Bioresource Technology 179 (2015) 249259

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

Enhanced enzymatic hydrolysis and ethanol production from cashew

apple bagasse pretreated with alkaline hydrogen peroxide
Jessyca Aline da Costa, Jos Edvan Marques Jr., Luciana Rocha Barros Gonalves,
Maria Valderez Ponte Rocha
Universidade Federal do Cear, Departamento de Engenharia Qumica, Campus do Pici, Bloco 709, 60455-760 Fortaleza, CE, Brazil

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Conversion of cashew apple bagasse

to fuel ethanol.
 The combination between different
enzymes was evaluated during
enzymatic hydrolysis.
 Cellulase and b-glucosidase showed
the more effective interactions.
 The study demonstrates an
insignicant impact of xylanase on
the hydrolysis yield.
 The ethanol production by SHF and
SSF processes showed promising
using CAB-AHP.

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

Article history: The effect of combinations and ratios between different enzymes has been investigated in order to assess
Received 25 August 2014 the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen perox-
Received in revised form 3 November 2014 ide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous sac-
Accepted 4 November 2014
charication and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic
Available online 3 December 2014
hydrolysis conducted with cellulase complex and b-glucosidase in a ratio of 0.61:0.39, enzyme loading of
30 FPU/gCAB-AHP and 66 CBU/gCAB-AHP, respectively, using 4% cellulose from CAB-AHP, turned out to be the
Keywords:
most effective conditions, with glucose and xylose yields of 511.68 mg/gCAB-AHP and 237.8 mg/gCAB-AHP,
Cellulase
Xylanase
respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an
b-Glucosidase ethanol yield of 61.8 kg/tonCAB, corresponding to 15 g/L ethanol and productivity of 3.75 g/(L h). The eth-
Ethanol anol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80%
Kluyveromyces marxianus yield and 74.2 kg/tonCAB.
2014 Elsevier Ltd. All rights reserved.

1. Introduction cashew apple juice industry, represents approximately 20% of the

total peduncle weight and is mainly composed of cellulose, hemi-
Ethanol production using lignocellulosic materials as substrates cellulose and lignin. The industrial peduncle processing for juice
represents one of the main routes for second-generation biofuel production results in 15% (w/w) of bagasse, which has essentially
production. Cashew apple bagasse (CAB), a by-product of the no commercial value and is usually discarded by local industries
(Rocha et al., 2011; Correia et al., 2013).
Cashew apple bagasse can be converted into bioethanol by per-
Corresponding author. Tel.: +55 85 3366 9611; fax: +55 85 3366 9610. forming the following operations: pretreatment, hydrolysis, fer-
E-mail addresses: valderez.rocha@ufc.br, valponterocha@yahoo.com.br
mentation, and distillation. Enzymatic hydrolysis is one of the
(M.V.P. Rocha). most common and effective methods employed to generate

http://dx.doi.org/10.1016/j.biortech.2014.11.010
0960-8524/ 2014 Elsevier Ltd. All rights reserved.
250 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259
fermentable sugars due to the high yields of sugars that can be
obtained, with subsequent fermentation of these sugars to produce
ethanol (Saha et al., 2011; Van-Dyk and Pletschke, 2012; Meng and
Ragauskas, 2014). For that to become possible, the raw material
needs to be pretreated so that the cellulose in the plant bers is
exposed to enzymatic action (Rocha et al., 2009).
Pretreatment is a crucial step to generate fermentable sugars. It
breaks open the polymeric structures of lignin and carbohydrates in
the lignocellulosic biomass and enhances the accessibility of
enzymes to the solid substrate during the enzymatic hydrolysis
step (Saha et al., 2011). A pretreatment that has been studied to pre-
pare lignocellulosic substrates from a wide range of raw materials
for subsequent bioconversion is performed with alkaline hydrogen
peroxide (AHP) (Correia et al., 2013; Karagz et al., 2012; Banerjee
et al., 2011). It is known to decrystallize cellulose and with the oxi-
dative action of the H2O2-derived radicals, it is also thought to con-
tribute to the depolymerization and high solubilization of lignin
(Correia et al., 2013; Karagz et al., 2012; Selig et al., 2009).
The effectiveness of hydrolysis in the polysaccharides present in
the lignocellulose substrates, therefore, is determined by an appro-
priate pretreatment, good selection of enzymatic complexes and
cellulose accessibility (Meng and Ragauskas, 2014).
Enzymatic hydrolysis is an environment-friendly process that
consists in the use of enzymes from groups of cellulases and hemi-
cellulases, which are capable of degrading polysaccharides (cellu-
lose and hemicellulose) with high-specicity catalyst activity
(Kinnarinen and Hkkinen, 2014). The optimization of the compo-
sition of enzymatic complexes aims to boost the yield and reduce
hydrolysis cost. But primarily, it should provide a high content of
glucose in the hydrolysate under the action of the available
enzymes combined (Zhou et al., 2009).
