Biochemical Engineering Journal 51 (2010) 7278

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Biochemical Engineering Journal

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Regular article

Cellulases, xylanases, -glucosidase and ferulic acid esterase produced by

Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane
bagasse
Leda Maria Fortes Gottschalk , Raul Alves Oliveira, Elba Pinto da Silva Bon
Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), Avenida Athos da Silveira Ramos, 149 Bloco A, 5 andar, CEP 21941-909, Cidade Universitria, Rio de Janeiro, RJ, Brazil

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

Article history: Trichoderma reesei and Aspergillus awamori enzymes were concentrated, pooled and assessed for the
Received 10 December 2009 hydrolysis of steam-pretreated sugarcane bagasse. The enzyme prole of T. reesei gave (IU/L): 1700
Received in revised form 26 March 2010 FPA, 20,000 CMCase, 340 -glucosidase and 12,600 xylanase. FPA and CMCase activities that were 4-
Accepted 5 May 2010
fold higher than those of A. awamori (420 and 4900 IU/L, respectively). However the -glucosidase and
xylanase activities were 134- and 6-fold lower than those of A. awamori (45,600 and 79,100 IU/L, respec-
tively). Furthermore, A. awamori produced ferulic acid esterase (160 IU/L) which acts synergistically with
Keywords:
cellulolyticxylanolytic enzymes in the hydrolysis of lignocellulosic materials. The FPA and CMCase activ-
Trichoderma reesei
Aspergillus awamori
ities in the T. reeseiA. awamori blends were enhanced synergistically by 2-fold. Moreover, the hydrolytic
Enzyme blends and synergy effectiveness of the blends was superior to the use of unblended T. reesei or A. awamori enzymes, under
Sugarcane bagasse hydrolysis corresponding conditions (10 FPU/g bagasse, 20 g bagasse/L and 50 C). Hydrolysis experiments, present-
Cellulases ing either 20 or 200 g/L bagasse, resulted in 3.9 or 40 g glucose/L, respectively. These values corresponded
Xylanases to 41% cellulose hydrolysis within 6 or 24 h, respectively. A. awamori enzymes hydrolyzed 91% (1.7 g/L
Ferulic acid esterase xylose) of the residual xylan in the bagasse within 6 h in experiments presenting 20 g/L bagasse.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Plant cell walls are a source of lignocellulosic materials
(biomass), the structure of which is represented primarily by the
physico-chemical interaction of cellulose (a linear glucose poly-
mer) with hemicellulose (a highly branched heteropolymer of
xylose, mannose, galactose, arabinose, glucose and various types
of uronic acids) and lignin (a very high molecular weight, cross-
linked aromatic molecule). Depending on the type of sugar that
predominates, hemicelluloses, which bridge cellulose and lignin,
are referred to as xylans, mannans or galactans [1].
Photosynthesis produces 200 billion tons of biomass per year,
from which 34% is used currently by man for food or non-food
purposes [2,3]. Worldwide attention has focused on the major
biotechnological uses of the carbohydrates in biomass as biomass
syrups that have sugars with ve or six carbons (derived from xylan
and glucan) can be used as carbon sources in industrial fermen-
tations that produce antibiotics, industrial enzymes, amino-acids
Corresponding author at: Biochemistry Department, Chemistry Institute,
Federal University of Rio de Janeiro, Avenida Athos da Silveira Ramos, 21941-909,
Rio de Janeiro, Brazil. Tel.: +55 21 25627358; fax: +55 21 25627356.
E-mail address: ledagott@gmail.com (L.M.F. Gottschalk).
and bulk chemicals, including ethanol. For these purposes, how-
ever, the polysaccharides in the biomass must rst be hydrolyzed.
Enzymatic hydrolysis is advantageous over chemical conversion,
as substrate is not lost due to chemical modications and moder-
ate and non-corrosive physicalchemical operating conditions can
be used. During enzymatic hydrolysis, the use of cellulases, hemi-
cellulases and auxiliary enzymes, such as ferulic acid esterases, is
necessary to break the lignocellulosic composite structure of the
biomass [4].
