What Is The Chemical Makeup Of Cork

Heliyon. 2019 December; 5(12): e02910.

Chemical characterization of cork, phloem and woods from different Quercus suber provenances and trees

Received 2019 Jul 31; Revised 2019 October eleven; Accustomed 2019 November xx.

Abstract

Sustainability of cork oak (Quercus suber) forests is threatened past biotic and abiotic factors and label of potentially differing genetic resources has therefore gained importance. This work addresses the chemical variation of the three tissues of cork oak stems – cork, phloem and wood – in relation to tree and provenance, looking for genetic chemical diversity and for physiological derived differences. The three tissues differ with cork clearly differentiating regarding summative composition, component ratios and monomeric limerick. Cork is the only tissue where suberin is nowadays (42.3% o.d. mass) as the main prison cell wall component, and information technology has a high content of extractives (11.vii%) with meaning proportion of lipophilic compounds. Phloem is more lignified than forest (38.0% vs. 23.iv%) and has less polysaccharides (49.1% vs. 64.six%) with glucose-to-other sugars relation of 1:1.3 in phloem and 1:0.7 in wood. Analytical pyrolysis showed that lignification is a heterogeneous process and the lignin monomeric composition depends on tissue and cell type: cork lignin has a H:Chiliad:S ratio of 1:2.5:0.3 and S/Chiliad ratio of 0.12, while phloem and woods lignins have mainly 1000 and S units with a Due south/G ratio of respectively 1.1 and two.three. No significant differences were plant betwixt the three provenances, but some chemical variation occurred between the trees within a provenance. NIR spectroscopy and principal component assay differentiated cork, phloem and woods, while the dispersion within each group highlighted the pregnant tree variability, while provenances were a not-significant factor of chemical variation.

Keywords: Chemical engineering, Analytical chemistry, Found biological science, Lignin, Suberin, S/G ratio, Cork oak, NIR, Py-GC/MS

1. Introduction

Cork oak (Quercus suber) forests are spread across the western Mediterranean areas of Southern Europe and North Africa, where they play a substantial ecological role. They are part of montado, a multifunctional agro-forestry-pastoral arrangement that is classified as a Loftier Nature Value Farming System past the European Environmental Bureau (Pinto-Correira et al., 2011) and listed in the Habitats Directive as conservation value habitats (Catry et al., 2012). Even so, this ecosystem is threatened by biotic and abiotic factors such as insect pests and wildfires (Catry et al., 2017). The importance of differing genetic resource for improving sustainability of cork oak forests is stressed and a multi-locality provenance trial started in 1998, every bit role of the European EUFORGEN Network (Almeida et al., 2005).

The cork oak is characterized past a substantial formation of cork in its periderm. The observation of a cross-section of a cork oak tree stem (Figure 1) shows distinctively the wood at the inside, and the bark located to the outside including the phloem and the cork tissues. Both are accumulated during tree growth by the functioning of two meristems: the cambium which forms the forest cells to the inside and phloem cells to the outside; and the phellogen which forms the periderm with phelloderm cells to the inside and cork cells to the exterior (Pereira, 2007). The cork oak phellogen has characteristics that are at the base of operations of the species exploitation for cork.

Stalk cantankerous-sections of the three 6-year-onetime Quercus suber trees of provenance P15.

Cork production is the major economical activity in this non-wood woods organisation. The cork concatenation from forest to consumer relies on the regular and sustainable production of cork with the quality required by the increasingly enervating consumers of cork products. Cork is a cellular material with a very interesting and unique set of physical, biological and chemic backdrop (Pereira, 2007; Fortes et al., 2004), known worldwide for the "not bad" of wine bottles. Applications as a thermal insulator and for vibrational and sound absorption take developed from archetype and historical uses to high-tech developments (e.yard. space ablative insulators, equipment sealants).

In this framework, many studies were done on cork eastward.m. formation (Graça and Pereira, 2004), construction (Pereira et al., 1987; Oliveira et al., 2016), chemical composition (Graça and Pereira, 1997; Pereira, 1988, 2013), and properties (Pereira et al., 1992; Oliveira et al., 2014) that have contributed to the technological innovation of the cork chain. Cork performance depends on structure and chemistry (Pereira, 2015), although the impacts of their variation are far from being well established, e.g. it is believed that the cell wall chemical variation related to contents in suberin (23.i%–54.two%) and lignin (17.1%–36.iv%) and the ratio suberin-to-lignin plays a determining office in properties namely in pinch (Pereira, 1988, 2013; Oliveira et al., 2014).

Cork oak woods was less studied despite its quality for shipbuilding, tools manufacturing and construction, and just a few works summarized several forest properties including chemical composition (Knapic et al., 2012; Leal et al., 2008a). On the other hand, phloem was studied for the get-go fourth dimension by Lourenço et al. (2016) who compared its chemic composition with that of wood and cork in one private tree.

The present written report addresses the chemic variation of the 3 tissues of cork oak stems – cork, phloem and wood – in relation to tree and provenance, looking for genetic chemic diversity and for physiological derived differences. Summative chemical composition methods were used as well as belittling pyrolysis for compositional analysis. NIR spectral measurements were performed, having in mind the potential applicability of NIR to determine chemic parameters and screen Q. suber samples.