Cellulose accessibility has been proposed as a key factor in the
effectiveness of bio-conversion of lignocellulosic biomass into fer-
mentable sugars. Different factors affecting the cellulose accessi-
bility, i.e., the substrate type, chemical composition (such as
lignin/hemicellulose content), and the biomass structure (Meng
and Ragauskas, 2014; Kinnarinen and Hkkinen, 2014; Van-Dyk
and Pletschke, 2012). Other factors affect the enzymes and the
optimization of the hydrolysis process, such as enzyme ratio, sub-
strate and enzyme loadings and inhibitors (Van-Dyk and Pletschke,
2012; Rocha et al., 2009). The enzyme loadings are determined by
nancial factors, such as the cost of enzymes and nal products,
and should be optimized (Kinnarinen and Hkkinen, 2014). In
addition to enzyme loadings, substrate loadings are a major factor.
It has to allow the bioconversion to be economical and, at the same
time, provide sufcient sugar levels for fermentation. Thus, opti-
mal enzyme and substrate loadings have to be identied for max-
imum efciency and economy (Van-Dyk and Pletschke, 2012). In
the case of industrial processes, in which a high sugar concentra-
tion must be obtained, the initial suspended solid concentration
in the hydrolysis should be high. However, high solid loadings
are known to reduce the obtainable yield (Rocha et al., 2009),
which adversely affects the process economy.
In this context, the aim of this study was to determine the fea-
sibility of employing several complexes of cellulolytic and hemicel-
lulolytic enzymatic preparations, the ratio of these complexes, and
the enzyme and cellulose loadings for efcient hydrolysis of poly-
saccharides in pretreated cashew apple bagasse. The effects of
hydrolysis were assessed based on the quantity of released sugars.
The pretreatment was conducted with CAB 5% (w/v), 4.3% (v/v)
AHP at 35 C and 250 rpm for 6 h, the solid fraction was then sep-
arated for experiments of enzymatic hydrolysis. Afterwards, etha-
nol production in both Separate Hydrolysis and Fermentation
(SHF) and Simultaneous Saccharication and Fermentation (SSF)
processes using the yeast Kluyveromyces marxianus ATCC36907
was also evaluated.
2. Methods
2.1. Lignocellulosic material
Cashew apple (Anacardium occidentalis L.) bagasse (CAB) was
kindly donated by Jandaia Juice Industry (Cear, Brazil). The initial
treatment of CAB was conducted of according to Correia et al.
(2013), in which CAB was washed three times with water, dried
at 60 C for 24 h and milled in a hammer mill in order to obtain
an average particle size of 2080 mesh (0.250.84 mm).
2.2. Pretreatment of cashew apple bagasse
The milled cashew apple bagasse was pretreated by alkaline
hydrogen peroxide according to the best conditions obtained in
the study of Correia et al. (2013). The cashew apple bagasse, with
a concentration of solids of 5% (w/v), was slurred in hydrogen per-
oxide H2O2 (4.3% v/v) with the H2O2 solution adjusted to pH 11.5
using 6 mol L1 NaOH. The pretreatment was conducted in an orbi-
tal shaker (Tecnal TE 422, SP, Brazil) at 35 C for 6 h and 250 rpm.
After the pretreatment, both solid and liquid fractions were sepa-
rated by ltration. The solid fraction was washed with distilled
water and oven dried at 60 C for 24 h. This solid fraction, named
CAB-AHP, was used as substrate for the subsequent enzymatic
hydrolysis.
2.3. Characterization of untreated and pretreated CAB
Compositional analyses (cellulose, hemicellulose and lignin) of
the CAB and CAB-AHP raw materials were determined according
to Gouveia et al. (2009). The extractables, total solids and ash were
analyzed according to the NREL Laboratory Analytical Procedures
LAP (Sluiter et al., 2008).
2.4. Lignin recovery
Lignin samples generated in the pretreatment were recovered
from the hydrolysate liquor by precipitation with acidication at
pH 2 using 50% v/v H2SO4. The mass of precipitated lignin was cal-
culated in dry mass basis.
2.5. Enzymes
The cellulase complex (NS22074), as well as the xylanase
(NS22036), hemicellulase (NS22002) and b-glucosidase
(NS50010) preparations were kindly supplied by Novozymes. The
enzymatic activities of the cellulase complex and b-glucosidase
enzymes were determined as recommended by Ghose (1987) and
expressed as follows: 1 FPU the quantity of enzyme releasing
1 lmol of glucose from blotting-paper Whatman No. 1 within
1 min and 1 CBU the quantity of enzyme transforming 1 lmol
of cellobiose into 2 lmol of glucose within 1 min (reaction condi-
tions: pH 4.8, temperature 50 C) for the cellulase complex and
the b-glucosidase, respectively. The enzymatic activities of xylan-
ase and hemicellulase were determined according to Bailey et al.
(1992) and the values were expressed as 1 U the quantity of
enzyme releasing 1 lmol of xylose from xylan within 1 min
(reaction conditions: pH 4.8, temperature 50 C). The protein con-
tent of the liquid enzyme preparations was determined using the
Bradford method (Bradford, 1976). The enzymatic activity and
the protein concentration of each of these enzymes are shown in
the Table 1.
 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259 251

