Unlocking the potential of undervalued forest residues: a comprehensive characterization of eucalypt stumps in the biorefinery context

doi:10.21203/rs.3.rs-4999430/v1

Eucalyptus globulusstumps were fractionated in three Discs, and their constituent tissues - heartwood, sapwood and bark - subjected to further chemical characterization by summative analysis, evaluation of the phytochemical profile and antioxidants activities, plus GC/MS and analytical pyrolysis. Wood density was similar between tissues and Disc level: values ranging from 0.652 to 0.705 g/cm³ (Disc 1) and 0.605 g/cm³ (Disc 5). Bark had high ash (3.5%), extractives (7.5%) and holocellulose (68.4%) but lower lignin contents (22.0%). Original heartwood contained 0.7% ash, 7.0% extractives, 27.1% lignin, and 67.3% holocellulose. Heartwood showed high extractives (12.1-15.8%), less lignin (23.9-24.5%), and high holocellulose (61.7-64.7%) than sapwood containing 3.9-5.4% extractives, 26.9-27.3% lignin and 68.6-71.5% holocellulose. Water extracts had poor antioxidant activity in contrast to ethanol extracts which activities were the highest in heartwood. All tissues presented GS lignin type with S/G ratios varying from 3.0-3.4 (heartwood), 3.2-3.4 (sapwood), bark (3.5) and 3.8 (original heartwood). In wood, fibers and vessels were highly lignified with SG and G-lignin respectively; while rays had low lignin with G-type. Light and fluorescence macroscopic observation of the tissues in Disc 1 revealed lower proportion and larger vessels in sapwood and high emission fluorescence at 488nm.

analytical pyrolysis

Eucalyptus globulus

fluorescence

lignin

macroscopic analysis

thioacidolysis.

Eucalypt stumps tissues: bark, sapwood and heartwood were fully characterized
Heartwood is rich in extractives and phenolics with high antioxidant activities
Lignin composition differ between tissues: bark has less S units comparatively to wood
Wood fibers (SG) and vessels (G) were highly lignified, ray had low lignin (G)
Sapwood presented larger but smaller number of vessels compared to heartwood

Eucalyptus globulus is a crucial species for the pulp and paper industry, prized for its rapid growth and superior wood quality, which make it ideal for producing high-quality bleached pulps. In Portugal, eucalypt is managed in short rotation coppice systems (8 to 14-year rotations) with three cutting cycles. However, other trees must be established at the end of the commercial exploitation. Thus, stumps must be removed from the field before soil preparation. Stumps are the basal part of the trees that remain in the soil after harvesting. In the case of eucalypts, stumps are uprooted and used as biofuel to produce heat and energy for the pulp and paper industry (Gominho et al., 2014; Angnes et al. 2021). Nevertheless, due to their chemical and anatomical characteristics, stumps have recently been used as feedstock for pulp and paper (Gominho et al., 2014), but more value-added products can be obtained from it within the concept of a biorefinery (Gominho et al., 2012; Luis et al. 2014; Pinto et al., 2017, Gominho et al., 2019). For instance, stumps extractives content can reach 15% (Gominho et al. 2012), higher than in eucalypt stemwood, with 3.1% (Morais and Pereira, 2012). Luis et al. (2014) reported high concentrations of phenolic compounds (> 200 mg GAE/g extract) and flavonoids (> 10 mg QE/g extract) in stump extracts with antioxidant and antimicrobial activities. Therefore, stumps can be previously extracted, and the bioactive molecules collected may be used for biomedical purposes. Also, isolated lignin from stumps can be used as an adsorbent for Cr(VI) to clean wastewater (Lourenço et al., 2022). In this context, lignin is an important chemical component that requires adequate valorization within the whole resource use concept. Lignin plays a central role in determining the heating value of biomass and offers opportunities for producing a variety of bio-based products such as antioxidants, resins, and polymers (Bajawa et al., 2019). Furthermore, its content and composition significantly impact fractionation and recovery processes (Faustino et al. 2010). Lignin characterization serves as fundamental groundwork for optimizing its utilization and designing tailored products. To date, the composition of lignin in various fractions of Eucalyptus globulus stumps (including sapwood, heartwood, and bark) remains unexplored. This presents a unique opportunity to obtain an initial insight into the extractives and lignin composition within these distinct stump components. Employing a comprehensive multi-analytical approach, encompassing traditional methods like summative analysis, gas chromatography, thioacidolysis, analytical pyrolysis, and FTIR spectroscopy, alongside light and fluorescence macroscopy, we aim to achieve a thorough characterization of stump constituents, particularly lignin. This holistic approach is essential for maximizing the valorization potential of stumps within the framework of biorefineries.

2.1. Collection of raw material

Eucalyptus globulus stumps were attained from a production site of a Portuguese pulp and paper Industry (ALTRI SGPS). The stumps were cut into Discs at five different levels (totalling about 1.5 m), transported to the laboratory, and left to dry under air conditions. After that, the bottom of each Disc was stained with methyl orange to identify the different tissues: heartwood (stained red) and sapwood (yellow-orange, Fig. 1). In Disc 1, methyl orange staining allowed to distinguish an older part corresponding to the first tree that was formed (original heartwood), and two trees that have grown after cutting the original wood (heartwood 1 and 2, sapwood 1 and 2). Discs 1, 3, and 5 were used the chemical analysis, while only Disc 1 was used for the macroscopical analysis. The bark was manually removed from the Disc and collected for both analyses.

Basic wood density was determined gravimetrically after water immersion using five aliquots; the average value and STDEV were calculated and presented here.

2.2. Chemical composition

The tissues from Discs 1, 3, and 5 were prepared for chemical analysis. In Disc 1, four tissues were selected: original heartwood, sapwood, heartwood, and bark; in Discs 3 and 5, just sapwood and heartwood. These tissues were milled in a knife mill, first sieved with a mesh of 6mm, then subjected to further grinding with a mesh of 1mm, and then sieved again to collect the 40–60 mesh fraction for chemical analysis. Summative analysis was made according to adaptations of Tappi standards: ashes (Tappi 15 os-58); total extractives were determined in the Soxhlet apparatus using sequential extraction with dichloromethane (6 h), ethanol (16 h), and water (16 h) following Tappi standard 211 om-02; total lignin was determined in extractives-free samples, as the sum of Klason lignin (Tappi T222 om-88) and soluble lignin (Tappi UM 250). Polysaccharides were determined by separating the hydrolyzate obtained from Klason lignin. Neutral monosaccharides and galacturonic and glucuronic acids were quantified by high-pressure ion chromatography (DIONEX ICS3000) as described in Costa et al. (2023). Holocellulose was also determined using the chlorite method according to Browning (1967) and after 240 min reaction time. In the bark sample, the amount of suberin was also determined by methanolysis, according to Pereira (2013). All analyses were made in duplicate.

2.3. Extracts composition

For GC-MS analysis, around 1 mg of the DCM extracts was derivatized with pyridine (Sigma-Aldrich) and BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, Sigma-Aldrich) to form trimethylsilyl ethers/esters (TMS). The mixture was heated at 60°C for 30 minutes and then cooled to room temperature. The TMS-extracts were injected into an Agilent 7890 A-5975C MSD GC-MS system equipped with a high-temperature capillary column (Zebron 5 HT, 30 m × 0.25 mm x 0.1 µm), using helium as the carrier gas at a constant flow (1 mL/min) in splitless mode. The detailed conditions can be seen in Gominho et al. (2020).

The ethanol and water extracts obtained from successive Soxhlet extractions were analysed as total phenolics content using the Folin-Ciocalteau method, total flavonoids content via the aluminium chloride colorimetric assay, and condensed tannins using the vanillin-sulfuric acid procedure (Ferreira et al., 2015). Phenolics content was reported as mg gallic acid equivalents, while flavonoids and condensed tannins as (+)-catechin equivalents through a calibration curve obtained with the same methodology. All tests were done in duplicate.

