Research Article pubs.acs.org/journal/ascecg
Morphology and Overall Chemical Characterization of Willow (Salix sp.) Inner Bark and Wood: Toward Controlled Deconstruction of Willow Biomass Jinze Dou,† Leonardo Galvis,† Ulla Holopainen-Mantila,‡ Mehedi Reza,§ Tarja Tamminen,‡ and Tapani Vuorinen*,† †
Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland ‡ VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, 02044 VTT, Finland § Department of Applied Physics, School of Science, Aalto University, P.O. Box 11100, 00076 Aalto, Finland S Supporting Information *
ABSTRACT: The morphology and chemical composition of the inner bark of four willow hybrids were analyzed as a step toward complete willow biomass valorization. The inner bark consisted of highly delignified bundles of thick-walled sclerenchyma fibers and nondelignified surrounding tissue of thin-walled parenchyma cells. In comparison with willow wood fibers, the sclerenchyma fibers were longer, they had a very narrow lumen and their walls were made of up to eight separate layers. One fourth of the dry mass of the inner bark was formed of ash and acetone extractable substances. Although the lignin-to-polysaccharide ratio was similar in the inner bark and wood, their polysaccharide compositions were different. While glucose and xylose were the main monomers in wood, the inner bark had also high arabinose and galactose contents. In addition, more rhamnose was present in the inner bark which was indicative of its higher pectin content. KEYWORDS: Chemical composition, Inner bark, Optical microscopy, Sclerenchyma fiber, Willow biomass
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INTRODUCTION
microorganism attack and dehydration, containing also valuable compounds such as resins and latex.3 The natural products present in bark can be classified into extractives and nonextractable compounds. Chemical compounds such as waxes and fatty acids (lipophilic extractives), terpenes, flavonoids, lignans, tannins and extractable carbohydrates are present in extractives, whereas nonextractable components include lignin, suberin, cellulose and inorganic elements.7 Unlike wood, bark contains condensed tannins, suberin and has higher content of extractives. The chemical distribution is also different from inner toward the outer bark, where extractives and polysaccharides content decrease but lignin and phenolic components increase.4 Several studies on chemical composition of bark of different wood species have been performed in order to develop innovative uses for this source of biomass from wood processing industries. For example, the extractives from bark of different several industrially important tree species,8 Pakistani conifer species,9 cork and phloem of oak bark rhytidome,10 inner and outer bark of Norway spruce11 and more recently Loblolly pine and Douglas fir bark have been studied.12 On the other hand, the research on willow bark has
Willow species (Salix sp.) are cultivated mainly for combined heat and power generation (CHP) due to their high growth rate on marginal forest land, where they can be up to 20 times more productive than other wood species.1 In Finland, this land represents 23% of the total forest area alone.2 To make the willow cultivation on this land even more attractive, its wood and bark can be alternatively used also in the production of valuable products such as fibers, biofuels and phenolic compounds. Although the overall chemical composition of some hardwood species has been studied, there is little knowledge available on the structure and composition of willow inner bark. Therefore, prior to the design of a biomass fractionation process, the chemical and structural analysis of willow bark is an important task to undertake. Bark comprises 10−20% of trunk, depending on the tree species and growing conditions. The bark is composed of two main zones, the inner bark (phloem) and outer bark (rhytidome). The inner bark consists mainly of parenchyma tissue.3,4 It also contains conducting elements called sieve tubes and sclerenchyma tissue consisting of highly lignified bast fibers organized in bundles. The function of sclerenchyma tissue is to provide mechanical strength.5 Bark extractives are traditionally used in the manufacture of medicines.6 The outer bark made of dead tissue has the function to protect trees against © XXXX American Chemical Society
Received: March 30, 2016 Revised: May 25, 2016
