Proceedings of the International Society of Sugar Cane Technologists, volume 30, 661–664, 2019
Poster paper
Cell-wall chemical-composition analysis of a
Saccharum spp. progeny for increased bagasse
digestibility
JM Garcia1,2, A Acevedo3, MP Silva4, L Gomez4 and LE Erazzú1
1
EEA Famaillá INTA, Ruta Provincial Nº 301, km 32, 4132, Tucumán, Argentina; [Link]@[Link]
2
CONICET, Buenos Aires, Argentina
3
Instituto de Suelos, CIRN-INTA, 1712 Castelar, Buenos Aires, Argentina
4
CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, UK
Abstract Knowledge of cell-wall composition in sugarcane hybrids could be used for the selection of
cultivars with high bagasse digestibility in order to improve sugar release in 2G ethanol
production. Cell-wall components of 76 hybrids of a sugarcane cross, their parents and two high-
fibre genotypes were analyzed. The Van Soest method was used to determine cellulose,
hemicellulose and acid detergent lignin contents (ADL). Spectroscopic acetyl bromide lignin
(ABS) was also determined. A correlation analysis was performed with major cell-wall polymers
and agronomic traits. Despite the minor differences observed between parental lines’ cell-wall
composition, genotypes with high cellulose and low lignin content were identified in the progeny.
Differences among them depended on the method used for lignin measurement. The ADL mean
was 43% lower than the ABS mean, in agreement with lignin losses during acid detergent
treatment previously reported. There was no correlation between ADL and ABS assays.
Bagasse of genotypes with higher stalk weights was associated with higher cellulose content and
lower ADL content. Saccharification studies are needed to validate lignin assessment
methodologies to be used as bioenergy cultivar selection tools.
Key words Lignin, cell wall, sugarcane, bagasse
INTRODUCTION
Sugarcane (Saccharum spp.) bagasse is a relevant lignocellulosic substrate for second-generation (2G) ethanol
production (Masarin et al. 2011). Large quantities of bagasse are obtained in the sugarcane industry that could be
used for this purpose, and this shares part of the infrastructure of first-generation (1G) ethanol production (Dias et
al. 2012).
Lignocellulose is the plant biomass formed mainly by cellulose, hemicellulosic polysaccharides and lignin (Vega-
Sanchez and Ronald 2010). In cellulosic ethanol production, costly pre-treatments are required to access to
monosaccharides (Vanholme et al. 2010) and one of the main hindrances in this process is the difficulties caused
by lignin (Siqueira et al. 2011).
Saccharification efficiency of sugarcane could be enhanced by improving the biomass quality through genotypes
with more cellulose and less lignin (Hoang et al. 2016). Despite this, the variability in the cell-wall composition of
sugarcane bagasse remains unexplored, and its study could provide valuable information for screening in breeding
programs.
The objectives of our study were: a) to analyze the bagasse cell-wall composition of a sugarcane progeny and
corresponding parents, and of two promising high-fibre genotypes; b) to compare two lignin determination
methodologies; and c) to assess bagasse cell-wall composition with agronomic breeding traits.
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MATERIALS AND METHODS
A trial was conducted at the experimental field of INTA Famaillá Experimental Station (27°03’S, 65°25’W, 363
masl.), Tucumán, Argentina. A set of 76 sugarcane siblings, their parents (NA 78-724 and LCP 85-384), and two
genotypes (INTA 05-3116 and INTA 05-3118) with high-fibre content were planted in a completely randomised
design without replicates. Each plot consisted of one row of 3 m length and 1.6 m inter-row spacing.
At the first ratoon, 10 stalks/plot were manually harvested, and tops and leaves were removed. Stalk diameter,
height and weight of each sample were recorded and internodes 5 to 7 (from the bottom of the stalk) were separated
and shredded. The sugar content was washed with water and stalks were dried in an oven at 100ºC until constant
weight. Two replicates of neutral detergent fibre (NDF), acid detergent fibre (ADF) (Van Soest et al. 1991) and
acid detergent lignin (ADL) (Van Soest 1973) were determined. Cellulose content was calculated as the difference
between ADF and ALD, and hemicellulose content as the difference between NDF and ADF. Three replicates of
spectroscopic acetyl bromide lignin (ABS) were determined according to Fukushima and Hatfield (2001). Total ash
content was determined (AOAC 1990).