The antioxidant activity of the extracts was determined by the ferric-reducing antioxidant power (FRAP) and free radical scavenging activity (DPPH) as described in Miranda et al. (2016). The FRAP measures the potential of the extracts to reduce Fe(III) to Fe(II) and results were reported as mmol Fe(II)/g extract. The DPPH was evaluated by the IC50 (concentration of extract necessary to achieve 50% DPPH inhibition) and by the antioxidant activity index AAI, which describes the antioxidant activity of extracts as poor if AAI < 0.5, moderate if 0.5 < AAI < 1, strong if 1 < AAI < 2 and very strong if AAI > 2 (Scherer and Godoy, 2009).

2.4. Analytical Pyrolysis (PY-GC/MS)

The samples were dried and finely ball-milled in a Retsch MM200 centrifugal ball mill for 10 min. Around 105 µg of each sample was pyrolyzed in a quartz boat at 550°C for 1min in a platinum coil Pyroprobe connected to a CDS 5150. The volatiles separation was made in a GC from Agilent (7890B) using a fused-silica capillary column (ZB-1701: 60m x 0.25 mm i.d. x 0.25 µm film thickness) and applying the chromatography conditions described by Lourenço et al. (2013). For mass spectra analysis (Agilent 5977B MS), electron impact ionization at 70 eV and damping helium gas (1 mL) were used. The total area of the chromatogram was attained by automatic integration (Mass Hunter software), and the lignin and carbohydrates derivatives were identified by comparing their mass spectra with NIST2014, Wiley databases and literature (Faix et al., 1990, 1991; Ralph and Hatfield 1991). The lignin derivatives represented by H, G, and S-lignin units were summed up, and the S/G ratio and the relation H:G:S were determined. Total carbohydrates were determined as the sum of all the carbohydrates derivatives, and the relation carbohydrates-to-lignin ratio was calculated (C/L).

2.5. Infrared analysis (FTIR)

Transmission FTIR analysis of the ground samples was carried out using a Nicolet 6700 spectrometer and Omnic software (Thermo Fisher Scientific, USA). KBr pellets were prepared by mixing the sample with potassium bromide. Spectra were obtained through 16 scans from 4000 to 400 cm^− 1 with 4 cm^− 1 spectral resolution while subtracting background spectra measured in the air. Spectra were then corrected by baseline subtraction and area normalization to compare samples.

2.6. Thioacidolysis analysis

The lignin monomeric composition was also determined by thioacidolysis, which allowed the quantification of the monomers involved in the labile ether linkages (Lapierre et al, 1986). Samples of sapwood, heartwood original, and bark free-extractives (40–60 mesh fraction) were incubated at 100°C under agitation in a solution of ethanethiol/BF3 etherate/dioxane (2.5/10/87.5v/v/v) with the addition of 1 mL from a mixture of standards (tetracosane (25.2 mg) and dodecosane (23.8 mg) both dissolved in 100L of dioxane). After 4h reaction, lignin compounds released by thioacidolysis were extracted using dichloromethane and then silylated before GC-FID analysis. The guaiacyl (G) and syringyl (S) thioethylated monomers were separated in a fused silica DB-1 column (30 m x 250 µm x 0.25 µm, J&W Scientific©) using He as carrier gas and 160–280°C temperature gradient at 2°C/min.

2.8. Macroscopic observations

The anatomical studies were made on the tissues collected from Disc 1: sapwood, heartwood, the original heartwood, and bark. Samples were cut into small blocks (1x1x1.5 cm), then fragments of about 20mm in length and 3mm in thickness were obtained using blades or saw. The original heartwood could not be cut at once and was conditioned under 85% relative humidity using a solution of KCl. Transverse sections of 30–40µm thickness were obtained using a semi-thin microtome equipped with disposable blades (Thermo scientific HM 340E semi-automated microtome).

2.8.1. Histochemical analysis (Wiesner and Mäule staining)

The Wiesner and Mäule reactions were used to visualize lignin in each tissue (Day et al. 2005). The Wiesner reaction was performed after staining the sections with phloroglucinol (2% in 96% ethanol) for 5 min and mounting in HCl (2N). For Mäule staining, sections were incubated with KMnO₄ (1% in water) for 5 min and washed with water before adding HCl (2N) until the section was partially decoloured. The section was finally mounted in ammonium hydroxide (28–30%). Samples stained with Wiesner and Maule reagents were imaged using a macroscope (AXIO Zoom.V16, Zeiss) and visible light. For each tissue, the images of different sections (5–9) acquired at 25x and 63x magnification were analysed by ImageJ software to determine the proportion and size of the vessels in each tissue.

2.8.2. Fluorescence determination

Sections were mounted in deionised water prior to fluorescence macroscopic observation (AXIO Zoom.V16, Zeiss). Fluorescence images of four sections were acquired at 63x magnification and two excitation/emission wavelengths conditions: 1) 365 nm excitation, 420–470 nm emission (blue fluorescence), and exposure time of 1000ms, 2000ms, and 3000ms; 2) 488 nm excitation, 500–550 nm emission (green fluorescence) and exposure time of 500ms, 1000ms, and 1500ms. The field of view, resolution, and depth of field for fluorescence image acquisition was 3.7 mm, 0.7 µm, and 9 µm. Fluorescence images were analysed using ImageJ software, following an automated procedure as described by Audibert et al. (2023). The procedure was applied to the stack of the four images acquired for each excitation and allowed the quantification of average fluorescence intensity. The stack image was converted to an 8-bit grey level, and an automated threshold called “Huang dark” was applied to detect the cell walls and extract a selection that is applied in the unmodified image stack to measure the average grey value.

3.1. Chemical analysis

The emergence of new feedstocks, such as biomass from eucalypt stumps, is a crucial development for biorefineries. Eucalypt stump biomass is gaining attention as a valuable raw material due to its unique physical and chemical properties, as illustrated in Tables 1 and 2. One of the key parameters for assessing wood quality is basic density, which generally increases with tree age and varies across species (Lima, 1995). In our findings, the basic density values did not show substantial differences between the tissues and Disc levels. For instance, Disc 1, which includes stumps above soil level, exhibited density values ranging from 0.652 to 0.705 g/cm³. In contrast, deeper samples, such as those from Disc 5, presented slightly lower values, around 0.605 g/cm³, for both heartwood and sapwood (Table 1). Notably, these values surpass those reported for E. globulus stemwood, with 0.478 g/cm³ (Carrillo et al., 2017) and 0.491 g/cm³ (Gominho et al., 2001), respectively, 6 and 5.6 years. The density of other eucalyptus species, such as E. nitens, was recorded at 0.490 g/cm³ with a six-year-old (Carrillo et al., 2017). When examining different tissues, Lourenço (2009) did not observe any variation in basic density between heartwood and sapwood from 18-year-old E. globulus, with a consistent density of 0.74 g/cm³. In contrast, S. mahogany heartwood presented lower basic density compared to sapwood (Arisandi et al., 2023), highlighting the complexity of this topic. The relationship between density, vessel frequency, and diameter adds another layer of complexity. As noted by Pfautsch et al. (2016), the basic density of sapwood tends to increase alongside vessel frequency; however, mean vessel diameter may decrease in trees growing in mesic environments. The implications of vessel diameter differences will be addressed in subsequent sections.

Table 1

Basic density values of the heartwood (Heart) and the sapwood (Sap) at different heights and of the bark of the *Eucalyptus globulus* stump. Mean values and standard deviation (STDEV) of five replicates.
	Disc 1			Disc 3		Disc 5
	O.Heart	Heart	Sap	Heart	Sap	Heart	Sap
Density (g/cm³)	0.678	0.695	0.657	0.620	0.654	0.605	0.605
STDEV	0.028	0.026	0.007	0.032	0.019	0.048	0.012

O.Heartwood = original heartwood, i.e. from the first tree grown.

