Interactions of Arabinoxylan and (1,3)(1,4)-β-Glucan with Cellulose

Article pubs.acs.org/Biomac

Interactions of Arabinoxylan and (1,3)(1,4)-β-Glucan with Cellulose Networks Deirdre Mikkelsen,† Bernadine M. Flanagan,† Sarah M. Wilson,‡ Antony Bacic,‡ and Michael J. Gidley*,† †

The University of Queensland, ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, Queensland 4072, Australia ‡ The University of Melbourne, ARC Centre of Excellence in Plant Cell Walls, School of Botany and Bio21 Molecular Science and Biotechnology Institute, Melbourne, Victoria 3010, Australia S Supporting Information *

ABSTRACT: To identify interactions of relevance to the structure and properties of the primary cell walls of cereals and grasses, we used arabinoxylan and (1,3)(1,4)-β-glucan, major polymers in cereal/grass primary cell walls, to construct composites with cellulose produced by Gluconacetobacter xylinus. Both polymers associated prolifically with cellulose without becoming rigid or altering the nature or extent of cellulose crystallinity. Mechanical properties were modestly affected compared with xyloglucan or pectin (characteristic components of nongrass primary cell walls) composites with cellulose. In situ depletion of arabinoxylan arabinose side chains within preformed cellulose composites resulted in phase separation, with only limited enhancement of xylan−cellulose interactions. These results suggest that arabinoxylan and (1 → 3)(1 → 4)-β-D-glucan are not functional homologues for either xyloglucan or pectin in the way they interact with cellulose networks. Association of cell-wall polymers with cellulose driven by entropic amelioration of high energy cellulose/water interfaces should be considered as a third type of interaction within cellulose-based cell walls, in addition to molecular binding (enthalpic driving force) exhibited by, for example, xyloglucans or mannans, and interpenetrating networks based on, for example, pectins.

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under a range of imposed stresses,15,16 both before and after the action of PCW proteins (expansins, xyloglucan endotransglycosylases, xyloglucanases) hypothesized to have a mechanical effect on native PCWs.17,18 Previous work studied composites of cellulose with xyloglucans (XG) or pectins, the major polysaccharides of the primary cell walls (“Type I” walls) of most dicots and nongraminaceous monocots but minor polysaccharides in commelinoid monocots. However, the primary cell walls (“Type II” walls) of cereals/grasses (Poaceae) and related commelinoid monocots differ in their polysaccharide composition, with cellulose being the only major conserved feature between the two PCW types.2,19−21 Instead, heteroxylans decorated with various amounts of arabinose (AXs) or glucuronic acid (GAXs/GXs) are the major noncellulosic components, while xyloglucan and pectin levels are low.1,2,20,21 In addition, (1 → 3)(1 → 4)-β-D-glucans (mixed linkage glucans or MLG) are deposited in certain tissues and at particular stages of development. Various studies have hypothesized that heteroxylans or MLGs are able to form

INTRODUCTION Cell walls provide the structural framework of plants, playing a critical role in their growth and development. Furthermore, they are an integral part of the human diet and a major source of renewal biomass. Therefore, structural features of plant cell wall (PCW) polymers have been the subject of research for decades and are now largely defined.1−6 Recently, research has intensified to identify genes responsible for the synthesis and assembly of individual PCW polymers.5−8 Despite these significant advances, there is still limited understanding of how individual polymers come together to form the PCW matrix and what the functional consequences of different matrix compositions and architectures are on cell-wall material properties. The assembly of PCWs has been modeled using an in vitro construction approach with the cellulose-producing bacterium Gluconacetobacter xylinus (formerly Acetobacter xylinus). Ga. xylinus produces extracellular cellulose via transmembrane synthesis and an extrusion process;9 similar to that which occurs in plants.10,11 Incorporating certain PCW polymers in the fermentation growth medium results in stable cellulose composites being produced through spontaneous self-assembly processes.12−14 The relatively homogeneous nature of these composites enables material testing studies to be performed © 2015 American Chemical Society