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DOI: 10.1021/acssuschemeng.6b00641 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Lignin in the sections was stained red with a freshly prepared mix (2:1) of 2% (w/v) phloroglucinol (Sigma, St. Louis, MO, USA) in 95% ethanol and 50% sulfuric acid. The samples were examined with a Zeiss AxioImager M.2 microscope (Carl Zeiss GmbH, Göttingen, Germany) in brightfield using 20X (Zeiss EC Plan-Neofluar, NA 0.50) and 100X (Zeiss EC Plan-Neofluar, NA 1.3) objectives. Micrographs were obtained using a Zeiss Axiocam 506 CCD color camera (Zeiss) and the Zen imaging software (Zeiss). Transmission Electron Microscopy (TEM). Willow inner bark samples were stained with 1% KMnO4 solution (w/v) for 45 min. KMnO4 stains specifically lignin rich regions, thus creating a contrast between regions with variable lignin content within the cell wall. Thereafter, the sections were dehydrated in an ascending series of ethanol (58%, 70%, 80%, 90%, and 99%). Stained inner bark samples were embedded in epoxy resin (SpeciFix). Embedded blocks were trimmed and sectioned with diamond knife on a Leica EM UC7 ultramicrotome. Ultrathin sections (∼200 nm) floating on the distilled water surface were collected on copper grids. Finally, the grids were examined with a TEM (FEI Tecnai 12) at an accelerating voltage of 120 kV using Gatan DigitalMicrograph. Scanning Electron Microscopy (SEM). Blocks of willow stems were polished with a diamond knife on a EM UC7 ultramicrotome (Leica, Germany) and coated with ultrathin layer of gold in a K100X (Emitech, France) sputtering device.17 The images were obtained with a Zeiss Sigma VP SEM device at an accelerating voltage of 3 kV. Fiber Dimension Analysis. To separate fibers for their morphological analysis, 1.5 g of inner bark or wood was treated with 0.5 mL of acetic acid (99.8%) and 1.5 g of sodium chlorite. Subsequently, 70 mL of distilled water was added and the sample was kept for 5 h in a water bath at 80 °C.18 Fiber analysis was carried out with FibreLab (Metso, Finland) after fiber disintegration (ISO 5263:1995 (E)). Extractives Content. The inner bark and wood powders were extracted with acetone in a Soxhlet setup following the standard SCAN-CM 49:03 (2003). 10 g of each sample was extracted with 300 mL of acetone for 6 h. After extraction, the solvent was evaporated by using a rotavapor (Büchi R-210/215, Switzerland). When the volume of the mixture was below 50 mL, it was transferred into a pointed shaped flask, concentrated further to a volume of 5 mL and finally transferred into a preweighted aluminum container. The extracts were then dried at 40 °C for 2 h according to SCAN-CM 49:03 and weighted for the dry mass. Carbohydrate and Lignin Contents. The carbohydrate and lignin contents of the willow wood and inner bark were determined on the solid residues after the acetone extraction according to NREL/TP510-42618. HPAEC-PAD (Dionex ICS-3000, CarboPac PA20 column, Sunnyvale, CA, USA) was used for the sugar analysis. Ash Content. The ash content of willow inner bark and wood was determined according to NREL/TP-510-42622. The samples were placed in a muffle-oven at 575 °C for 180 min for slow carbonization until white ash was obtained. Crucibles with ash were left in a desiccator for cooling down before weighing the crucibles.
focused on the study of phenolic glucosides in different species,13 the extractive composition in desert willow and also cellulose fiber production from unbarked willow (Salix viminalis).14,15 Recently, we started to explore the bark of some hardwood and softwood species growing natively in Finland for uses other than combined heat and power generation. In these studies, we found the inner bark fibers of goat willow (Salix caprea) to possess unique characteristics for materials production. In this paper, we report on some of these initial observations. In the present study, the morphology and overall chemical composition of willow inner bark in four different planted willow hybrids were analyzed, including the basic characterization of the fibers present in the bark sclerenchyma tissue. In doing so, several microscopic techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and optical microscopy of stained histological sections were used. Overall, chemical analyses such as lignin, sugars, and hydrophilic extractives were performed in willow inner bark and wood as the first step to increase knowledge on valorization of willow stem within a new biorefinery concept. In the new fractionation concept, we suggest that the bark of willow biomass should be processed for extractives and fibers whereas the debarked wood should be hydrolyzed for sugars and the solid lignin residue used as a polymer or as a source of chemicals (Figure 1).
Figure 1. Fractionation scheme of a conceptual willow biorefinery.
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MATERIALS AND METHODS
Plant Material. Four year old trees of Salix myrsinofolia, willow hybrids “Karin” and “Klara” (SalixEnergi Europa AB), and Salix schwerinii were obtained from the plantation of VTT Technical Research Centre of Finland Ltd. located in Central Finland (Kyyjärvi) on October 17, 2014. Plant material was classified from first to four year of growth by visually selecting and cutting the tree joint sections. After cutting the willow stems were stored at −20 °C. Prior to debarking, the stems were immersed in water at 20 °C for overnight.16 Wood, inner and outer bark were separated manually with scalpel and stored at 20 °C until their use. For chemical analyses, the willow inner bark and wood fractions were grinded to small particle size (
