Bioresource Technology 147 (2013) 645–648

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Influence of the alkaline delignification on the simultaneous

saccharification and fermentation (SSF) of sugar cane bagasse
Mariana Lucena Soares, Ester Ribeiro Gouveia ⇑
Department of Antibiotics, Federal University of Pernambuco, Cidade Universitária, CEP 50670-901, Recife, PE, Brazil

h i g h l i g h t s

 Ethanol production from steam explosion alkaline delignified bagasse was investigated.
 Alkaline delignification increased the ethanol production despite decreased cellulose.
 Production of glycerol and acetate were decreased in SSF versus SHF.

a r t i c l e i n f o a b s t r a c t

Article history: Ethanol production from steam explosion alkaline delignified bagasse was investigated by saccharifica-
Received 16 July 2013 tion and simultaneous fermentation. Non delignified bagasse (ND) contained 25% lignin, and after alka-
Received in revised form 11 August 2013 line delignification, materials with 6% (D1 – NaOH 1% w/v) and 12% (D05 – NaOH 0.5% w/v) lignin,
Accepted 13 August 2013
respectively, were obtained. Ethanol production increased 450% and 733% in relation to ND, when D05
Available online 26 August 2013
and D1 material, respectively, were used. Higher productivity and EtOH/bagasse were observed for D1.
However, higher enzymatic convertibility of cellulose was obtained with 0.5% w/v NaOH. Alkaline delig-
Keywords:
nification increased the ethanol production despite decreased cellulose.
Delignification
Ethanol
Ó 2013 Elsevier Ltd. All rights reserved.
Lignin
Saccharomyces cerevisiae

1. Introduction known to hinder the accessibility of cellulases to the substrate

Lignocellulosic biomass generally contains 75–80% cellulose
and hemicellulose, which cannot be easily converted to simple
monomeric sugars due to their recalcitrant nature (Adsul et al.,
2005). The pretreatment is a necessary step to alter some struc-
tural characteristics of lignocellulose, increasing glucan and xylan
accessibility to the enzymatic attack (Alvira et al., 2010). A number
of pretreatment options are available: acid pretreatment, alkaline
treatment, steam explosion, wet oxidation, organic solvent
pretreatment, and hot water (Adsul et al., 2005). Steam explosion
is the most widely employed physic-chemical pretreatment for lig-
nocellulosic biomass. The objective of a steam pretreatment/steam
explosion is to solubilize the hemicellulose to make the cellulose
more accessible for enzymatic hydrolysis (Hendriks and Zeeman,
2009).
An optional step after the pretreatment by steam explosion is
alkaline delignification (Wanderley et al., 2013) that solubilizes
lignin and a small percentage of the hemicelluloses (Beukes and
Pletschke, 2010). The presence of lignin in the vegetal cell wall is
⇑ Corresponding author. Tel.: +55 8121268949.
E-mail address: estergouveia@gmail.com (E.R. Gouveia).
(Mooney et al., 1998). Some authors (Santos et al., 2012; Wander-
ley et al., 2013; Oliveira et al., 2013) have used 1% w/v NaOH at
100 °C and 60 min in the delignification of sugar cane bagasse after
pretreatment by steam explosion. However, lower values of NaOH
concentration were not found for sugar cane bagasse. Furthermore,
these authors not used simultaneous saccharification and fermen-
tation (SSF) of sugar cane bagasse only pretreated by steam explo-
sion. The aim of this work was to compare the ethanol production
from steam explosion pretreated sugar cane bagasse in SSF using
three conditions: without and with alkaline delignification at
0.5% w/v or 1% w/v NaOH, 100 °C and 30 min.
2. Methods
2.1. Steam-pretreated sugarcane bagasse and delignification
Sugarcane bagasse, pretreated by steam explosion at 200 °C for
7 min on the pilot scale, was kindly provided by Department of
Biotechnology of Engineering College of Lorena (University of Sao
Paulo). A portion of the pretreated material was delignified at
100 °C during 30 min and 0.5% w/v (D05) or 1% w/v NaOH (D1).
The delignification reaction took place in a 20 L rotary reactor