2. Material and methods

Six-year-quondam Quercus suber copse were sampled from a provenance trial located in Santiago do Cacém, Southern Portugal. This provenance trial was established in March 1998 as part of an European project (Off-white 1 CT 95–0202) with cork oak seeds collected from 35 provenances from Portugal, Spain, Italia, France, Morocco, Tunisia and Algeria (Sampaio et al., 2017). The present study used three provenances from Portugal, from the same cork producing region where the trial was established: Alcácer do Sal (P14), Azeitão (P15) and Santiago do Cacém (P19). The location, and climatic and soil label of the seed provenance sites as well as of the trial site are presented in Table i.

Table 1

Information on the cork oak provenances (P14, P15, P19) regarding location and main ecological features. Tm – long-term annual average air temperature; PPT – long-term almanac boilerplate precipitation; sPPT – long-term annual summer atmospheric precipitation.

Provenances	P14	P15	P19	Stablished trial site
Site	Herdade da Palma	Quinta da Serra	Monte Branco	Monte da Fava
Nearest locality	Alcácer do Sal	Azeitão	Santiago practise Cacém	Santiago exercise Cacém
Latitude	38°29′Northward	38°30′North	38°01′Due north	37°56′Due north
Longitude	8°35′W	9°02′W	8°42′W	8°27′Due west
Altitude (m)	30	120	140	79
Tm (°C)	16.3	xiv.3	15.6	fifteen.eight
PPT (mm)	577	681	736	557
Soil blazon	Sedimentary of silica			Sandy

Three trees were sampled from each provenance. From each tree, a disc was taken between ane.0 and 1.iii m of stem summit (Effigy 1). The wood, phloem and cork areas were determined using image assay of the discs, caused by a Kaiser RS1 board connected to a reckoner using AnalySIS® image processing software (Federal republic of germany, version 3.1). The woods diameter, phloem thickness and cork proportion in the total cross-exclusive area was calculated and the results are included in Tabular array 2.

Tabular array 2

Cork oak cross-sectional dimensions of the trees from each of the three provenances (P14, P15, P19) (mean values and standard departure).

Tree cross-department dimensions	P14	P15	P19
Wood area (cm²)	21.4 (eleven.3)	25.1 (1.5)	13.i (7.eight)
Phloem area (cm^two)	5.5 (iii.2)	four.four (eight.0)	3.3 (7.8)
Cork expanse (cm²)	24.2 (13.2)	21.2 (six.2)	17.6 (8.eight)
Total stem area (cm ² )	51.1 (27.7)	fifty.seven (8.4)	34.0 (xviii.5)
Woods diameter (mm)	51.1 (5.1)	56.5 (5.vi)	39.2 (iii.9)
Phloem thickness (mm)	3.1 (0.9)	two.4 (0.4)	2.4 (0.7)
Cork proportion (%)	47.3 (one.i)	41.4 (four.ix)	53.ii (iv.1)

The wood, phloem and cork were manually separated with a chisel from each disc. The samples were milled in a cut mill from Retsch (SM2000), first passing through a six × 6 mm sieve and then past a ane × 1 mm sieve. The samples were dried in an oven at lx °C and kept for analysis.

2.1. Chemical analysis

The summative chemic analysis was fabricated in ii aliquots of the wood, phloem and cork samples (after manual separation with a chisel) using procedures adjusted from TAPPI standards: ash content by incineration (TAPPI T15 bone-58), extractives by solvent extraction with a sequence of dichloromethane, ethanol and water during xvi h for each solvent (TAPPI T204 cm-07), Klason lignin (TAPPI T222 om-11) and soluble lignin (TAPPI UM 205 om-93). The composition of monosaccharides, acetic acid and uronic acids were determined in the hydrolysate obtained from lignin analysis using a Dionex ICS-3000 system in HPIC-PAD and an Aminotrap plus Carbopac PA10 column (250 × 4 mm) for monosaccharides and uronic acids, and by HIPCE-UV using a Waters 600 system with a Biorad Aminex 87H cavalcade (300 × 7.8 mm) for acerb acid.

For the cork samples, the suberin content was determined in the extractive-gratuitous material by methanolysis, as presented by Pereira (2013). The subsequent determinations of total lignin, monosaccharides, uronic acids and acerb acid were made in the suberin-free fabric, equally to a higher place described. All the chemical results were calculated and reported as percentage of the initial fabric.

two.2. Analytical pyrolysis

The extractive-costless samples were stale and powdered in a Retsch MM200 mixer ball during 10 min. Approximately 100 μg were weighted and pyrolysed at 650 °C during 10 south in a CDS platinum ringlet pyroprobe linked to a 5150 CDS apparatus, and connected to a GC (Agilent 7890B) equipped with a mass detector (Agilent 5977B, EI at 70 eV). The injector and the GC/MS interface were kept at 270 °C and 280 °C, respectively. The oven program and the pyrolysis products identification are described in more item by Lourenço et al., (2017). The percentage of each compound was calculated using the corresponding peak area in relation to the total area of the chromatogram. The per centum of hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin-derived products were separately summed, thus allowing calculation of the South/G ratio and the H:G:South relation.

ii.3. NIR spectroscopy

Near-infrared spectra were obtained for the wood, phloem and cork samples (Figure 3a). The samples were scanned using a Near Infrared Reflectance Accessory (NIRA) attached to a Perkin Elmer Frontier TM model FT-NIR spectrometer and the spectra collected over a wavenumber range of thousand–2500 nm, with 8 cm^−ane spectral resolution and 32 scans per spectrum.