Table 1 In the second process, different ratios of the best combination of

Enzymes complexes used in the study of enzymatic hydrolysis of cashew apple enzymes obtained in the previous study were evaluated. The ratio
bagasse pretreated by alkaline hydrogen peroxide.
was calculated based on the amount of protein in the assay
Enzyme Enzymatic activity Protein mg/mlEXTRACT (mgPROTEIN/gCELLULOSE), taking into consideration the content of
Cellulase complex 108.12 FPU/ml 28.47 protein in 1 ml for each enzyme preparation (see Table 3).
b-glucosidase 384.28 CBU/ml 28.29
Xylanase 4943.17 U/ml 6.50
2.6.2. Effect of the enzyme loading
Hemicellulase 824.92 U/ml 11.77
The effect of enzyme loading on the enzymatic hydrolysis was
studied with the best combination and ratios of enzymes. In these
assays, the enzyme loadings ranged from 5 FPU/gCAB-AHP cellulase
2.6. Enzymatic hydrolysis complex and 11 CBU/gCAB-AHP b-glucosidase to 35 FPU/gCAB-AHP
cellulase complex and 77 CBU/gCAB-AHP b-glucosidase.
The effect of combining various complexes of cellulolytic and
hemicellulolytic enzymatic preparations and their ratio, as well 2.6.3. Effect of the cellulose loading from CAB-AHP
as enzyme and cellulose loadings, were evaluated for enhancing In addition to enzyme loadings, substrate loadings are an
sugar yields in the enzymatic hydrolysis. The hydrolysis was con- important factor in allowing bioconversions to be economical
ducted in 150 mL asks containing 15.0 mL of 0.1 mol L1 sodium and should provide sufcient sugar levels for fermentation. Thus,
citrate buffer at pH 4.8 and 80 lL of 10 mg/mL tetracycline in after optimizing the conditions for the enzymatic hydrolysis, the
70% v/v ethanol. Tetracycline was added to prevent the growth of effect of substrate loadings of 1%, 2%, 3% and 4% w/v cellulose,
organisms during the saccharication. The cellulose loading was corresponding to 2.26%, 4.52%, 6.79% and 9.04% w/v CAB-AHP,
xed at 1 g/100 mL, except in the study of cellulose loading. Dis- respectively, were evaluated for yields of released sugars.
tilled water was added to each ask to bring the total reaction vol-
ume of 20 mL. The hydrolysis was conducted in orbital shaker
2.7. Study of ethanol production using CAB-AHP
(Tecnal TE 422, SP, Brazil) at 45 C and 150 rpm. Samples
(1 mL) were withdrawn and centrifuged at 10,000g for 15 min,
2.7.1. Yeast cultivation and inoculum preparation
and the supernatants used for sugar analysis.
The yeast K. marxianus ATCC36907 was used in the study of the
The enzymatic yields (%) of glucose and xylose were calculated
ethanol production in both Separate Hydrolysis and Fermentation
using Eqs. (1) and (2), respectively:
(SHF) and Simultaneous Saccharication and Fermentation (SSF)
Glucoseg=L  v processes. The stock culture was inoculated on agar YEPD (20 g/L
Glucoseyields  100 1 glucose, 10 g/L yeast extract, 20 g/L peptone and 20 g/L agar) and
Celluloseg  0:9
incubated at 30 C for 48 h. For the inoculum preparation, cells
were transferred to 250 mL Erlenmeyer ask with 50 mL of YEPD
Xyloseg=L  v medium (containing 20 g/L glucose, 10 g/L yeast extract and 20 g/
Xyloseyields  100 2
Hemicelluloseg  0:88 L peptone) at pH 5.0 and the growth was carried out at 30 C and
150 rpm on a rotary shaker (Tecnal TE 422, SP, Brazil) for 24 h.
where 0.9 and 0.88 are conversion factors of cellulose to glucose Afterwards, the medium was centrifuged (10,000g, 10 min) and
and hemicellulose to xylose, respectively, and v is the total solution the cells were used as inoculums in both SFS and SSF processes.
volume. The amount of cellulose and hemicellulose cited in Eqs. (1)
and (2) refers to polymers present in the pretreated cashew apple 2.7.2. Separate hydrolysis and fermentation (SHF)
bagasse. Ethanol production was conducted using the liquid fraction of
the CAB-AHP hydrolysate, without any nutritional supplements,
2.6.1. Combination of various enzymatic complexes preparations obtained after an enzymatic saccharication using the best condi-
Two studies were conducted to evaluate the effect of different tions determined in the experiments. An inoculum of 10 g/L bio-
combinations of four enzymes preparations (cellulase complex, mass was used and batch fermentation experiments were carried
xylanase, hemicellulase and b-glucosidase). Initially, the rst pro- out on a rotary shaker (Tecnal TE 422, SP, Brazil) at 30 C,
cess was performed with different combinations of enzymes using 150 rpm and pH 5.0 using 100 mL of medium.
the following enzymatic loading: 5 FPU/gCAB-AHP cellulase, 873
U/gCAB-AHP xylanase, 43 U/gCAB-AHP hemicellulase and 11 2.7.3. Simultaneous Saccharication and Fermentation (SSF)
CBU/gCAB-AHP b-glucosidase. As shown in Table 2, this loading Experiments were conducted in 250 mL asks using a CAB-AHP
corresponds to 30 mgPROTEIN/gCAB-AHP. loading of 4 g cellulose/100 mL and 50 mM citrate buffer at pH 4.5
5.0 supplemented with 5 g L1 yeast extract and 1 g L1 (NH4)2SO4.
The culture media was sterilized at 110 C for 10 min. The enzyme
Table 2 loadings were 30 FPU/gCAB-AHP cellulase and 66 CBU/gCAB-AHP b-glu-
Assays conducted with different combinations of four enzymes for enzymatic
cosidase. The yeast K. marxianus ATCC 36907 was inoculated
saccharication (45 C, pH 4.8 and 150 rpm) of pretreated cashew apple bagasse
(CAB-AHP). immediately after enzyme addition with an initial cell concentra-
tion of 5 g/L and SSF was performed at 45 C and 150 rpm for
Run Enzymes
72 h. The theoretical yield of ethanol production was calculated
Cellulase complex Xylanase Hemicellulase b-Glucosidase as follows (Eq. (3)):
(FPU/gCAB-AHP) (U/gCAB-AHP) (U/gCAB-AHP) (CBU/gCAB-AHP)
01 5.0 43.0 Ethanolt g L1  Ethanol0 g L1
% Theoreticalyields
02 5.0 873.0 0:511xCelluloseinitial %xCAB-AHP g L1 x1:11
03 5.0 11.0
04 873.0 43.0
3
05 43.0 11.0
where Ethanolt is the concentration of ethanol at time t, Ethanol0 is
06 5.0 873.0 11.0
the initial ethanol concentration, CAB-AHP is the dry biomass
() Enzyme did not use in the run. concentration at the start of fermentation (g L1), 0.511 is the
252 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259

Table 3
Effect of enzymes proportions (cellulase complex, xylanase and b-glucosidase) on the release of sugars in the enzymatic hydrolysis of CAB-AHP at 45 C, pH 4.8 for 72 h.

Run Enzyme proportions (%) Glucose yield (%) Glucose concentration (g/L) Xylose yield (%) Xylose concentration (g/L)
Cellulase Xylanase b-Glucosidase
02 0.54 0.46 0.00 68.5 0.1 6.2 0.1 11.9 0.1 1.1 0.0
03 0.61 0.00 0.39 76.2 0.9 6.9 0.1 33.1 0.1 1.6 0.0
06 0.40 0.35 0.25 79.9 0.0 7.2 0.2 26.0 0.0 2.3 0.0
07 1.00 0.00 0.00 63.7 0.7 5.73 0.6 9.0 0.2 0.8 0.0
08 0.00 0.00 1.00 31.5 0.3 2.8 0.0 0.0 0.0 0.0 0.0
09 0.00 1.00 0.00 7.8 0.0 0.7 0.0 12.5 0.1 1.8 0.0
10 0.26 0.53 0.21 47.8 3.0 4.31 0.4 39.4 1.2 3.5 0.1
11 0.26 0.21 0.53 69.2 0.4 6.2 0.1 25.9 4.0 2.3 0.5