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.08.103
646 M.L. Soares, E.R. Gouveia / Bioresource Technology 147 (2013) 645–648
fitted with mixing and heating systems (Regmed AUE/20, Regmed
Indústria Técnica Ltda., Brazil), using a solid:liquid ratio 1:10 w/v.
The pre-treated and delignified bagasse was filtered through a
cloth, and washed seven times to remove the remaining lignin
and to reduce the pH. The pulp was dried at 50 °C and stored for
subsequent chemical analyses and simultaneous saccharification
and fermentation.
2.2. Microorganism and inoculum
An industrial strain (UFPEDA 1238) of Saccharomyces cerevisiae,
kindly provided by the Culture Collection of the Department of
Antibiotics of the Federal University of Pernambuco, Brazil, was
used. The strain was maintained in a solid medium containing
(in g/L) glucose (20), yeast extract (4), peptone (3) and agar (15),
at pH 7.0. Pure yeast culture growth in culture medium described
above, was added to a 500 mL Erlenmeyer flask, which contained
100 mL of following medium: glucose (20 g/L), yeast extract (4 g/
L) and peptone (3 g/L) at pH 7.0. The Erlenmeyer flask was incu-
bated in a rotary shaker at 30 °C and 250 rpm. After 12 h, the sus-
pended cells were filtered through a 0.45 lm filter. The residue
was discarded and the cells were re-suspended in 10 mL sterile
water.
2.3. Simultaneous saccharification and fermentation
Simultaneous saccharification and fermentation was performed
in 250 mL Erlenmeyer flasks, contained 90 mL of fermentation
medium (nutrients dissolved in a sodium citrate buffer at 50 mM
and pH 4.8), 8 g of solids and enzyme loads of 10 FPU/g cellulose
(Celluclast 1.5L; 69.50 FPU and 13.70 CBU) and 5% v/v (of the vol-
umetric Celluclast 1.5L addition) b-glucosidase (1340 CBU) prepa-
ration (Novozym 188), both from Novozymes A/S (Bagsværd,
Denmark). b-glucosidase was added in all SSF. The Erlenmeyer
flasks were incubated in a rotary shaker at 50 °C and 150 rpm. After
a 6 h pre-hydrolysis, each Erlenmeyer flask was inoculated with
yeast cells and incubated at 37 °C and 80 rpm. Initial cell concen-
tration was 1 g/L. Nutrients added were (NH4)2SO4 (2 g/L); KH2PO4
(2 g/L); MgSO47H2O (0.75 g/L); yeast extract (4 g/L). Samples
withdrawn at regular intervals were filtered (0.45 lm). All the
experiments were performed in duplicate. The enzymatic convert-
ibility of cellulose (ECC) was calculated based in ethanol concen-
tration (Martín et al., 2008).
Ef  Ei
ECC ¼  100%
C i 0:57
where Ef is the final ethanol concentration (g/L); Ei is the initial eth-
anol concentration (g/L); Ci, initial cellulose concentration (g/L). The
factor 0.57 is the stoichiometric yield of ethanol from cellulose.
2.4. Analytical methods
The content of the polysaccharides and lignin in the raw mate-
rial was determined by two-step analytical acid hydrolysis, accord-
ing to the analytical procedure validated for sugarcane bagasse by
Gouveia et al. (2009). Sugars, carboxylic acids, ethanol and furan
aldehydes were quantified by HPLC (Agilent HP 1100, Germany)
on an Aminex HPX-87H+ (Bio-Rad, Hercules, CA, USA) column at
60 °C, using 5 mM H2SO4 at a flow rate of 0.6 mL/min as mobile
phase, and detected using an RI-detector (Agilent). The total
content of phenolic compounds was determined colorimetrically
Folin–Ciocalteu (Singleton et al., 1999) in a HP 8453 spectropho-
tometer (Agilent, Germany). Gallic acid was used as the calibration
standard.
Table 1
Cellulose, hemicellulose and lignin content in the bagasse steam explosion pretreated
(ND, D05 and D1) and solubilization of cellulose, hemicellulose and lignin.