Efcient cellulose hydrolysis requires the cooperative action
of endoglucanases (E.C. 3.2.1.4), which hydrolyze the cellulose
polymer internally, exposing reducing and non-reducing ends,
and exoglucanases or cellobiohydrolases (E.C. 3.2.1.91), which act
on the reducing and non-reducing ends, releasing cellobiose and
cellooligosaccharides. The cellulose hydrolysis process is nal-
ized through the action of -glucosidase (E.C. 3.2.1.21), which
cleaves cellobiose, liberating two molecules of glucosethe end
product [5,6]. Xylan is degraded by the depolimerizing endo-
acting xylanase (E.C. 3.2.1.8), and exo-acting -xylosidase (E.C.
3.2.1.37) and debranching enzymes such as acetyl-xylan esterase
(E.C. 3.1.1.72), -arabinofuranosidase (E.C. 3.2.1.55), acetyl mannan
esterase (E.C. 3.1.1.6), feruloyl esterases (E.C. 3.2.1.73) p-coumaroyl
esterases (E.C. 3.2.1.73) and -glucuronidase (E.C. 3.2.1.139). Fer-
ulic acid esterase (FAE) can release ferulic acid (FA) bound to the

1369-703X/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2010.05.003
 L.M.F. Gottschalk et al. / Biochemical Engineering Journal 51 (2010) 7278
arabinose side chains of hemicellulose [7]. Enzyme blends that
present cellulases, xylanases and also FAEs improve the degrada-
tion of plant cell walls [8,9] due to increased accessibility.
Enzymes for the degradation of the polysaccharide part of
biomass have been produced, mostly by fungi belonging to the
genus Trichoderma [10]. Even though Trichoderma reesei has been
considered the best microorganism to carry out cellulose degrada-
tion, several works have claimed that this fungus is inefcient for
breaking down lignocellulosic substrates into glucose molecules,
due to its low -glucosidase titre (0.1 IU/mL) [11,12]. To over-
come the Trichoderma enzyme pool deciency studies have been
carried out on the supplementation of the Trichoderma enzymes
by Aspergillus enzymes that typically present high levels of -
glucosidase. As such, Trichoderma viride and Aspergillus ustus
enzymes were separately produced, blended and used for the sac-
charication of sugarcane bagasse [13]. Moreover Trichoderma and
Aspergillus enzymes were produced by mixed fungal cultures of T.
reesei and Aspergillus niger in solid substrate fermentation [14] and
in a stirred tank bioreactor [15]. Submerged shake asks co-culture
of T. reesei and Aspergillus phoenicis are also reported [12].
Sugars syrups, which are derived from the hydrolysis of biomass,
can be used for the production of ethanol. This is currently a large-
scale option that can be used by the transportation sector to ght
climate change through the decrease of greenhouse gas (GHG)
emissions. Ethanol is currently produced through the conversion of
carbohydrates from dedicated crops such as corn (USA), sugarcane
(Brazil) and sugar beet (Europe). However, the use of land for these
fuels competes with the food supply and environmental preserva-
tion. As such, efforts have been made towards the development and
implementation of advanced process technologies to produce cel-
lulosic ethanol from low value agricultural co-products or wastes
such as corn stover, wheat straw, sugarcane bagasse and straw, or
wood wastes [16]. The potential use of these residues worldwide
is indeed signicant; in Brazil the agroindustry of corn, sugarcane,
rice, cassava, wheat, citrus, coconut and grass collectively generate
597 million tons of residues per year [17].
Brazilian sugar and alcohol industries generate 195 million tons
of sugarcane bagasse per year [17], and these have been burnt in
mills inefciently for energy cogeneration and as a way to reduce
the bagasse disposal problem. Despite this, a 12% surplus remains
unexploited and could be used as a raw material for the produc-
tion of lignocellulosic ethanol, thereby increasing fuel production
per planted area [18]. Moreover, an increase of from 12% to 50% in
the bagasse surplus has been forecasted, upon the increase in the
efciency of boilers and in the electricity generation system [19].
