IAWA Bulletin n. s., Vol. 9 (4), 1988: 337-345

THE EFFECT OF CHEMICAL TREATMENTS ON THE CELLULAR STRUCTURE OF CORK

by

Helena Pereira* and A. Velez Marques**

Summary The effect of chemical treatments on the pressibility (Ross & Krahmer 1971). An ob• cellular structure of cork from Quercus suber vious example is given by the cork of the L. was studied using SEM. Treatments in• cork-oak (Quercus suber L.), as it forms a cluded successive solvent extractions with thick layer of several centimetres with out• petroleum ether, ethanol and water; desuber• standing properties of impermeability to liq• inisation with NaOCH3 in methanol; and de• uids, compressibility and resilience, and en• lignification with HNOJ in CH3COOH. ergy absorbing capacity (Gibson et al. 1981). Solvents swell cork anisotropically, with The cellular structure of Q. subercork has considerably larger dimensional increases in been shown to be regular and homogeneous the radial direction, but removal of extractives with rare intercellular voids apart from the does not change its cellular structure. The presence of lenticular channels. The cells are properties of cork are affected by the chemi• approximately prismatic, mostly pentagonal cal treatments that react with and remove the and hexagonal , stacked in rows in the radial main cell wall structural components. The direction of the tree, and have corrugations in removal of suberin leaves holed cell walls the radial and transverse walls (Pereira et ai. and reveals a loose ribbonlike network; lignin 1987). These structural characteristics are acts as a supporting framework and deligni• generally invoked when explaining some of fied cell walls collapse. In both cases, cork the properties of cork (Gibson et ai. 1981, looses its low density, compressibility and Rosa & Fortes 1987a, b). However, it may resilience. be assumed that chemical composition and Key words: Quercus suber; chemical treat• wall topochemistry will also contribute to es• ments; cell walls; suberin; lignin; solvent tablish these properties. extractions. The role of the different cell wall compo• nents is already well understood in relation to Introduction the properties of wood. However, cork cells Cork cells in tree barks are thin-walled differ from wood cells in their chemical com• cells, unpitted or with very infrequent pits. position, with their most typical chemical fea• They are observed in sections with an ap• ture being the presence of suberin and waxes. proximate polygonal form and they show In Q. suber, cork is made up mainly of sub• corrugations or wrinkling of the cell walls erin and lignin (respectively, 40% and 22%); (Esau 1965; Krahmer & Wellons 1973; Ho• cellulose only represents approximately 9% ward 1977). Cork cells have physical and of cork (Pereira 1988a). The comparison of mechanical properties that differ from the these values with the average chemical com• other bark and wood cells, namely in relation position of wood shows a similar lignifica• to density, dimensional variation and com- tion but very different cellulose contents,

* Departamento de Engenharia Florestal, Instituto Superior de Agronomia, Universidade Tee• nica de Lisboa, 1399 Lisboa Codex, Portugal. ** Departamento de Qufmica Organica, Instituto Superior de Engenharia de Lisboa, 1900 Lis• boa, Portugal.

Downloaded from Brill.com10/07/2021 05:40:44PM via free access 338 IAWA Bulletin n.s ., Vol. 9 (4),1988 suggesting that the role of main cell wall After each treatment the cubes were air• component played by cellulose in wood is dried, and dimensional variation and mass taken by suberin in the case of cork. yield were measured. For the solvent extrac• Some aspects of the role of the different tions, the absorption of solvent was cal• chemical components in establishing cell culated from the mass increase after each ex• structure were investigated by observing cork traction and the extracted material was gravi• samples of Q. suberafter the removal of cell metrically determined in the solvent. wall components with different chemical Samples for observation in the scanning treatments. This paper reports the results ob• electron microscope were cut from the cubes tained by using scanning electron microscopy and coated with gold (approximately 200 A on the chemically treated cork samples. thickness). The surface and the interior ofthe cubes were observed in radial, transverse and tangential surfaces. Material and Methods Reproduction cork from Quercus suberL. Results and Discussion with an average thickness of 2.4 cm and of The chemical treatments of the cork cubes good quality (low porosity and absence of under the conditions used in this study defects) was used in this study . Samples with should only affect the superficial layers of the approximate dimensions of 2 x 2 x 2 em were samples, since reactants and reaction times prepared by cutting cubes from the same cork were similar to those used when attacking plank. All the dried phelloderm and phloem cork samples of much smaller dimension, tissues from the outer surface of cork were e. g. 40-60 mesh fractions, as in the course removed prior to the cutting. The cubes were of chemical analysis (Pereira 1988a). This measured, and weighed and subjected to the was in fact the case, as seen macroscopically following chemical treatments: by the extent of penetration of reagents and 1. Successive solvent extractions, in soxhlet, the colour change of the cork, and sub• with petroleum ether, ethanol and water sequently observed in the scanning electron during 10 h for each solvent. microscope. This allowed the comparison 2. Desuberinisation of the pre-extracted cork between chemically changed and unchanged cubes with 3% NaOCH3 in methanol in cells, respectively at the surface and in the reflux for 6 h. interior of each cork cube. 3. Treatment of the pre-extracted cork cubes The results of the successive solvent ex• with a solution of HN03 and CH3COOH tractions are shown in Table 1 in relation to (aqueous solution of 90 ml HN03 and dimensional variations and solvent absorp• 732 ml CH3COOH per liter) in reflux for tion. Absorption of solvent and swelling of 1 h. the cork cubes occurred in variable extents