Component/solubilization ND (%) D05 (%) D1 (%)
Cellulose 45.46 ± 1.39 64.79 ± 2.99 87.30 ± 2.22
Csol 27.77 ± 3.12 44.41 ± 3.63 30.65 ± 2.49
Hemicellulose 15.33 ± 1.64 13.72 ± 0.34 6.88 ± 0.69
Hsol 59.59 ± 6.11 80.47 ± 0.68 90.02 ± 1.28
Lignin 24.67 ± 0.91 11.88 ± 0.55 5.68 ± 0.13
Lsol 24.09 ± 3.96 70.45 ± 1.16 91.26 ± 0.28
Ash 5.46 ± 0.39 5.53 ± 0.12 3.19 ± 0.31
Total 90.92 ± 6.12 96.15 ± 5.33 103.05 ± 4.74
3. Results and discussion
Table 1 presents cellulose, hemicellulose and lignin content in
the bagasse steam explosion pretreated, with (D05 and D1) and
without (ND) alkaline delignification. The alkaline delignification
increased cellulose content and decreased hemicelluloses and
lignin contents in all cases. Higher cellulose content was found in
the delignified bagasse with 1% w/v NaOH (D1; 87.30%), followed
by 0.5% w/v NaOH (D05; 64.79%). The hemicellulose contents de-
creased 10.50% (D05) and 55.12% (D1) after delignification with
0.5% w/v and 1% w/v NaOH, respectively. In delignified bagasse,
when the lignin contents decreased (51.84% and 76.98% to D05
and D1, respectively), the cellulose contents increased 42.52%
(D05) and 92.04% (D1).
Barcelos et al. (2013), using 4% w/v NaOH, observed a 50%
decreasing lignin in sugar cane bagasse pretreated with dilute
sulfuric acid and by autoclaving. The steam explosion before the
alkaline delignification (D05 and D1) resulted in lower lignin
content, probably due to removal of hemicelluloses and the lignin
redistribution which occurs in steam explosion pretreatment
(Alvira et al., 2010). On the other hand, Santos et al. (2012), using
1% w/v NaOH and Oliveira et al. (2013), using 1.5% w/v, both at
100 °C and 60 min obtained 91.10% and 66.50%, respectively,
decreasing lignin when sugar cane bagasse pretreated by steam
explosion at 200 °C was delignified. Comparing these two works
can be observed that lignin content was 24.60% lower with increas-
ing NaOH concentration of 1% w/v to 1.5% w/v. In the present work,
alkaline delignification with 1% NaOH (D1) was 12% lower than
that obtained by Santos et al. (2012). However, was employed half
the time (30 min) than Santos et al. (2012) used.
Around 54% and 50% w/w of the raw material was recovery,
respectively, in the experiments at 0.5% and 1% w/v NaOH, respec-
tively. Higher recovery was obtained in steam explosion pretreat-
ment (68% w/w). These yields and the cellulose, hemicellulose
and lignin contents were used to calculate the percentage of solu-
bilization (Table 1). Hemicellulose was the main solubilized
(59.50%) component in steam explosion pretreatment only (ND).
However, the maximum solubilization hemicellulose was observed
in alkaline delignification with 1% w/v NaOH (90.93%). Lignin
solubilization was above 70% for the delignified materials, and
the maximum (91.26%) was reached for the material pretreated
at 1% w/v NaOH (D1). Lower values solubilization were observed
for cellulose in three pretreatments, with the lowest value for D1
(30.65%). Oliveira et al. (2013), found values between 88.5% and
93.7% for hemicellulose, above 80% for lignin and 43.5% for
cellulose.
Fig. 1 shows ECC (%), QP (productivities at 48 h for D05 and D1
and 12 h for ND) and EtOH/Bagasse (ethanol volume in litre in rela-
tion to bagasse mass in ton). EtOH/Bagasse was calculated consid-
ering the recovering of solids after the alkaline delignification (50%
for D1 and 54% for D05) and after the steam explosion (68%). Under
optimum conditions ethanol production of 32.45 g/L was obtained
 M.L. Soares, E.R. Gouveia / Bioresource Technology 147 (2013) 645–648 647

