Chemical-Anatomical Characterization of Stems of Asparagaceae Species with Potential Use for Lignocellulosic Fibers and Biofuels

: During the last decades, the possibility of using species resistant to droughts and extreme temperatures has been analyzed for use in the production of lignocellulosic materials and biofuels. Succulent species are considered to identify their potential use; however, little is known about Asparagaceae species. Therefore, this work aimed to characterize chemically-anatomically the stems of Asparagaceae species. Stems of 10 representative species of Asparagaceae were collected


Introduction
Asparagaceae is one of the most important families in Mexico due to its biological, economic, and cultural importance [1]. Several species are used in the production of fibers, intoxicating drinks, food preparation, and the consumption of plant parts (flower, stem, and leaf) [2]. Asparagaceae has several subfamilies, including Agavoideae with Agave, Furcraea, Manfreda, and Polianthes; Yuccoideae including Yucca, Hesperaloae, and Hesperoyucca [3], and the subfamily Nolinoideae that includes the genera Beaucarnea and Nolina [4]. Most of the species of Agavoideae and Nolinoideae are distributed in arid and semiarid, and warm temperate regions of North and Central America [3] and in other parts of the world naturally or introduced. Several species of Agave have been used and studied the most because ethanol is produced in the form of intoxicating beverages such as mezcal and tequila [5].
In recent years, mainly the fibers and bagasse waste of several agave species, mainly A. tequilana [6,7], A. angustifolia [8], and A. salmiana [9] have been studied because a large amount of waste is produced annually from the production of tequila and mezcal. In addition, the subfamilies Agavoideae and Nolinoideae present acid metabolism of the crassulacean (CAM), which is considered raw material for the production of biofuel [10,11]. Furthermore, these species are part of the second generation of plants focused on biofuels. They are not part of the plants essential for human consumption [12], and they tolerate drought conditions and high temperatures [13]. The samples were analyzed in triplicate based on the TAPPI T-222 om-02 standard and based on the method proposed by Maceda et al. [20,21]. From each ground sample, 2 g were taken, which were placed in filter paper cartridges to carry out successive extractions for six hours in a Soxhlet with ethanol-benzene (1:2 v/v) and subsequently in ethanol (96%). After each extraction, the cartridges were allowed to dry for 24 h at 70 • C to record their constant weight.
Subsequently, the cartridges were discarded, and the samples were kept in a reflux system for 1 h in water at 90 • C. The samples were filtered through a medium pore Büchner filter and dried at 70 • C for 24 h to record constant dry weight. The formula used was the following: Total extractives (%) = [(A + B + C)/W 0 ] × 100 where A is the weight lost (g) after extraction with ethanol:benzene, B is the weight lost (g) after extraction with ethanol (96%), C is the weight lost (g) after extraction with water at Forests 2022, 13, 1853 3 of 18 90 • C, and W 0 is the initial weight of each sample. The percentage of extractive-free lignocellulose was obtained by subtracting from 100% the initial weight of the total percentage of extractives.