(a) NIR raw spectra and (b) 2d derivative of the three tissues cork, phloem and forest from 6-year-old Quercus suber trees. Black and grayness solid line indicate forest and phloem tissues, respectively, and dash line signal cork tissue.

A blank spectrum was acquired subsequently every 10 samples to remove whatever random furnishings associated with the instrument or environment (due east.g., room temperature or humidity). Two spectra per sample were recorded at room temperature (20–22 °C).

The employ of chemometric techniques has been the choice for extracting information from NIR spectra in which absorption bands correspond mainly to overtones and combinations of central vibrations, and are relatively weak in intensity (Blanco and Villarroya, 2002). Since NIR spectra of solid samples are susceptible to scattering effects, such as offset, slope, and non-linear effects, spectral pre-treatments were performed to minimize those furnishings (Blanco and Villarroya, 2002; Rinnan et al., 2009). In this study the second-order derivative was selected, resulting in a spectral pattern display of assimilation peaks in the negative management, without faux peaks (Schwanninger et al., 2011).

2.iv. Statistical analysis

The chemical variability between tissues and provenances assessed by NIR spectroscopy was performed with main component analysis (PCA) using The Unscrambler® (CAMO Equally, Norway) software, version 10.5. The variation of chemical features within each tissue regarding the dissimilar cork oak provenances was statistically analysed using an assay of variance with a nested blueprint of trees within provenances. The furnishings were considered equally statistically significant when the p-value was less than or equal to 0.05. All the statistical analysis was performed using SPSS® statistical software (version 25.0; SPSS Inc., Chicago IL).

3. Results and discussion

This study focused on young Quercus suber trees of different provenances, aiming at ascertaining chemical differences between the stem components i.eastward. cork, phloem and wood and the potential impact of provenance. A previous report (Lourenço et al., 2016) indicated that there were chemical differences betwixt the 3 tissues just the limited sampling of only i tree did not allow to appraise between tree variability. Here, a more robust sampling was made of three trees from each of three provenances with the elimination of the potential influence of tree age and growth conditions past taking 6-year-erstwhile plants from the same trial (Table ane).

The trees adult differently both within provenance every bit between provenances, with mean total stem cross-sectional areas of 51.1 cm^two, 50.7 cm² and 34.0 cm² for P14, P15 and P19 respectively (Table 2). The dimensional measurements taken at the breast top cantankerous-department showed between tree variation of radial growth on all tissues. The boilerplate annual wood growth was 4.1 mm (varying from iii.3 to 4.7 mm/twelvemonth for P19 and P15 respectively). This compares with the 2.vii mm/yr reported for copse with 35 years, while cork oak wood annual growth is lower (i.half-dozen mm/year) for lx-year-sometime copse (Leal et al., 2008b). Phloem growth was on average 0.43 mm per year, meaning that the cambial meristematic action had a radial formation ratio of wood-to-phloem of iv.1:0.4.

It is striking to encounter that in that location was high action of the phellogen during these get-go half dozen years of the tree life, which was comparable to the cambial activity in terms of cellular volume production: cork represented on average 47.3 % of the cantankerous-sectional area, where wood and phloem represented respectively 43.3% and ix.7% (Table 2). This large amount of cork confers an increased protection to the tree, namely regarding insulation confronting high temperatures and the lowering of the probability of a tree being killed by burn down and the increase of crown regeneration (Catry et al., 2012).

3.1. Chemic composition of cork, phloem and woods

The summative limerick of cork, phloem and forest in the Q. suber samples is presented in Table 3, by provenance and in average, including the monomeric composition of polysaccharides regarding neutral and acid monosaccharides, and acetyl groups. The data obtained evidence that cork, phloem and forest differ chemically, with cork clearly differentiating itself regarding summative composition likewise as component ratios and monomeric composition.

Tabular array iii

Chemical summative composition (% of o.d. mass) and monomeric limerick of polysaccharides (% of total monomeric units) of Quercus suber cork, phloem and wood from three provenances (P14, P15, P19) as mean and standard difference of 3 trees and ii aliquots, and their hateful standard deviation (3 trees, three provenances, two aliquots).