Chemical analysis revealed distinct characteristics among tissues and across the three stump levels (Table 2). Bark had elevated ash content (3.5%) and substantial extractives (7.5%), but a lower lignin content (22.0%) compared to sapwood and heartwood (23.9–27.3%). Stump bark contained 0.3% suberin, typical for E. globulus species (0.98% Miranda et al. 2013), yet lower than other barks like Q. faginea (2.9%) and B. pendula (5.9%, Miranda et al. 2013). Holocellulose content in bark (68.4%) fell within the range for eucalypt stem bark (62.6%) but was higher compared to birch (49.8%, Miranda et al. 2013), indicating potential for valorization beyond combustion for power generation. Bark presented the highest concentration of glucose (70.1% of polysaccharides), while displaying the lowest content of xylose (20.7%), as compared to the wood tissues.

Heartwood exhibited a notable abundance of extractives, particularly in Discs 3 and 5 (15.6% and 15.8%), possibly indicating a defensive response or leaching due to rain after tree removal. Interestingly, the original heartwood showed a lower level of extractives (7.0%), likely due to seasoning from previous tree removal (Gutiérrez et al. 1998), allowing the regrowth of the plant. Sapwood consistently had a higher lignin content (26.9–27.3%) compared to heartwood (23.9–24.5%), whatever the Disc analysed. However, original heartwood (27.1%) displayed lignin levels similar to sapwood. Lignin variation between heartwood and sapwood has also been found in a hybrid eucalypt E. urophylla X E. grandis, but in this case, heartwood presented higher values compared to sapwood, reaching values of 33.8 and 26.0% respectively (Xiao et al. 2019), while other studies have reported higher lignin content in heartwood of Michelia macclurei (Ren et al. 2023). In Disc 1, sapwood exhibited the highest holocellulose content (mean 68.6%), akin to the original heartwood (67.3%), whereas heartwood ranked behind at 64.7%. This trend persisted in Discs 3 and 5, with sapwood exceeding 70% of dry matter. Generally, sapwood across various species displays higher total carbohydrate content than heartwood. For instance, E. urophylla, E. globulus, A. mangium and the hybrid E. urophylla x E. grandis sapwood samples have reported total carbohydrate values of 51.5%, 54.4%, 55.5% and 61.9% respectively (Çetinkol et al. 2012; Xiao et al. 2020). Overall, xylose and glucose predominate among tissues, consistent with the glucuronoxylan type of hemicelluloses found in hardwoods like E. globulus (Rowell et al. 2012). Xylose content prevailed in heartwood polysaccharides (23.1–26.6%) while constant values were reported in sapwood (22.7–22.9%). On the opposite, glucose content was not clearly consistent between wood tissues (Disc 1: heartwood had the lowest value, but in Disc 3 and 5 the tissues presented similar values). Hemicelluloses isolated from the heartwood, sapwood, and bark of a eucalypt hybrid revealed the highest xylan content mainly in heartwood (73.0%, 58.5%, and 69.9%, respectively), followed by galactose that prevailed in sapwood (3.0%, 14.0% and 3.2%, Xiao et al. 2020). Furthermore, the presence of acetic acid and galacturonic acid, with values exceeding 5% and around 1.5% of total sugars, respectively, suggests partial acetylation of hemicelluloses.

Table 2

Chemical composition of sapwood and heartwood at different heights and of the bark of *Eucalyptus globulus* stump (mean values of 2 replicates).
	Disc 1			Disc 3		Disc 5
Component (% dry matter)	O.Heart	Heart	Sap	Heart	Sap	Heart	Sap	Bark
Ash	0.7	0.3	0.7	0.2	0.3	0.2	0.3	3.5
Extractives	7.0	12.1	5.4	15.6	4.5	15.8	3.9	7.5
Dichloromethane	0.5	0.6	0.3	1.0	0.3	1.2	0.2	0.6
Ethanol	2.1	8.4	1.6	11.7	0.8	11.6	0.6	2.8
Water	4.4	3.1	3.5	2.8	3.4	3.0	3.0	4.1
Total Lignin	27.1	24.5	27.3	24.0	26.0	23.9	26.4	22.0
Klason Lignin	23.5	21.4	24.1	21.3	22.9	21.1	23.1	19.3
Soluble Lignin	3.6	3.1	3.2	2.7	3.1	2.8	3.3	2.7
Holocellulose	67.3	64.7	68.6	61.7	71.5	62.5	71.5	68.4
Suberin	-	-	-	-	-	-	-	0.3
Lignin-to-Holocellulose ratio	0.40	0.37	0.39	0.38	0.36	0.38	0.37	0.32
Sugars (% of Total monosaccharides)
Rhamnose	0.7	0.7	0.5	0.4	0.4	0.4	0.5	0.2
Arabinose	0.6	0.8	0.8	0.4	0.8	0.5	0.5	0.9
Galactose	1.9	2.2	2.6	1.6	1.5	1.4	1.3	1.9
Glucose	63.6	60.8	64.9	66.2	66.1	67.4	66.6	70.1
Xylose	25.6	26.6	22.9	24.6	22.7	23.1	22.8	20.7
Galacturonic acid	1.6	1.6	1.5	1.3	1.2	1.2	1.1	1.2
Acetic acid	6.0	7.5	7.0	5.5	7.2	6.0	7.1	5.0

O.Heart = original heartwood, i.e., from the first tree. Heart = heartwood; Sap = sapwood; In bark, the total lignin value was corrected to extractives and ashes contents.

3.2. FTIR analysis

FTIR spectra of the samples from Disc 1 (Fig. 2) contain the typical carbohydrates and lignin-associated bands. Generally, a broader band appears in all samples at 3416 cm^− 1, which corresponds to O-H absorption bands from carbohydrates and cellulose, followed by the band at 2915 cm^− 1 corresponding to C-H stretching (asymmetric and symmetric stretching methyl and methylene, Popescu et al. 2009). In the fingerprint region (1800 − 800 cm^− 1), more bands were identified: the band associated with carbonyl groups from hemicelluloses at 1740 cm^− 1 and absorbed water at 1682 cm^− 1 (Popescu et al. 2009). The bands assigned to cellulose were: 1377 (C-H deformation in polysaccharides), 1246 (C-O stretch and O-H in plane), 1054 (C-O stretch in polysaccharides), and 897 cm^− 1 (C-H deformation in cellulose, Popescu et al. 2009; Rodrigues et al. 1998; Gominho et al. 2019; Rubio-Valle et al. 2024). The bands assigned to lignin were 1595 and 1505 (aromatic skeleton vibration in lignin), 1331 (C_aryl-O vibration in syringyl derivatives), 1124 cm^− 1 (aromatic skeletal and C-O stretch), while the bands 1737 and 1265 cm^− 1 (guaiacyl ring breathing or C-O linkage in guaiacyl aromatic methoxyl groups, Schwanniger et al., 2004; Popescu et al., 2009) were not dominating. All samples have a GS lignin type since the band 1465 is higher than 1505 cm^− 1 (Faix, 1991). There were also bands common to lignin and cellulose such as: 1465 and 1428 cm^− 1 (C-H deformation, Popescu et al., 2009). Only bark presented some differences compared to wood tissues, being some bands higher, in particular 1628 cm^− 1 assigned to C = O stretching in flavones and calcium oxalate (Zhang et al. 2016; Javier-Astete et al., 2021), while some bands were assigned to polysaccharides (1246, 1162, and 1054 cm^− 1).

