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Culm cell-wall compositions of tribes bambuseae and olyreae (subfamily bambusoideae; Family poaceae) from the Brazilian Atlantic Forest
Marco Aurelio´ Tine,´ Michele Silva, Maria Tereza Grombone-Guaratini
PII: S0367-2530(20)30060-8 DOI: https://doi.org/10.1016/j.flora.2020.151596 Reference: FLORA 151596
To appear in: Flora
Received Date: 25 April 2019 Revised Date: 26 March 2020 Accepted Date: 2 April 2020
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© 2020 Published by Elsevier. 1
Culm cell-wall compositions of tribes Bambuseae and Olyreae (subfamily Bambusoideae; family Poaceae) from the Brazilian Atlantic Forest
Marco Aurélio Tiné1*, Michele Silva2 and Maria Tereza Grombone-Guaratini2*
1Núcleo de Pesquisa em Fisiologia e Bioquímica, Instituto de Botânica, São Paulo, Brasil, C.P. 68041, 04301-902, São Paulo SP, Brasil 2Núcleo de Pesquisa em Ecologia, Instituto de Botânica, São Paulo, Brasil, C.P. 68041, 04301-902, São Paulo SP, Brasil.
* Corresponding author: E-mail: [email protected]; [email protected]
Telephone number 55 11 50676164 Fax number 55 11 5073 3678
Highlights
The cell wall of the culms of six species of tropical bamboos were examined. Arabinoxylans were the main hemicellulose in the cell walls. Cell walls of the herbaceous species P. micrantha had the lowest content of lignin. The culms of tropical bamboo species could be a valuable resource for biotechnology.
Abstract:Journal Pre-proof
Brazil has the greatest diversity of bamboos in the neotropics. This biodiversity is reflected in the diversity of plant architectures, ranging from trees to herbs. As cell walls constitute the main mechanical component of plant tissues and organs, the compositions 2
of these walls may differ depending on the mechanical properties required for different plant life strategies. The present work examines the polysaccharide composition of the culm cell walls of six neotropical bamboo species from different habits and biomes. It also compares the percentage of monosaccharide compositions with other grasses studied as feedstock. The polysaccharide fractions were composed of small amounts of pectin, 1,3;1,4--glucans and the main hemicellulose was arabinoxylan, consistent with grasses in other subfamilies. Comparatively, the amount of glucose in the cell wall is higher in sugarcane, followed by bamboo and miscanthus. Different habits are not associated with different cell wall compositions. Tropical bamboo species could be a valuable resource with quite interesting possibilities for biotechnology.
Key words: bamboo, cell wall, Chusquea, Guadua, Filgueirasia, Merostachys, Pardyolyra.
1. Introduction
Brazil has the greatest bamboo biodiversity in the New World (Judziewicz et al., 1999). The Atlantic Forest, the Cerrado and the Amazon encompass 89% of the genera and 65% of all known bamboo species (36 genera and 254 species) (Filgueiras et al., 2016). The true bamboo group (subfamily Bambusoideae of the Poaceae) includes two tribes: Bambuseae woody (lignified) and Olyreae herbaceous (or bambusoid grasses) (Filgueiras and Gonçalves, 2004). The first group, the tribe Bambuseae, is represented by a large number of bamboo genera seen as components of shrub and arboreal strata found in forests and Cerrado, among which the following are the predominant genera: Aulonemia Goudot, Merostachys Spreng., Guadua Kunth, Chusquea Kunth and Apoclada McClure (Judziewicz et al., 1999; BPG, 2012). The second group, the Olyreae tribe, encompasses bamboos that are important components of the herbaceous stratum of neotropical forests (Filgueiras and Gonçalves, 2004; Oliveira and Longui-Wagner, 2005). JournalIn tropical and subtropical areas, bamboos Pre-proof represent approximately 20 – 25% of the total biomass. This contributes to their status as one of the most important renewable resources, as they offer an alternative to traditional wood products (Banzal and Zoolagud, 2002; Nguyen et al., 2010). In spite of this, information about their geographic distribution, evolutionary history and taxonomy are scarce (Triplett et al., 2014). Considered a rapid atmospheric carbon sink, bamboos (Grombone-Guaratini et al., 2013) 3