	Cork				Phloem				Wood
	P14	P15	P19	Mean	P14	P15	P19	Mean	P14	P15	P19	Hateful
Ash	0.7 (0.1)	0.6 (0.i)	0.6 (0.1)	0.6 (0.2)	3.1 (0.7)	2.vii (ane.0)	two.viii (0.2)	ii.7 (1.0)	1.1 (0.2)	1.two (0.1)	1.ane (0.1)	1.1 (0.1)
Total extractives	10.iv (0.4)	12.6 (0.7)	12.1 (1.8)	11.7 (one.4)	3.9 (0.five)	four.8 (one.iv)	5.0 (0.8)	4.v (1.0)	v.0 (0.7)	five.eight (0.iii)	5.ix (0.7)	5.6 (0.7)
CH_twoCl₂	iv.eight (0.three)	5.iii (0.five)	5.i (0.half-dozen)	5.i (0.v)	0.i (0.03)	0.two (0.05)	0.2 (0.04)	0.two (0.04)	0.3 (0.05)	0.iii (0.03)	0.4 (0.07)	0.three (0.06)
EtOH	2.1 (0.6)	iv.ane (0.8)	three.1 (0.7)	iii.ane (1.0)	one.5 (0.4)	1.vii (0.4)	1.iv (0.iii)	1.5 (0.4)	1.2 (0.5)	one.9 (0.2)	1.five (0.5)	1.half dozen (0.5)
H₂O	three.4 (0.4)	3.three (0.half-dozen)	iii.8 (0.7)	3.5 (0.6)	two.iii (0.ii)	ii.nine (1.6)	three.4 (one.0)	2.viii (1.1)	3.4 (1.one)	iii.five (0.4)	iv.0 (0.7)	3.7 (0.8)
Suberin	42.7 (2.eight)	43.iii (6.3)	41.0 (iv.seven)	42.3 (4.7)	-	-	-	-	-	-	-	-
Full lignin	24.9 (1.3)	23.4 (2.8)	23.8 (2.1)	23.iii (2.1)	38.iv (ane.2)	37.eight (3.0)	37.9 (1.0)	38.0 (1.9)	22.6 (0.6)	24.five (1.3)	23.i (0.9)	23.4 (1.2)
Klason	24.ii (1.3)	22.7 (2.7)	23.0 (two.1)	24.ane (2.i)	35.9 (1.3)	35.3 (three.2)	35.5 (0.8)	35.6 (ane.9)	19.7 (0.8)	21.half-dozen (1.four)	twenty.4 (0.7)	20.vi (1.3)
Soluble	0.seven (0.09)	0.seven (0.1)	0.9 (0.08)	0.7 (0.1)	2.5 (0.ii)	ii.four (0.three)	2.4 (0.iii)	2.5 (0.3)	ii.9 (0.3)	two.nine (0.2)	2.6 (0.4)	2.8 (0.3)
Polysaccharides	16.8 (ane.6)	xv.two (iv.4)	16.half dozen (4.one)	xvi.ii (3.2)	48.v (v.3)	51.6 (2.one)	47.iii (1.1)	49.one (two.3)	66.nine (1.seven)	64.8 (five.1)	62.1 (2.3)	64.6 (iii.six)
Monosaccharides (% total monosaccharides)
Arabinose	17.1 (2.one)	19.0 (6.9)	17.0 (three.0)	17.7 (iv.0)	2.3 (0.2)	2.6 (0.6)	2.iii (0.five)	ii.4 (0.4)	1.5 (0.two)	1.four (0.3)	ane.vii (0.ii)	i.5 (0.2)
Xylose	29.3 (1.8)	27.1 (seven.one)	30.5 (3.5)	29.0 (4.3)	38.5 (1.five)	37.5 (iii.ane)	41.0 (two.8)	39.0 (2.vii)	25.vi (1.5)	27.8 (1.five)	29.vii (1.3)	27.seven (2.two)
Galactose	6.4 (1.0)	seven.0 (one.eight)	6.5 (1.3)	6.7 (1.3)	ane.8 (0.01)	2.three (0.v)	1.7 (0.two)	ii.0 (0.4)	two.4 (0.6)	two.iv (0.vii)	two.2 (0.three)	ii.3 (0.5)
Glucose	43.seven (1.6)	43.4 (2.6)	42.9 (0.viii)	43.3 (one.6)	43.seven (3.3)	43.8 (4.4)	42.8 (iii.iii)	43.iv (3.ii)	lx.iv (1.1)	57.nine (0.v)	58.5 (2.ane)	58.9 (1.vi)
Galacturonic acid	3.1 (0.3)	iii.i (0.vii)	ii.vii (0.2)	3.0 (0.46)	two.ane (0.six)	2.5 (0.7)	1.8 (0.4)	ii.one (0.6)	1.7 (0.ii)	ane.8 (0.3)	1.4 (0.1)	i.vii (0.iii)
Glucuronic acid	0.three (0.0)	0.3 (0.1)	0.4 (0.one)	0.34 (0.0)	0.three (0.iii)	0.5 (0.3)	0.1 (0.0)	0.3 (0.3)	0.four (0.2)	0.3 (0.2)	0.1 (0.0)	0.3 (0.ii)
Acetic acid	-	-	-	-	xi.2 (1.3)	10.8 (one.8)	x.ii (ane.4)	ten.7 (i.iv)	8.0 (0.seven)	8.2 (i.7)	half-dozen.four (1.ane)	seven.5 (ane.four)

It is well known that suberin is the major structural component of cork and that it is not present in phloem and wood (Pereira, 2007). The chemical composition of cork (Table 3) with 44.8 % suberin is in accordance with published values (Pereira, 2013). The suberin-to-lignin ratio was 1.8 similar to the reported value of 2.0, and the relation cellulose-to-hemicelluloses, determined by the ratio glucose-to-other-sugars was 1:ane.three, like to the reported 1:ane.2 (Pereira, 2013, 2015). The cellulose and hemicelluloses totalled 16.2% of the cell wall structural components (Tabular array 3). Hemicelluloses were mainly composed by arabinoxylans with a meaning proportion of galactose (6.7% of the total monomeric units) and including uronic acids (3.3% of the full) but without acetyl groups. Similar predominance of arabinoxylans in virgin cork hemicelluloses is reported in the literature east.g. xylose and arabinose representing xl.4% of total sugars (Pereira, 2007).