conversion factor of glucose to ethanol, and 1.11 is the conversion
factor of cellulose to glucose.
For SHS and SSF processes, samples were withdrawn for analy-
sis of glucose, xylose, cellobiose, arabinose, inhibitors (organic
acids, furfural and hydroxymethyl-furfural (HMF)) and ethanol, at
preset time intervals. Cell concentration was also analyzed in the
SHS process. The samples were centrifuged at 10,000g for 15 min
and the supernatant was ltered through 0.45 lm membrane
lters and then analyzed. All experiments were conducted in
triplicate.
2.8. Analytical methods
Biomass Cell growth was monitored by measuring the optical
density at 600 nm in the SHF process. The calibration curve was
determined in function of dry weight. Samples were taken from
the cell growth media at certain time intervals and centrifuged at
10,000g for 15 min. The pellet was dried at 60 C until constant
weight. Glucose, xylose, cellobiose, arabinose, inhibitors (organic
acids, furfural and hydroxymethyl-furfural (HMF)) and ethanol
were determined by HPLC used the procedure described by
Rocha et al. (2011).
2.9. Statistical analysis
All the experiments were carried out with three independent
replicates. Data were analyzed for statistical signicance by a
one-way analysis of variance (ANOVA) at 95% condence level
(p < 0.05). Tukeys multiple comparison tests (p < 0.05) were used
to analyze the signicances of different hydrolysis enzymatic con-
dition, available in Microcal Origin 8.1 software (Microcal Software
Inc, Northampton. MA. USA).
3. Results and discussion
3.1. Characterization of untreated and pretreated cashew apple
bagasse
The composition of cashew apple bagasse used in the study was
20.6 2.2% cellulose, 10.2 0.9% hemicellulose, 35.3 0.9% lignin,
7.8 0.6% extractable and 1.6 0.1% ash on dry weight basis. The
chemical composition of cashew apple bagasse is in good agree-
ment with other values reported in the literature (Rocha et al.,
2011; Correia et al., 2013). The cellulose content makes bagasse a
very promising substrate for ethanol production (Rocha et al.,
2009). The cashew apple bagasse at a solid loading of 5% was pre-
treated using alkaline peroxide hydrogen AHP (4.3% v/v AHP at
pH 11.5, 35 C for 6 h). The yield of the pretreated solid (CAB-
AHP) was 37.3%, composed by 44.2 0.3% cellulose, 18.3 0.9%
hemicellulose, 2.9 0.1% lignin, 4.9 0.3% extractable and
5.3 0.9% ash. The AHP pretreatment affects predominantly lignin
components, with 96.9% of solubilization of the original lignin,
resulting in a low solid yield. Lignin contents of solids decreased
from 35.3% to 2.9%, which caused cellulose and hemicellulose con-
tents to increase from 20.6% to 44.2% and from 10.2% to 18.3%,
respectively. On the other hand, regarding the content of the
untreated CAB (100 g), a loss of 4.1 g and 3.4 g of cellulose and
hemicellulose, respectively, was observed. Nevertheless, the
removal or disruption of lignin has been established as essential
for efcient bioconversion of lignocellulose into sugars (Van-Dyk
and Pletschke, 2012) and the pretreatment with alkaline hydrogen
peroxide improves the enzymatic hydrolysis by delignication
(Correia et al., 2013). AHP pretreatment is more effective for lignin
solubilization than alkali pretreatment (Karagz et al., 2012; Chen
et al., 2008).
3.2. Study of enzymatic hydrolysis of CAB-AHP
3.2.1. Effect of combination of commercial enzymes for glucose and
xylose release on the enzymatic hydrolysis of CAB-AHP
A large variety of enzymes of different specicities, such as cel-
lulase, xylanase, b-glucosidase and hemicellulase, is required to
degrade all the components of lignocellulose. When selecting
enzymes for degradation of lignocellulose substrates, the initial
choice has to be made between customized cocktails of individual
enzymes or using commercial, crude mixtures of enzymes. Some
cellulolytic enzymes mixtures may contain different enzymes with
the exact composition thereof being unknown, a factor that is
heavily criticized by Banerjee et al. (2011). These commercial mix-
tures are therefore not optimized for all types of biomass or pre-
treatment and further puried enzymes or mixtures may have to
be added to achieve optimum combinations. Then, in order to test
the effectiveness of AHP pretreatment in combination with a
superior enzyme cocktail, different combinations of four enzymes
preparations (cellulase complex, xylanase, b-glucosidase and
hemicellulase) were tested.
Glucose and xylose yields obtained in the enzymatic hydrolysis
in CAB-AHP using the different combinations of the enzymes are
shown in Fig. 1A and B, respectively.
It can be observed that glucose and xylose were released within
24 h, for all conditions investigated (Run 16). The yields for both
glucose and xylose increased with hydrolysis time in the runs, indi-
cating that there are difculties to the access of the enzyme to the
substrate and that during the disruption of the bonds of cellulose
and hemicellulose molecules and shrink in size, facilitating the
access to the enzymes.
The lowest glucose concentrations were obtained in the assays
conducted without the cellulase complex. 1.8 g L1 and 2.2 g L1 of
glucose was released in the experiments 04 and 05, respectively,
and these values remained relatively constant after 24 h
(Fig. 1A). It is generally accepted that three types of enzymes are
required to hydrolyze cellulose into glucose monomers: exo-1,4-
b-glucanases, EC 3.2.1.91 and EC 3.2.1.176 (cellobiohydrolase),
endo-1,4-b-glucanases, EC 3.2.1.4 and b-glucosidases, EC 3.2.1.21
 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259 253

cleave the xylan backbone into shorter oligosaccharides and b-

(A) 100 9
xylosidase, which in turn cleaves short xylo-oligosaccharides into
90 8
xylose (Van-Dyk and Pletschke, 2012) and xylanase preparation
80 7 (Novozymes NS22036) used in this study is a puried endo-xylan-
ase with a high specicity. This result indicates that the degree of
Glucose yield, %

70
6

Glucose (g/L)
60 interaction between cellulase and xylanase was higher when com-
5 pared with the effect obtained using cellulase and hemicellulase.
50
4 The effect of combination depends on the ratio of the enzymes
40 involved (Nidetzky et al., 1994), as well as on the specic charac-
3
30 teristics of enzymes and substrates (Van-Dyk and Pletschke, 2012).
20 2 Comparing the assays using hemicellulase and xylanase
enzymes (Run 04) and hemicellulase and b-glucosidase enzymes
10 1
(Run 05), xylose yield (8%) and concentration (0.65 g/L) were
0 0 higher in Run 05. This result indicates that the degree of interac-
0 10 20 30 40 50 60 70 80 tion was lower when combining hemicellulase and xylanase com-
Time (h) pared with hemicellulase and b-glucosidase. Regarding the
hemicellulolytic enzymes, three types of synergies have be identi-
(B) 30 2.4 ed, named homeosynergy, heterosynergy and anti-synergy
2.2 (Kovacs, 2009). In the present study, a probable anti-synergism
25 2.0 between hemicellulase and xylanase enzymes occurred. Anti-syn-
1.8 ergism is the term used when one enzyme inhibits the action of
Xylose yield, %

20 1.6 another, for example, when a main-chain cleaving enzyme requires

Xylose (g/L)