Lignocellulosic Purification
Klason lignin. From the extractive-free lignocellulose of each species, 0.2 g were taken, and 15 mL of concentrated sulfuric acid (72%) was added at a temperature of 2 • C. The mixture was kept under constant stirring and at room temperature (18 • C) for 2 h. Then, 560 mL of distilled water was added, and the mixture was refluxed and boiled for 4 h. The samples were filtered through a fine-pore Büchner filter and dried at 105 • C for 24 h to record constant dry weight. Lignin was quantified as follows: where W L is the obtained weight of lignin (g), and W W is the extractives-free lignocellulose (g).
Cellulose. From the extractive-free lignocellulose, 0.2 g were taken to purify the cellulose using the Kûshner-Höffer method [21]. Twenty-five mL of HNO 3 /ethanol (1:4 v/v) were added to each sample and kept in a reflux system, and boiled for one hour. The sample was allowed to decant to discard the HNO 3 /ethanol solution, and another 25 mL was added again. This cycle was repeated three more times, and in the last cycle, 25 mL of an aqueous solution of KOH at 1% was added and kept for 30 min at reflux and boiling to finally filter the sample through a fine-pore Büchner filter. The sample was left to dry at 70 • C for 12 h to record the constant dry weight and obtain the percentage of cellulose based on the following formula: where W C is the obtained weight of cellulose(g), and /W W is the extractives-free lignocellulose (g).
Hemicellulose. The purification was carried out based on the methodology proposed by Li. et al. [22]. From the extractive-free lignocellulose, 0.5 g were taken and placed in a reflux system with 10 mL of water for 3 h (solid-to-liquid ratio 1:20 g/mL). The system was cooled to room temperature and filtered. The filtrate was concentrated at 1.25 mL and purified into 3.75 mL of ethanol (95%) with stirring. The mixture was held for 1 h without stirring, and the hemicellulose precipitated. In order to obtain the dry weight (H 0 ), the sample was centrifuged at 4500× g for 4 min, and then lyophilized. The residue insoluble in water was dried at 60 • C for 16 h, then successive extractions were performed with different concentrations of KOH (0.6, 1.0, 1.5, 2.0, and 2.5%) in a ratio of 1:20 (g/mL) at 75 • C for 3 h in each extraction. In the last concentration of 2.5% KOH, ethanol (99.7%) was added in a ratio of 2:3. The five mixtures were filtered and acidified to pH 5.5 with glacial acetic acid and concentrated to 1.25 mL. The mixtures were poured into 3.75 mL of ethanol (95%) with constant stirring. The mixtures were kept for 1 h and finally were centrifuged (4500× g for 4 min) and lyophilized. The constant dry weight was recorded in each extraction stage (H 0.6 , H 1.0 , H 1.5 , H 2.0 , H 2.5 ), and the percentage of cellulose was obtained with the following formula: where W H is the sum of H 0.6 to H 2.5 and W W is the extractive-free lignocellulose (g).

Fourier Transform Infrared Spectroscopy Analysis
Lignin analysis. The ratio of syringyl/guaiacyl monomers (S/G) was obtained by Fourier transform infrared spectroscopy (FTIR) analysis. Klason lignin samples were kept dry until analyzed by FTIR (30 scans with a resolution of 4 cm −1 , 15 s per repeat). Three FTIR readings (Agilent Cary 630 FTIR) were made from each sample, and then the baseline correction was performed to separate the peaks of the fingerprints (wavelength of 800-1800 cm −1 ) [23] in the MicroLab PC program (Agilent Technologies). The peaks of 1269 to 1272 cm −1 and 1328 to 1330 cm −1 were used to quantify the proportion of guaiacyl (G) and syringyl (S), respectively [24]. The value of each peak was obtained by drawing a line connecting the lowest values and a similar line for the highest values of each peak. A vertical line was drawn from the base of the X−axis to the highest part of the peak. The portion of the line between the top and the base is the value of each peak, so the S/G ratio was calculated by dividing the values of each peak [24].
Cellulose analysis. The proportion of crystalline cellulose was obtained by analyzing the dried samples with FTIR [21]. From each sample and doing the analyzes in triplicate, a small portion of the sample was placed in the FTIR Spectrometer (Agilent Cary 630 FTIR), and the spectrum was obtained in a range of 400-650 cm −1 (30 scans with a resolution of 4 cm −1 , 15 s per repeat). Samples were converted from transmittance to absorbance, and spectra were averaged using the Resolution Pro FTIR Software program (Agilent Technologies, Santa Clara, CA, United States).
The crystallinity indices used were: Total crystallinity index (TCI) proposed by Nelson and O'Connor [25] or also called the crystallinity ratio [26,27]. The lateral order index (LOI) [25,27] or second proportion of crystallinity [26]; and hydrogen bonding intensity (HBI) [28]. TCI was calculated with the ratio between the absorption intensity of the peaks 1370 cm −1 and 2900 cm −1 [27], LOI was calculated from the ratio between the absorption intensity of the peaks 1430 cm −1 and 893 cm −1 [26], while HBI was calculated with the ratio between 3350 cm −1 and 1315 cm −1 [28].

Statistical Analysis
The data obtained from the percentages of extractives and lignocellulosic components were analyzed with the non-parametric Kruskal-Wallis test and Dunn's post hoc analysis since the values did not present normality based on the results of Kolmogorov-Smirnov and Shapiro−Wilk, even when they were transformed with the square root of the arc sine. In addition, a multivariate principal component analysis was performed to separate the groups based on the values of the structural components.