also have physical and mechanical properties that make them suitable for use in the development of products normally produced with native wood or wood from reforestation; these include construction components, furniture, handles for agricultural tools, panels and dishes, among others (Dwivedi et al., 2019). In addition, the ever- increasing interest in replacing fossil fuel with sustainable sources, has led to research showing that some plants, such as the grasses bamboos, miscanthus, and switchgrass, could be considered promising feedstock for biomass energy due to their fast growth (He et al., 2014; Zhang et al. 2013; Yu et al., 2016). The total bamboo culm comprises about 50% parenchyma, 40% fibers, and 10% conducting tissue (Sánchez-Echiverri et al., 2014). The main constituents of bamboo culms are cellulose (more than 50%), hemicelluloses (mainly heteroxylans) and lignin (Liese and Weiner, 1996). This is consistent with the cell wall compositions of other grasses (Family Poaceae) (Carpita and Gibeaut, 1993). In the cell wall of growing tissues in the bamboo Phyllostachys edulis the following hemicelluloses were identified: arabinoxylan, 1,3;1,4--glucans (mixed-linked-glucans) and some xyloglucan (Edashige and Ishii, 1998). Heteroxylans in the form of glucuronoarabinoxylans (xylose 43.9– 91.0%; arabinose 4.9–19.3%; glucuronic acid 2.4–11.4%) were also found in the hemicellulosic fractions of the cell walls of the hemicellulosic fractions of Sinocalamus affinis and Phyllostachys aurea (Guan et al., 2015; Zelaya et al., 2017). In Phyllostachys edulis some of the hemicelluloses may be feruloylated or p-coumaroylated (Ishii et al., 1990; Ishii, 1991). Evidence for this was obtained by treating cell walls with a fungal enzyme preparation containing a mixture of endo- and exo- glycanases, which released two feruloyl-arabinoxylan oligosaccharides (Ishii and Hiroi, 1990), a diferuloyl- arabinoxylan hexosaccharide (Ishii, 1991), a p-coumaroyl-arabinoxylan trisaccharide (Ishii et al, 1990, Ishii and Hiroi, 1990) and a feruloyl-xyloglucan disaccharide (Ishii and Hiroi, 1990). Some studies have shown that the composition of chemical constituents varies according to the species, age, phenophase, part of the culm and growing conditions of the plant (LieseJournal and Weiner, 1996; Scurlock et al.,Pre-proof 2000). Additionally, studies have shown a progressive increase in lignification of fiber walls during growth (Itoh, 1990) and maturation (Murphy et al., 1997; Lin et al., 2002; Li et al. 2007). Differences in lignin content were also observed between species from temperate and tropical climates (Lybeer and Koch, 2005 a and b). 4
Based on the multifunctional structure of cell walls and their importance to the mechanical properties and anatomy of plants (Taiz, 1984; Wolf et al., 2012), it is possible to relate their composition to the evolutionary distance between plant species (Silva et al., 2011). Studies about the chemical and structural composition of shoots or culms of woody bamboo are limited to Asian bamboos; data on neotropical species are scarce (Lybeer and Koch 2005b). Different plant anatomies entail issues with different biomechanical properties (Rowe and Speck, 2005). Given this and the fact that plant cell walls are one of the main components contributing to the mechanical properties of the organs, it is expected that this would be reflected in cell wall compositions. Species like lianas and herbs are expected to have less demand for support tissues and, therefore, should have less rigid tissues than woody (tree) bamboos. This study aims, firstly, to compare the monosaccharide compositions of culm cell walls among five neotropical bamboo species belonging to three different subtribes (Arthrostylidiinae, Chusqueinae and Guadinae) pertaining to the tribe Bambuseae were examined and one species belonging to the subtribe Olyrinae (tribe Olyreae) with different habits. From the Bambusae tribe, species with shrub, tree and climbing habits were examined. From the Olyreae tribe, a species with an herb habit was studied. Secondly, the study aimed to compare the monosaccharide compositions of the fractions from the cell walls of those species studied to those of other bamboos and major grasses. We believe that further study can provide a better understanding of neotropical bamboo cell walls which in turn could reveal the species with the best potential for biotechnology.
2. Material and methods
2.1. Plant material
Whole plants were collected from different populations in São Paulo and Goiás. The species collected, their habitats and sampling sites are described in Table 1. To avoid tissues Journalunder expansion or in different stages Pre-proof of development, the middle third of mature culms were collected as the standard sample from each species. The collected plant material was then identified and taxonomically confirmed by Dr. Tarciso Filgueiras and Regina Tomoko Shirasuna MSc., both from the Instituto de Botânica de São Paulo (São Paulo Botanical Institute) (São Paulo, SP, Brazil).Voucher 5
specimens were deposited at the Maria Eneyda Kauffmann P. Fidalgo and UFG Herbarium at the Botanical Institute and Federal University of Goiania (Table 1). Individuals collected, in triplicate, in the field, were brought to the laboratory, fragmented (to accelerate drying), dried at 60°C in a forced air oven for three days and grounded to a powder in a knife mill (Willye type TE 650, Tecnal, Piracicaba).
