Dehydrogenative Polymerization of Coniferyl Alcohol in Artificial

Article pubs.acs.org/JAFC

Dehydrogenative Polymerization of Coniferyl Alcohol in Artificial Polysaccharides Matrices: Effects of Xylan on the Polymerization Qiang Li,† Keiichi Koda,‡ Arata Yoshinaga,§ Keiji Takabe,§ Masatsugu Shimomura,⊥ Yuji Hirai,⊥ Yutaka Tamai,‡ and Yasumitsu Uraki*,‡ †

Graduate School of Agriculture and ‡Research Faculty of Agriculture,Hokkaido University, Kita-ku, Sapporo 060-8589, Japan Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan ⊥ Faculty of Photonic Science, Chitose Institute of Science and Technology, 758-65 Bibi, Chitose 066-8655, Japan §

S Supporting Information *

ABSTRACT: To elucidate the influence of wood polysaccharide components on lignin formation in vitro, models for polysaccharide matrix in wood secondary cell wall were fabricated from two types of bacterial cellulosic films, flat film (FBC) and honeycomb-patterned film (HPBC), as basic frameworks by depositing xylan onto the films. An endwise type of dehydrogenative polymerization, “Zutropfverfahren”, of coniferyl alcohol was attempted in the films with/without xylan. The resultant dehydrogenation polymer (DHP) was generated inside and outside xylan-deposited films, whereas DHP was deposited only outside the films without xylan. The amount of the generated DHP in the xylan-deposited films was larger than that in the films without xylan. The frequency of 8-O-4′ interunitary linkage in DHP was also increased by the xylan deposition. These results suggest that xylan acts as a scaffold for DHP deposition in polysaccharides matrix and as a structure regulator for the formation of the 8-O-4′ linkage. In addition, mechanical properties, i.e., tensile strength and modulus of elasticity (MOE), of both cellulosic films were found to be augmented by the deposition of xylan and DHP. Especially, DHP deposition remarkably enhanced MOE. Such effects of xylan on DHP formation and augmentation of mechanical strength were clearly observed for HPBC, revealing that HPBC is a promising framework model to investigate wood cell wall formation in vitro. KEYWORDS: honeycomb-patterned bacterial cellulose film, xylan, dehydrogenation polymer, 8-O-4′ linkage, mechanical property

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Salmén and Burgert8 reported that highly substituted xylan was closely associated with lignin with less condensed structure in hardwood. The 8-O-4′ frequency of DHP was increased by the presence of xylan.19 Thus, xylan is assumed to contribute to lignin deposition in cell wall and steric regulation of lignin polymerization. On the other hand, Donaldson and Knox20 reported that xylan was not closely related to the degree of lignification in radiata pine. In Eucalyptus globulus, a xylan-bonded lignin fraction had fewer 8-O-4′ linkages than other hemicellulosebonded lignin.21,22 In spruce, the lignin−xylan fraction was also rich in condensed structure (fewer 8-O-4′ linkages) in comparison with the lignin−glucomannan fraction.23,24 There were, thus, opposite findings and hypotheses in the previous reports. The first objective in this investigation was to elucidate the effects of xylan on lignin formation in vitro, using solid models for wood polysaccharide matrix. Wood has excellent mechanical strength. Of course, this characteristic is mainly attributed to a crystalline material, cellulose.25−27 However, the contribution of other components, hemicellulose and lignin, to the mechanical strength of wood cell wall is also unclear. Our second objective was to clarify the