Lignocellulosic Anatomical Distribution
Stem fragments were saved from each sample and were fixed, embedded, and cut based on the procedures of Arias and Terrazas [29] for succulent hardwood species. The transverse sections were stained with acridine orange and calcofluor [30] to observe the distribution of cellulose and lignin in the stem, in addition to comparing the anatomical results with the chemical ones.

Extractives and Lignocellulosic Structural Compounds
The Asparagaceae species had significant differences between species (Table 2) in the variables of extractives and lignocellulosic components. The percentages of extractives were heterogeneous between the species of the same genus; however, the differences occurred mainly between Agave and Yucca-Nolina. In Tables 3 and 4, the means and standard deviation of the extractives are presented, and the different superscript capital letters show the species that are significantly different. In the ethanol extractives, A. striata had the lowest percentage and was significantly different from Y. gigantea, which had the highest percentage (Table 3). In hot water extractives, a similar situation was shown; A. convallis had the highest percentage, while N. excelsa and Y. periculosa had the lowest percentage. In the ethanol:benzene extractives, the statistical differences were presented between N. excelsa, with the lowest percentage, and F. longaeva, with the highest percentage. The species with the highest content of extractives and the lowest content of lignified tissue were A. convallis and Y. gigantea. On the contrary, the species with the lowest content of extractives and the highest amount of lignified tissue were N. excelsa and Y. periculosa.
Different letters in each column indicate significant differences (p < 0.05). Mean ± standard deviation (SD). The percentages of lignocellulosic components had significant differences in the percentages of lignin between Y. filifera and F. longaeva (Table 4). In cellulose, the species that had the lowest percentage were A. celsii and A. convallis, and N. excelsa was the species with the highest percentage. Finally, in the hemicelluloses, Y. gigantea presented the least quantity and Y. filifera the largest.
In the Principal Component (PC) analysis, the first two PCs had eigenvalues above 1, while PC3 was less than 1; however, PC3 was considered in the analysis due that it explained 11% of the variance, so the three PCs explained 84.5% of the total variation (Table 5). In each PC, the highest negative or positive values were those that influenced the separation of each species, as shown in Figure 1. For PC1, the variables that determined the separation of the different groups were hot water extractives and extractive-free lignocellulose. In PC2, the determinant variables of the variation were hemicelluloses and ethanol extractives, while in PC3 was the cellulose percentage (Table 5). separation of each species, as shown in Figure 1. For PC1, the variables that determined the separation of the different groups were hot water extractives and extractive−free lignocellulose. In PC2, the determinant variables of the variation were hemicelluloses and ethanol extractives, while in PC3 was the cellulose percentage (Table 5).   When plotting the species based on the first three PCs and the variables that influenced each PC (Figure 1), the species were separated into four groups. The first group (blue points) included the species of N. excelsa and Y. periculosa, which were the species with the lowest content of hot water extractives and higher extractive−free lignocellulose content. The second group (green point), represented by Y. filifera had the highest percentage of hemicelluloses. The third group (pink point), conformed by Y. gigantea was separated from the other Yuccas and the other species because they had lower percentages of hemicelluloses, but it was one of the species with the highest percentage of cellulose and percentage of total extractives. In the fourth group (orange points), the remaining species that belong to Agave, F. longaeva, and B. gracilis were clustered (Figure 1).

Cellulose Crystallinity
In the cellulose spectra ( Figure 2) and Table 6, the main cellulose peaks are observed. In order to determine the purity of the cellulose, the absence of xylans and hemicellulose was obtained by not detecting the peak at 1735 cm −1 . Lignin was not detected with the peaks 1595 cm −1 , 1512 cm −1 , and 1463 cm −1 . Only weak lignin signals were observed in A. striata and Y. periculosa. In addition, the absence of hemicellulose was observed without the presence of the 1269 cm −1 peak, except for A. celsii, which had a weak peak.
percentage of total extractives. In the fourth group (orange points), the remaining species that belong to Agave, F. longaeva, and B. gracilis were clustered (Figure 1).