Using the FTIR bands intensity, it was possible to calculate different indexes: i) Lateral order index (LOI): an empirical crystallinity index = A1428/A897, where 1428 cm^− 1 band is associated with the crystalline structure of cellulose, while the band at 897 cm^− 1 corresponds to the amorphous region of cellulose (Nelson and O’Connor, 1964); ii) Total crystalline index (TCI) = A1377/A2915; the band 1377 cm^− 1 is associated with glycosidic bond β-(1,4) in cellulose and the band 2915 cm^− 1 is related to the crystalline structure of cellulose (Nelson and O’Connor, 1964); iii) Cross-linked lignin (CLL) indicates the proportion of condensed lignin and cross-linked structures = A1505/A1595; where both bands 1505 cm^− 1 and 1595 cm^− 1 relate to aromatic skeletal vibration of lignin (Auxenfans et al., 2017); iv) Lignin-to-cellulose index (L/C) was determined between A1505/A897, where the 1505 cm^− 1 band corresponds to the lignin stretching of C = C, and 897 cm^− 1 is assigned to C-H deformation in cellulose (Gaur et al., 2015). Original heartwood and heartwood attained similar values for LOI and TCI; the LOI values were 3.24 and 3.00, respectively, while their TCI values were 1.61 and 1.62. In bark tissue: the lateral order index (LOI) total crystalline index (TCI) was the lowest, suggesting that cellulose in bark has a lower degree of crystallinity compared to wood tissues. The lignin-to-cellulose index (L/C) was also lower in bark (2.03) compared to heartwood and sapwood (2.34–2.62), which recorded the highest value (2.62). This is consistent with the chemical analysis, where the lignin-to-holocellulose ratio was the lowest in bark (0.32, Table 2). The lowest value of the LCC ratio was obtained in the bark suggesting that lignin is more condensed with crosslinked G-type lignin structures. These infrared data were in the range of values reported in the literature. For instance, industrial chips derived from E. globulus stumps exhibited a high degree of crystallinity, with LOI and TCI values reaching 4.31 and 1.49, respectively. Additionally, the CLL stood at 0.87, and the L/C ratio was 3.29 (Gominho et al., 2019). Other hardwoods such as poplar presented a LOI of 1.77, and CLL of 1.32 (Auxenfans et al. 2017).

3.3. Extractives composition

Figure 3 illustrates the distribution of the primary compound families in the dichloromethane (DCM) extracts, and a detailed list of compounds identified is available in Supplementary material (Table S1). On average, approximately 76% of the total chromatogram area was identifiable, with compounds primarily belonging to phytosterols, triterpenes, fatty acids, and aromatics families. Sapwood exhibits a higher concentration of triterpenes, with asiatic and arjunolic acids being the most prominent, followed by phytosterols (predominantly β-sitosterol) and fatty acids (notably hexadecanoic acid and cis 9-octadecenoic acid). Conversely, aromatics and monoglycerides are more prevalent in heartwood. This composition pattern of the DCM extract mirrors findings from Gominho et al. (2020) in mature E. globulus trees, indicating that heartwood tends to be richer in triterpenes and aromatic compounds. These compounds, which accumulated in the heartwood region, play a crucial role in providing protection against biotic agents. In turn, the bark is rich in pentacyclic triterpenes (53.6%, with betulinic acid as the main compound), followed by fatty acids (17%) and phytosterols (13%). According to Gominho et al. (2021), this type of biomass could be a good source of bioactive compounds with a wide range of applications.

Table 3 presents the phytochemical profile of ethanol and water extracts, encompassing total phenolics (TP), total flavonoids (TF), and condensed tannins (CT), which total values were calculated having in account the percentage of each extract (Table 2). Alongside, the antioxidant activities were determined using the FRAP and DPPH methods. Notably, ethanol extracts generally exhibited a superior phytochemical profile compared to water extracts across all samples. Heartwood samples exhibited phenolic levels ranging from 866.7 to 1 066.2 mg GAE/g extract for ethanol extracts and 177.4 to 280.8 mg GAE/g extract for water extracts. Similarly, sapwood ethanol extracts ranged from 758.7 to 860.7 mg GAE/g extract, while water extracts ranged from 149.1 to 231.9 mg GAE/g extract. This variation can be attributed to the higher content of phenolics in ethanol extracts compared to water extracts in heartwood (8.4% vs. 3.1%, as shown in Table 2), whereas the opposite is observed in sapwood (1.6% vs. 3.5%). Interestingly, the original heartwood mimics the behavior of sapwood tissue, exhibiting a TP value of 983.3 and 243.6 mg GAE/g extract, respectively, ethanol and water extracts.

In terms of total flavonoids (TF) and condensed tannins (CT) compositions, both heartwood and sapwood displayed a similar pattern to that of total phenolics (TP). For instance, heartwood TF levels in the ethanol extract ranged from 106.8 mg CE/g extract (disk 5) to 143.9 mg CE/g extract (disk 1). Conversely, bark exhibited intermediate values for TP of the ethanolic extract (431.4 mg GAE/g ethanolic extract) falling between those of heartwood and sapwood, but with generally higher levels for TF and CT (135.3 and 149.1 mg CE/g extract, respectively). Luís et al. (2014) also studied the phenolic compounds of wood, bark, and stumps from E. globulus, attaining in the ethanol extracts the values 460.0 (stumps) and 253.9 mg GAE/g extract (bark). These authors measured flavonoids by using a different standard (quercetin), and the values reached 33.6 and 8.3 mg QE/g extract for stump wood and bark, respectively (Luís et al., 2014).

Table 3

Phytochemical profile and determination of the antioxidant activities of the phenolic extracts from heartwood, sapwood, and bark of eucalypt stumps (mean values of 2 replicates).
	Disc 1				Disc 3		Disc 5
	O. Heart		Heart	Sap	Heart	Sap	Heart	Sap	Bark
Phytochemical Profile
Total Phenolics (TP, mg GAE/g extract)
Ethanol	983.3		1 066.2	758.7	866.7	820.2	884.7	860.7	431.4
Water	243.6		280.8	231.9	177.4	149.1	252.0	174.8	201.0
Flavonoids (TF, mg CE/g extract)
Ethanol		145.5	143.9	154.9	108.9	221.7	106.8	173.8	135.3
Water		3.0	11.1	2.4	12.5	1.6	11.5	1.3	72.2
Condensed Tannins (CT, mg CE/g extract)
Ethanol		22.3	21.9	47.5	17.7	45.2	20.3	42.1	149.1
Water		0.5	1.7	0.8	2.0	0.8	2.2	0.3	51.9
Antioxidant Activity
FRAP (mmol Fe(II)/g extract)
Ethanol		12.6	12.1	7.6	10.8	7.5	8.8	7.8	4.4
Water		0.3	0.9	0.1	1.2	0.1	1.0	0.1	2.0
DPPH
IC50 Ethanol (mg/L)		2.6	1.3	2.3	1.9	3.0	2.1	2.5	6.8
AAI Ethanol		9.2	18.3	10.4	12.5	7.9	10.9	9.5	3.5
Water IC50 (mg/L)		12.7	7.3	10.8	17.8	18.8	8.0	13.7	148.4
AAI Water		1.8	3.2	2.6	1.3	1.2	2.9	1.7	0.1

O. Heart – original heartwood; *values reported to the percentage of each extract attained from chemical composition.

Ethanol extracts from heartwood exhibited superior antioxidant activity compared to water-soluble extracts. FRAP values ranged from 8.8 to 12.6 mmol Fe(II)/g extract in heartwood ethanol extracts and 7.5 to 7.8 mmol Fe(II)/g extract in sapwood ethanol extracts. Conversely, bark showed higher antioxidant activity in water extracts (2.0 Fe(II)/g extract) than in ethanol extracts (4.4 Fe(II)/g extract). Regarding the DPPH method, both heartwood and sapwood ethanol extracts displayed strong antioxidant activity, with AAI values exceeding 2. Heartwood ranged from 9.2 to 18.3, while sapwood ranged from 7.9 to 10.4. Bark ethanol extracts exhibited significant antioxidant activity (AAI of 2.4), whereas water extracts showed poorer activity (AAI of 0.1). Studies by Luís et al. (2014) corroborated these findings, showing higher antioxidant activity in ethanol extracts (AAI 7.39) compared to other solvents, such as n-hexane extracts (AAI 0.25) when analyzing stump wood.