1.4 a substituent for its activity and a desbranching enzyme removes

15
10
5 0.4
0 0.0
0 10 20 30 40 50 60 70
Time (h)
Fig. 1. Time course of glucose (A) and xylose (B) released by enzymatic sacchar-
ication at 45 C, 150 rpm and pH 4.8 from pretreated CAB using six enzyme
combinations. (j) Run 01 (cellulase + hemicellulase); (d) Run 02 (cellulase + xylan-
ase); (N) Run 03 (cellulase + b-glucosidase); (.) Run 04 (xylanase + hemicellulase);
(h) Run 05 (hemicellulase + b-glucosidase) and (s) Run 06 (cellulase + xylan-
ase + b-glucosidase).
(cellobiases) (Wilson, 2011). Cellobiohydrolases attack the ends of
cellulose chains while endo-glucanases cleave cellulose chains in
the middle and reduce the degree of polymerization, generating
lower molecular weight oligosaccharides, called celodextrinas
and cellobioses. Cellobiohydrolases may have a preference for
attacking the reducing or the nonreducing ends of cellulose chains
(Van-Dyk and Pletschke, 2012).
In assay 04, conducted only with hemicellulolytic enzymes, the
formation of glucose probably occurred from the hemicellulose
chain, which is a molecule composed of several sugar units such
as pentoses (xylose and arabinose) and hexoses (glucose, mannose
and galactose) and may also provide varying amounts of uronic
acids and deoxyhexose in some types of vegetables (Fengel and
Wegener, 1989). Another possible explanation is the presence of
cellulolytic enzymes in xylanase and/or hemicellulase enzymes
(Saha et al., 2011). As to assay 05, the presence of b-glucosidase
enzyme only catalyzes the breakdown of the amorphous portion
of cellulose, and there was no breakage of the crystalline portion.
The amorphous portion is attacked or digested more easily than
the crystalline portion of the cellulose (Fan et al., 1980).
The combination of cellulase complex with hemicellulase (Run
01) and cellulase complex with xylanase (Run 02) showed the
same glucose yield (70%). However, the xylose yield was higher
in the assay conducted with xylanase, probably due to the fact that
this preparation has higher enzymatic activity. The core enzymes
for degradation of xylan to monomers are endo-xylanases, which
1.2 that substituent (Kovacs, 2009).
1.0 In Run 03, b-glucosidase was added to the cellulose complex
and this supplementation favored glucose and xylose release,
0.8
compared to Run 01 and 02 (combinations of hemicellulase and
0.6
xylanase, respectively). It is probable that supplementation of
b-glucosidases during hydrolysis decreases this inhibition, since
0.2
cellulase activity is hindered by cellobiose and, to a lesser extent,
80
by glucose (Selig et al., 2009; Sun and Cheng, 2002). However,
the best results were obtained in Run 06.
The optimal mixture that maximized the sugar yields was the
combination of cellulase complex, xylanase and b-glucosidase
enzymes (Run 06), with yields of 79% (319.7 mg/gCAB-AHP) and
25% (101.7 mg/gCAB-AHP) of glucose and xylose, respectively
(Fig. 1A and B). It has been suggested that increasing cellulose
accessibility depends not only on how much biomass was removed
but also what component with a specic structure and where it
was removed from (Leu and Zhu, 2013). Hemicellulose, which is
generally found on the outer surface of cellulose bers and also dif-
fused into the interbrillar space through ber pores, has been pro-
posed to act as a physical barrier that limits the cellulose
accessibility. It is well documented that hemicellulose (xylan) acts
as a physical barrier around the cellulose restricting cellulase
access and decreasing hydrolysis efciency (Van-Dyk and
Pletschke, 2012). Therefore, the addition of accessory enzymes
such as xylanase during enzymatic hydrolysis increased cellulose
accessibility as a result of xylan solubilization.
Similar results were obtained by Mcintosh and Vancov (2011),
that described the effect of enzyme mixtures and obtained the
highest yield with the combination of three enzymes (cellulase,
xylanase and b-glucosidase) in enzymatic saccharication of wheat
straw pretreated with diluted alkaline. However, total sugar yields
were lower.
In enzymatic hydrolysates obtained with different combina-
tions of enzyme preparations, also achieved with other carbohy-
drates as arabinose (Runs 03, 05 and 06) and cellobiose (Runs 01,
04 and 06), data not show. Therefore, the enzymatic hydrolysis
from CAB-AHP conducted by the cellulase complex, xylanase and
b-glucosidase enzymes improved glucose and xylose yields, and
it was used in all subsequent steps.
3.2.2. Ratio variation in the best combination of commercial enzymes
The yields and concentrations of glucose and xylose obtained
in the study of enzyme ratios (cellulase complex, xylanase and
254 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259
b-glucosidase) are shown in Table 3. A better understanding of
which enzymes to employ and their proportions is important for lig-
nocellulosic degradation, what could eventually lead to the rational
design of more efcient, and hence less expensive, enzyme mixtures.
The process conducted with a ratio of 0.61 cellulase: 0.39
b-glucosidase (Run 03) promoted substantial sugar release and
yielded better values than the ones in the process conducted with
sole cellulase (Run 07). The addition of b-glucosidase in the hydro-
lysis increased glucose yield to 76.2% and xylose yield to 33.1%, pro-
ducing 6.9 g/L and 1.6 g/L of glucose and xylose in the hydrolysate,
respectively (Table 3). It has been well documented that cellulases
are inhibited by the products of their reactions and by cellobiose
(Meng and Ragauskas, 2014), while b-glucosidase is inhibited by
glucose (Andric et al., 2010). For this reason, b-glucosidase is gener-
ally added to cellulases in bioconversion processes to prevent inhi-
bition of cellulases or to avoid the use of high concentrations of
enzymes in the supplementation of b-glucosidases during hydroly-
sis (Sun and Cheng, 2002; Van-Dyk and Pletschke, 2012).
The lowest glucose yield (approximately 8%) was achieved in
Run 09 (proportion of 1.00 xylanase). The released glucose in this
assay may have been derived from the breakdown of the hemicel-
lulose chain. Hemicellulose is more diverse in structure and com-
position than cellulose and includes xylan, mannan, galactan and
arabinan polymers. Mannan contains sugar such as mannose, gal-