Cellulose Crystallinity
In the cellulose spectra ( Figure 2) and Table 6, the main cellulose peaks are observed. In order to determine the purity of the cellulose, the absence of xylans and hemicellulose was obtained by not detecting the peak at 1735 cm −1 . Lignin was not detected with the peaks 1595 cm −1 , 1512 cm −1 , and 1463 cm −1 . Only weak lignin signals were observed in A. striata and Y. periculosa. In addition, the absence of hemicellulose was observed without the presence of the 1269 cm −1 peak, except for A. celsii, which had a weak peak.  In the crystallinity indexes (Table 7), the TCI values showed that most of the species had values above one because they had a higher percentage of crystalline cellulose, except A. striata, B. gracilis, and Y. periculosa, which presented a higher percentage of amorphous  In the crystallinity indexes (Table 7), the TCI values showed that most of the species had values above one because they had a higher percentage of crystalline cellulose, except A. striata, B. gracilis, and Y. periculosa, which presented a higher percentage of amorphous cellulose. In LOI, the species that had the highest value was A. striata, while the species that had the lowest value was F. longaeva, which had the highest value in TCI. In HBI, similarly, A. striata had the lowest value, while B. gracilis had the highest value.  Figure 3 shows the FTIR spectra for the 10 species of Asparagaceae. Representative peaks of lignin were 1501 cm −1 which showed the C=C aromatic ring vibration of syringyl and guaiacyl monomers. The 1325 cm −1 peak reflected the breathing of the ring of syringyl in addition to C-O stretching. The 1271 cm −1 and 1225 cm −1 peaks reflected the symmetric vibration of C-O and the glucopyranose cycle of guaiacyl and syringyl, respectively. The 1030 cm −1 peak reflected the C-H in-plane deformation of guaiacyl and C-O deformation in primary alcohol; finally, the 913 cm −1 peaks showed the =CH out-of-plane deformation in the aromatic ring of syringyl and guaiacyl monomers (Figure 3).

Yucca periculosa
0.78 ± 0.05 0.59 ± 0.02 1.03 ± 0.05 Mean ± standard deviation (SD). Figure 3 shows the FTIR spectra for the 10 species of Asparagaceae. Representative peaks of lignin were 1501 cm −1 which showed the C=C aromatic ring vibration of syringyl and guaiacyl monomers. The 1325 cm −1 peak reflected the breathing of the ring of syringyl in addition to C−O stretching. The 1271 cm −1 and 1225 cm −1 peaks reflected the symmetric vibration of C−O and the glucopyranose cycle of guaiacyl and syringyl, respectively. The 1030 cm −1 peak reflected the C−H in−plane deformation of guaiacyl and C−O deformation in primary alcohol; finally, the 913 cm −1 peaks showed the =CH out−of−plane deformation in the aromatic ring of syringyl and guaiacyl monomers (Figure 3).

Lignin S/G Ratio
Whit the peaks 1325 and 1271 cm −1 the S/G ratio was calculated ( Table 8). The species with the lowest proportion of S/G was A. attenuata because it had a higher percentage of guaiacyl in its structure, while in the genus Yucca, the three species presented high values of syringyl, for which the proportion of S/G was in the range of 2.8 to 3.9. The other species had similar proportions, so the syringyl monomer prevailed except in A. convallis.   Whit the peaks 1325 and 1271 cm −1 the S/G ratio was calculated ( Table 8). The species with the lowest proportion of S/G was A. attenuata because it had a higher percentage of guaiacyl in its structure, while in the genus Yucca, the three species presented high values of syringyl, for which the proportion of S/G was in the range of 2.8 to 3.9. The other species had similar proportions, so the syringyl monomer prevailed except in A. convallis. Table 8. Percentages of syringyl and guaiacyl, and S/G ratio of Asparagaceae species.

Anatomical Distribution
The species of Asparagaceae studied showed the same type of vascular tissue, with closed vascular bundles forming isolated patches or two or more patches joined through lignified parenchyma. In the vascular bundle, the presence of tracheary elements with mainly reticular type of secondary wall thickenings was observed, with completely lignified fibers and non-lignified phloem with contents inside with fluorescence emission in bluish  (Figure 4). In most species, patches of non-lignified parenchyma were observed on the edges of the stem, while in the center, the parenchyma was completely lignified. In the species A. attenuata and A. convallis, the fluorescence tones of the tracheary elements and fibers predominated in yellow-green tones, while in the Yucca species, the fluorescence emission was observed in green to bluish-green tones. In all species, the presence of crystals was observed, mainly raphides and prisms. Cellulose fluoresced in bluish tones but differed from lignin due to the intensity of fluorescence emission since the cellulose had lower intensity compared to lignin.