3.4. Pyrolysis analysis

Analytical pyrolysis is a robust methodology for characterizing both wood and bark, offering a rapid analysis that enables a relative quantification of carbohydrates and lignin (Lourenço et al., 2019; Rencoret et al., 2011). While versatile for insights into both components, its main interest lies in determining lignin composition. Therefore, we present in more detail the data for lignin characterization and briefly those of carbohydrates. Based on the pyrolysis findings (refer to Table 4, Table S2, and Figure S1), carbohydrate derivatives accounted for 54.6% of the original heartwood composition, comparable to sapwood (ranging from 52.3–54%). However, heartwood displayed slightly lower values, ranging from 48.9% (Disc 5) to 51.1% (Disc 1). For a detailed identification of the derived compounds and their relative quantification, please refer to the supplementary information (Table S2). Lignin content remained consistent across all tissues, with heartwood registering 27.8% (Disc 1) and sapwood slightly higher at 29.7% (Disc 1), while bark exhibited a notably lower percentage at 20.1%. Minimal differences were observed among the other two levels. All these values are in the range of those attained by chemical analysis (Table 2). In terms of lignin composition, all tissues showed a prevalence of syringyl (S) over guaiacyl units (G), with a minor presence of p-hydroxyphenyl units (H). Consequently, S/G ratios ranged from 3.1 (heartwood) to 3.5 (bark), with slightly lower values recorded in the wood of Disc 5. These findings align with the range of 1.9–5.5 reported for eucalypt stemwood as determined by Py-GC/MS analysis (del Río et al. 2005; Rencoret et al., 2011; Lourenço et al., 2019). Also, when studying E. globulus stemwood with 1 month, 18 months, and 9 years, Rencoret et al. (2011) identified more H units like methylphenols and dimethylphenol, particularly in the young wood (9%), decreasing in oldest wood (2%). This is in line with our findings here, where only phenol was identified, maybe because stumps are an older material, and during wood maturation, there is an increase of G and S-units being deposited in the cells (Terashima et al., 1986).

Table 4

Resume of the results from the pyrolysis analysis (values as % of total chromatogram area).
	Disc 1			Disc 3		Disc 5		Bark
	O. Heart	Heart	Sap	Heart	Sap	Heart	Sap	Bark
Total carbohydrates	54.6	51.1	52.3	49.4	54.0	48.9	52.0	58.9
Total lignin	28.6	27.8	29.7	28.5	28.2	28.2	27.3	20.1
S	22.6	21.3	23.1	21.6	21.7	21.1	20.7	15.4
G	5.9	6.4	6.4	6.9	6.3	7.0	6.5	4.4
H	0.2	0.2	0.2	0.1	0.2	0.1	0.1	0.3
Others	0.8	0.7	0.7	0.9	1.0	0.9	0.9	0.9
S/G ratio	3.8	3.4	3.4	3.1	3.4	3.0	3.2	3.5
H:G:S relation	1:20:79	1:22:77	1:21:78	0:24:76	1:22:77	0:25:75	0:24:76	2:21:77

Data from Disc one: Heartwood values are the mean values of Heart 1 and Heart 2, the same for Sapwood.

3.5. Thioacidolysis analysis

Even though pyrolysis is an interesting technique for attaining the lignin monomeric composition of the whole polymer, thioacidolysis can allow the detection of the monolignols that are linked only through labile aryl ether linkages, thereby providing an estimation of the proportion of non-condensed lignin structure (Lapierre, 1993). In agreement with pyrolysis data, the β-O-4´ linked lignin structures in all stump tissues contained both G and S units with S/G molar ratio ranging from 3.3-4.0, whereas H units were at trace levels (data not shown). This result agrees with a previous study reporting an S/G value close to 3.3 in mature wood of Eucalyptus. Xiao et al. (2019) found that the lignin of sapwood of Eucalyptus displayed increasing S/G ratios of lignin from heartwood to sapwood. The weak variation shown for stump tissues indicates that sapwood and heartwood reached quite similar maturity in terms of lignin as the S units increased during lignification (Lourenço et al., 2016).

Based on the total recovery yields from thioacidolysis and an average monolignols molecular weight of around 200, the fraction of non-condensed lignin structure was estimated and accounted for 39 to 53% of Klason lignin (Lapierre et al., 1993). The lowest frequency was found in the bark tissue, indicating that lignin is more condensed in the bark than other tissues. This result thus confirmed that the FTIR analysis showed the lowest CLL ratio in the bark. However, a higher proportion of β–O–4′ linkages have been reported in lignin isolated from the wood bark of Eucalyptus urophylla × E. grandis (Xiao et al., 2019). A deeper investigation using spectroscopic analysis could allow a more detailed characterization of the bonding pattern of lignin in the different tissues, as shown by 2D NMR analysis of Eucalyptus wood and the constitutive tissues (Rencoret et al., 2010; Xiao et al., 2019).

Table 5

Monolignols quantification after thioacidolysis (as µmol/g Klason lignin ± STDEV).
Sample	G	S	G + S	S/G
Original heartwood	455.6 ± 29.9	1 807.3 ± 107.1	2 262.8 ± 135.3	4.0 ± 0.1
Heartwood	557.1 ± 4.2	2 082.9 ± 21.2	2 640.1 ± 25.2	3.7 ± 0.0
Sapwood	554.6 ± 17.9	1 832.4 ± 7.0	2 387.1 ± 22.1	3.3 ± 0.1
Bark	432.8 ± 31.9	1 518.4 ± 198.4	1 951.2 ± 230.3	3.5 ± 0.2

3.6. Histochemical analysis

Lignin was visualized in the stump tissues using Mäule and Wiesner staining techniques (Fig. 5). Wiesner staining imparted a red or dark rose in the cell walls rich in lignin cinnamaldehyde groups (Clifford, 1974). This staining method unveiled extensive lignification in both heartwood and sapwood, notably in fibers (F) and vessels (V), whereas rays (R) exhibited a darker brown coloration, indicating lower lignification levels (Fig. 5.a-c). Conversely, sclerenchyma (S) exhibited a deep rose coloration in bark, indicative of lignification, particularly notable in the middle lamella and cell corners (Fig. 5.d). However, other cell types, such as parenchyma cells, showed less pronounced lignification. Obtaining high-quality microscope sections from original heartwood and bark posed challenges due to their tougher texture, necessitating additional effort during sample preparation.

Following Mäule staining (Fig. 5.e-h) that gives a purple-red coloration with syringyl unit (Meshitsuka and Nakano, 1979), the prevalence of syringyl and guaiacyl lignin types in the cell walls of fibers (F) was evident in original heartwood, heartwood, and sapwood, while vessels (V) and ray cells (R) displayed a G-type lignin, appearing orange to brown as already reported in hardwood vessels (Fergus and Goring, 1970; Donaldson, 2001). In the bark, sclereids (S) stained red and exhibited lignin of the SG type. Parenchyma cells (P), appearing pink-rose, were at a distinct stage of lignification, while the rays, displaying a brown coloration, were primarily composed of G-lignin. The tyloses observed in vessels of both original and heartwoods (blue arrow) displayed pale staining with Maule reagent. Using an improved Mäule reaction for fluorescence observation, Yamashita et al. (2016) suggested that tylose would differ from vessels in their syringyl proportion.

Image analysis revealed differences between different wood. Both original heartwood and heartwood had a higher proportion of vessels (7.8 ± 0.2 and 7.2 ± 2.8 vessels/mm² respectively) then sapwood (3.3 ± 0.6 vessels/mm²). Vessels in sapwood were larger (9064 ± 1362 µm²) than in original heartwood (5825 ± 472 µm²) and in heartwood (4786 ± 1246 µm²). This result suggests that stump anatomy is very similar to stemwood, for which different studies have reported that vessel diameter increased from heartwood to sapwood (Longui et al., 2014).