Brix and pol in juice (%) were obtained from the remainder of the stalk internodes and pol in cane (%), purity (%)
and sugar content (%) were calculated as described by Acreche et al. (2015).
RESULTS AND DISCUSSION
Cultivar LCP 85-384 had slightly higher mean values of cellulose (+1.7%) and ABS (+ 0.9%) contents and lower
hemicellulose (-1.2%) and ADL (-0.6%) contents than NA 78-724 (Table 1). The main component of the NDF mean
in the progeny was cellulose (51.4%), followed by hemicellulose (+35.2%) and ADL (+13.4%). Hemicellulose was
the cell-wall component with the lowest percentage of variability in the progeny with a range of 9.4%. Higher ranges
for cellulose (+19.9%), ADL (+19.7%) and ABS (+17.1%) were observed (Table 1).
Even though small differences were obtained when comparing cell-wall composition of both parents these results
demonstrate a significant variability for cell-wall components in the progeny. Genotypes with low ADL and high
cellulose contents were identified in the progeny as potential candidates for better bagasse digestibility.
High-fibre genotypes showed higher cellulose and lower hemicellulose and ADL content than the mean of parents
and progeny. Interestingly, progeny maximum values were the highest for all the fibre components.
Table 1. Chemical composition of bagasse of sugarcane parental lines, progeny (N= 76), and two high-fibre
sugarcane genotypes.
Parents Progeny High-fibre genotypes
Component NA LCP INTA 05- INTA 05-
Mean Min. Max. Range
78-724 85-384 3116 3118
Cellulose
52.0±0.8 53.7±0.1 51.4 41.6 61.5 19.9 58.2±2.0 58.3±1.7
(%NDFa)
Hemicellulose
33.0±0.3 31.8±1.1 35.2 31.0 40.4 9.4 29.0±1.2 29.6±1.3
(%NDF a)
b
ADL(%NDF ) 15.0±1.1 14.4±1.0 13.4 4.8 24.5 19.7 12.8±0.9 12.0±0.4
ABS (%DM c) 23.1±0.2 24.0±1.2 23.5 16.1 33.2 17.1 23.2±1.0 24.1±0.0
Ash (%DM) 2.1±0.0 2.0±0.1 2.4 1.8 3.1 1.3 2.7±0.0 3.0±0.2
a
Van Soest (1991), bVan Soest (1973), c Fukushima and Hatfield (2001). NDF= Neutral Detergent Fibre, ADL= Acid Detergent
Lignin, ABS= Acetyl Bromide Lignin, DM= Dry Matter.
ADL mean value of parents, progenies and high fibre genotypes was 43% lower than ABS mean. This difference
could be attributed to the acid detergent action that dissolves more than 50% of the lignin content in grasses
(Hatfield and Fukushima 2005).
Cellulose content in the progeny was positively correlated with stalk weight (r=0.23, p˂0.05) and negatively
correlated with Brix (r=-0.35, p˂0.01). Nevertheless, hemicellulose content was negatively correlated with stalk
height (r=-0.26, p˂0.05) and though low Brix (r=0.28, p˂0.01), while ADL was negatively correlated with stalk weight
(r=-0.23, p˂0.05). These results suggest that bagasse of genotypes with greater stalk weight were associated with
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a higher cellulose content and lower ADL content. Although cellulose was negatively correlated with Brix, no
correlation with quality sugar parameters (pol in juice%, pol in cane%, purity% and sugar content %) was observed.
Spectroscopic acetyl bromide lignin showed no correlation with any agronomic trait evaluated. Similar results were
obtained by Baffa et al. (2014) for lignin content determined by Klason’s method. It is worth noting, that no
correlation between ADL and ABS was found (r=0.04, p=0.7).