Discussion
The Asparagaceae family presented heterogeneity in the percentages of the structural components that allow its grouping based on the values of extractive compounds, hemi-

Discussion
The Asparagaceae family presented heterogeneity in the percentages of the structural components that allow its grouping based on the values of extractive compounds, hemicellulose, and cellulose. In addition, the presence of high percentages of cellulose, a majority proportion of crystalline cellulose, and the presence of S/G ratios greater than one make it possible to use in biofuels and cellulosic compounds.

Extractives and Lignocellulose
Asparagaceae species had similar percentages of extractives when extracted with ethanol: benzene and ethanol. However, hot water extractives were low except for A. convallis (Table 3). Compared with the literature on other Agave species (Table 9), the extractive percentages in water were similar to the values reported here. A. tequilana, A. angustifolia, and A. salmiana have values between 4.4%-6.0% (Table 9). On the contrary, the values reported in Agave for the percentages of extractives in ethanol: benzene and ethanol (Table 9) were low (1.5%-4.0% in ethanol: benzene and 1.3%-5.0% in ethanol) compared to the values reported here (Table 3). For the genera Beaucarnea, Furcraea, and Nolina, there are no reports of extractive percentages in their stems or leaves. In the Yucca genus, the literature report that Y. gloriosa presents a percentage of total extractives of 1.1 [31], which is low compared to the percentages obtained here (Table 3).
Although extractives in ethanol: benzene and in ethanol are hardly reported in the literature for the species of the genera analyzed here, they have been reported for other succulent species such as cacti [20,21], which had lower percentages of ethanol: benzene (2.5%-4.2%), similar in ethanol (1.0%-9.1%), higher in hot water (8.2%-44.5%) and total extractives (16.7-49.2 %).
In the percentages of lignocellulosic compounds, Table 4 shows that there was heterogeneity in the results within the same genus. In the species that are considered arborescent, it was obtained that the percentages of lignin were low. However, the percentage of cellulose was higher (37.3%-52.2%) and in herbaceous species was lower (31.6%-37.1%). In the hemicelluloses, there is homogeneity between the agaves (20.9%-27.1%), while in the Yucca genus, notable differences are observed between Y. gigantea (5.7%) concerning the other two species of Yucca (21.0% and 30.7%). The differences possibly were due to the type of environment in which they live; Y. gigantea is distributed in regions with higher humidity [32], while Y. filifera [33,34] and Y. periculosa [35] grow in arid zones and semi-desert, respectively. In celluloses, the percentages reported in the literature (Table 9) were high compared to those obtained here (Table 4), possibly due to the differences in the species or the conditions in which they have been developed [36,37].
When comparing the results obtained with succulent species such as cacti, it was observed that the percentages of lignocellulose, in general, are higher in agave species than in non-fibrous wood cacti species [20], while in fibrous species, the percentages were similar [38].    The differences in the extractive and lignocellulosic compounds between species allowed us to identify four groups through the principal component analysis. The first group with N. excelsa and Y. periculosa presented a lower amount of hot water and total extractives. This could have implications for their use because various authors consider the hot water and total extractives to represent the nonstructural sugars that are used in fermentation processes for the production of ethanol [8,48,51]. However, in the case of N. excelsa, the percentage of cellulose presented in the stem would allow it to be used to obtain cellulosic products ( Table 4). The characteristic that distinguishes Y. filifera from the other species studied was the presence of a large amount of hemicellulose. This hemicellulose can be transformed into usable sugars either through enzymatic hydrolysis processes or with temperature [9], in addition to the fact that Y. filifera presented lower percentages of lignin so that the purification of cellulose would be more efficient [64]. The main characteristic of Y. gigantea was the lower amount of hemicelluloses; however, it had a high percentage of cellulose that could be used to obtain cellulosic fibers. The three species of Yucca studied here revealed the higher differences in the extractives and lignocellulosic compounds between them, and other species of Yucca should be studied in the future to support these findings. All agave species plus Beaucarnea and Furcraea were grouped together by similar values in the extractive compounds (Table 4). In the lignocellulosic components, the agaves had low percentages of cellulose and lignin; however, they had a higher percentage of hemicelluloses. Finally, chromatographic analyzes can be carried out on all the extractives compounds to identify components with potential use, such as flavonoids and triterpenes, which have the antioxidant capacity and can be used mainly in the food and cosmetic industries [65]. In addition, lignocellulose can be treated in different ways to obtain various derivatives, as mentioned by Palomo-Briones et al. [66] for Agave tequilana: through the elimination of lignin by alkaline, organosolv or enzymatic methods, lignin can be solubilized and fermented to obtain ethanolic derivatives. Hemicelluloses and celluloses can be degraded by acids or enzymes to obtain insoluble fractions and use microcrystalline and nanocrystalline cellulose, while the solubilized carbohydrates can be used in the fermentation to obtain ethanol. Solubilized carbohydrates can also be fermented by anaerobic digestion or dark fermentation to obtain biogas and H 2 which can be used to obtain energy [66]. Therefore, the species analyzed in this work could be used in different ways, both energetically and in the production of paper or cellulose components [21].