INTRODUCTION Lignin is one of the three major components in wood secondary cell wall and has a complex structure due to a variety of interunitary linkages resulting from radical coupling of monolignols initiated by hydrogen abstraction with oxidase and/or peroxidase.1 A main interunitary linkage is 8-O-4′ bonding. Its frequency in native lignin is much greater than that of dehydrogenation polymer (DHP) as an artificial lignin, even though DHP is prepared by the endwise polymerization method “Zutropfverfahren,” which yields DHP richer in 8-O-4′ bonding than the bulk polymerization does.2−5 The radical coupling of monolignols in a living tree, namely lignification, always occurs in the polysaccharides matrix composed of cellulose and hemicelluloses,6,7 whereas DHP is normally prepared in an aqueous medium. Thus, it is plausible to suppose that the polysaccharides play a significant role in the lignin formation.8−10 In fact, the frequency of 8-O-4′ linkages in DHP was increased by adding polysaccharides, such as pectin,11−14 extracted hemicellulose,13 and cyclodextrin,15 to the reaction media. However, such effects of polysaccharides addition on lignification in solid media are still unclear. Xylan, as a major hemicellulose, accounts for 15−30% of wood components in hardwood and for 5−10% in softwood,16 and its influence on lignification is in debate. When xylan is fully deposited in S1 and S2 layers of phloem fibers in Mallotus japonicus, lignin deposition is spatially associated with xylan location.17 In addition, Reis and Vian18 reported that xylan acted as a “host structure” for the regulation of lignin structure. © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4613

December March 13, March 16, March 16,

24, 2014 2015 2015 2015 DOI: 10.1021/acs.jafc.5b01070 J. Agric. Food Chem. 2015, 63, 4613−4620

Article

Journal of Agricultural and Food Chemistry

three solutions were prepared.4 Solution I: 100 mg of coniferyl alcohol was dissolved in 2 mL of acetone and then mixed with 48 mL of phosphate buffer saline (PBS) solution (pH 6.1, 0.01 M phosphate containing 0.8 w/v% of NaCl and 0.02 w/v% of KCl). Solution II: 5 mg of horseradish peroxidase (100 units/mg) (Wako Pure Chemicals, Osaka, Japan) was dissolved in 50 mL of PBS solution. Solution III: 0.25 mL of hydrogen peroxide (30 wt % in aqueous solution, 14.6 mmol) was mixed with 50 mL of PBS solution. Both HPBC and FBC with/without adsorption of xylan were immersed in solution II, and solutions I and III were injected dropwise into solution II separately from two syringes each using a YSP-101 syringe pump (YMC Keyboard Chemistry, Kyoto, Japan) at a flow rate of 5 mL/h for 10 h, and then the mixture was kept for a further 16 h. The resultant films were picked up and immersed in distilled water two times for 5 min each and then air-dried. The remaining DHP not deposited on the film but suspended in the buffer solution was collected by centrifugation (2000g, 10 min) and washed two times with distilled water and then lyophilized. The collected DHP was used as a lignin standard for the acetyl bromide method. The morphologies of the obtained films were observed under a VK9500 violet-laser color 3D profile microscope (Keyence, Osaka, Japan) or a JSM-6301F field emission scanning electron microscope (FESEM) (JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 5 kV. The samples for SEM observation were coated with gold− palladium by cathodic spreading with an E101 ion sputter (Hitachi, Tokyo, Japan). Lignin Bromination and SEM−EDXA Analysis. DHP on the cellulosic films was brominated with bromine vapor in a sealed desiccator. The cellulosic films and bromine (5 mL, 99% purity) in a Petri dish were separately placed in the desiccator and kept for 1 d at room temperature after being tightly sealed in the desiccator with a glass cap. The brominated films were placed in air for half a day and then embedded in epoxy resin. After polymerizing in an oven (35 °C for 1 d, followed by 45 °C for 1 d, and then 65 °C for 3 d), the resins were trimmed with a RM2255 microtome (Leica Biosystems, Nussloch, Germany) to obtain the cross sections of films. The location of bromine on the cross sections of films was analyzed on a S800 FE-SEM (Hitachi, Tokyo, Japan) equipped with an EMAX-2000 energy dispersive X-ray analyzer (EDXA) (Horiba, Tokyo, Japan) after specimens were coated with carbon under a VC-100 carbon coater (Vacuum-Device Ltd., Ibaraki, Japan). SEM analysis was completed at an accelerating voltage of 10 kV. The EDXA images were represented as an inversion mode of black and white using Microsoft Powerpoint 2007 software. Immunofluorescence Labeling. Immunofluorescence labeling of xylan was preformed according to the method of Kim et al.35 Two kinds of primary antibodies, LM 10 and LM 11 (PlantProbes, Leeds, UK), were used to label xylan. A secondary antibody, goat anti-rat IgG Alexa Fluor 568 (Invitrogen, Carlsbad, CA), was used to recognize the primary antibodies. As control experiments, the specimens were incubated with the secondary antibody only. Micrographs of immunolabeling specimens were taken with a BX50 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a Semrock TxRed-4040C filter set (Opto-Line Inc., Tokyo, Japan). Characterization of DHP. All specimens were washed with 5 mL of 90% 1,4-dioxane aqueous solution (aq dioxane) once, and dried in vacuo at 50 °C. DHP contents in DHP-deposited cellulosic films before and after washing were determined by the acetyl bromide method.36 The absorptivity of acetylated DHP at 280 nm was found to be 19.9 L/g cm, which was obtained by using the DHP not deposited on the film but recovered from the suspension by centrifugation, as mentioned above. Alkaline nitrobenzene oxidation for the specimens washed with aq dioxane was conducted according to the method reported by Chen.37 Reaction products (vanillin and vanillic acid) were determined by a GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) under the following conditions: detector, FID; column, InertCap 1701 (30 m, 0.25 mm i.d., 0.25 μm film thickness) (GL Science, Tokyo, Japan); split ratio, 5.0; carrier gas, He (110 mL/min); injector temperature,