2.2. Cell wall fractionation The freeze-dried samples (each 200 mg) were extracted three times with 80% ethanol at 80°C for 20 minutes to remove soluble sugars. Dimethyl-sulfoxide (DMSO) 90% aqueous (20 mL) was added to the residue, and the suspension was stirred at 60 °C for 24 hours to remove starch. All chemical analyses were performed with three biological and three technical replicates. The residue was washed with deionized water twice and freeze dried. The freeze-dried material (alcohol-insoluble residue AIR) was extracted twice in 20 mL of aqueous 0.5% ammonium oxalate (pH 7.0) at 80°C for 3 hour each.
The soluble fractions were pooled, dialyzed and freeze-dried, reaching a state of pectin (Gorshkova et al., 1996). To extract lignin from the residue, 30 mL of an aqueous solution containing 3% sodium chlorite (w/v) and 0.3% acetic acid (v/v) were added to the pellet and extraction was performed as described by Carpita (1984) and summarized by Peng et al. (2000). The remaining cell-wall material was extracted sequentially with 0.1, 1.0 and
4 M NaOH solutions (containing 3mg/mL NaBH4). At each stage, the material was pelleted via centrifugation at 2500g for 15 minutes and washed twice with distilled water. Insoluble material was neutralized with glacial acetic acid, washed twice with water, freeze dried and weighed. Crystalline cellulose content was determined gravimetrically after digestion of non-cellulosic material in acetic-nitric acid at 100°C for 1 hour in a boiling water bath (Updegraff, 1969). The cellulose was then washed three times with distilled water, freeze dried and weighed.
2.3. Lignin determination Journal“Klason” lignin content was determined Pre-proof according to Hatfield et al. (1994), with some modifications. Samples (50 mg residue from ethanolic extraction) were incubated with 72% sulfuric acid for 45min at 30°C to hydrolyze and solubilize the carbohydrates. After incubation, the samples were diluted to 4% with distilled water and autoclaved for 1h at 121°C (Carrier et al., 2011). The hydrolysate was centrifuged at 2000g, the 6
supernatant discarded, and the residue washed with hot water five times. The material was dried and weighed for yield calculation.
2.4. Monosaccharide composition and polysaccharide digestion with xylanase To determine the monosaccharide composition, two milligrams of each fraction were hydrolysed with 2M trifluoroacetic acid for 1 hour at 120oC. After hydrolysis, the content was dried under vacuum and solubilized in deionized water. The proportion of monosaccharides was determined by anion exchange chromatography ICS 3000 Dionex (Thermo Scientific) using a PA1 Carbopak column with isocratic elution (16 mM NaOH, 0.9 mL. min-1) (Tiné et al., 2006). The monosaccharides were determined by comparison with commercial standards. The arabinoxylan was profiled via the digestion of the 4 M NaOH fraction with 1,4-β-xylanase from Aspergillus niger (Megazyme) (Souza et al., 2013). For the digestion, 5 mg of polysaccharides were dissolved in 500 µL of Na acetate buffer (50 mM) and 10 µL of xylanase (diluted to 0.85 mg.mL-1) and the system was incubated for 24 hours at 30oC. The oligosaccharides were analyzed by ion exchange chromatography ICS 3000 Dionex (Thermo Scientific) using a PA1 Carbopak column with isocratic elution (88 mM NaOH at 0.9 mL. min-1). The main oligosaccharides were identified by comparing their retention times with a sugar cane standard (Souza et al., 2013). The oligosaccharides responsible for most of the differences, however, were the less abundant ones that could not be identified by ion chromatography. Because of that, thirty peaks in the chromatograms, corresponding to unidentified oligosaccharides in different samples, were aligned according to their retention time and the Chromatogram Integration Table was corrected for a total area of 1000. The area of the thirty peaks was used to build a similarity tree in the Statistica software (Statsoft) using Manhattan distance to reduce the effect of extreme values (Lo et al., 2007).