Table 1. Successive solvent extraction of cork cubes with petroleum ether, ethanol and water .

% of initial cork weight % of initial linear dimension Extracted Weight Dimensional swelling material increase* Radial Tangential Axial

Petroleum ether 0.2 3.0 2.5 0.0 0.3 Ethanol 0.9 71.2 4.8 1.8 2.3 Water 4.0 70.4 9.5 1.9 2.5

* After extraction, before drying .

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Fig. 1. SEM photographs of the surface of cork cubes after solvent extraction . - a: Petroleum ether extracted; transverse section showing the general structure of the cork tissue, namely the corrugations of cell walls and late cork cells at the end of an annual growth ring (arrow); bar = 100 urn. - b: Water extracted; radial section showing the flattening of cell wall corrugations; bar = 20 urn, - c: Petroleum ether extracted; tangential section showing cell wall voids and lamellation (arrows); bar = 10 urn.

with the different solvents. Swelling was ob• of swelling with much larger increases in the served after each solvent treatment but the ef• radial direction. This is probably the result of fect of petroleum ether was smaller than that the cell geometry and corresponds to the ef• of ethanol or water: the increase in volume fect of flattening of the cell wall corrugations; corresponded to 2.8% when using petroleum this will cause a larger increase in the cellu• ether, 9.1% after the ethanol treatment and lar prism height and therefore an enhanced 14.4% with the final extraction with water. swelling in the radial direction. Petroleum The dimensional variations show anisotropy ether was not significantly absorbed by cork,

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Fig. 2. SEM photographs of the pre-extracted cork cubes after desuberinisation. - a: The sur• face of the sample in radial section, showing transition between two annual growth rings with failure between growth rings; bar = 100 11m. - b: Radial section of the interior of one sample, from the surface (arrow) inwards; bar = 100 11m. - c: As b, showing the transition between the area of chemical attack and the interior of sample; bar = 20 11m. - d: Radial section; holed cell walls at the surface of sample; bar = 111m. - e: Transverse section showing fragments of terti• ary walls; bar = 20 11m. - f: Detail of e, showing a loose intercrossed cell wall structure; bar = 5 11m. - g: Cells where desuberinisation is beginning; bar = 11m. - h: Amorphous material in the desuberinised region; bar =211m.

as was the case with ethanol and water. Ab• traction (Fig. la, b). The removal of extrac• sorption of the latter accounted for approxi• tives does not alter the physical aspect of the mately 70% weight variation in the samples. cell wall and its thickness remains un• Absorption into the cork cell wall seems to changed. However, extraction of waxes by require a polar and H-bonding solvent such petroleum ether in some cases causes voids in as ethanol or water. A similar effect in di• the cell wall and seems to facilitate the sepa• mensional variations and solvent swelling ration of the cell wall into lamellae, as ob• characteristics has been reported for cork served in Figure 1c both in transverse and cells in Douglas-fir bark (Krahmer & Wel• tangential sections of the cell wall. lons 1973). This is in agreement with the localisation The SEM observations of the extracted of soluble waxes between the suberin lamel• samples did not show remarkable differences lae in the secondary wall of cork, as pro• in the structure of cork after the solvent ex• posed by Sitte (1962, 1975). The extractable traction and the cellular form was maintained waxes in Quercussuber cork amount to only in all cases . The flattening of the cell wall approximately 7% (Pereira 1988a) and their corrugations, as discussed previously, could removal does not cause a noticeable decrease be noticed, especially after the hot water ex- of cell wall thickness. This might not be