actose and glucose (Van Zyl et al., 2010). Therefore, when only
xylanolytic enzymes were used on the hydrolysis of CAB-AHP, con-
siderable hemicellulose chain breakings occurred. Xylose conver-
sion was 12.5%, being the highest yield in all the assays performed.
The ratio 0.40 cellulase: 0.35 xylanase: 0.25 b-glucosidase
(0.25) (Run 06) showed the highest glucose yield (79.9% corre-
sponding to 7.2 g/L concentration of glucose in the hydrolysate).
The improvement in conversion is highlighted when the xylanolyt-
ic enzymes are present on the hydrolysis of CAB-AHP. A recent
study by Hu et al. (2011) also reported this signicant improve-
ment in cellulose accessibility indicated by Simons stain (Yu
et al., 1995) is due to the increase in ber swelling and porosity
caused by the interaction between xylanase and cellulase. Hemi-
cellulose, which is generally found on the outer surface of cellulose
bers and also diffused into the inter-brillar space through ber
pores, has been proposed to act as a physical barrier that limits cel-
lulose accessibility. Therefore, the addition of accessory enzymes
such as xylanase during enzymatic hydrolysis can increase cellu-
lose accessibility (Meng and Ragauskas, 2014).
The ternary diagrams for optimized ratio of the three commer-
cial enzymes on hydrolysis of CAB-AHP are shown in Fig. 2A and B,
for glucose and xylose yields, respectively. Compared to the glu-
cose ternary diagrams, the xylose diagrams (Fig. 2B) evidenced a
relatively contrary topology, that is, conditions for optimizing the
glucose yield are different from the ones used to optimize xylose
yield. For example, to obtain high yields of glucose, small percent-
ages of xylanase should be used, however for better yields of
xylose, a higher percentage of xylanase should be used in combina-
tion with a small percentage of cellulase.
Digestion with the addition of xylanase and b-glucosidase to the
low cellulase proportion are shown in Table 3 and Fig. 2A. Despite
the conversion being slightly higher than in the experiments using
cellulase only, digestion with more enzyme mixtures resulted in
mild improvements in extending glucose conversion.
In hydrolysis using individual enzymes, a decrease in the yield
and concentration of xylose could be observed, proles that are
represented by light colors areas in Fig. 2B1. This behavior was
not observed regarding the yield and concentration of glucose
(Fig. 2A1 and Table 3). It is observed that using only cellulase com-
plex as a biocatalyst, high yields and glucose concentrations were
obtained. However, glucose yield increased with the addition of
b-glucosidase to the hydrolysis (Run 03). The area shown in
Fig. 2A gets redder with the increase of the enzyme proportion
(until 0.50) in the hydrolysis. The cellulose chain is attacked by cel-
lulases with the formation of cellobiose. Cellobiose cleavage to glu-
cose is catalyzed by b-glucosidase, which also reduces inhibition of
cellulases by cellobiose (Van-Dyk and Pletschke, 2012). Current
market offers many cellulase complexes that contain low levels
of b-glucosidase, leading to a high accumulation of cellobiose in
hydrolysates (Banerjee et al., 2011). Thus, the presence of sufcient
b-glucosidase is important to obtain high yields of glucose in the
biomass-to-ethanol process.
The equations of the surface curves obtained for yields of glu-
cose and xylose yields are shown in Eqs. (4) and (5), respectively.
In these equations (Eqs. (4) and (5)) X, Y and Z represent the pro-
portion of cellulase, xylanase and b-glucosidase, respectively.
Glucose yield % 63:42X 6:14Y 32:05Z 126:38XY
118:99XZ 69:22YZ 4
Xylose yield % 9:3X 14:14Y 0:78Z 8:64XY
99:90XZ 113:96YZ 5
Using Eqs. (4) and (5), the optimized proportion of enzymes to
obtain the highest yield and concentration of glucose is
0.62:0.03:0.34 (cellulase: xylanase: b-glucosidase), and presented
78.5% of maximum glucose release at 72 h. With this optimized
proportion, the xylose yield was 28.8%. However, in the sacchari-
cation process conducted with the enzymes in a ratio of 0.61 cellu-
lase: 0.39 b-glucosidase (Run 03), the conversion of cellulose and
hemicellulose to their respective monomers, glucose and xylose,
were 76.2% (6.9 g/L glucose) and 33.1% (1.6 g/L glucose), respec-
tively. These values correspond to a difference of 2.9% (negative)
and 8.0% (positive) for glucose and xylose yields, respectively, com-
pared to the results obtained in the optimization. With a statistical
analysis, it was observed that addition of xylanase did not show a
signicant difference in the yield of glucose. As glucose is the main
carbohydrate feedstock for the production of ethanol, the yield is
already satisfactory without the supplementation with xylanase
(the amount of enzyme extract used is greater than the volume
necessary to increase the proportion of b-glucosidase of 0.34
0.39), since the addition of this enzyme would increase the cost
of the process, the main factor in the production of second-gener-
ation ethanol (Gnansounou and Dauriat, 2010).
Therefore, the proportion 0.61 cellulase: 0.39 b-glucosidase
improved the glucose yield, and was used in all subsequent steps
of enzymatic hydrolysis from CAB-AHP.
3.2.3. Effect of enzyme loading in enzymatic hydrolysis
Enzyme loading is considered one of the most important factors
in ethanol production of lignocellulosic materials (Meng and
Ragauskas, 2014; Van-Dyk and Pletschke, 2012) and a critical fea-
ture in an effective pretreatment is high sugar yield with low
enzyme loadings (Rocha et al., 2009). The enzyme cost is still a
major hurdle for commercial production of ethanol from any ligno-
cellulosic feedstock.
A study on enzyme loading was done, using a cocktail of two
commercial enzyme preparations at a proportion of 0.61 cellulase
to 0.39 b-glucosidase (condition selected in the previous step). The
sugar yields from CAB-AHP obtained in the study are shown in the
Table 4.
The sugar yield was greater with the increase of enzyme loading
and the highest glucose releases were obtained in assays 14, 15 and
16. In assay 16 (35 FPU/gCAB-AHP cellulose, 77 CBU/gCAB-AHP), the
yield of total sugars was 564 mg per gCAB-AHP, corresponding at
413 mg 15 mg glucose and 151 mg 6 mg xylose (Table 4) at
72 h of hydrolysis, with cellulose and hemicellulose digestibility of
 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259 255