Received: January 6, 2015 Revised: March 6, 2015 Published: March 10, 2015 1232

DOI: 10.1021/acs.biomac.5b00009 Biomacromolecules 2015, 16, 1232−1239

Biomacromolecules

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noncovalent cross-linked networks either alone or with cellulose.2,9,19 However, there is little direct evidence that such structures form in cell walls in muro. To date, the most detailed study of cereal primary PCW architecture has been conducted on the elongating maize hypocotyl,22 where a deconstruction approach was adopted. Sequential chemical extraction of specific polysaccharides, together with enzymatic digestion of MLGs allowed two distinct domains in the grass primary PCW to be proposed. In the first proposed domain, polymers were tightly associated with cellulose, and were enriched in MLG and infrequently substituted heteroxylans and glucomannans.22 The second domain was composed of more frequently substituted heteroxylans and additional glucomannan, which was proposed to be interstitial with respect to cellulose, and to interconnect with the first domain.22 The adsorption of GAX, isolated from barley husks (Hordeum vulgare), on the properties of cellulose fibers has been investigated23 to demonstrate the utilization of hemicelluloses in cellulose fiber modification to develop materials (e.g., paper) with increased tensile strength. Other research has studied the adsorption of heteroxylans, extracted from birch and aspen woody tissues, on the properties of cellulose fibers to understand the mechanism of xylan retention on cellulose fibers under pulping conditions and for preparation of new composite materials with increased flexibility and tensile strength from wood polysaccharides.24−27 One study has investigated films of either native or enzymatically debranched rye AX reinforced with bacterial cellulose, with the focus on producing environmentally friendly packaging materials based on renewable polymers.28 The principles underlying binding of heteroxylans to cellulose surfaces have been studied in vitro and in silico, with a general consensus that less substituted heteroxylans have a higher affinity for cellulose surfaces.29−31 In plants, it is likely that heteroxylans are synthesized with sufficient substitution by arabinose, acetyl, or glucuronic acid to maintain solubility prior to incorporation into the cell wall, where it is possible that modification enzymes, such as arabinoxylan arabinofuranoside hydrolase, remove side-chain substituents.32−34 Recently, slow, irreversible but limited (99% glucose composition. When cellulose was produced in the presence of wheat endosperm AX, the levels of AX incorporated into the composite depended on the method used to dissolve the AX prior to incorporation into the fermentation medium. Incorporation levels were relatively low (8.5 to 12% w/w dry weight) for AX dissolved by the addition of the polysaccharide to boiling water with vigorous stirring for 2 h but as high as 50% dry weight for AX dissolved by vigorous overnight stirring of AX added to boiling water in a 90 °C oil bath. When barley endosperm MLG was added to the fermentation medium, 28 to 29% MLG was consistently incorporated into the composite. For Cell-AX and Cell-MLG composites harvested after 72 h, the percentage incorporation was the same as at 48 h, although the total composite yield was higher, suggesting that the ratio of cellulose to either AX or MLG is controlled by the self-assembly process and is not limited by either barrier or diffusion effects. This phenomenon was also observed for cellulose-XG (Cell-XG) networks.12 Cell-XG and cellulose-pectin (Cell-P) composites were constructed to provide a point of comparison with previously published work. For Cell-XG, a Cell/XG ratio of 1:0.41 was determined by monosaccharide analysis, similar to the ratio of 1:0.38 previously reported.12 Pectin incorporation into cellulose composites was determined to be ∼30% (w/w) by a colorimetric assay for galacturonic acid, also in agreement with past results.14 Microstructure of Cellulose Produced in the Presence of AX or MLG. Field-emission scanning electron microscopy (FESEM) allowed visualization of the microstructure within

Native cellulose pellicles contained a densely packed network of cellulose fibrils, in apparently random orientation on the micron length scale, with no indication of fibril directionality (Figure 1A). In contrast with the extensive molecular binding exhibited by Cell-XG, resulting in the formation of noncovalent cross bridges between cellulose microfibrils (Figure 1B), neither Cell-AX (Figure 1C) nor Cell-MLG (Figure 1D) exhibited such features. In these latter composites, “nodules” of ca. 100 nm size were visible in the micrographs. Larger size “nodules” were also observed in cryo-SEM micrographs of cellulose composites with 12% AX or with 27% MLG incorporation. (See the Supporting Information.) It is likely that the smaller size of “nodules” in FESEM images is due to in situ precipitation by methanol during the sample preparation protocol. Transmission electron microscopy (TEM) of ultrathin sections incubated with immunogold-labeled monoclonal antibodies for AX and MLG showed the apparently even distribution of these polymers in the respective composites (Figure 2B,C), consistent with a relatively homogeneous composite microstructure. Molecular Organization in Cellulose Composites with AX or MLG. 13C NMR spectroscopy was used to investigate molecular structure and mobility within composites. Under cross-polarization (CP) and magic-angle spinning (MAS) conditions, relatively rigid segments are detected (Figure 3). Integration of signals at 88 to 92 ppm and 83 to 86 ppm corresponding to crystalline and noncrystalline cellulose C4 sites46 gave a % crystallinity of ∼80% for hydrated cellulose (Figure 3). From this region, the ratio of Iα to Iβ polymorphic form was calculated to be 62:38, consistent with values previously reported for bacterial cellulose.12,14,46 This level of cellulose crystallinity as well as the Iα/Iβ ratios did not change markedly due to the presence of either AX or MLG in the composite materials. For hydrated Cell-AX and Cell-MLG 1234