To bioconvert lignocellulosic biomass into sugars customized,
effective and economical pretreatments need to be developed such
that a high yield of fermentable sugars can be obtained in the sub-
sequent enzymatic hydrolysis step; pretreatment conditions must
also be well balanced to avoid the formation of inhibitors of the
hydrolysis and fermentation processes of the biomass [20]. More-
over, low cost, stable and efcient enzymes are also of paramount
importance, as the high cost of enzymes restricts their use in large-
scale applications for the conversion of lignocellulosic materials.
The present work studied the effectiveness of enzyme prepara-
tions produced by either T. reesei or Aspergillus awamori cultures,
or that of different enzyme blends from both fungi, to hydrolyze
steam-treated sugarcane bagasse, which is produced industrially
in Brazil to be used as cattle feed. The enzymes were concentrated
using ultraltration and blended to obtain preparations with dif-
ferent levels of exo- and endoglucanases, -glucosidase, xylanase
and ferulic acid esterase. The hydrolytic efciency of the enzyme
preparations was quantied using HPLC measurements of glu-
cose, cellobiose and xylose released from the bagasse. It was also
investigated the synergism between the T. reesei and A. awamori
enzymes.
73
2. Materials and methods
2.1. Pretreated sugarcane bagasse
Pretreated sugarcane bagasse was kindly provided by the sugar
and ethanol industry, Usina Vale do Rosrio (Santa Elisa Vale Con-
glomerate), located in Morro Agudo, So Paulo State, Brazil. This
plant processes 250 tons of bagasse per day, during the harvest
season, to be used as cattle feed. The bagasse is pretreated with
steam at 200 C for 7 min (average pressure of 15 atm), followed by
gradual decompression. In our laboratory, the bagasse was washed
with warm distilled water to remove residual soluble sugars and
dried overnight at 60 C. The carbohydrate and lignin content of
the unpretreated and pretreated bagasse samples were determined
using the NREL procedure [21].
2.2. Fungal propagation and enzyme production
Stock cultures of T. reesei RUT-C30 (ATCC 56765) and A. awamori
2B.361 U2/1 [22,23] were propagated on potato dextrose agar
(PDA) plates at 30 C for 7 days. To prepare the seed culture, the
conidia from sporulating cultures were suspended in 5 mL of sterile
water, and 1 mL of the suspension (106 spores) was transferred to a
500 mL Erlenmeyer ask containing 100 mL of the growth medium,
which was incubated in an orbital shaker for up to 3 days at 30 C
and 200 rpm. A total volume of 30 mL of the mycelium suspen-
sion (obtained from the seed cultures) was used to initiate cell
growth in 1 L asks, containing 300 mL of the growth medium. The
growth media composition used for both, the seed culture and for
enzyme production by T. reesei (A) and A. awamori (B) were as fol-
lows: medium A (g/L): 30 lactose, 6 yeast extract, 0.3 urea, and
0.6% (v/v) corn steep liquor; plus salts (g/L): 1.4 (NH4 )2 SO4 , 2.0
KH2 PO4 , 0.3 CaCl2 and 0.3 MgSO4 7H2 O; and trace elements (mg/L):
5 FeSO4 7H2 O, 20 CoCl2 , 1.6 MnSO4 and 1.4 ZnSO4 [24]. Medium
B (g/L): 30 wheat bran, 6 yeast extract, 1.2 NaNO3 , 3 KH2 PO4 ,
6 K2 HPO4 , 0.2 MgSO4 7H2 O and 0.05 CaCl2 2H2 O. The asks were
incubated for up to 7 days, and the production of enzymes was
monitored by periodical sampling. The cells were separated by l-
tration using glass lter membranes, and the culture supernatants
were concentrated by ultraltration (Stirred Cells, Amicon) using
a 10-kDa cut-off membrane. T. reesei and A. Awamori supernatants
were concentrated 2- and 3-fold, respectively.
2.3. Enzyme activity assays
The lter paper (FPU), endo-1-4--glucanase (CMCase) and
-glucosidase (BGU) activities were determined using standard
IUPAC procedures and expressed in international units (IU) [25].