influence of such components on the mechanical properties of cellulose. To achieve the objectives, we prepared models for polysaccharide matrices from bacterial cellulose (BC) secreted by Gluconacetobacter xylinus as a basic framework. The bacterium produces pellicles in Hestrin−Schramm (HS) liquid media.28 The pellicle produced for a short time can be used as a thin film. However, most cellulose fibrils of the film are not exposed outside, indicating low response of cellulose filaments to guest molecules or external stimuli. To overcome the problem, we fabricated honeycomb-patterned bacterial cellulose film (HPBC), in addition to flat bacterial cellulose film (FBC), as a BC pellicle. Most fibrils of HPBC are assumed to be exposed to the outer circumstance, and thus, they would have high accessibility to external guests. Its hexagonal alignment mimicked the cross-section of native wood cell wall arrangement. Therefore, we consider HPBC as a better model framework than FBC to investigate the effect of xylan on lignification and the contribution of xylan and DHP to the mechanical property of cellulose.

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MATERIALS AND METHODS

Fabrication of HPBC and FBC. HPBC was prepared by our method reported previously.29,30 Briefly, a precultured G. xylinus (ATCC53582) was cultured on a template of concave honeycombpatterned agarose film containing the nutrients of HS medium. After 48 h incubation, HPBC was obtained by dissolving the agarose film with hot, distilled water at 70 °C. FBC as a normal BC pellicle was prepared by culture of the bacterium in HS medium for 12 h. Both HPBC and FBC were soaked in 1 M aqueous sodium hydroxide at 65 °C for 1 h and then washed by distilled water repeatedly until the pH of the washings became 7. Afterward, the films were air-dried and kept in a refrigerator until use. Xylan Adsorption Analysis. Beech xylan was purchased from Sigma-Aldrich (Steinheim, Germany), and used as received. Xylan was subjected to acid hydrolysis bythe Klason method for measuring its lignin content, and neutral sugar compositions in the filtrate by this method were analyzed.31 Uronic acid content of xylan was determined according to the method reported by Blumenkranz and AsboeHansen,32 using a calibration line.33 A sheet of cellulosic film (2.5 mg of FBC and 0.8 mg of HPBC with 90 mm in diameter) was immersed in 10 mL of aqueous xylan solution at different concentrations of 0.1−1.0 mg/mL for 1 d at room temperature and then washed with 1 mL of distilled water. After the adsorption, the residual xylan solutions were subjected to HPLC to measure the xylan concentration. The column used was a 300 × 7.5 mm i.d., Shodex Asahipak GF 7 M HQ, with a 50 mm × 7.5 mm i.d. guard column of the same material (Showa Denko K.K., Tokyo, Japan). Column oven temperature was set at 40 °C, and Milli-Q water as an eluent and a Corona detector (ESA Bioscience Inc., Chelmsford, MA) were used. The xylan concentration was calculated from the total area of the xylan peak using a calibration line. The adsorption of xylan was expressed as the following equation