3. ResultsJournal and Discussion Pre-proof 3.1. Fractionation yield The yield of the oxalate fractions (Table 2) was very low, as expected for type II cell walls which are poor in pectin (Carpita and Gibeaut, 1993). Uronic acids were quantified in these samples, but only trace amounts were measured (data not shown). The sodium chlorite fraction should be a delignification step, but the low yield compared to 7
the lignin content (Table 3), suggests that not all the lignin was extracted from the bamboos using the usual fractionation methodology. In general, the amount of lignin in Asian bamboos is about 25–30% compared with the 11–27% reported for other species of Poaceae (Bagby et al., 1971), which more closely resembles the ranges reported for softwoods (24–37%) and hardwoods (17–30%) (Wen et al., 2015). Our results showed that the tropical bamboo studied (tribes Olyreae and Bambuseae), independent of habit (tree, shrub, liana), had lignin values similar to those reported for other Poaceae, and lower than that reported for Asian bamboos (Bagby et al., 1971). According to Lybeer and Kock, (2005a) bamboos from tropical areas have less lignin than those reported for other Poaceae. They state that it is lower than those reported for Asian bamboos because they grow in a more stable climate than the latter. In tropical bamboos the lignin content could possibly be associated with other functions like UV protection (Souza et al., 2013) or herbivory defense (Fukushima and Savioli, 2001), as shown in other species. In our study, the significantly lower content of lignin presented in P. micrantha (Table 3), would provide an association between tropical climate and an herbaceous habit without stages of wall hardening in the life cycle. The most abundant fraction was the 4M NaOH fraction, which extracted most of the wall hemicelluloses (Table 2). The residue consisted almost entirely of crystalline cellulose (more than 95%), except in C. capituliflora, were the hydrolysis rate was 60%. But even then, the content of the residue was more than 97% glucose, indicating almost pure cellulose (Table 4). This result suggests a difference in the crystallinity of cellulose in C. capituliflora, since the method used hydrolyzes all the amorphous material (Harris et al., 2010).
3.2. Cell-wall monosaccharide compositions and oligosaccharide profiling The neutral monosaccharide composition of the oxalate fraction (Table 4) shows a significant amount of glucose, particularly in Guadua tagoara, Merostachys riedeliana and Parodiolyra micrantha, with contents as high as 56% glucose. In these species, the presenceJournal of mixed-linked glucan (MLG) sePre-proofems to be more prominent, despite their completely different architecture. In the other three species, the MLG is less abundant, being replaced by arabinoxylan, as these other three species have the highest amounts of xylose and arabinose. This separation into two groups was not maintained in the 0.1M NaOH fraction. Apparently, this pattern exists only for loosely bound polysaccharides, like pectin. Rhamnose and galactose are very conspicuous in the oxalate fraction, 8
probably as pectins, such as rhamnogalacturonan 1, despite there being only trace amounts of uronic acids (data not shown). The main monosaccharide in the 0.1M NaOH fraction was xylose in all species, emphasizing the low quantity of pectins in type II cell walls. The amount of glucose was lower in the 0.1M and 1M NaOH fractions but higher in the 4M NaOH fraction, indicating the presence of two distinct glucose-rich polysaccharides: one loosely bound to the wall (MLG) and the other tightly bound (probably xyloglucan). The presence of xyloglucan in the 4M NaOH fraction would be consistent with the higher amount of galactose. In sugarcane cell walls, xylan was also reported as the main hemicellulose but approximately 3% xyloglucan was identified in the 4M NaOH fraction (Crivellari, 2012). Studies on other bamboo species have also found xylose, glucose and arabinose to be the main neutral monosaccharides and arabinoxylan is the main hemicellulose (Fengel and Shao, 1984; Wen et al., 2011). The residue consisted mostly of cellulose, except in the case of Chusquea capituliflora, where the residues contained about 12% non-cellulosic polymers. The small amount of hemicellulose in the residue indicates that, despite the low yield of the chlorite fraction, when compared to the lignin content, the extraction of the hemicelluloses was not hindered by the limited extraction of lignin. In Guadua tagoara, almost half of the AIR is preserved in the residue. In this case, the yield of the 4M NaOH fraction is the lowest among all the species. The xyl/ara ratio shows a gradual increase along the fractions. The ratio in the initial fractions could be lower either due to: (1) the presence of polymers rich in arabinose other than arabinoxylan or (2) due to highly ramificated xylans in the fraction that are loosely bound to the wall and can be removed under low stringency conditions. In the main arabinoxylan fractions (1M and 4M NaOH), the ratio of P. micrantha shows unusual behavior with the highest ratio in the 1M NaOH fraction. The extraction of this arabinoxylan under this condition suggests that this polymer is not tightly bound to other polymers as expected for such a low degree of branching. This could be due to the lower levels ofJournal lignification in the tissue. Pre-proof Comparison between the bamboo culm and other grasses studied as feedstock (Table 5) showed that bamboo has a higher content of arabinose and rhamnose in the initial fractions, suggesting a larger amount of pectins, despite the low detection of uronic acids. Comparison of the xylose and glucose content shows clearly that the arabinoxylan is replaced with xyloglucan in some species. In the 1M and 4M NaOH fractions, the 9