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Fig. 3. SEM photographs of pre-extracted cork cubes after delignification. - a: Sample surface in the tangential section showing cell collapse; bar = 20 urn, - b: Sample surface in the radial section showing cell collapse; bar =20 urn. - c: Radial section of the interior of one sample, from the surface (arrow), inwards; bar = 50 urn. - d: As c, but transverse section; bar = 10 um, - e: Surface of sample, tangential section, showing separation of cells; bar = 20 urn. • f: Cross section of delignified cell walls; bar = 1 urn, - g: Delignified cells showing walllamel• lation; bar =2 urn,

the case in species with higher wax contents, acquired a soft and spongy surface; on dry• and especially in cases where an individual• ing, they shrank in all directions , approxima• ised layer of soluble waxes is present in the tely 12% from the initial dimensions and cell wall, as in Douglas-fir cork (Litvay & hardened. The light brown colour of cork Krahmer 1977). turned to a dark greenish brown. The annual The effect of suberin removal from the growth rings became clearly marked by dif• cork cells was studied by observation of ferentiated swelling which after drying caus• samples submitted to treatment with NaOCH3 ed failure between growth rings (Fig. 2a). 'methanol'. This chemical treatment brings The reaction only proceeded in the outer part about the desuberinisation of cork as a result of the cork cubes and the interior remained of the transesterification of the ester bonds in unchanged, as the SEM observations show• the suberin molecule followed by solubilisa• ed. Figure 2b was taken from a section cut tion of the monomers. Desuberinisation of through one of the cork cubes from the the samples was far from carried to comple• exterior surface inwards. The change in the tion: the treated cubes weighed 89% of the cork structure affected only an outside layer initial weight, while suberin in reproduction with a depth of approximately 30-40 cells, cork amounts to approximately 40% (Pereira maintained rather uniformly along the sam• 1988a). The treatment dramatically affected ple. An abrupt transition was found between cork. The cubes swelled considerably, and chemically affected and seemingly unaffected

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cells (Fig. 2c). This shows the slow pene• and shrinkage period, cells may smoothly tration of solvents, like methanol, inside the separate by their middle lamella, as in Figure cork tissue and was again verified for the 3e. During delignification, the middle lamella case of the delignification, as discussed be• is solubilised and material is also removed low. from the secondary wall, where voids may be The removal of suberin gave way to im• seen in cross section (Fig. 3f). As a conse• portant changes in the cellular structure and in quence, the cell wall thickness of delignified the cell walls, as summarised by Figures 2d cells is appreciably smaller than in untreated to 2h. A considerable amount of material was cells. A lamellation of the cell wall may also removed from the cell wall, substantially be found, as in Figure 3g. holing the walls (Fig. 2d). The inside layer of These observations support some specu• the cell wall, the tertiary wall, was separated lative hypotheses on the contribution of the from the cell wall and could be observed re• different chemical components to cork cell maining in more or less fragmented form structure. The removal of extractives does not (Fig. 2e). In some cases, a loose network change the structure of cork nor cell wall or• was revealed inside the cell wall, as in Figure ganisation. These , as well as the resulting 2f. Figure 2g possibly shows suberin begin• properties of cork, will depend on the pres• ning to be removed from the cork cells. It ence of both main cell wall components, represents a cross section of cell walls in the suberin and lignin. transition region where chemical attack was Lignin provides the rigid framework that incipient. The tertiary wall is absent and the maintains the cell form, much in the role it cell wall material starts to be removed from has in wood cells. Therefore, upon delignifi• the inside of the cells; the cell walls show an cation cells totally collapse and cork becomes irregular surface and their thickness de• a succession of superposed cell walls. In creased. In the area where full chemical attack the process of collapse, delignified cell walls occurred, the material remainder from the cell fold, without wall failure . This seems to in• walls appeared globular in many places (Fig. dicate that the material remaining in the cell 2h). wall, i.e., mostly suberin, possesses high The samples submitted to treatment 3 flexibility and the possibility of molecular (HN03 and CH3COOH in water) show the slippage. This is in agreement with the pro• effect of the removal of lignin and easily hy• posed macromolecular structure of suberin drolysed polysaccharides (hemicelluloses) on with its long and straight aliphatic chains the cell wall structure. The major part of the (Kolattukudy 1978; Holloway 1972). Sub• suberin will remain, although some material erin therefore acts as a flexible material that may be solubilised by acid hydrolysis. The easily adapts to surrounding contours, and cork was bleached by this treatment into a thus allows important dimensional and form light yellow colour and shrank upon drying. variations without failure. The large compres• The SEM observations showed that with• sibility and resilience shown by cork seem to out their lignin skeleton, the cell walls col• require the presence of suberin in important lapsed, like a structure which weakens when amounts, as is the case for the cork of Q. its supporting elements are removed (Fig. 3a, suber . Such properties are lacking , for in• b). The cell collapse may be clearly seen in stance, in cork cells of other species with low sections cut through the cubes (Fig. 3c, d), suberin contents (Pereira 1988b). where a layer of cells that totally collaps• In the model proposed by Sitte (1957, ed is found at the surface of the cube s, 1962) for Q. suber cork, the cell wall con• while cells maintained their form in the in• sists of a thin primary wall, a thick secondary terior. However, even in delignified and col• wall and a tertiary wall which may not always lapsed regions, the cellular structure of cork be present. In the primary wall a network of is still recognised. The cells lack the binding cellulose microfibrils may be observed (Sitte through the middle lamella as seen especially 1957); the secondary wall is free of cellulose at cell corners where voids develop; as a re• and consists of a succession of alternating sult of tensions developed during the drying lamellae of suberin and waxes (Sitte 1962,

