u20098.129(7) Å, bu2009=u20098.347(6) Å, cu2009=u200910.311(7) Å, space group P212121] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains.

The initial structure of cellulose during ammonia pretreatment

AbstractA protocol was developed to freeze-trap (at 150xa0K) cellulose as it is undergoing liquid ammonia pretreatment, and then to collect X-ray diffraction data from the freeze-trapped reactants as the reaction is allowed to proceed and ammonia is allowed to melt and then evaporate, leaving ammonia-cellulose I. Cellulose adopts a new two-chain crystal form, which we call low temperature phase ammonia-cellulose I (two-chains and ~ten ammonia molecules within a unit cell of axa0=xa015.49xa0Å, bxa0=xa011.35xa0Å, cxa0=xa010.42xa0Å and γxa0=xa0143.5°). A schematic model was developed that is characterized by sheets of hydrophobically stacked cellulose-chains with hydrophilic channels between them that are filled with ammonia molecules. Neighboring chains in these sheets have either different conformations or are staggered with respect to each other. As ammonia is allowed to evaporate, the unit cell size is reduced by a factor of two as the two independent chains become identical.n

Structure and dynamics of a complex of cellulose with EDA: insights into the action of amines on cellulose

The neutron structure of a complex of EDA with cellulose has been determined to reveal the location of hydrogen atoms involved in hydrogen-bonding. EDA disrupts the hydrogen-bonding pattern of naturally occurring cellulose by accepting a strong hydrogen-bond from the O6 hydroxymethyl group as the conformation of this group is rotated from tg to gt. The O3-H·O5 intrachain hydrogen-bond commonly found in cellulose allomorphs is observed to be disordered in the neutron structure, and quantum chemistry and molecular dynamics calculations show that O3 prefers to donate to EDA. The hydrogen-bonding arrangement is highly dynamic with bonds continually being formed and broken thus explaining the difficulty in locating all of the hydrogen atoms in the neutron scattering density maps. Comparison with other polysaccharide-amino complexes supports a common underlying mechanism for amine disruption of cellulose.

Tension wood structure and morphology conducive for better enzymatic digestion

BackgroundTension wood is a type of reaction wood in response to bending or leaning stem as a corrective growth process. Tension wood is formed by both natural and man-made processes. Most attractively, tension wood contains higher glucan content and undergoes higher enzymatic conversion to fermentable sugars. Here, we have employed structural techniques, small-angle neutron scattering (SANS) and wide-angle X-ray diffraction (WAXD) to elucidate structural and morphological aspects of tension wood conducive to higher sugar yields.ResultsSmall-angle neutron scattering data exhibited a tri-modal distribution of the fibril cross-sectional dimension. The smallest size, 22xa0Å observed in all samples concurred with the WAXD results of the control and opposite side samples. This smallest and the most abundant occurring size was interpreted as the cellulose elementary microfibril diameter. The intermediate size of 45xa0Å, which is most pronounced in the tension side sample and consistent with WAXD results for tension side sample, indicates association of neighboring elementary microfibrils to form larger crystallite bundles. The largest size 61xa0Å observed by SANS was however not observed by WAXD and therefore associated to mesopores.ConclusionsStructure and morphology of tension wood is different from control wood. Cellulose crystallinity increases, lignin content is lower and the appearance of mesopores with 61xa0Å diameter is observed. Despite the presence of higher crystalline cellulose content in tension side, the lower lignin content and may be combined with the abundance of mesopores, substantially improves enzyme accessibility leading to higher yields in cellulose digestion.

Solid–solvent molecular interactions observed in crystal structures of β-chitin complexes

AbstractnThree β-chitin structures [anhydrous, di-hydrate, mono-ethylenediamine (EDA)] recently determined by synchrotron X-ray and neutron fiber diffraction were reviewed from the viewpoint of molecular interactions. Both water and EDA molecules interact with the chitin chains through multiple hydrogen bonds. When water complexes with chitin, the hydrogen bonding pattern rearranges with the replacement of an intrachain chitin hydrogen bond by a stronger hydrogen bond between chitin and water, with an associated reduction in the degrees of freedom; the water oxygen is a much stronger acceptor than the O5 ring atom. The behavior of hydrogen exchange by deuterium supports this interpretation. EDA-molecules change the conformation of hydroxymethyl group from gg to gt, accompanied by changes in hydrogen bonds due to the strong accepting ability of the EDA nitrogen atoms. Some important interactions are in common with experimental crystallographic results of cellulosic crystals and of molecular dynamics studies. These new insights into solid–solvent interactions are valuable in understanding molecular interactions in other polysaccharides-solvents system in solution or on surface.

Distinguishing Surface Versus Bulk Hydroxyl Groups of Cellulose Nanocrystals using Vibrational Sum Frequency Generation Spectroscopy

In plant cell walls and cellulose-containing composites, nanocrystalline cellulose interacts with water molecules or matrix polymers through hydrogen bonding of the hydroxyl groups at the cellulose surface. These interactions play key roles in cellulose assembly in plant cell walls and mechanical properties of cellulose composites; however, they could not be studied properly due to the spectroscopic difficulty of selectively detecting the surface hydroxyl groups of nanocrystalline domains. This study employed the sum frequency scattering principle to distinguish the hydroxyl groups inside of the crystalline nanodomain of cellulose and those exposed at the surface of crystalline domains. The comparison of the spectra at various scattering angles revealed that the OH peak near ∼3450 cm-1 comes from the weakly hydrogen-bonded OH groups at the surface of crystalline cellulose. Also, a time delay measurement found that the sharp vibrational features observed near 3700 cm-1 can be attributed to isolated OH groups not accessible by ambient water molecules. These findings allow the distinction of surface versus bulk OH groups in sum frequency generation vibrational spectroscopy.

Crystalline alignment of metal ions templated by β-chitin ester

AbstractThe highly crystalline β-chitin from diatom Thalassiosira weissflogii was esterified via intercalation with succinic anhydride followed by simple heating, maintaining the original crystalline order. Due to the introduced free carboxyl groups, the chitin ester crystal showed ion exchange ability for metal cations in aq. solution. Heavy metal cations such as Pb2+ bound to the β-chitin succinate gave characteristic X-ray diffraction patterns, indicating regular alignment of metal ions. Such materials represent a new type of organometallic architecture, possibly leading to novel functionalities.n

The Shape of Native Plant Cellulose Microfibrils

Determining the shape of plant cellulose microfibrils is critical for understanding plant cell wall molecular architecture and conversion of cellulose into biofuels. Only recently has it been determined that these cellulose microfibrils are composed of 18 cellulose chains rather than 36 polymers arranged in a diamond-shaped pattern. This study uses density functional theory calculations to model three possible habits for the 18-chain microfibril and compares the calculated energies, structures, 13C NMR chemical shifts and WAXS diffractograms of each to evaluate which shape is most probable. Each model is capable of reproducing experimentally-observed data to some extent, but based on relative theoretical energies and reasonable reproduction of all variables considered, a microfibril based on 5 layers in a 34443 arrangement is predicted to be the most probable. A habit based on a 234432 arrangement is slightly less favored, and a 6u2009×u20093 arrangement is considered improbable.