Cork contains a high content of extractives corresponding to xi.vii%, of which a significant proportion of 44% are lipophilic compounds. This is in accord with the reported range of values constitute in cork (e.one thousand. Pereira, 2013; Bento et al., 2001; Sen et al., 2016). The chemical composition of cork from the cork oak is in full general similar to that of cork from other species (Leite and Pereira, 2017) although chemical differences between species arise eastward.yard. in extractives content and composition (for instance in Quercus cerris, Sen et al., 2010; or in Pseudotsuga menziesii, Cardoso et al., 2018).

Phloem and woods are lignocellulosic tissues produced by the cambium that accept transport and mechanical support functions and which are specialized into different prison cell types e.g. respectively sieve elements and vessels for transport, and sclereids and fibers for support (Lourenço et al., 2016; Sousa et al., 2009). Phloem and forest have a moderate content of extractives (4.five% vs. v.6%), mostly polar compounds respective to respectively 4.iii% and 5.three% (Table 3). Phloem is more lignified than wood (38.0% vs. 23.4%) and has less polysaccharides (49.1% vs. 64.half dozen%) with some differences in limerick: the relation glucose-to-other sugars was one:1.3 in phloem and 1:0.7 in wood. The cork oak wood chemical limerick is in line with reported values (Leal et al., 2008a).

The chemic difference between the tissues is also noticeable past near-infrared spectroscopy. NIR spectra of cork present distinctive bands (1730, 1762, 2310 and 2349 nm) allowing a articulate bigotry betwixt the cork and the forest and phloem tissues (Figure 3 and Figure 4). The 1730 nm band is probably due to the first overtone of C–H stretching from CH₂ groups and was assigned by Schwanninger et al. (2011) to cellulose because it was but found in spruce cellulose, although they likewise refer that, combination bands of C–H stretching vibration and the 1^st overtone of C–H deformation vibration of methylene and methyl groups tin can also appear in this range. Workman and Weyer (2008) assigned the 1762 nm ring to the 1^st overtone of C–H stretching due to the symmetric C–H methylene (CH₂).

Principal component analysis of NIR raw spectra for cork, phloem and wood from 6-twelvemonth-former Quercus suber trees from iii provenances (P14, P15, P19).

The assignment in the spectral region of the combination bands, that include the 2310 and 2350 nm bands, is hard due to the high number of possibilities for the coupling of vibrations (Schwanninger et al., 2011). Nonetheless, Prades et al. (2012, 2014) assigned the 2300–2360 nm region to CH combination bands of antisymmetric and symmetric stretching plus one deformation manner in the CH and CH_ii structures. Workman and Weyer (2008) assigned the 2310 nm band to the 2^nd overtone of C–H fundamental bending due to lipids, and the 2349 nm band to the functional group of C–H methylene C–H, associated with linear aliphatic R(CH₂)_NR, due to aliphatic hydrocarbons as representative of cork suberin chemical construction (Pereira, 2007).

The differences between phloem and xylem are observed in NIR spectra with bands around 1428 nm in the ii^nd derivative (1450 nm in raw spectra) and 1722 nm. In asymmetric bands of the NIR spectrum (Effigy 3a), the position of the two^nd derivative can deviate from the position of the band in the spectrum. The ring around 1428 nm (1450 nm) is due to bond vibrations from the 1^st overtone of O–H stretching, and it was assigned to baggy polysaccharides of wood, including gratis and weakly H-bonded OH of cellulose, gratuitous and weakly H-bonded OH: O(six)–H(vi) of cellulose, glucomannan, and O(2)–H(2) of cellulose and xylan (Fackler and Schwanninger, 2010). Recently, Liang et al. (2020) highlighted the existence of a strong absorption signal at 1454 nm corresponding to the 1^st overtone of O–H stretching from phenolic groups nowadays in lignin. The ring around 1722 nm that helps to differentiate phloem and xylem tissues, was assign by Fackler and Schwanninger (2010) to 1^st overtone of C–H stretching in furanoses and pyranoses of hemicelluloses (xylan and glucomannan).

The band at 2267 nm is present in the NIR spectra of all tissues and tin contribute to differentiate between the tissues; yet, information technology is difficult to assign this band equally several bands can be seen in 2^nd derivatives of the spectra of milled wood lignin and in celluloses and hemicelluloses (Schwanninger et al., 2011). Sandak et al. (2013) assigned the 2270 nm band to semi-crystalline and/or crystalline regions in cellulose, particularly to CH₂ stretching and deformation in cellulose. Liang et al. (2020) assigned bands at 2267 nm and 2383 nm to combination bands of O–H stretching and C–O stretching, likewise as C–H stretching and C=C stretching. In this work we tentatively assigned this ring to lignin because it appears in the lignin Klason spectra from the aforementioned samples (non shown). Toscano et al. (2017) already highlighted this pinnacle equally one of the almost relevant wavelengths for the bigotry betwixt bark and forest samples.