Reducing sugars were estimated according to Miller [26], using
glucose as standard. Glucose concentration was measured using
a Biochemistry Analyzer YSI 2700 Select. Xylanase activity was
determined by mixing 50 l of the enzyme preparation to 100 l
of soluble oat spelts xylan (1%, w/v) in 100 mM sodium acetate
buffer, pH 5.0 at 50 C for 30 min. Reducing sugars were deter-
mined according to Miller [26], using xylose as the standard. One
unit of xylanase activity was dened by the formation of 1 mol
of reducing sugar per minute. Ferulic acid esterase (FAE) activity
was assayed by measuring the release of ferulic acid in a reaction
mixture containing 10 l of the enzyme preparation, 20 l of 1%
ethyl ferulate in dimethylsulphoxide (DMSO), 100 l of 1 M acetate
buffer (pH 5.0), and 870 l of water. After incubation at 50 C for
20 min, the reaction was terminated by boiling the mixture for
5 min, followed by the quantication of ferulic acid by HPLC. One
unit of FAE corresponded to the formation of 1 mol of ferulic acid
per minute.
74 L.M.F. Gottschalk et al. / Biochemical Engineering Journal 51 (2010) 7278
2.4. Preparation of T. reesei and A. awamori enzyme blends and
enzyme activity synergism
Enzyme blends were obtained by mixing the concentrated
supernatants of the two fungi cultures at the following T. reesei:A.
awamori ratios: 25:75, 40:60, 50:50, 60:40 and 75:25. The FPA,
CMCase, -glucosidase, xylanase and FAE activities were measured
in the individual T. reesei and A. awamori concentrated supernatants
as well as in all enzyme blends. To investigate the existence of
activity synergism in the blends, the different theoretical activities,
calculated as follows: (T. reesei ratio T. reesei enzyme activity) + (A.
awamori ratio A. awamori enzyme activity), were compared to the
actual measured activity. Synergism was expressed as percentage
of the theoretical activity.
2.5. Sugarcane bagasse hydrolysis experiments
Hydrolyses were carried out in 250 mL Erlenmeyer asks con-
taining 100 mL of the reaction mixture (20 g bagasse/L, suspended
in 50 mM sodium citrate buffer, pH 4.8), which were incubated at
50 C and 200 rpm for 72 h in an orbital shaker. The experiments
used either T. reesei or A. awamori enzyme concentrates, or blends
of T. reesei and A. awamori enzyme at ratios of 25:75, 50:50 or 75:25.
The hydrolysis experiments were designed to present, in all cases,
10 FPU/g of pretreated bagasse. All experiments were performed
in triplicate, and the average values (standard deviations were less
than 4%) were reported.
The blend presenting T. reesei:A. awamori in the ratio 75:25
(10 FPU/g and 5 BGU/g) was used in hydrolysis experiments pre-
senting 200 g bagasse/L, which were carried out in an agitated
reactor BIOSTAT BPLUS 1 L MO. Experiments were initiated
with 100 g bagasse/L (750 mL, 50 C and 100 rpm) followed, after
4 h, by a continuous fresh bagasse supply, up to 100 g, parallel
to the supply of the corresponding enzyme load. In all of the
hydrolysis experiments, samples were withdrawn regularly and
centrifuged to separate lignin plus residual polysaccharides from
the glucosexylose rich supernatant (i.e., the biomass syrup).
2.6. HPLC analysis
Glucose, cellobiose and xylose were analyzed using high-
performance liquid chromatography (HPLC) equipped with a
Lichorospher 100 NH2 5 m column at 35 C. The samples were
eluted using a mobile phase of acetonitrile:water (80:20) at a ow
rate of 1.5 mL/min. A refraction index detector was used to mea-
sure the sugars. Ferulic acid was analyzed using HPLC equipped
with a Lichrocart-250-4.6 PurospherStar RP-18e (5 m) column
at 25 C. Samples were eluted using a mobile phase of acetoni-
trile:0.5% methanol in 0.01 M H3 PO4 (with an gradient of 96.5%
acetonitrile at 0 min; 90% acetonitrile at 11 min; 80% acetonitrile
at 22.5 min and 96.5% acetonitrile at 23.5 min), using a ow rate
of 1.0 mL/min until 24.5 min. A diode array detector was used for
measurements at 320 nm.