Stump tissues were also observed using fluorescence macroscopy at two different excitation wavelength, 353 nm which can be attributed mainly to lignin fluorescence and 488 nm that may correspond to the presence of phenolic extractives such as flavonoids (Donaldson, 2020). All the cell walls showing positive reaction with Wiesner and Maule reagents displayed intense blue and green fluorescence when illuminated with 353 and 488 nm respectively (Fig. 6). In contrast to the blue fluorescence, a higher green fluorescence could be observed in ray cells and vessels cell walls as compared to fibers. This observation is consistent with studies reporting that extractives accumulate in rays and impregnate cell walls (Watanabe et al, 2004; Koch and Kleist, 2001; Mishra et al, 2018). Image analysis allowed to quantify the emission fluorescence, thus enabling us to compare samples (Table 6).

Fluorescence quantification in stump samples was performed at a common exposure time and similar magnification. The results indicate stronger fluorescence at 488nm whatever the stump tissues. For both excitation wavelength, the highest fluorescence intensity was obtained for original heartwood which contain more lignin than heartwood in Disc 1 and almost similar content of phenolic extractives. Lignin would mainly contribute to the blue fluorescence with maximum excitation at UV wavelength but can still have weak fluorescence at 488 nm excitation and some phenolic extractives also display autofluorescence under 355 nm excitation (Donaldson, 2000). Thus, stump wood autofluorescence can include both lignin and extractives (Donaldson, 2019). Nevertheless, the lowest autofluorescence of bark at both excitation wavelengths is consistent with the lowest content in lignin and extractives. Further investigation combining confocal spectral characterization and chemical extraction of extractives would give a more detailed information on the distribution of lignin and extractives in stump tissues as reported for Eucalyptus stemwood (Speranza et al., 2009).

Table 6

Values of the fluorescence emission determined using the images acquired at 63x magnification. Mean ± STDEV of three to four images. (mean grey value ± STDEV)
63x magnification; 353 nm excitation
Exposition time (ms)	O. Heartwood	Heartwood	Sapwood	Bark
1000	15.4 ± 0.6	14.2 ± 1.4	12.8 ± 0.4	7.8 ± 0.9
2000	31.4 ± 1.2	28.9 ± 2.9	26.3 ± 0.8	16.0 ± 1.8
3000	43.7 ± 2.1	41.3 ± 3.2	39.4 ± 1.3	24.2 ± 2.8
63x magnification; 488 nm excitation
500	19.5 ± 4.4	8.6 ± 1.9	10.4 ± 0.6	12.4 ± 2.1
1000	39.7 ± 8.8	17.8 ± 3.6	21.5 ± 1.3	25.1 ± 3.8
1500	56.7 ± 9.5	27.2 ± 5.4	32.9 ± 2.0	37.6 ± 5.2

Eucalypt stumps, a by-product of eucalypt wood exploitation valued primarily for pulp production, represent a significant opportunity for biomass valorization. In this study, we comprehensively characterized the stump tissues, including original heartwood (from the first tree), heartwood, sapwood, and bark, to uncover their potential applications in biorefineries. Our findings revealed chemical differences among these tissues. Specifically, heartwood exhibited high levels of extractives and holocellulose while containing less lignin compared to sapwood. Conversely, bark was found to have increased ash content alongside higher holocellulose levels. Moreover, the ethanolic and water extracts derived from these wood tissues demonstrated promising antioxidant activities, indicating potential for utilization in health-related applications. Upon analyzing the lignin composition, we observed a predominance of S-units over G-units, with a minor presence of H-units across all sampled tissues. Notably, the bark exhibited a higher degree of condensed lignin compared to the wood components. Histochemical analyses further elucidated that fibers and vessels were highly lignified, in contrast to ray cells, which displayed lower lignin content. Our research also showed that fibers contained both S- and G-lignin types, while vessels and rays primarily exhibited G-lignin. In the bark, sclereids manifested an SG lignin type whereas rays were predominantly composed of G-lignin. Morphologically, sapwood had fewer but larger vessels, whereas heartwood displayed a greater number of smaller vessels. These combined results not only enhance our understanding of Eucalyptus globulus stump anatomy and chemistry but also propose valuable avenues for their industrial application. The insights gained from this comprehensive characterization pave the way for the effective integration of these various stump regions into biorefinery processes, ultimately contributing to sustainable biomass valorization and waste reduction.

Acknowledgments

The authors thank Edwige Audibert, David Crônier, and François Gaudard for their support during the macroscopy, thioacidolysis, and FTIR analyses respectively.

Funding: This work was supported by FCT (Fundação para a Ciência e Tecnologia, Portugal) by financing the Forest Research Center (UIDB/00239/2020 and UIDP/00239/2021), Ana Lourenço through a research contract (https://doi.org/10.54499/DL57/2016/CP1382/CT0007), and Ricardo Costa by a doctoral fellowship with reference 2020.07451.BD. Ana Lourenço thanks for her STSM attributed by the COST Action CA17128 (https://lignocost.eu/), supported by COST (European Cooperation in Science and Technology). Open access funding provided by FCT/FCCN (b-on).

CRediT authorship contribution statement

AL: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Writing - original draft. JG: Conceptualization; Formal analysis; Funding acquisition; Investigation; Writing - review & editing. BC: Investigation; Supervision; Formal analysis; Writing - review & editing. RAC: Formal analysis; Writing - original draft. AH: Formal analysis; Investigation; Writing - review & editing.

Declaration of Competing Interest

The authors have no relevant financial or non-financial interests to Disclose.

Data Availability

Data will be available on request.