160 0.7 1.0

Glycerol; Acetic Acid (g/L)

Glycerol ND
140 0.8 Acetic Acid ND
0.6
ECC (%); EtOH/Bg (L/ton)

Glycerol D05
120 Acetic Acid D05
0.6 Glycerol D1
0.5
Acetic Acid D1

100
80
60
40
EtOH/Bg = -5.70 * Lignin + 176.47
20 QP = -0.02 * Lignin + 0.65
2
ECC = -0.40 * Lignin + 8.31 * lignin + 62.39
0
3 6
Fig. 1. Enzymatic convertibility of cellulose in ethanol (ECC; square), productivity
(QP; triangle) and ethanol volume in relation to bagasse mass in ton (EtOH/Bagasse;
circle) as a function of lignin content.
from alkali (4% w/v NaOH) treated sugarcane bagasse (Asgher et al.,
2013). However, QP was 10% lower than that with 1% w/v NaOH.
Within the range of the lignin contents evaluated in this work,
there was a linear correlation between lignin content and QP
(R2 = 0.9965) and between lignin content and EtOH/Bagasse
(R2 = 0.9971). Negatives values of linear coefficient indicate that
lignin content and productivity or EtOH/Bagasse is inversely pro-
portional. This suggests that higher NaOH content would increase
ethanol concentration. However, maximum ECC was found for
D05, due to polynomial fit obtained in this case (R2 = 1). All data
in Figs. 2a and 2b were obtained with 48 h of SSF, since the data
found in 72 h were stable for three cases studied.
The concentration of xylose increased slightly (Fig. 2a) during
SSF, probably due to the activity of xylanase in Celluclast. However,
due to the inability of Saccharomyces cerevisiae to consume this su-
gar, the concentration remained constant after 18 h for ND SSF and
24 h for D1 SSF and D05 SSF. The concentrations of xylose in all SSF
were significantly different in their analysis of variance (p < 0.05),
but not so with regard to the delignified materials. The concentra-
tion of cellobiose in ND SSF was approximately zero throughout
the time. However, the concentrations of cellobiose in the D1 SSF
and D05 SSF were considerably higher, but remained below 1 g/L
during the entire time in the D05 SSF and after 6 h in the D1 SSF.
Furfural and hydroxymethylfurfural were not found, probably
due to wash of the pulp after the pretreatment. Acetic acid produc-
tion occurred during the fermentation (Fig. 2b). Higher acetic acid
concentration was observed in SSF without delignification (ND).
Glycerol concentration increased during the SSF in three cases
(ND, D05 and D1). Maximum glycerol concentration was observed
5 EtOH/Bagasse, but no increase was detected in ECC due to losses
Xylose (g/L); Cellobiose (g/L)
QP (g/L.h)
0.4
0.4
0.2
0.3
0.0
0.2
0 6 12 18 24 30
0.1 Time (h)
9 12 15 18 21 24 27 Fig. 2b. Time of course of acetic acid and glycerol production during the SSF of
pretreated sugar cane bagasse.
Lignin (%)
in D1 SSF (about 0.8 g/L). Wanderley et al. (2013) in SHF obtained
values between 0.7 and 2.8 g/L of glycerol, when sugar cane ba-
gasse non-delignified and delignified (1% w/v NaOH, 60 min and
100 °C), respectively, were used. Higher glycerol concentration is
observed with increased carbon source. The glycerol concentration
obtained in this work was much lower that obtained by Wanderley
et al. (2013), due to the lower glucose concentration. Glucose is
generated during the SSF process, unlike Wanderley et al. (2013),
in which the initial glucose concentration was 59 g/L in SHF
process.
The total content of phenolic compounds in early SSF was about
two times higher (52 mg/L) in ND SSF than in D05 (26 mg/L) or D1
(28 mg/L) SSF, due to higher lignin content in the ND bagasse (Ta-
ble 1). Some authors have chosen 4-hydroxybenzoic acid as a mod-
el phenolic compound because of its abundance in hardwood
hydrolysates (Palmqvist and Hahn-Hägerdal, 2000). These authors
have reported that 1 g/L caused a 30% decrease in ethanol yield for
Sacchromyces cerevisiae. In the present work, the phenolics com-
pound concentrations were much lower and, probably, were not
the reason for the lower ethanol production in ND SSF.
4. Conclusion
The steam explosion pretreatment increased the alkaline delig-
nification due to removal of hemicelluloses and the lignin redistri-
bution which occurs in this pretreatment. Results with 1% w/v
NaOH indicated that the delignification can be with half time than
that is utilized generally in literature. High values hemicellulose
and lignin solubilization and lower values cellulose solubilization
were obtained in delignified material. Low cellobiose concentra-
tion did not allowed inhibition b-glucosidade by glucose. Produc-
tion of glycerol and acetate were decreased in SSF versus SHF.
Higher NaOH content increased ethanol concentration, QP and
cellulose.

Xylose ND
4 Cellobiose ND
Xylose D05
Cellobiose D05 Acknowledgement
3 Xylose D1
Cellobiose D1 The authors acknowledge the financial support from Conselho
2 Nacional de Desenvolvimento Científico e Tecnológico, Brasilia
DF, Brazil (CNPq).
1
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