All spectra were analysed by principal component analysis and Figure 4 presents their projection on the aeroplane defined by the first two principal components, cumulatively representing 98% of the total original information variance. Three separated clusters are observed in the score plot, showing high clustering tendency of the NIR data from the three tissues. The dispersion of the observations within each group highlights the meaning variability associated with the tree and that provenances were a not-pregnant cistron of chemical variation.

The results obtained here for the chemical differences between cork, phloem and woods confirm the values obtained in the previous study that analysed the three tissues from one cork oak tree (Lourenço et al., 2016). The nowadays piece of work with a more aggressive sampling of three provenances and 3 trees per provenance, thereby allows the consolidation of the findings and an insight into the factors of chemical variation (provenance and tree).

There are very few studies comparing the chemic composition of phloem and cork in the bark of other species that are in accordance with the results obtained in this study due east.grand. in Pseudotsuga menziesii (Ferreira et al., 2016; Cardoso et al., 2019) and in Quercus cerris (Sen et al., 2010).

Regarding the chemical variation related to the provenances, no statistically significant differences were found for about all the chemic features. The results show that simply ethanol extractives in cork have provenances as a cistron for chemic variation (p = 0.026). The studied provenances belong to the same broad cork production region, corresponding to a coastal region south of the river Tagus (e.k. the maximal distance betwixt the provenances locations is around 125 km). For cork, the lack of chemical differences betwixt regions was already reported in a large-scale study with sampling of 29 sites in half dozen regions of Portugal (Pereira, 2013) likewise every bit in a report with 7 provenances in Espana (Conde et al., 1998). Notwithstanding, some chemical variation was found between the copse within a provenance (Table 3). This between-tree variation was also noticed for cork chemical limerick, including suberin monomeric composition (Bento et al., 2001).

three.2. Lignin composition

Belittling pyrolysis is a powerful tool to evaluate lignin composition of plant tissues allowing lignin chemical classification based on its precursors proportion (Lourenço et al., 2019). The results obtained in the present study testify that cork, phloem and wood have different types of lignin (Tabular array four): cork lignin is mainly constituted by Chiliad units with also an important proportion of H units, simply with few S units (H:G:S 1:2.5:0.3, Southward/G 0.12); while phloem and wood lignins have mainly G and Southward units, just contain also H units; forest has more syringyl units (H:G:S of one:2.0:4.5 and S/One thousand of ii.three).

Table 4

Py-GC/MS results obtained for Quercus suber cork, phloem and forest from three provenances (P14, P15, P19) classified by chemical families (% of total area), besides as South/G and H:G:S lignin ratios.

Pyrolysis	Cork				Phloem				Wood
compounds	P14	P15	P19	Mean (STDEV)	P14	P15	P19	Mean (STDEV)	P14	P15	P19	Hateful (STDEV)
Lignin	13.2	12.4	12.1	12.6 (1.0)	fifteen.0	fourteen.6	13.5	14.4 (i.half-dozen)	10.4	12.0	9.9	10.7 (i.9)
Due south	1.0	0.eight	0.5	0.8 (0.two)	6.3	5.6	4.half dozen	5.v (1.2)	6.3	7.2	5.four	6.3 (1.seven)
G	seven.0	vi.5	five.iv	6.3 (1.0)	five.v	5.2	4.ix	v.2 (0.seven)	2.5	3.0	2.6	ii.7 (one.7)
H	2.5	ii.5	2.6	2.5 (0.5)	one.viii	2.i	2.3	2.0 (0.three)	1.2	i.3	1.6	ane.4 (0.4)
Others	2.7	two.seven	3.6	three.0 (0.5)	1.4	1.6	one.7	i.6 (0.3)	0.three	0.4	0.iii	0.4 (0.1)
S/G	0.14	0.12	0.09	0.12	1.ii	1.one	0.92	1.1	2.5	two.4	ii.1	2.iii
H:G:S	1:2.nine:0.4	1:two.6:0.3	ane:2.0:0.2	1:ii.5:0.3	one:three.1:3.5	1:ii.v:ii.7	1:ii.two:ii.0	1:2.6:two.seven	i:2.1:5.i	1:2.3:five.4	1:1.6:3.3	1:ii.0:4.5
Carbohydrates	26.ii	24.3	25.half-dozen	25.iv (4.two)	57.7	57.iv	60.8	58.6 (two.1)	64.ane	62.vii	62.4	63.1 (1.half-dozen)
Pyran	seven.7	7.0	6.8	7.two (1.6)	17.3	fifteen.7	17.vii	16.9 (ii.2)	24.ane	xx.9	22.2	22.iv (2.1)
Furan	4.5	iv.ii	4.5	four.4 (0.vii)	5.six	5.7	5.9	5.vii (0.3)	half dozen.0	six.2	6.0	6.one (0.ii)
Low molecular	xi.0	10.3	11.0	10.eight (one.7)	27.1	28.5	28.eight	28.1 (ii.0)	25.9	27.two	26.0	26.iii (1.5)
Others	3.i	2.8	iii.2	iii.0 (0.seven)	7.8	7.5	8.4	7.9 (0.6)	viii.1	8.3	8.three	8.2 (0.vii)
Suberin	33.ii	33.0	32.nine	33.0 (ii.4)	-	-	-	-	-	-	-	-
Fatty acid	7.four	7.4	seven.ix	7.vi (0.7)	-	-	-	-	-	-	-	-
Alkane	one.ix	ii.1	1.6	1.9 (0.3)	-	-	-	-	-	-	-	-
Alkene	xviii.1	17.v	17.v	17.seven (1.iii)	-	-	-	-	-	-	-	-
Alkadiene	iv.2	4.4	4.4	4.3 (0.5)
Non identified	i.vii	1.6	1.5	ane.vi (0.3)	-	-	-	-	-	-	-	-