amounts of glucose and xylose are inversely proportional. Souza et al. (2013) detected a considerable amount of glucose-containing polysaccharides, such as xylogucan and mixed-linked glucan. When the walls are compared, sugarcane has the highest amount of glucose, followed by bamboo and miscanthus. From a bioethanol point of view, hydrolysates rich in hexoses are easier to ferment, which points to bamboo’s real biotechnological potential. It is important to consider that the data shown in Table 5 is the average of six species. Further studies of bamboo walls should reveal the species with the best potential for biotechnology. Since the monosaccharide analyses indicate that the main hemicellulose in all the species analysed is an arabinoxylan, the presence of xylan was analysed for by digesting the fractions with a pure 1,4-β-xylanase and analyzing the products by ion chromatography (Figure 1). The main oligosaccharides released were xylobiose and xylotriose, confirming the presence of xylan. Very small additional peaks were also found but the structures of the compounds resulting in these peaks are unknown. To our knowledge, this is the first study on the structure of bamboo xylan structure. For this reason, there were no commercial standards or references available to be used for oligosaccharides identification. A sugarcane standard was used to identify the xylobiose and xylotriose (not shown), but some minor components, which were specific to bamboo, were not identified. The main oligosaccharides were the same for all bamboo species but there were small differences in the proportions and in the less abundant fragments of the polymer’s structure. Different from the previous analysis where it was not possible to determine the precise origin of the monosaccharides, the specificity of the enzyme assures that only arabinoxylan was considered in this analysis. Of the thirty oligosaccharides used, seven were common to all the species, but most of them had their occurrence restricted to a few species (see Data in Brief). The integration Table of oligosaccharide profiles was used to build a similarity tree (Figure 2) that allowed comparison of the profiles despite the lack of ability to identify some of the oligosaccharides. Three main groups could be identified: Chusquea capituliflora/Guadua tagoaraJournal, Merostachys riedeliana/Parodiolyra Pre-proof micrantha and the Filgueirasia species. The two species of Filgueirasia grouped together, but the other member of their subtribe (Merostachys) was more similar to Parodiolyra. Guadua and Chusquea were grouped together, although the subtribe Guaduinae is phylogenetically closer to Arthrostylidinae than to Chusqueinae. When the structure of the hemicellulose is considered, therefore, there is little similarity with the accepted phylogeny of bamboos (Kelchner et al., 2013). 10
The categorization of the species by habit (shrub, climbing, herb or tree) was not reflected in the groups formed in the similarity tree. Two clear examples are the high similarity of the hemicellulose from G. taguara and C. capituliflora, the first being a tree and the last having a climbing habit and the grouping of M. riedeliana (a tree) and P. micrantha (a herb). The separation into biomes did not reflect the grouping in the similarity tree either. The analysis of the cell wall composition and hemicellulose structure in six neotropical bamboos did not reflect the differences in their habits or the biome where they occur.
5. Concluding remarks
Although the studied species had habits ranging from shrub to tree the difference in the cell wall composition was very subtle. The type II cell walls obtained from all the bamboo species analyzed presented very little variation, with arabinoxylan as being the main hemicellulose. Of note are the results showing, that in comparison to other major grasses, tropical bamboo species could be a valuable resource, with potential for biotechnology.
Credit Author Statement
Maria Tereza Grombone-Guaratini and Marco Aurélio Tiné devised the project, the main conceptual ideas and proof outline. Michele Silva, Marco Aurélio Tiné and Maria Tereza Grombone-Guaratini worked out almost all of the technical details, and performed the numerical calculations for the suggested experiment. Maria Tereza Grombone-Guaratini and Marco Aurélio Tiné wrote the manuscript.
Conflict of Interest Statement
The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent- licensingJournal arrangements), or non-financial interestPre-proof (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. *Maria Tereza Grombone-Guaratini - Núcleo de Pesquisa em Ecologia, Instituto de Botânica, São Paulo, Brasil, C.P. 68041, 04301-902, São Paulo SP, Brasil.