W=

10(C0 − C1) M

where W (g/g of cellulose) is the adsorption of xylan on a BC film, 10 (mL) is the volume of xylan solution, C0 (mg/mL) is xylan concentration before adsorption, C1 (mg/mL) is xylan concentration after adsorption, and M (mg) is the weight of a BC film. The cellulosic films, which were soaked in xylan solution at 1 mg/ mL, washed once with 1 mL of distilled water, and air-dried, were subjected to the following experiment. Dehydrogenative Polymerization of Coniferyl Alcohol with Enzyme in the Presence of Cellulosic Films. Coniferyl alcohol was synthesized according to the procedure reported previously.34 For its polymerization with enzyme (horseradish peroxidase), the following 4614

DOI: 10.1021/acs.jafc.5b01070 J. Agric. Food Chem. 2015, 63, 4613−4620

Article

Journal of Agricultural and Food Chemistry 250 °C; detector temperature, 280 °C; column temperature program, 150 °C (5 min), 5 °C/min to 230 °C (1 min), 10 °C/min to 250 °C (10 min). All experiments described above were performed in duplicate, and the average value was shown as a result. Mechanical Test. Films were gently pressed under a 300 g load for 10 min, and then cut into 1−2 mm in width and 5 mm in length. The cut specimens were allowed to stand at 50% relative humidity and 22 °C for 1 d and subjected to tensile testing under ambient conditions. Tensile strength, modulus of elasticity (MOE), and elongation were measured on a specially designed testing apparatus reported previously.38 Film thickness was measured for at least five different positions under the aforementioned 3D profile microscope. The mechanical test was carried out for at least 20 specimens cut from four different films.

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RESULTS AND DISCUSSION Deposition of Xylan onto HPBC and FBC. Beech xylan used in this study contained 1.3% of lignin as Klason lignin. The lignin-free part of this xylan was found to give 86.0% xylose, 1.2% galactose, 0.5% mannose, 0.5% glucose, and 11.8% glucuronic acid. Adsorption isotherms of the xylan on HPBC and FBC are shown in Figure 1. The adsorption of xylan on HPBC was

Figure 2. 3D microscopic images of the HPBC series (A, C, and E) and SEM images of the FBC series (B, D, and F). A and B are intact cellulosic films. C and D are images of both cellulosic films after DHP preparation. E and F are images of xylan-deposited films after DHP preparation. White arrows show the “white region” in HPBC.