Fig. 2. Ternary diagrams for optimization of mixtures of three enzymes (cellulase, xylanase, b-glucosidase) for release of glucose (A13) or xylose (B13) of the hydrolysis
from CAB-AHP at 45 C, pH 4.8 and 150 rpm. A1 and B1 represent the ternary diagrams and contoured areas. A2 and B2 the surface of the yield obtained for glucose and
xylose, respectively. A3 and B3 represent the ternary diagrams with contours and lines, with the X axes (n) the proportion of cellulase, Y axes (/) proportion of xylanase and Z
axes () the proportion of b-glucosidase. The experimental data represent the ternary diagrams are based in the same experiment shown in Table 3.

100% and 38%, respectively. This corresponds to 70% of conversion of
the total sugar obtained from cellulose and hemicellulose present in
the cashew apple bagasse. The highly reactive nature of the alkaline-
pretreated CAB is demonstrated by its near-complete digestion into
monomeric sugars.
Rocha et al. (2009) also observed an increase in the digestibility
by increasing the enzyme Celluclast 1.5 L loading on the enzymatic
hydrolysis from cashew apple bagasse pretreated with diluted sul-
furic acid. The authors Kinnarinen and Hkkinen (2014) evaluated
the inuence of enzyme loading on enzymatic hydrolysis of
256 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259

Table 4
Effect of enzyme loadings of two commercial enzymes (cellulase complex and b-glucosidase) on the release glucose and xylose in the enzymatic hydrolysis of CAB-AHP at 45 C,
150 rpm for 72 h. Values with different letters represent statistically signicant differences (p < 0.05).

Assays Enzymes load Glucose yield (%) Glucose concentration (g/L) Xylose yield (%) Xylose concentration (g/L)
Cellulase (FPU/gBC-PHA) b-glucosidase (CBU/gBC-PHA)
12A 5.0 11.0 76.2 0.9 6.9 0.1 33.1 0.1 1.6 0.0
13B 15.0 33.0 85.8 0.7 7.7 0.3 54.8 0.3 2.8 0.2
14C 20.0 44.0 98.15 0.6 8.8 0.2 75.7 0.1 4.0 0.1
15C 30.0 66.0 103.0 1.7 9.3 0.0 82.3 2.1 4.2 0.3
16C 35.0 77.0 103.8 1.1 10.0 0.1 83.7 1.6 4.5 0.2

40 40
Glucose D
35 Xylose 35

30 30
Concentration (g/L)

Concentration (g/L)
25 25

C 20
20
d 15
15
A B 10
10 c
b 5
5
a
0
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
1.0 2.0 3.0 4.0
Time (h)
Cellulose loading %
Fig. 4. Ethanol production and substrate consumption proles in SHF process from
Fig. 3. Effect of cellulose loading on sugar release in the enzymatic hydrolysis MCAB-AHP hydrolysate by K. marxianus ATCC 36907. The enzymatic hydrolysis was
(30 FPU/gCAB-AHP cellulase, 66 CBU/gCAB-AHP b-glucosidase, 45 C, 150 rpm, 72 h) conducted at 45 C and 150 rpm for 72 h using loading enzyme of 30 FPU/gCAB-AHP
from cashew apple bagasse pretreated with alkaline hydrogen peroxide (4.3%. v/v; cellulase and 66 CBU/gCAB-AHP b-glucosidase) and the SHF was performed at 30 C
pH 11.5; 35 C, 6 h). Values with different letters represent statistically signicant and 150 rpm for 12 h. (d) Glucose, (.) xylose and (j) ethanol.
differences (p < 0.05).

cardboard waste and the higher cellulase loading largely increased 22

the amount of sugars produced, similar prole to the one obtained 20
in this study.
18
Increasing the enzyme loading by 0.2-fold (of 30 FPU/66 CBU to
35 FPU/77 CBU) failed to improve the nal sugar yield, suggesting 16
Concentration (g/L)

that cellulase loading may have reached its saturation point. 14

Disparities in cellulase saturation loading amongst bagasse, wheat 12
and other herbaceous straw saccharications are well documented
10
and reported to result from variations in enzyme activities and
substrate composition/structure (Vsquez et al., 2007; Chen 8
et al., 2008). 6
Although a greater enzyme loading provides a higher sugar 4
yield, the glucose yields obtained for those three assays (Runs
2
14, 15 and 16) were not signicantly different according to
ANOVA (p < 0.05). Therefore, experiment 15 (30 FPU/gCAB-AHP cel- 0
lulase, 66 CBU/gCAB-AHP b-glucosidase) was chosen and subse- 0 10 20 30 40 50 60 70 80 90 100 110 120
quently used in other steps of study. Increasing cellulose
loading in the process can enhance the yield and rate of hydroly-
Time (h)
sis (Run 16), but increase the cost of the process. Cellulase dosage Fig. 5. Glucose, xylose and ethanol proles in the SSF process of CAB-AHP using
of 1530 FPU/g cellulose is often used in laboratory studies enzyme loadings of 30 FPU/gCAB-AHP cellulase and 66 CBU/gCAB-AHP b-glucosidase
because it provides a hydrolysis prole with high levels of glucose and K. marxianus ATTC 36907 (5 g/L) at 45 C and 150 rpm. (d) Glucose, (.) xylose
yield within a reasonable time (4872 h) at a reasonable enzyme and (j) ethanol.

cost (Sun and Cheng, 2002).