DOI: 10.1021/acs.biomac.5b00009 Biomacromolecules 2015, 16, 1232−1239

Article

Biomacromolecules

Figure 2. Immunogold labeling of AX and MLG in composite material. Ultrathin sections (80 nm) of cellulose (A), Cell-AX (B), and Cell-MLG (C) material with 12% AX and 27% MLG incorporated, respectively, were immunogold-labeled using AX (B) or MLG (C) monoclonal antibodies (mAb). For each composite, distribution of gold labeling was observed (black dots in TEM micrographs).

composites, a % crystallinity of about 78 and 73% was calculated, respectively (Figure 3). Iα/Iβ ratios of 66:34 were calculated for Cell-AX, while Cell-MLG composites had Iα/Iβ ratios of 64:36. There were no signals for either AX or MLG in CP/MAS spectra (Figure 3), suggesting that these polymers are not in a rigid/solid phase in hydrated composites, but signals for these molecules were detected in the mobile phase (DP/ MAS spectra) (Figure 3). Thus, there was no evidence of extensive molecular associations between cellulose and wheat endosperm AX or barley endosperm MLG either at the point of cellulose chain aggregation (resulting in altered cellulose crystallinity) or subsequently (resulting in immobilization of bound AX or MLG), unlike the case for either XG12,47 or some mannan-family13 polysaccharides. Mechanical Properties of Cellulose Hydrogels with Hemicellulose or Pectin. Uniaxial tensile testing was used to assess the inherent mechanical properties of cellulose composites. Between 7 and 20 replicates per sample were tested to account for heterogeneity both within and between pellicles. Figure 4 shows representative stress/strain curves for cellulose, Cell-AX, and Cell-MLG from several fermentation experiments, with Cell-P included as a point of comparison with previous work. Overall, the curve shapes observed were characteristic of a viscoelastic material that did not exhibit linear stress/strain behavior at low deformations. Cellulose exhibited yield strains between 10 and 30% before brittle failure, covering slightly higher values than those previously reported of between 10 and 20%, but maximum stress values of ∼2.2 MPa were comparable (Table 1, Figure 4).16,44 Cell-AX with 8.5 to 12% AX incorporation exhibited yield strains between 10 and 20%, while Cell-MLG exhibited yield strains between 10 and 25%, before brittle failure (Figure 4), with little effect on the extensibility of these composites. For

Figure 3. 13C CP/MAS and DP/MAS NMR spectra of hydrated cellulose and composites Cell-AX amd Cell-MLG showing the C4 region used to calculate crystallinity and ratios of Iα and Iβ. Incorporation of AX or MLG polysaccharide into the cellulose pellicle did not markedly alter the % crystallinity or the Iα and Iβ ratios. This suggests the lack of extensive molecular associations between cellulose and AX or MLG, either at the point of cellulose chain aggregation (resulting in altered cellulose crystallinity) or subsequently (resulting in immobilization of bound AX or MLG). The spectra demonstrated that there was no detectable rigid AX (A) or MLG (B) in the CP/ MAS (solid phase). However, segmentally flexible AX (A) and MLG (B) were present in the DP/MAS spectra (mobile phase).

Figure 4. Effects of AX and MLG on the uniaxial extension of bacterial cellulose composites. Apparent stress/stain curves for cellulose, CellAX, and Cell-MLG composites under uniaxial tension. Results shown are representative stress/strain curves from several independent Ga. xylinus fermentation experiments. AX content of materials ranged from 8.5 to 50%, while MLG content of composites averaged 28%. The Cell-P composite was included as a point of comparison. 1235

DOI: 10.1021/acs.biomac.5b00009 Biomacromolecules 2015, 16, 1232−1239

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Biomacromolecules Table 1. Uniaxial Tensile Mechanical Properties of Cellulose Composite Materials samples cellulose cellulose-8.5% AX cellulose-12% AX cellulose-50% AX cellulose-27% MLG cellulose-28% MLG cellulose-29% MLG cellulose-30% P probability MSD

N

maximum tensile stress (MPa)

maximum tensile strain (%)

apparent Young’s modulus (MPa)

relative density (mg/cm3)

15 17 20 11 11 7 15 17

2.22 ± 0.11 0.86 ± 0.04d 0.92 ± 0.03d 0.88 ± 0.04d 1.04 ± 0.03c,d 1.24 ± 0.09c 1.26 ± 0.05c 0.11 ± 0.02e