As far equally we know, only Lourenço et al. (2016) studied in detail the lignin limerick in these tissues after lignin isolation and reported a lignin compositional profile similar to the one reported here, e.g. the S/G ratio was 0.ten in cork, 0.62 in phloem and 1.66 in wood. These results bear witness that lignification is a heterogeneous process and the lignin monomeric limerick depends on tissue and cell type (Barros et al., 2015; Ruel et al., 1999). Lignification has implications upon the protection and strength of the individual cells and therefore on the tissues. For case, tracheary elements present cell walls that are able to support negative force per unit area of sap ascent which can be provided by the presence of 1000-units; whereas fibers and sclereids offer mechanical strength and are typically constituted past Southward-units (Terashima and Fukushima, 1989; Higuchi, 1990). This might explain the different monomeric composition of cork, phloem and wood found in Q. suber: cork constitutes a homogeneous tissue of phellem cells that possess more M-lignin, while phloem has a large proportion of sclereids and woods more fibers, both mainly constituted by S-units (Barceló, 1997).

Another justification for the differences could exist the time of lignification i.e., the deposition of the lignin units in the cell wall is believed to be successive, first the H-units, followed by the G-units and at concluding the Due south-units (Terashima et al., 1986; Chesson et al., 1997). The incorporation of G-units is in the early on to late stages of cell formation, whereas the Southward-units are deposited mainly during the middle and late stages (Terashima et al., 1986; Fukushima and Terashima, 1991; Rencoret et al., 2011). Chesson et al. (1997) also refer for the later phase a variation in the degradation of G and S units with cell type.

Information technology is known that plants are able to incorporate substantial amounts of dissimilar components to respond to external conditions every bit an adaptive survival strategy. For example, in poplar after wounding the xylem cells became thicker with more guaiacyl lignin, a mechanism associated with a plant response to increment resistance (Schmitt et al., 2006). However, it is difficult to establish the relation between lignin content, composition and localization with the specific cell type and function (Neutelings, 2011).

3.three. Belittling pyrolysis

Table 4 presents a summary of the main derived group of compounds obtained by Py-CG/MS analysis of cork, phloem and woods. The Supplementary data includes more detailed information (east.chiliad. identification of pyrolysis compounds, their origin and hateful value in percentage of the total chromatographic area by provenance).

In addition to lignin composition (as mentioned to a higher place), analytical pyrolysis as well allows insight into the composition of the other structural components (Figure two). The pyrolytic degradation of suberin is dominated by the presence in the pyrolysis products of dissimilar families belonging to aliphatic products, which include fat acids (vii.4%), alkanes (1.9%), alkenes (18.ane%), alkadienes (four.ii%) and other unidentified aliphatic products (i.vii%), as summarised in Table 4. These aliphatic compounds included homologous serial with different chain lengths from 6 to 22 carbons. The alkenes and alkadiene carbon chains ranged from 9 to 22 carbons, the main compounds existence 1-hexene (C6:one), ane-heptene (C7:ane), one-octene (C8:1), 1,8-nonadiene (C8:ii) and ane,fifteen-hexadiene (C6:two). The identification of long concatenation fatty acids was difficult due to the impossibility to identify the molecular ion, and some peaks (peaks 142–164, Supplementary data) were not identified accurately. Thus, only a small amount of fatty acids with brusque carbon chains was identified such as C8:0 (1.5%, meridian 66), C7:1 (ane.three%, acme 55) and C8:1 (i.0%, superlative 69). Marques and Pereira (2014) did not identify fatty acids from the pyrolysis of different corks, due to decarboxylation and decarbonylation reactions, and instead found alkenes, alkadienes and alkanes. The pyrolysis conditions, namely temperature and period rates certainly influence the thermochemical deposition reactions and therefore the pyrograms (Marques and Pereira, 2014).