3.2.4. Inuence of the solid loadings in enzymatic hydrolysis hydrolysis leads to low sugar concentration in the hydrolysis liquor
One other main factor that affects the yield and initial rate of and, consequently, to low ethanol concentration in the fermenta-
enzymatic hydrolysis is the substrate (cellulose and/or hemicellu- tion, thus increasing distillation costs. On the other hand, high sub-
lose) concentration in the slurry solution. Low solid loadings in strate concentration can cause inhibition, which substantially
 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259
Fig. 6. Mass balance for pretreatment, hydrolysis and ethanol production using cashew apple bagasse as feedstock.
impacts catalysis rate. Then, in this section, the inuence of
increasing solid (cellulose) loadings was evaluated by varying it
from 1% to 4% w/v cellulose, corresponding at 2.269.04% of CAB-
AHP, with enzymes loadings set on 30 FPU/gCAB-AHP cellulase and
66 CBU/gCAB-AHP b-glucosidase. The higher cellulose loading evalu-
ated was 4% w/v cellulose, since CAB-AHP particles experienced
swelling during the process. In the previous sections, the hydroly-
sis solid concentration was 1% w/v cellulose. The results of the
hydrolysis processes are show in Fig. 3.
As expected, glucose and xylose concentration increased with
increasing cellulose loading, i.e., 36 g/L glucose and 12.5 g/L xylose
were obtained using 4% w/v cellulose, an increase of 75% (glucose)
and 68% (xylose) on sugar concentration, compared to the results
obtained using 1% w/v cellulose (Fig. 3). Other authors also
observed that increasing solid percentage had a positive effect on
glucose production when using sugarcane bagasse (Vsquez
et al., 2007), cashew apple bagasse pretreated with diluted sulfuric
acid (Rocha et al., 2009) and straw (Lpez-Linares et al., 2014).
Glucose and xylose yields for the four assays are signicantly dif-
ferent according to ANOVA (p < 0.05) and Tukey Test. Thus, cellulose
load of 4% w/v cellulose was chosen for this stage of the process.
257
3.3. Ethanol production by SHF and SSF processes using
AHP-pretreated cashew apple bagasse as feedstock
The enzymatic hydrolysate (MCAB-AHP) obtained in the best
conditions evaluated was used as the culture medium for the pro-
duction of ethanol by K. marxianus ATCC 36907 in Separation
Hydrolysis and Fermentation (SHF). The composition of the
MCAB-AHP hydrolysate was 35.7 g/L 0.5 g/L glucose, 13.1
g/L 1.9 g/L xylose and the presence of cellobiose and inhibitors
(formic acid, acetic acid, furfural and hydroxymethylfurfural) was
not identied.
Fig. 4 shows the time proles for glucose, xylose and ethanol
concentrations in the SHF process. The yeast used the glucose
and xylose present in MCAB-AHP medium at 30 C. Glucose was
completely consumed before 6 h of fermentation. Xylose, on the
other hand, presented a slow uptake rate while glucose was avail-
able. After glucose in the medium was exhausted, xylose was con-
sumed and reached 3.7 g/L 0.2 g/L at 12 h. K. marxianus ATCC
36907 produced 15 g/L 0.2 g/L of ethanol at 6 h of fermentation,
giving an ethanol yield based in the consumption of glucose
(YE/G) and total sugar (YE/S) of 0.41 0.06 gethanol/gglucose and
258 J.A.da. Costa et al. / Bioresource Technology 179 (2015) 249259
0.33 0.04 gethanol/gtotal sugar, respectively, and a productivity of
3.75 g/(L h). These results are superior to the ones obtained in
other studies (Rocha et al., 2011).
Integration of process steps is very important in lowering the
cost of the ethanol production from any lignocellulosic feedstock
(Saha et al., 2011). Simultaneous Saccharication and Fermentation
(SSF) process combine enzymatic hydrolysis of cellulose with fer-
mentation of the glucose obtained to ethanol. Thus, the presence
of yeasts along with cellulose reduces the accumulation of this
sugar, thereby increasing saccharication rate and ethanol yields
(Chen et al., 2008). Therefore, ethanol production by K. marxianus
ATCC 36907 was also evaluated for SSF process. This process was
conducted at 45 C using pretreated CAB-AHP (9.04% w/v, corre-
sponding at 4% w/v cellulose) and an enzyme loading of 30 FPU/
gCAB-AHP cellulase and 66 CBU/g CAB-AHP b-glucosidase. Fig. 5 shows
the ethanol, glucose and xylose proles obtained in the SSF process.
The glucose produced was quickly converted to ethanol with no
accumulation in the rst hours of process, while xylose was uti-
lized slower than glucose, a result similar to those obtained by
other authors (Lane and Morrissey, 2010; Lee et al., 2013). A trend
seen throughout this study was the accumulation of glucose in SSF
at 45 C from 54 h to 120 h. This trend is similar to trends seen in
previous studies on SSF with other K. marxianus strains (Pessani
et al., 2011). The reason for this accumulation is not clear, but it
is likely that heat and/or ethanol stress over time may lead to ceas-
ing of fermentation after 72 h.
The highest ethanol concentration (18 g/L 0.41 g/L) was
achieved at 48 h with ethanol yields (based in the CAB-AHP mass)
and productivity of 0.2 gethanol/gCAB-AHP and 0.375 g/(L h), respec-
tively. The efciency of ethanol production was approximately
80% based on the available glucose content from pretreated CAB-
AHP. The experiment conducted up to 80 h did not increase the
level of ethanol production. Comparing the combined time
(72 + 12 = 84 h) required for separate enzymatic hydrolysis (72 h)
and time of fermentation from hydrolysate (12 h), the SSF process
offers advantages within a shorter time (71%, 48 h) required for
obtained higher ethanol concentration.
Thermotolerance of K. marxianus strains has been explored in
many studies (Lane and Morrissey, 2010; Pessani et al., 2011),
but the yield and productivity varies among and within species,
making this search for better strains still needed. The selected
strain here (K. marxianus ATCC 36907) is in agreement with the
most studied strains on SSF process for bioethanol production.
3.4. Overall balance
The Fig. 6 shows the mass balance starting from 100 g of cashew
apple bagasse, performing the pretreatment using alkaline hydro-
gen peroxide H2O2 (4.3% v/v), solid concentration of 5% (w/v) at
35 C, 250 rpm for 6 h, and hydrolysate fermentation after studying
different enzymes and solid loadings. It can be seen that 79.9% of
the cellulose is recovered in the solid fraction and the pretreatment
yield is 37.3 g of pretreated solid and the lignin mass precipitated
was of 12.69 g. Considering the enzymatic hydrolysis, 14.84 g of
glucose is obtained from 100 g CAB, thus reaching a concentration
of 36 g/L in 242.6 g/L of raw bagasse. The bioprocess for the pro-
duction of ethanol using the hydrolysate enzymatic from CAB-
AHP and K. marxianus resulted in 61.8 kg/tonCAB and ethanol yield
of 81.7%. The result was compared with the SSF process and
74.2 kg/tonCAB was obtained.
4. Conclusion
The study demonstrated that preparations with cellulase and
b-glucosidase were the most effective combinations for enzymatic
hydrolysis from cashew apple bagasse pretreated with alkaline
hydrogen peroxide (CAB-AHP). The highest yields attained with
the polysaccharide hydrolyses was obtained at 0.61:0.39 cellulase:
b-glucosidase, enzyme loadings of 30 FPU/gCAB-AHP and 66 CBU/
gCAB-AHP, respectively, and at 4% cellulose in CAB-AHP. The SHF
and SSF processes are promising for application from CAB-AHP in
second-generation ethanol production by K. marxianus ATCC36907,
achieving yields of 61.8 kg/tonCAB and 74.2 kg/tonCAB, respectively.
Acknowledgements
The authors are grateful for the nancial support provided by
the Brazilian research agencies FUNCAP (Process number PJP-
0072-00125.01.00/12) and CAPES. The authors would like to thank
the Novozymes Group for providing the cellulase employed.
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