Cellulose Crystallinity
The crystallinity indexes allowed identifying the species that had the highest proportion of crystalline cellulose. The purity of the cellulose could affect these indexes by presenting hemicellulose or lignin residues that alter the peaks [67]. The FTIR spectra can determine the purity of the samples and calculate the proportion of crystalline cellulose [68,69] by the absence of peaks belonging to hemicelluloses [70] and lignin [71].
The most commonly used indexes in conjunction with FTIR are TCI, LOI, and HBI [68,72]. The TCI index provides information about the amount of crystalline or amorphous cellulose in a sample. The peak indicating the presence of crystalline cellulose is 1370 cm −1 , while the peak of 2900 cm −1 [27] indicates the presence of amorphous cellulose. Therefore, if the value of the ratio is greater than one, there will be a greater amount of crystalline cellulose [28]. The LOI index is related to the order of crystalline cellulose, in addition to the fact that the 1430 cm −1 peak shows the presence of crystalline cellulose of type I and the peak 893 cm −1 the presence of type II crystalline cellulose and cellulose amorphous [73]. The order of the crystalline cellulose and LOI peaks can be altered by the type of chemical extraction, and the type of purification used [74]. The HBI index reflects the crystallinity of the sample and its water absorption. Low values reflect a greater amount of crystalline cellulose, while high values indicate the presence of cellulose II or amorphous [27], however, TCI and HBI values are related in terms of cellulose structure and stability, so if they are similar, the cellulose structure has greater stability [28].
Generally, the samples had a higher proportion of crystalline cellulose; however, the type of extraction modifies the order of the cellulose, so the low LOI values reflect the presence of type II crystalline cellulose [75], in addition to possibly the presence of NaOH during purification and the temperature used would alter the order of the crystalline [73]. Furthermore, LOI is correlated with the overall degree of cellulose order, while TCI is directly proportional to the percentage of crystalline cellulose [28]. The similar values of HBI with TCI confirm that the highest proportion of crystalline cellulose predominates among the Asparagaceae species, except the species A. striata, B. gracilis, and Y. periculosa, which had lower crystalline cellulose, and this is reflected in the LOI values that were also the highest [76].
The presence of a greater amount of crystalline cellulose (between 50 and 56% so that the proportion of TCI is 1 to 1.27) in most of the Asparagaceae species agrees with that reported for stems (Table 10). Furthermore, the percentages were also similar for other structures such as fibers in Agave (50.07 [46]), Furcraea (52.6% [62]), and Yucca spp. (55-56% [77]). The other structures have a higher percentage, possibly due to the species analyzed, the type of sample, such as bagasse that no longer has extractives, and some structural sugars, such as hemicelluloses. However, in future studies, X−ray diffraction analysis [78] could be used to confirm the proportion of crystalline cellulose in samples of Asparagaceae species.