Py-GC/MC pyrograms of cork, phloem and wood from 6-year-former Quercus suber trees. 1: two-oxo-propanal; 2: 1-hexene (C6:1); 3: i-heptene (C7:1); v: hydroxyacetaldehyde; vi: acetic acrid; seven: 1-octene (C8:one); 8: acetol; 9: toluene; xiv: 3-hydroxypropanal; 19: CH₃–CO–CHOH–CHO; 20: CHO–CH_two–CH_ii–CHO; 22: furfural; 23: 2-cyclopenten-one-one; 31: 2-hydroxy-2-cyclopenten-1-ane; 33: 1-undecene (C11:i); 36: Not identified carbohydrate; 42: 4-hydroxy-5,6-dihydro-(twoH)-pyran-2-ane; 46: methyl-dihydro-(2H)-pyran-ii-one; 50: one-dodecene (C12:1); 55: vi-heptenoic acrid (C7:one); 66:octanoic acrid (C8:0); 69: vii-octanoic acid (C8:i); 72: one-tetradecene (C14:one); 75: Not identified carbohydrate; 78: 8-nonenoic acid (C9:one); 80: 1,five-anhydro-arabinofuranose; 81: 2,three-dihydrobenzofuran; 82: four-vinylguaiacol; 87: v-hydroxymethylfurfural; 95: 2-hydroxymethyl-5-hydroxy-2,iii-dihydro-(ivH)-pyran-4-one; 96: trans-isoeugenol; 97: similar to 1,five-anhydro-arabinofuranose; 99: vanillin; 107: 4-vinylsyringol; 121: levoglucosan; 123: syringaldehyde; 126: i-eicosene (C20:ane); 127: 1,nineteen-eicosadiene (C20:2); 130: acetosyringone; 132: trans-coniferaldehyde; 137: 1-heneicosene (C21:two); 140: one-docosene (C22:1); 142–144: Non identified suberin derivatives.

The pyrolysis products derived from carbohydrates also showed differences between cork, phloem and wood: the ratio of pyran:furan structures was 1.half dozen, 3.0 and 3.7 respectively, while the low molecular compounds represented a substantial proportion of the total sugar-derived compounds (42.5%, 48.0% and 41.7% respectively, Table 4). The quantification of hexose and pentose type of compounds cannot exist made past pyrolysis data, since their degradation produces the aforementioned compounds, except for levoglucosan (elevation 121) that is derived exclusively from cellulose, and for iv-hydroxy-5,6-dihydro-iiH-pyran-ii-one (top 42) that is a pentosan marking (Faix et al., 1991). Other hexose markers were divers (peaks 5, viii, 46, 79, 87, 95, 121) and for pentoses, peaks 42 and 80 (Marques et al., 1994). Under these assumptions, the ratio hexoses/pentoses was 3.7, seven.8, 11.v respectively for cork, phloem and forest; Marques et al., (1994) reported for cork an fifty-fifty lower ratio of one.3. This shows the importance of pentoses in the polysaccharides of cork corresponding to the chief proportion of arabinoxylans (Pereira, 1988).

Analytical pyrolysis has been proposed for chemical quantification, e.thousand. lignin content (Lourenço et al., 2019; Meier and Faix, 1992). However, in the conditions used in this work, namely a pyrolysis temperature of 650 °C, an important amount of low mass pyrolytic compounds was generated, that could not be assigned equally originating from a specific component (near thirty% of the chromatogram area). Therefore, the specific components are under or overestimated in accordance with the intensity of their thermal degradation. For instance, cork thermal behaviour showed that suberin is more thermally resistant (Sen et al., 2012, 2014; Pereira, 2015). The effect of pyrolysis temperature was besides discussed past Marques and Pereira (2014) who proposed 650 °C for the pyrolysis of cork-containing materials. In consequence, lignin content determined by pyrolysis is by far lower than the results attained by chemical analysis (12.vi% vs. 26.4% of extractive-free cork, Table 4 vs. Table 3) while the nether-estimation of suberin content was of smaller magnitude (33.1% vs. 37.4% of extractive-free cork). Therefore, the quantification past pyrolysis of structural components of plant materials, namely those of complex nature, should exist made with circumspection.

The comparison of the analytical pyrolysis information regarding provenances showed no meaning differences due east.g. the compositional pyrolytic contour regarding lignin and suberin was like (Table 4).

4. Conclusions

The focus of this study was to evaluate chemical differences in young Quercus suber trees between the three stem tissues (cork, phloem and forest) corresponding to physiological derived differences and between provenances and trees growing nether the same edaphoclimatic conditions, therefore respective to genetic variation.

The chemical limerick of cork was remarkably unlike from both phloem and forest. Cork has suberin every bit the major structural component followed by a lignin with a great proportion of G units but with few S units, while phloem and woods are mainly constituted by polysaccharides and lignin which was characterized by increasing amounts of S units from phloem to wood.

No chemical differences were found between provenances but amongst tree variation was present, showing genetic stardom at the private tree level. NIR spectroscopy and principal component analysis allowed to differentiate cork, phloem and wood, with loftier clustering tendency of the NIR information from the iii tissues while the dispersion within each grouping highlighted the significant variability associated with the tree and that provenances were a non-significant factor of chemical variation.

Declarations

Author contribution statement

Ana Lourenco: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Ricardo Costa: Performed the experiments; compile the data.

Vanda Oliveira: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Helena Pereira: Conceived and designed the experiments; Wrote the paper.

Funding statement

This work was supported by FCT (Fundação para a Ciência e Tecnologia, Portugal) past financing the Forest Research Center (UID/AGR/00239/2019). Vanda Oliveira was supported by FCT through a postdoctoral grant (SFRH/BPD/118037/2016). Ana Lourenço was supported by FCT through a enquiry contract (DL 57/2016/CP1382/CT0007).

Competing interest argument

The authors declare no conflict of interest.

Additional information

No additional data is available for this paper.

Acknowledgements

A word of appreciation to Duarte M. Neiva and Jorge Gominho for their assistance in the chemical assay and discussion of the results.

Appendix A. Supplementary information

The following is the supplementary data related to this article:

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