SEM-EDXA.39 Figure 3 shows the SEM-EDXA images of Figure 2 at high magnification. The location of bromine dots in parts a3, b3, c3, and d3 of Figure 3 was consistent with the location of white regions in parts A3, B3, C3, and D3 of Figure 3, respectively. Consequently, DHP deposition on the surface of cellulosic films was confirmed by a combined analysis of bromination and SEM-EDXA. Afterward, films were identified as follows: DHP-deposited HPBC and FBC (DHP−HPBC and DHP−FBC, respectively) and DHP-deposited Xyl−HPBC and Xyl−FBC (DHP−Xyl−HPBC and DHP−Xyl−FBC, respectively). Effects of Xylan on DHP Formation. To further investigate the DHP location inside the films, the Br-stained films were embedded into epoxy resins, and then the specimens were trimmed and microtomed after polymerizing the resins. Figure 4 shows the microtomed positions for HPBC and FBC series. Two cross sections from the first and the second micotome cutlines were analyzed under SEM-EDXA. Figure 5 shows the SEM-EDXA images of the cross sections of DHPdeposited cellulosic films at the first cutline (line 1) (Figure 4). In Figure 5a3 of DHP−FBC without xylan, Br dots (or DHP) were observed only at the outside of the film, but not in the inside. However, when xylan was adsorbed on FBC (DHP− Xyl−FBC), DHP was located on both sides of the film (Figure 5b3). Identical results (Figure S1, Supporting Information) were obtained from the cross sections at the second cutline (line 2) (Figure 4). From these images, we anticipated that xylan induced DHP formation into the inside of the FBC films. To confirm this assumption, xylan distribution in the films was observed by a combination of immunolabeling and fluorescent microscopy. LM 10 can recognize low-substituted and/or unsubstituted xylan, while LM 11 can recognize not only low-substituted and/or unsubstituted xylan but also arabinoxylan.40 Both antibodies have been widely used for immunolabeling xylan in both hardwood and softwood, for instance, birch,40 poplar,41 pine,20 and cedar.35 The control specimens (with the secondary antibody only) omitting

Figure 1. Adsorption isotherms of xylan on HPBC and FBC films.

much higher than that on FBC at any concentration. This result indicated that HPBC had a larger accessible area to xylan than FBC did. The reason may be attributed to the difference in cellulose fibrils: most cellulose fibrils in HPBC are exposed outward (facing air and solvent), whereas most of the fibrils in FBC face inside the pellicle and only the fibrils at the surface are exposed outward. Thereby, xylan could easily interact with most of the cellulose microfibrils in HPBC as compared with those in FBC. We thus considered that HPBC was a suitable platform for the evaluation of the interaction between cellulose microfibrils and guest molecules. Dehydrogenative Polymerization of Coniferyl Alcohol in the Presence of Cellulosic Films with/without Xylan. The endwise polymerization of coniferyl alcohol4 was attempted in the presence of cellulosic films to obtain DHPdeposited cellulosic films. Surface images of the resultant films are shown in Figure 2. Before DHP deposition, no white region was observed for HPBC and FBC, respectively (Figure 2A,B). After DHP deposition, white regions were observed on the surfaces of all HPBC and FBC, respectively (Figure 2C−F). The number of white regions in xylan-deposited FBC (Xyl− FBC) (Figure 2F) was much larger than that in FBC without xylan (Figure 2D). It was presumed that white regions of HPBC and FBC were deposited DHP. To prove this assumption, bromination of the specimens was conducted in order to stain and visualize DHP with bromine. The bromine distribution on the surface of the films was observed under a 4615


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Figure 3. SEM-EDXA images of the surface of DHP-deposited FBC and HPBC films. A series, FBC after DHP preparation without xylan; B series, xylan-deposited FBC after DHP preparation; C series, HPBC after DHP preparation without xylan; and D series, xylan-deposited HPBC after DHP preparation. The magnification is shown by the bar below each panel. The area in the dotted square was magnified under SEM and is shown in the photo for which the numerical label is increased by one (e.g., A1 to A2). The lower case letters, a, b, c, and d, are Br-mapping images by EDXA for the corresponding images labeled with upper case letters.

incubation of primary antibodies (LM 10 and LM 11) did not show obvious fluorescence, including self-fluorescence of lignin (Figure 6A1−D1), whereas the other test specimens clearly indicated red fluorescence, which revealed the existence of xylan. In Figure 6A2, and A3, xylan distributes in the whole inside of Xyl−FBC. In addition, the xylan distribution was also confirmed in the whole inside of DHP−Xyl−FBC (Figure 6B2,B3). From Figures 5b3 and 6B2,B3, DHP existed with xylan. These phenomena suggest the following DHP formation in the matrix; xylan first penetrates into the cellulosic film. Coniferyl alcohol attaches to the xylan-deposited film, and then it migrates inside the film because of its high affinity for xylan.33 Finally, the coniferyl alcohol is polymerized with peroxidase to form DHP in the polysaccharide matrix. In the case of HPBC, Br dots or DHP were observed (Figure 5c3,d3). The density of dots was increased by the xylan adsorption. In addition, xylan deposition inside the film was confirmed in Figure 6D2,D3. These observations suggest that coniferyl alcohol can easily penetrate inside HPBC because most bundles or fibrils composed of several microfibrils in