11
*Marco Aurélio Tiné - Núcleo de Pesquisa em Fisiologia e Bioquímica, Instituto de Botânica, São Paulo, Brasil, C.P. 68041, 04301-902, São Paulo SP, Brasil
*Michele Silva - Núcleo de Pesquisa em Ecologia, Instituto de Botânica, São Paulo, Brasil, C.P. 68041, 04301-902, São Paulo SP, Brasil.
Acknowledgments
This research was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant N. 2011/51099-1) awarded to M. T. Grombone-Guaratini. The authors are grateful to the two anonymous reviewers whose contributions improved the quality of this manuscript considerably.
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Figure 1. Arabinoxylan oligosaccharides produced by the digestion with xylanase of the 4M NaOH fractions extracted from cell walls from Chusquea capituliflora, Filgueirasia arenicola, Filgueirasia cannavieira, Guadua tagoara, Merostachys riedeliana and Parodiolyra micrantha. X=xylose, X2=xylobiose; X3=xylotriose.
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Figure 2. Similarity diagram built from the oligosaccharide profile obtained from the digestion of the 4M NAOH fractions with β-xylanase from Aspergillus niger. Species: C – ChusqueaJournal capituliflora, FA – Filgueirasia arenicolaPre-proof, FC – Filgueirasia cannavieira, G – Guadua tagoara, M – Merostachys riedeliana, P – Parodiolyra micrantha.
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Table 1. Species collected, their habitats and sampling sites. Tribe Subtribe Species Habit Biome site
Bambuseae Arthrostylidiinae Filgueirasia arenicola Shrub Savanna Parque Nacional das Emas, GO (McClure) G.F. Guala (18°6’23’’S; 52°55’40’’W)
Bambuseae Arthrostylidiinae F. cannavieira (McClure) Shrub Savanna Reserva Quilombola no município de G.F. Guala Cavalcante, GO (13°41’968”S; 47°27,979”W)
Bambuseae Chusqueinae Chusquea capituliflora Trin. Climbing Tropical Forest Parque Estadual Fontes do Ipiranga var. pubescens McClure & (PEFI), SP (23°39’25,40”S; L.B. 46°37’41,95”W).
Bambuseae Guaduinae Guadua tagoara (Ness) Tree Tropical Forest Guarulhos, SP (23°23'28,8"S; Kunth 46°25'24,4"W)
Bambuseae Arthrostylidiinae Merostachys riedeliana Tree Tropical Forest Parque Estadual Fontes do Ipiranga Rupr. ex Döll (PEFI), SP (23°39’25,40”S; 46°37’41,95”W). Olyreae Olyrinae Parodiolyra micrantha Herb Tropical Forest/Savanna Parque Estadual Fontes do Ipiranga (Kunth) Davidse & Zuloaga (PEFI), SP (23°39’25,40”S; 46°37’41,95”W).
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Table 2. Yield of the cell wall fractions from different species (in percentage of the AIR). The values in parenthesis represent the cellulose content of the residue. Average of 3 biological samples and their standard deviation.
Oxalate Chlorite NaOH 0.1M NaOH 1M NaOH 4M Residue C. capituliflora 0.8 ± 0.2a 7.0 ± 0.5ab 6.6 ± 1.2ac 9.3 ± 1.0ac 44.9 ± 1.7a 31.5 ± 0.7a(19.1a) F. arenicola 1.0 ± 0.01ab 7.8 ± 0.8ab 6.9 ± 0.3ac 10.2 ± 0.2ac 42.6 ± 0.6ac 31.6 ± 0.1a (29.7ab) b b a ac ac a ab Yield F. cannavieira 1.3 ± 0.2 7.8 ± 0.8 5.3 ± 0.3 10.8 ± 0.3 43.5 ± 1.4 31.2 ± 0.4 (30.8 ) (%) G. tagoara 0.6 ± 0.2a 8.3 ± 0.2b 6.2 ± 0.4b 6.4 ± 0.03b 29.0 ± 1.1b 49.5 ± 0.6a (48.9b) M. riedeliana 1.3 ± 0.1b 5.2 ± 0.4a 6.6 ± 0.9ac 7.0 ± 0.3a 45.7 ± 1.6a 34.6 ± 2.5a (33.2ab) P. micrantha 0.7 ± 0.1a 9.5 ± 0.5b 10.2 ± 0.03ac 8.6 ± 0.2c 37.8 ± 0.9c 33.2 ± 0.2a (32.0ab)
*Different letters represent samples with significant differences between the species for each fraction. ANOVA< 0.05.