CONCLUSIONS
Genotypes with cell-wall composition with improved biomass digestibility (high cellulose and low lignin contents) in
a sugarcane biparental population were identified. Because ADL and ABS methods showed no correlation,
different genotypes could be selected according to the lignin method considered. These divergences indicate that
further research is needed to validate lignin assessment as a cultivar selection tool, e.g. complementary
saccharification analyses.
Few significant correlations between cell-wall components and agronomic traits were found. Stalk weight was
associated with higher cellulose and lower ADL content.
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Analyse de la composition chimique de la paroi cellulaire d'une progéniture de Saccharum spp.
pour une digestibilité accrue de la bagasse
Résumé. La connaissance sur la composition des parois cellulaires des hybrides de canne à sucre pourrait être utilisées dans
la sélection de variété à haute digestibilité de la bagasse afin d'améliorer la libération de sucre dans la production d'éthanol 2G.
Dans cette étude, les composantes de la paroi cellulaire de 76 hybrides d'une progéniture de canne, leurs progéniteurs et deux
génotypes à haute teneur en fibre ont été analysés. La méthode Van Soest a été utilisée pour déterminer la teneur en cellulose,
en hémicellulose et en lignine au détergent acide (ADL). Le pourcentage de lignine soluble dans le bromure d'acétyle (ABS) fut
déterminé. Les corrélations entre les principaux polymères des parois cellulaires et les caractères agronomiques ont été
effectuées. Malgré les différences mineures observées entre la composition de la paroi cellulaire des géniteurs, des génotypes
à forte teneur en cellulose et à faible teneur en lignine ont été identifiés dans la progéniture. Les différences entre eux
dépendaient de la méthode utilisée pour déterminer la teneur en lignine. La moyenne de l’ADL était inférieure de 43% à la
moyenne de l'ABS, en accord avec les pertes en lignine pendant le traitement aux détergents acides. Aucune corrélation n'a été
observée entre les dosages ADL et ABS. La bagasse de génotypes ayant un poids de tiges plus élevé était associée à une
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teneur plus élevée en cellulose et à une teneur plus faible en ADL. D'autres analyses de saccharification sont nécessaires afin
d’évaluer la libération de sucre de la biomasse et améliorer la digestibilité de la bagasse dans des génotypes de canne à sucre.
Mots-clés: Lignine, paroi cellulaire, bagasse de canne à sucre
Análisis de la composición química de la pared celular de una progenie de Saccharum spp. para
el incremento de la digestibilidad del bagazo
Resumen. El conocimiento de la composición de la pared celular en híbridos de caña de azúcar puede ser utilizado en la
selección de cultivares de elevada digestibilidad del bagazo, orientados a incrementar la liberación de azucares en la producción
de etanol 2G. Se analizaron los componentes de la pared celular de 76 híbridos obtenidos de un cruzamiento de caña de azúcar,
sus progenitores y dos genotipos de alta fibra. Se utilizó el método de Van Soest para determinar los contenidos de celulosa,
hemicelulosa y lignina detergente ácido (LDA); además se determinó el contenido de lignina mediante espectroscopía utilizando
brumuro de acetilo (EBA). Se realizó un análisis de correlaciones con los componentes de la pared celular y características
agronómicas. A pesar de las diferencias mínimas en la composición de la pared celular observadas entre progenitores, fueron
identificados en la progenie genotipos con contenido elevado de celulosa y bajo de lignina. Sin embargo, estos genotipos fueron
distintos dependiendo del método empleado en la determinación de lignina. El promedio calculado para LDA fue un 43% más
bajo que el promedio de EBA, en coincidencia con pérdidas previamente reportadas en la bibliografía debido al uso del
detergente ácido. Se observó una ausencia de correlación entre LDA y EBA, mientras que el peso de tallos estuvo asociado a
un contenido mayor de celulosa y menor de LDA. Futuros análisis de sacarificación son necesarios para evaluar la liberación
de azúcares de la biomasa y validar los métodos de determinación de lignina como herramientas de selección.
Palabras clave: Lignina, pared celular, bagazo, caña de azúcar
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