HPBC are exposed outward. By xylan deposition, migration of coniferyl alcohol was accelerated. Consequently, more DHP was observed in Figure 5d3 than in Figure 5c3. Effects of Xylan on Lignin Content and Frequency of 8-O-4′ Linkage. To further examine the effects of xylan on DHP formation quantitatively, DHP content in the films and the frequency of interunitary linkage in DHP were estimated by the acetyl bromide method and alkaline nitrobenzene oxidation, respectively. Parts A and B of Figure 7 show DHP contents in the cellulosic films based on the total weight of DHP-deposited films and the initial weight of cellulose before and after washing with aq dioxane, respectively. The DHP contents of FBC and HPBC, based on the initial weight of cellulose, were increased by the xylan adsorption on cellulosic films in both parts A and B of Figure 7. This result also suggests that xylan enhances the amount of DHP deposition in the films and contradicts the previous findings,13,14 where hemicelluloses (pectin, pectic acid, and dilute alkali-extracted hemicellulose) did not affect the 4616


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DHP formation in both solid and liquid media. Consequently, xylan affected DHP formation as a scaffold. DHP content was remarkably decreased by washing with aq dioxane. However, a significant amount of DHP remained in the films, for HPBC in particular (Figure 7B). The remained DHP fraction was tightly associated with the polysaccharide matrix, suggesting that this aq-dioxane-unwashable DHP fraction might be a lignin−carbohydrate complex. The DHP-deposited films, after washing with aq dioxane, were then subjected to nitrobenzene oxidation. The oxidation products, vanillin and vanillic acid, are generated from the noncondensed structure of lignin, and therefore, the yield of the oxidation products is one of the measures to estimate the frequency of aryl ether interunitary linkages in lignin, mainly the 8-O-4′ linkage.37 The yield of the oxidation products from DHP prepared in liquid media (without cellulosic films) was increased by the addition of xylan into the buffered media, as shown in Figure 8. Similarly, the yield from DHP formed in the HPBC and FBC films was increased by the xylan adsorption. These results

Figure 4. Cutting positions for SEM-EDXA observation at the cross section of (A) FBC and (B) HPBC. Images were taken under the violet-laser color 3D profile microscope.

Figure 5. SEM-EDXA images of DHP-deposited FBC and HPBC films at the cross section from the first cutline. A series, FBC after DHP preparation without xylan; B series, xylan-deposited FBC after DHP preparation; C series, HPBC after DHP preparation without xylan; D series, xylan-deposited HPBC after DHP preparation. The magnification is shown by the bar below each panel. The area in the dotted square was magnified under SEM and is shown in the photo for which the numerical label is increased by one (e.g., A1 to A2). The lower case letters, a, b, c, and d, are Brmapping images by EDXA for the corresponding images labeled with upper case letters. 4617


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Figure 6. Immunofluoresence labeling of xylan in the cross sections of the xylan−FBC series (A1−A3), DHP−xylan−FBC series (B1−B3), xylan− HPBC series (C1−C3), and DHP−xylan−HPBC series (D1−D3). A1−D1, controls without primary antibodies labeling; A2−D2, immunolabeling with LM 10; A3−D3, immunolabeling with LM 11. Bars in magnified images are 2 μm.