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Table 3. Lignin content (%) in the alcohol insoluble residue of 6 bamboo species.
Chusquea capituliflora 19.0 ± 0.1a Filgueirasia arenicola 19.5 ± 0.3a Filgueirasia cannavieira 19.0 ± 0.4a Lignin (%) Guadua tagoara 19.2 ± 0.3a Merostachys riedeliana 19.2 ± 0.1a Parodiolyra micrantha 15.5 ± 0.3b
*Different letters represent samples with significant differences between the species for each fraction. ANOVA< 0.05.
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Table 4. Mole percentages of neutral monosaccharides in hydrolysates of cell wall fractions from the six native bamboo species analyzed by PHAEC-PAD. Average of three biological samples and their standard deviation.
PROPORTION %
Fucose Rhamnose Arabinose Galactose Glucose Xylose Xyl/Ara ratio
C. capituliflora 2.0 ± 0.8 10.2 ± 1.8a 17.5 ± 0.7ª 19.5 ± 2.9a 21.1 ± 1.5ª 29.7 ± 6.7ab 1.7 F. arenicola 0.6 ± 0.1 5.1 ± 0.4bc 19.5 ± 0.3ª 15.4 ± 1.1ab 14.9 ± 0.4ª 44.3 ± 1.6a 2.3 F. cannavieira 1.3 ± 0.4 7.2 ± 0.5ab 17.2 ± 0.9ª 11.2 ± 0.6ab 22.2 ± 3.9ª 40.9 ± 3.8ab 2.4 Oxalate G. tagoara 0.8 ± 0.2 6.2 ± 0.3abc 12.7 ± 2.0ª 9.5 ± 1.5ab 44.2 ± 12.4ª 26.6 ± 8.9ab 2.1 M. riedeliana 0.5 ± 0.2 1.4 ± 0.3c 10.4 ± 2.9ª 7.9 ± 1.9ab 52.8 ± 14.5ª 26.9 ± 9.3ab 2.6 P. micrantha 1.1 ± 0.3 6.5 ± 1.6ab 10.5 ± 2.9ª 13.9 ± 3.7b 56.4 ± 11.5ª 11.6 ± 3.2b 1.1 C. capituliflora 0.5 ± 0.1a 1.5 ± 0.4bb 17.1 ± 2.1 5.7 ± 0.5ab 9.2 ± 6.1ab 66.1 ± 4.3ab 3.9 F. arenicola 0.5 ± 0.1a 0.7 ± 0.04a 19.5 ± 0.9 6.5 ± 0.7ab 2.4 ± 0.1ab 70.5 ± 1.7ab 3.6
ab ab ab ab ab NaOH F. cannavieira 0.7 ± 0.03 0.8 ± 0.1 20.8 ± 0.9 5.5 ± 0.4 2.5 ± 0.1 69.8 ± 1.5 3.4 0.1M G. tagoara 0.8 ± 0.03ab 0.7 ± 0.04ba 14.2 ± 0.9 4.8 ± 0.3a 3.7 ± 0.2a 75.8 ± 0.5 a 5.3 M. riedeliana 3.9 ± 1.5b 2.8 ± 0.8b 17.9 ± 1.8 7.4 ± 0.8b 7.9 ± 3.1b 59.9 ± 4.6 b 3.4 P. micrantha 0.7 ± 0.02ab 0.8 ± 0.06ab 17.5 ± 0.4 4.9 ± 0.02a 2.2 ± 0.2ab 73.9 ± 0.7ab 4.2 C. capituliflora 1.6 ± 0.3 1.4 ± 0.5 11.3 ± 1.4a 2.9 ± 0.6ab 4.5 ± 0.9ª 78.2 ± 2.7ª 6.9 F. arenicola 1.9 ± 0.1 2.2 ± 0.1 10.6 ± 0.6a 5.7 ± 0.6b 3.9 ± 0.6ª 78.7 ± 0.7ª 7.5
F. cannavieira 2.8 ± 0.01 2.4 ± 0.3 10.5 ± 0.3a 1.8 ± 0.1a 4.5 ± 0.9ª 78.6 ± 1.4ª 7.5 NaOH 1M G. tagoara 2.1 ± 0.1 2.3 ± 0.2 10.8 ± 0.2a 1.7 ± 0.1a 3.8 ± 0.2ª 79.4 ± 0.5ª 7.4
M. riedeliana 1.9 ± 0.01 2.3 ± 0.1 10.8 ± 0.4a 2.5 ± 0.6b 3.37 ± 0.8ª 79.2 ± 0.6ª 7.3