Lignin S/G Ratio
The proportion of S/G obtained for the 10 species reflected that most of the species presented high percentages of syringyl monomers, except for A. attenuata (36.9%) and A. convallis (48%). The species with the highest percentage was of the Yucca genus since it had percentages of 73.9 to 79.5%. The presence of higher percentages of syringyl in species with stem potential allows the purification of cellulose and the degradation of lignin by hydrolytic processes to be more efficient [80] by presenting more bonds of the β-O-4 type and being less condensed [81]. In species with higher percentages of guaiacyl, the stem tissue is hard, and the lignin is difficult to degrade, which is why it is called recalcitrant lignin [82].
Therefore, in the Yucca species and generally in the other species of Agave, Beaucarnea, Furcraea, and Nolina, by presenting a higher percentage of syringyl, the hydrolyzation process with Kraft would be more efficient [83]. In the literature, there are few reports on the proportion of S/G for A. fourcroydes, proportions are reported in fibers (1.05) and spines (1.2) [84], in leaves of A. sisalana (2.0) [85], and leaf fibers (3.0-3.5) [86]. In stems, S/G values have been reported for A. americana (1.27), A. angustifolia (1.29), A. fourcroydes (1.40), A. salmiana (1.33) y A. tequilana (1.57) in untreated samples [11], while for A. tequilana S/G values of 4.3 have also been reported in untreated samples [87] while in A. sisalana the proportion of S/G in stem fibers is 3.6 [88]. In the other genera analyzed here, there are no reports on the composition of lignin. In general, it is observed that the values obtained are similar to those reported by the aforementioned authors.
The presence of high percentages of syringyl in lignin has also been reported in various fibrous species where it provides resistance to and cellular support [89][90][91]. However, in succulent species such as cacti, the presence of syringyl monomers is more associated with tissues with non−lignified parenchyma and not fibers [38,92,93], so the presence of syringyl could be associated with a defense mechanism against pathogens [93] as it has been reported for bryophytes, conifers and angiosperms [94][95][96][97][98].

Cellulose and Lignin Anatomical Distribution
Analyzing the anatomical distribution of lignin and cellulose in the vascular bundles of Asparagaceae species was useful in identifying the location of the main structural components, in addition to explaining the results obtained both in the percentages of lignocellulosic components and in the proportion of S/G [49,99].
In Figure 4, the fluorescence emitted by the tracheary elements, the fibers, and the non−lignified and lignified parenchyma showed that lignin had different emissions in its fluorescence since it ranged from bluish tones to yellow tones. In the species of A. attenuata and A. convallis, the presence of yellow tones was observed, which would be related to the type of lignin present in the lignified walls. As proposed by Maceda et al. [93], based on a tone scale, yellow to green tones would reflect the presence of guaiacyl-type lignin, while syringyl−type lignin would have shades of lime-green to blue. The difference in the tones in the emission of fluorescence has already been reported for various species with similar tones [100]. The presence of blue tones in fibers in species with S/G ratios above two [93], as for Ferocactus hamatacanthus and F. pilosus, whose S/G ratio is 11.7 and 3.5, respectively [38]. Therefore, in the species of Yuccas, whose proportion ranges from 2.8 to 3.9, the presence of fibers and tissue in blue tones responds to the presence of lignin of the syringyl type.
The presence of lignified parenchyma in the stems of the Yucca species (Figure 4c-f) agrees with the lignin percentages shown in Table 4 since these species, except for B. gracilis, had high lignin values, in contrast to the species of A. convallis and A. attenuata (Figure 4a,b). Therefore, the presence of lignin is a structural support factor for species of tall size and arborescent shape. However, these species also had high percentages of cellulose (Table 4), with a predominance of crystalline cellulose (Table 7). Cellulose and lignin not only provide structural rigidity but also the proportion of S/G, and the presence of crystalline cellulose could improve water conduction in tracheary elements [101] in addition to protecting against pathogens [91,102]. The species analyzed in this study are naturally distributed in Mexico and Y. guatemalensis in Mexico and Guatemala, so it would be interesting to expand the number of genera to determine if there is heterogeneity in a greater number of species of the same genus or if they present homogeneity in the composition of cellulose and lignin.

Conclusions
The presence of high percentages of cellulose, the predominance of syringyl−type lignin, percentages above 10% of total extractive components, and the high percentages of holocellulose (structural sugars) show that Asparagaceae species have potential in the use of both productions as biofuels as in the production of paper. In addition, the chemical composition that Asparagaceae species present would be related to biological implications such as conduction, support, and protection against pathogens.