Angnes G, Almeida BO, Milan M, Romanelli TL. 2021. Energy and economic performances of stump and roots removal of eucalyptus for bioenergy. Biomass Bioenerg. 153, 106229. DOI: https://doi.org/10.1016/j.biombioe.2021.106229
Arisandi R, Marsoem SN, Sutapa JPG, Lukmandaru G. 2023. Heartwood formation and its relationship with basic density and green moisture content of young small-leaf mahogany trees. South. For.: J. For. Sci. 85(1), 26-39. https://doi.org/10.2989/20702620.2023.2180453
Audibert E, Lebas B, Spriet C, Habrant A, Chabbert B, Paës G. 2023. Automated quantification of fluorescence and morphological changes in pretreated wood cells by fluorescence macroscopy. Plant Methods 19, 16. https://doi.org/10.1186/s13007-023-00991-6
Auxenfans T, Crônier D, Chabbert B, Paës G, Guo C, Zhou L, Lv J. 2017. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. Biotechnol. Biofuels 10, 36. DOI: https://doi.org/10.1186/s13068-017-0718-z
Bajwa DS, Pourhashem G, Ullah AH, Bajwa SG. 2019. A concise review of current lignin production, applications, products and their environmental impact. Ind. Crop. Prod. 139, 111526. https://doi.org/10.1016/j.indcrop.2019.111526
Browning BL. 1967. In: John Wiley & Sons (Ed.), Methods of wood chemistry, vol. 2. Institute of paper chemistry. Interscience Publishers, New York, pp. 415–427.
Carrillo I, Valenzuela S, Elissetche JP. 2017. Comparative evaluation of Eucalyptus globulus and E. nitens wood and fibre quality. IAWA J 38(1), 105-116. DOI: https://doi.org/10.1163/22941932-20170160
Çetinkol ÖP, Smith-Moritz AM, Cheng G, Lao J, Geroge A, Hong K, Henry R, Simmons BA, Heazlewood JL, Holmes BM. 2012. Structural and chemical characterization of hardwood from tree species with applications as bioenergy feedstocks. Plos One 7(12), e52820. https://doi.org/10.1371/journal.pone.0052820
Clifford MN. 1974. Specificity of acidic phloroglucinol reagents. J. Chromatogr. 94, 321–324.
Costa RA, Lourenço A, Patrício H, Quilhó T, Gominho J. 2023. Valorization of pine nut residues on a biorefinery context. Waste Biomass Valori. 14, 4081-4099. DOI: https://doi.org/10.1007/s12649-023-02068-w
Day A, Ruel K, Neutelings G, Crônier D, David H, Hawkins S, Chabbert B. 2005. Lignification in the flax stem: evidence for an unusual lignin in bast fibers. Planta 222, 234-245. DOI: https://doi.org/10.1007/s00425-005-1537-1
del Rio JC, Gutierrez A, Hernando M, Landín P, Romero J, Martinez AT. 2005. Determining the influence of eucalypt lignin composition in paper pulp yield using Py-GC/MS. J. Anal. Appl. Pyrolysis. 74, 110-115. DOI: https://doi.org/10.1016/j.jaap.2004.10.010
Donaldson L. 2020. Autofluorescence in Plants. Molecules 25, 2393. DOI: https://doi.org/10.3390/molecules25102393
Donaldson LA. 2001. Lignification and lignin topochemistry — an ultrastructural view. Phytochem. 57(6), 859-873. DOI: https://doi.org/10.1016/S0031-9422(01)00049-8
Donaldson L, Singh A, Raymond L, Hill S, Schmitt U. 2019. Extractive distribution in Pseudotsuga menziesii: effects on cell wall. AWA J. 40, 721-740. DOI: https://doi.org/10.1163/22941932-40190248
Faix O. 1991. Classifications of lignins from different botanical origin by FT-IR spectroscopy. Holzforschung 45, 21–27. https://doi.org/10.1515/hfsg.1991.45.s1.21
Faix O, Fortman I, Bremer J, Meier D. 1990. Thermal degradation products of wood. A collection of electron-impact (EI) mass spectra of monomeric lignin derived products. Holz als Roh-und Werkstoff 48, 351–354. https://doi.org/10.1007/BF02639897
Faix O, Fortman I, Bremer J, Meier D. 1991. Thermal degradation products of wood. A collection of electron-impact (EI) mass spectra of polysaccharide derived products. Holz als Roh-und Werkstoff 49, 299–304. https://doi.org/10.1007/BF02613278
Faustino H, Gil N, Baptista C, Duarte AP. 2010. Antioxidant activity of lignin phenolic compounds extracted from kraft and sulfite black liquors. Molecules 15, 9308-9322. DOI: https://doi.org/10.3390/molecules15129308
Fergus BJ, Goring DAI. 1970. The distribution of lignin in birch wood as determined by ultraviolet microscopy. Holzforschung 24(4), 118–124. DOI: https://doi.org/10.1515/hfsg.1970.24.4.118
Ferreira JP, Miranda I, Gominho J, Pereira H. 2015. Selective fractioning of Pseudotsuga menziesii bark and chemical characterization in view of an integrated valorization. Ind. Crop. Prod. 74, 998-1007. DOI: https://doi.org/10.1016/j.indcrop.2015.05.065
Gaur R, Agrawal R, Kumar RR, Ramu E, Bansal VR, Gupta RP, Kumar RR, Tuli DK, Das B. 2015. Evaluation of recalcitrant features impacting enzymatic saccharification of diverse agricultural residues treated by steam explosion and dilute acid. RSC Adv. 5, 60754–60762. DOI: https://doi.org/10.1039/C5RA12475A
Gominho J, Costa R, Lourenço A, Neiva DM, Pereira H. 2019. The effect of different pre-treatments to improve delignification of eucalypt stumps in a biorefinery context. Biores. Techno.l Report 6, 89-95. DOI: https://doi.org/10.1016/j.biteb.2019.02.004
Gominho J, Costa RA, Lourenço A, Quilhó T, Pereira H. 2021. Eucalyptus globulus stumps bark: chemical and anatomical characterization under a valorization prespectives. Waste Biomass Valorization 12, 1253-1265. DOI: https://doi.org/10.1007/s12649-020-01098-y
Gominho J, Figueira J, Rodrigues JC, Pereira H. 2001. Within-tree variation of heartwood, extractives and wood density in the eucalypt hybrid urograndis (Eucalyptus grandis x E. urophylla). Wood Fiber Science 33(1): 3-8. DOI: https://wfs.swst.org/index.php/wfs/article/view/1393
Gominho J, Lopes C, Lourenço A, Simões R, Pereira H. 2014. Eucalyptus globulus stumpwood as a raw material for pulping. BioResources 9 (3), 4038-4049. DOI: https://doi.org/10.15376/biores.9.3.4038-4049
Gominho J, Lourenço A, Marques AV, Pereira H. 2020. Na extensive study on the chemical diversity of lipophilic extractives from Eucalyptus globulus wood. Phytochemistry 180, 112520. https://doi.org/10.1016/j.phytochem.2020.112520
Gominho J, Lourenço A, Miranda I, Pereira H. 2012. Chemical and fuel properties of stumps biomass from Eucalyptus globulus plantations. Ind. Crop. Prod. 39, 12-16. DOI: https://doi.org/10.1016/j.indcrop.2012.01.026
Gutiérrez A, del Río JC, González-Vila FJ, Romero J. 1998. Variation in the composition of wood extractives from Eucalyptus globulus during seasoning. J. Wood Chem. Technol. 18(4), 439-446. DOI: https://doi.org/10.1080/02773819809349591
Javier-Astete R, Jimenez-Davalos J, Zolla G. 2021. Determination of hemicellulose, cellulose, holocellulose and lignin content using FTIR in Calycophyllum spruceanum (Benth.) K. Schum. and Guazuma crinita Lam. Plos One 16(10): e0256559. DOI: https://doi.org/10.1371/journal.pone.0256559
Koch G, Kleist G. 2001. Application of scanning UV microspectrophotometry to localise lignins and extractives in plant cell walls. Holzforschung 55, 563-567. DOI: https://doi.org/10.1515/HF.2001.091
Lapierre C, Monties B, Rolando C. 1986. Thioacidolysis of poplar lignins: identification of monomeric syringyl products and characterization of guaiacyl-syringyl lignin fractions. Holzforschung 40(2), 113-118. DOI: https://doi.org/10.1515/hfsg.1986.40.2.113
Lapierre C. 1993. Application of new methods for the investigation of lignin structure in forage cell wall structure and digestibility. Chaper 6, Forage Cell Wall Structure and Digestability. Editor(s): Jung HG, Buxton DR, Hatfield RD, Ralph J. ACSESS Publications. pp133-166. https://doi.org/10.2134/1993.foragecellwall.c6
Lima JT. 1995. The wood density of 3 Eucalyptus saligna Smith clones in relation to age. Ann. Sci. For. 52, 347-352. DOI: https://doi.org/10.1051/forest:19950404
Longui EL, Romeiro D, Pfleger P, Lima IL, Silva FG, Garcia JN, Bortoletto G, Freire Neto AOL, Florsheim SMB. 2014. Radial variation of anatomical features, physicomechanical properties and chemical constituents and their potential influence on the wood quality of 45-year-old Eucalyptus propinqua. Aust. Forestry 77 (2), 78–85. DOI: https://doi.org/10.1080/00049158.2014.905739