suggested that xylan enhanced the frequency of the aryl ether linkage in DHP, probably resulting in the predominant formation of the 8-O-4′ linkage. This observation is consistent with the findings by Barakat et al.,19 where the higher the concentration of arabinoxylan added to the DHP reactant, the higher the yield of thioacidolysis products obtained. Therefore, we conclude that xylan can act not only as a scaffold for lignin deposition but also as a structure regulator of lignin, especially formation of the aryl ether linkage. Figures 7 and 8 also demonstrated the difference of the two cellulosic models in the deposition of DHP. A larger amount of DHP was deposited on/in the HPBC film, but a lower frequency of the 8-O-4′ linkage was formed than on the FBC film. It is supposed that HPBC is a promising model for the adsorption of wood components on cellulose, while FBC gives a good framework to increase the frequency of the 8-O-4′ linkage in DHP in artificial polysaccharide matrix. Our next target is to elucidate the effect of glucomannan on the cell wall formation using the similar models of polysaccharides matrices. Effects of Xylan and DHP on the Mechanical Properties of Cellulosic Films. In general, hemicellulose and lignin influence the mechanical properties of wood cell wall or cellulosic framework.8,25,42 To confirm these hypotheses, the

Figure 7. DHP content in the cellulosic films (A) before and (B) after washing with 90% 1,4-dioxane solution: blue, based on the total weight of DHP-deposited films; orange, based on the initial weight of cellulose.

Figure 8. Yields of nitrobenzene oxidation products (vanillin and vanillic acid) from DHP deposited on cellulosic films after washing with 90% 1,4dioxane solution: Blue, vanillin; orange, vanillic acid; columns with profiles of solid lines, based on the weight of DHP (left axis); columns with profiles of dashed lines, based on the total weight of DHP-deposited films (right axis). 4618


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Journal of Agricultural and Food Chemistry Table 1. Mechanical Properties of BC Films after Xylan and DHP Deposition tensile strength (MPa) FBC Xyl−FBC DHP−FBC DHP−Xyl−FBC HPBC Xyl−HPBC DHP−HPBC DHP−Xyl−HPBC a

55.1 72.7 71.6 90.4 19.4 23.6 31.9 36.6

± ± ± ± ± ± ± ±

22.9 26.4 22.7 26.6 6.9 5.7 9.2 14.3

MOEa (MPa) 574 1058 965 1434 278 352 486 833

± ± ± ± ± ± ± ±

215 359 233 331 100 110 128 256

elongation (%) 8.5 8.3 8.6 8.6 6.3 6.9 7.1 6.4

± ± ± ± ± ± ± ±

2.4 4.4 2.8 3.4 1.4 3.2 3.1 3.8

film thickness (μrn) 2.0 2.0 2.1 1.9 0.77 0.79 0.76 0.77

± ± ± ± ± ± ± ±

0.6 0.4 0.3 0.4 0.03 0.05 0.06 0.05

Modulus of elasticity.

Hokkaido University, for their kind help and discussion about microtome cutting and SEM-EDXA analysis.

mechanical properties, tensile strength, and modulus of elasticity (MOE) were measured for the specimens prepared in this study. Table 1 summarizes the mechanical properties of cellulosic films after deposition of xylan and/or DHP. The tensile strength and MOE of HPBC were smaller than those of FBC, which was attributed to the lower density of HPBC (0.17 mg/mm3) than that of FBC (0.36 mg/mm3). The amount of cellulose in HPBC is much smaller than that in FBC with same thickness. The smaller amount of cellulose in HPBC caused the weaker mechanical strength.38 The tensile strength of FBC and HPBC was increased 1.6 and 1.9 times, respectively, by xylan deposition followed by DHP deposition. MOE of FBC and HPBC was augmented 2.4 and 3.0 times, respectively, by the deposition of xylan and DHP. Thus, both materials strengthen the cellulosic films. If the increment between tensile strength and MOE was compared, the MOE increment was larger than that of tensile strength upon the deposition of both xylan and DHP on FBC and HPBC, especially DHP deposition on HPBC. It seems that DHP gives cellulose more rigidity. In addition, the increments of tensile strength and MOE were clearly observed for HPBC. Therefore, we suppose that HPBC is a better model for the investigation of the mechanical effect of wood components than FBC.