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1975); finally , the lumen-lining tertiary wall Gibson, L.J., K.E. Easterling & M.F. Ash• includes carbohydrates and other materials by. 1981. The structure and mechanics of (Sitte 1962) . Although lignin is mostly ig• cork. Proc. R. Soc. Lond. A377: 99 -117. nored in descriptions of cork topochemistry, Holloway, P.J. 1972. The composition of Sitte (1957) suggested that it should not be a suberin from the corks of Quercus suber component of the secondary wall. However, L. and Betula pendula Roth. Chern. Phys. it may be argued whether lignin is restricted Lipids 9: 158-170. to the middle lamella and primary wall, con• Howard, E.T. 1977. Bark structure of south• sidering that the large amount of lignin in ern upland oaks. Wood and Fiber 9: 172• cork (Pereira 1988a) would not fit easily in 183. the compound middle lamella only. The de• Kolattukudy, P.E. 1978 . Chemistry and bio• crea se of wall thickness in delignified cells , chemistry of the aliphatic components of as well as the aspect shown by both the delig• suberin. In: Biochemistry of wounded nified and the desuberinised cells, lead to the plant tissues: 43 -83. W. de Gruyter & conclusion that lignin is present not only in Co., Berlin, New York. the middle lamella but also in the secondary Krahmer, R. L. & J.D. Wellons. 1973. Some wall where it probably surrounds the suberin anatomical and chemical characteristics of layered deposits. Douglas-fir cork. Wood Science 6: 97• The removal of suberin destroys the cel• 105. lular structure of cork, leaving substantially Litvay, J. D. & R. L. Krahmer. 1977. Wall holed cells and a mix of wall fragments and layering in Douglas-fir cork cells. Wood amorphous material. This material shows a Sci. 9: 167-173. globular pattern, as characteristic of lignin, Pereira, H. 1988a. Chemical composition and it is interesting to notice that the de• and variability of cork from Quercu s suber suberinised material was hard and brittle. Cell L. Wood Sci. Techn . 22: 211-218. coll apse in the way it was observed during - 1988b. Structure and chemical composi• delignification did not occur, testifying the tion of cork from Calotropis procera Ail. very different properties imparted by suberin IAWA Bull. n.s. 9: 53-58. (as in the delignified cork) and by lignin (as - , M. E. Rosa & M.A. Fortes. 1987. The in the desuberinised cork) . cellular structure of cork from Quercus In summary, the cellular structure and the suber L. IAWA Bull. n.s. 8: 213 -218. properties of cork are decisively affected by Rosa, M.C. & M.A. Fortes. 1987a. Stre ss chemical treatments that react with and re• relaxation and creep of cork. J. Mater. move the main cell wall components. In spite Sci. 23: 35-42. of very different roles, both suberin and -&- 1987b. Rate effects on the compres• lignin contribute to give cork cells the ability sion and recovery of dimensions of cork . to corrugate the walls and to expand, while 1. Mater. Sci. 23: 879-885. maintaining a closed cellular structure, which Ross, W.D. & L. Krahmer. 1971. Some are requirements for the low density and the sources ofvariation in structural character• large compressibility and resilience of cork. istics of Douglas-fir bark . Wood and Fiber 3: 35-46. Acknowledgements Sitte, P. 1957. Der Feinbau der Kork -Zell• The research was financially supported by wand. In: Die Chemie der Pflanzenzell• Instituto de Ciencia e Tecnologia dos Materi• wand (ed. E. Treiber): 421-432. Sprin• ais (ICTM), Lisbon , Portugal. ger, Berlin. The second author thanks the Instituto - 1962. Zum Feinb au der Suberinschichten Nacional de Inve stigacao Cientffica (rNIC), in Flaschenkork. Protoplasma 54: 555 • Lisbon , for a research scholarship. 559 . - 1975 . Die Bedeutung der molekularen References Lamellenbauweise von Korkzellwanden , Esau, K. 1965. Plant anatomy. 2nd Ed. Biochem. Physiol. Pflanzen 168: 287• Wiley & Sons Inc., New York. 297.

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