Lourenço A, Gominho J, Marques AV, Pereira H. 2013. Variation of lignin monomeric composition during kraft delignification of Eucalyptus globulus heartwood and sapwood. J. Wood Chem. Technol. 33, 1-18. DOI: https://doi.org/10.1080/02773813.2012.703284
Lourenço A, Gominho J, Pereira H. 2019. Chemical characterization of lignocellulosic materials by ana-lytical pyrolysis. Chapter 2 of the Book “Analytical pyrolysis”. Peter Kusch (Editor). Publisher: InTechOpen, pp 9-30. ISBN 978-1-78984-958-5. DOI: 10.5772/intechopen.80556
Lourenço A, Kukić D, Vasić V, Costa RA, Antov M, Šćiban M, Gominho J. 2022. Valorisation of lignocellulosic wastes, the case study of eucalypt stumps lignin as bioadsorbent for the removal of Cr(VI). Molecules 27(19), 6246. https://doi.org/10.3390/molecules27196246
Lourenço ACS. 2009. The influence of Eucalyptus globulus heartwood in pulp production. Ms.D. Engenharia Florestal e dos Recursos Naturais. Instituto Superior de Agronomia (ISA), Universidade Técnica de Lisboa (UTL).
Lourenço A, Rencoret J, Chemetova C, Gominho J, Gutiérrez A, del Río JC, Pereira H. 2016. Lignin composition and structure differs between xylem, phloem and phellem in Quercus suber L. Front. Plant Sci. 7, 1612. DOI: https://doi.org/10.3389/fpls.2016.01612
Luís A, Neiva D, Pereira H, Gominho J, Domingues F, Duarte AP. 2014. Stumps of Eucalyptus globulus as a source of antioxidant and antimicrobial polyphenols. Molecules 19:16428 16446. DOI: https://doi.org/10.3390/molecules191016428
Meshitsuka G, Nakano J. 1979. Studies on the mechanism of lignin color reaction (XIII): Mäule color reaction (9). Mokuzai Gakkaishi 25: 588–594.
Miranda I, Gominho J, Mirra I, Pereira H. 2013. Fractioning and chemical characterization of bars of Betula pendula and Eucalyptus globulus. Ind. Crop. Prod. 41, 299-305. DOI: https://doi.org/10.1016/j.indcrop.2012.04.024
Miranda I, Lima L, Quilhó T, Knapic S, Pereira H. 2016. The bark of Eucalyptus sideroxylon as a source of phenolic extracts with anti-oxidant properties. Ind. Crop. Prod. 82, 81-87. DOI: https://doi.org/10.1016/j.indcrop.2015.12.003
Mishra G, Collings DA, Altaner CM. 2018. Cell organelles and fluorescence of parenchyma cells in Eucalyptus bosistoana sapwood and heartwood investigated by microscopy. N.Z. j. For. Sci. 48, 13. DOI: https://doi.org/10.1186/s40490-018-0118-6
Morais C, Pereira H. 2012. Variation of extractives content in heartwood and sapwood of Eucalyptus globulus trees. Wood Sci. Technol. 46 (4): 709-719. DOI: https://doi.org/10.1007/s00226-011-0438-7
Nelson ML, O'Connor RT. 1964. Relation of certain infrared bands to cellulose crystallinity and crystal latticed type. Part I. Spectra of lattice types I, II, III and of amorphous cellulose. J. Appl. Polym. Sci. 8, 1311–1324. DOI: https://doi.org/10.1002/app.1964.070080322
Pereira H. 2013. Variability of the chemical composition of cork. Bioresources 8, 2246–2256. DOI: https://doi.org/10.15376/biores.8.2.2246-2256
Pfautsch S, Harbusch M, Wesolowski A, Smith R, Macfarlane C, Tjoelker MG, Reich PB, Adams MA. 2016. Climate determines vascular traits in the ecologically diverse genus Eucalyptus. Ecol. Lett. 19(3), 240-248. DOI: https://doi.org/10.1111/ele.12559
Pinto F, Gominho J, André RN, Gonçalves D, Miranda M, Varela F, Neves D, Santos J, Lourenço A, Pereira H. 2017. Improvement of gasification performance of Eucalyptus globulus stumps with torrefaction and densification pre-treatments. Fuel 206, 289-299. DOI: https://doi.org/10.1016/j.fuel.2017.06.008
Popescu CM, Singurel G, Popescu MC, Vasile C, Argyropoulos DS, Willfor S. 2009. Vibrational spectroscopy and X-ray diffraction methods to establish the differences between hardwood and softwood. Carbohydr. Polym. 77, 851–857. DOI: https://doi.org/10.1016/j.carbpol.2009.03.011
Ralph J, Hatfield RD. 1991. Pyrolysis-GC-MS characterization of forage materials. J. Agric. Food Chem. 39, 1426–1437. DOI: https://doi.org/10.1021/jf00008a014
Ren S, Wang Z, Yan L, Feng Q, Chen Z, Zhao R. 2023. Comparison of anatomical characteristics and chemical compositions between sapwood and heartwood of Michelia macclurei. Ind. Crop. Prod. 193, 116190 DOI: https://doi.org/10.1016/j.indcrop.2022.116190
Rencoret J, Gutierrez A, Nieto L, Jimenez-Barbero J, Faulds CB, Kim H, Ralph J, Martinez AT, del Rio JC. 2011. Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiol. 155(2), 667–682. DOI: https://doi.org/10.1104/pp.110.167254
Rodrigues J, Faix O, Pereira H. 1998. Determination of lignin content of Eucalyptus globulus wood using FTIR spectroscopy. Holzforschung 52, 46-50. DOI: https://doi.org/10.1515/hfsg.1998.52.1.46
Rowell RM, Pattersen R, Han JS, Rowell S, Tshabalala MA. 2012. Cell wall chemistry. In Handbook of wood chemistry and wood composites. Rowell RM (ed). CRC press.
Rubio-Valle JF, Martín-Alfonso JE, Eugenio ME, Ibarra I, Oliva JM, Manzanares P, Valencia C. 2024. Bioethanol lignin-rich residue from olive stones for electrospun nanostructures development and castor oil structuring. Int. J. Biol. Macromol. 255, 18042. DOI: https://doi.org/10.1016/j.ijbiomac.2023.128042
Scherer R, Godoy HT. 2009. Antioxidant activity index (AAI) by the 2, 2-diphenyl-1-picrylhydrazyl method. Food Chem. 112(3), 654-658. DOI: https://doi.org/10.1016/j.foodchem.2008.06.026
Schwanninger M, Rodrigues JC, Pereira H, Hinterstoisser B. 2004. Effects of short-time vibratory ball milling on the shape of FT-IR spectra of wood and cellulose. Vibr. Spectrosc. 36, 23-40. DOI: https://doi.org/10.1016/j.vibspec.2004.02.003
Speranza M, Gutiérrez A, del Río JC, Bettucci L, Martínez AT, Martínez MJ. 2009. Sterols and lignin in Eucalyptus globulus Labill. wood: Spatial distribution and fungal removal as revealed by microscopy and chemical analyses. Holzforschung, 63(3), 362-370. DOI: https://doi.org/10.1515/HF.2009.041
Terashima N, Fukushima K, Takabe K. 1986. Heterogeneity information of lignin. VIII: an autoradiographic study on the formation of guaiacyl and syringyl lignin in Magnolia kobus DC. Holzforschung 40, 101–105.
Watanabe Y, Kojima Y, Ona T, Asada T, Sano Y, Fukazawa K, Funada R. 2004. Histochemical study on heterogeneity of lignin in eucalyptus species ii. The distribution of lignins and polyphenols in the walls of various cell types. IAWA Journal 25, 283-295. DOI: https://doi.org/10.1163/22941932-90000366
Xiao M, Chen W, Cao X, Chen Y, Zhao B, Jian Z, Yuan T, Sun R. 2020 Unmasking the heterogeneity of carbohydrates in heartwood, sapwood, and bark of Eucalyptus. Carbohydr. Polym. 238, 116212. DOI: https://doi.org/10.1016/j.carbpol.2020.116212
Xiao M-Z, Chen W-J, Hong S, Pang B, Cao X-F, Wang Y-Y, Yuan T-Q, Sun R-C. 2019. Structural characterization of lignin in heartwood, sapwood, and bark of eucalyptus. Int. J. Biol. Macromol. 138, 519-527. DOI: https://doi.org/10.1016/j.ijbiomac.2019.07.137
Yamashita D, Kimura S, Wada M, Takabe K. 2016. Improved Mäule color reaction provides more detailed information on syringyl lignin distribution in hardwood. Journal of Wood Science 62(2), 131–137. DOI: https://doi.org/10.1007/s10086-016-1536-9
Zhang, FD., Xu, CH., Li, MY, Chen XD, Zhou Q, Huang AM. 2016. Identification of Dalbergia cochinchinensis (CITES Appendix II) from other three Dalbergia species using FT-IR and 2D correlation IR spectroscopy. Wood Sci. Technol. 50, 693–704. DOI: https://doi.org/10.1007/s00226-016-0815-3

No competing interests reported.

Supplementarymaterial.pdf

Unlocking the potential of undervalued forest residues: a comprehensive characterization of eucalypt stumps in the biorefinery context

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials and methods