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ABBREVIATIONS USED PDMS, polydimethylsiloxane; DHP, dehydrogenation polymer; BC, bacterial cellulose; HPBC, honeycomb-patterned bacterial cellulose film; FBC, flat bacterial cellulose film; DHP−FBC, DHP-deposited FBC; DHP−Xyl−FBC, DHP- and xylandeposited FBC; DHP−HPBC, DHP-deposited HPBC; DHP−Xyl−HPBC, DHP- and xylan-deposited HPBC; MOE, modulus of elasticity

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental SEM-EDXA images of the DHP-deposited FBC and HPBC films at the cross section from the second cutline. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01070.

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REFERENCES

(1) Higuchi, T. Biosynthesis of lignin. In Biosynthesis and Biodegradation of Wood Components. Higuchi, T., Ed.; Academic Press: Orlando, FL, 1985; pp 141−160. (2) Sarkanen, K. V. Precursors and their polymerization. In Lignins: Occurrence, Formation, Structure and Reactions; Sarkanen, K. V., Ludwig, G. H., Eds.; John Wiley & Sons, Inc.: New York, 1971; pp 95−163. (3) Jacquet, G.; Pollet, B.; Lapierre, C.; Francesch, C.; Rolando, C.; Faix, O. Thioacidolysis of enzymatic dehydrogenation polymers from p-hydroxylphenyl, guaiacyl, and syringyl precursors. Holzforschung 1997, 51, 349−354. (4) Cathala, B.; Saake, B.; Faix, O.; Monties, B. Evaluation of the reproducibility of the synthesis of dehydrogenation polymer models of lignin. Polym. Degrad. Stab. 1998, 59, 65−69. (5) Cathala, B.; Aguié-Béghin, V.; Douillard, R.; Monties, B. Properties of model compounds of lignin (dehydrogenation polymers = DHPs) at the air/water interface. Polym. Degrad. Stab. 1998, 59, 77− 80. (6) Northcote, D. H. Chemistry of the plant cell wall. Annu. Rev. Plant Physiol. 1972, 23, 113−132. (7) Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Physiol. 2003, 54, 519−546. (8) Salmén, L.; Burgert, I. Cell wall features with regard to mechanical performance. A review COST Action E35 2004−2008: Wood machining− micromechanics and fracture. Holzforschung 2009, 63, 121−129. (9) Houtman, C. J.; Atalla, R. H. Cellulose−lignin interactions. Plant Physiol. 1995, 107, 977−984. (10) Atalla, R. H. Cellulose and hemicelluloses: Patterns for the assembly of lignin. In Lignin and Lignan Biosynthesis; ACS Symposium Series 697; Lewis, N. G., Sarkanen, S., Eds.; American Chemical Society: Washington, DC, 1996; pp 172−179. (11) Terashima, N.; Atalla, R. H.; Ralph, S. A.; Landucci, L. L.; Lapierre, C.; Monties, B. New preparations of lignin polymer models under conditions that approximate cell wall lignifications II. Structure characterization of the models by thioacidolysis. Holzforschung 1996, 50, 9−14. (12) Terashima, N.; Atalla, R. H.; Ralph, S. A.; Landucci, L. L.; Lapierre, C.; Monties, B. New preparations of lignin polymer models under conditions that approximate cell wall lignifications I. Synthesis

AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +81-11-706-2817. E-mail: [email protected]. ac.jp. Funding

This research work was financially supported by JSPS KAKENHI [Grant-in-Aid for Scientific Research (A), Grant No. 2625202204]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Yuzo Sano and Hiromi Shibui from the Laboratory of Woody Plant Biology, Hokkaido University, for their kind help with embedding cellulose films with epoxy resin. Gratitude also goes to Toshiaki Ito, Masanori Yasui, and Manabu Nagao at the Laboratory of Electron Microscopy, 4619


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Dehydrogenative Polymerization of Coniferyl Alcohol in Artificial

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