P. micrantha 1.2 ± 0.2 1.5 ± 0.6 7.6 ± 0.2b 4.0 ± 0.3a 4.3 ± 0.5ª 81.3 ± 0.9ª 10.6
Journal Pre-proof a C. capituliflora 0.6 ± 0.1 1.0 ± 0.1 8.9 ± 1.2 4.5 ± 0.4 25.4 ± 1.2 59.5 ± 2.6 6.7 NaOH 4M F.arenicola 0.5 ± 0.02 1.2 ± 0.2 7.8 ± 0.5 5.6 ± 0.3 24.3 ± 2.2ª 60.6 ± 2.7 7.8 24
F. cannavieira 0.7 ± 0.1 1.9 ± 0.4 8.6 ± 1.4 4.8 ± 0.6 22.6 ± 1.6ª 61.5 ± 1.6 7.2 G. tagoara 0.6 ± 0.02 1.4 ± 0.1 11.1 ± 1.4 5.6 ± 0.2 18.2 ± 3.8ª 63.2 ± 5.0 5.7 M. riedeliana 0.7 ± 0.1 1.4 ± 0.2 9.4 ± 0.6 5.9 ± 0.4 15.7 ± 0.7b 67.0 ± 1.8 7.1
P. micrantha 0.9 ± 0.1 1.3 ± 0.1ª 10.6 ± 0.5 5.8 ± 0.5 24.1 ± 2.9ª 57.4 ± 1.8 5.4
C. capituliflora 0.1 ± 0.01a 0.06 ± 0.01a 0.6 ± 0.01ª 0.1 ± 0.01a 97.8 ± 0.1 1.3 ± 0.1a 2.3
F. arenicola 0.06 ± 0.01a 0.03 ± 0.0b 0.3 ± 0.05ª 0.06 ± 0.01b 98.8 ± 0.2 0.8 ± 0.1b 2.6
F. cannavieira 0.05 ± 0.0a 0.03 ± 0.0b 0.2 ± 0.05ª 0.05 ± 0.0b 98.9 ± 0.1 0.8 ± 0.1b 3.3 Residue G. tagoara 0.05 ± 0.0a 0.03 ± 0.0b 0.2 ± 0.05ª 0.05 ± 0.0b 98.9 ± 0.1 0.8 ± 0.1b 3.2
M. riedeliana 0.05 ± 0.0a 0.03 ± 0.0b 0.2 ± 0.03ª 0.05 ± 0.0b 98.8 ± 0.1 0.9 ± 0.1b 4.4
P. micrantha 0.05 ± 0.0a 0.03 ± 0.0b 0.2 ± 0.02ª 0.05 ± 0.0b 98.8 ± 0.1 0.8 ± 0.1b 4.1
*Different letters represent samples with significant differences between the species for each fraction. ANOVA p < 0.1.
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Table 5. Comparison between bamboo and the culm cell wall of other grasses studied as feedstock Percentage of monosaccharide composition in the fractions. Bamboo Sugar cane Miscanthus* Miscanthus sinensis Oxalate Fuc 1.0 0.2 0.9 Rham 6.1 n.d. 0.1 Ara 14.6 8.8 8.9 Gal 12.9 14.8 3.0 Glc 35.3 61.9 29.9 Xyl 30.0 8.2 60.4 NaOH 0.1M Fuc 1.2 0.1 0.1 Rham 1.2 0.1 0.1 Ara 17.8 13.8 13.0 Gal 5.8 0.7 1.3 Glc 4.6 10.5 10.7 Xyl 69.3 68.7 84.3 NaOH 1M Fuc 1.8 n.d. 0.1 Rham 2.0 n.d. 0.1 Ara 10.3 7.3 7.4 Gal 2.6 0.7 0.4 Glc 4.1 14.3 0.9 Xyl 79.2 73.7 91.0 NaOH 4M Fuc 0.6 n.d. trace Rham 1.4 n.d. trace Ara 9.4 2.0 6.4 Gal 5.4 1.2 0.8 Glc 21.7 63.6 4.1 Xyl 61.5 26.3 88.3 References Average of the Souza et al. 2013. Souza et al. 2015. species from this manuscript. * Average of three genotypes calculated from data in the reference. n.d. = not detected. n.p= extraction not performed in this methodology Journal Pre-proof 26
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