3. Results and discussion
3.1. Effect of steam treatment on sugarcane bagasse
The normalized composition of unpretreated and pretreated
bagasse (Table 1) indicates that the steam treatment alters the ini-
tial composition of the biomass such that the xylan content was
decreased by 54% and the glucan and lignin content was increased
by 17% and 54%, respectively. Furthermore, the pretreatment have
either fully removed the galactan, arabinan and mannan content in
the bagasse or have decreased these components to undetectable
levels.
Table 1
The dry matter-based composition of the biomass components (%) of steam-treated
bagasse.
Biomass component Unpretreated bagasse Pretreated bagasse
Glucan 41.4 0.1 48.4 0.3
Xylan 22.5 0.2 10.3 0.1
Galactan 1.3 0.05 0
Arabinan 1.3 0.04 0
Mannan 3.4 0.04 0
Lignin + ash 23.6 34
3.2. T. reesei and A. awamori enzyme production and activity
proles
In this study the T. reesei and A. awamori enzymes were produced
separately in spite of previous reports regarding enzyme produc-
tion by mixed cultures of T. reeseiA. niger [14,15], and T. reeseiA.
phoenicis [12] aiming mostly to complement the Trichoderma cel-
lulase enzymes with the Aspergillus -glucosidase. However, as the
A. awamori strain used in this work was found to produce, besides
high levels of -glucosidase and xylanase, cellulases and ferulic
acid esterase, it was carried out, beforehand, a medium optimiza-
tion study to maximize the A. awamori enzyme pool accumulation.
Culture conditions for cellulases accumulation by T. reesei, which
have been very much studied, were used in this work [27]. As a
result maxima enzyme accumulation by A. awamori and T. reesei
was achieved in growth media with different compositions, mostly
regarding the main carbon source that was found to be wheat bran
for A. awamori and lactose for T. reesei. Moreover, whereas lac-
tose induced T. reesei cellulases, as expected [27], it repressed A.
awamori enzymes (unpublished data from our laboratory). It has
been reported, though, the use of lactose fed batch fermentation
on mixed culture of T. reesei RUT-C30 and Aspergillus niger LMA
[15] that may have lessened the lactose repressive effect on the
Aspergillus culture.
In the T. reesei cultures, peak enzyme activities (IU/L): FPA
1200; CMCase 15,000; -glucosidase 150 and xylanase 10,000,
were detected within 5 days incubation, whereas in the A. awamori
cultures, peak activities (IU/L): FPAse 150; CMCase 2500; -
glucosidase 18,800; xylanase 25,000 and FAE 160 were detected
within 7 days.
The activity prole of the T. reesei enzymes preparation,
which were ultraltrate (IU/L): FPA 1700; CMCase 20,000; -
glucosidase 340; xylanase 12,600, showed activities of FPA and
CMCase that were 4 times higher than those in A. awamori
(420 and 4900 IU/L, respectively) as it is shown in Table 2. The
activities of -glucosidase and xylanase were 134 and 6 times
lower than those in A. awamori (45,600 and 79,100 IU/L, respec-
tively). As such, the activity proles of T. reesei RUT-C30 and
A. awamori 2B.361 U2/1 were quite complementary concerning
the main enzymatic activities that are required to hydrolyze cel-
lulose and xylan. Moreover, and in accordance to a previous
work, which reports FAE as an Aspergillus exoenzyme [28], A.
awamori produced high levels of ferulic acid esterase (160 IU/L),
an enzymatic activity that was not detected in the Trichoderma
supernatant. As FAE cleaves the linkages between xylan and lignin,
it creates more sites for the hydrolysis of glucan and xylan,
enhancing the effectiveness of the cellulase and xylanase enzyme
blends.
3.3. The activity prole of the T. reesei and A. awamori blends and
the synergistic activity of the enzyme blends
Table 2 presents the levels of FPA, CMCase, -glucosidase and
xylanase in the A. awamori and T. reesei concentrated enzyme
 L.M.F. Gottschalk et al. / Biochemical Engineering Journal 51 (2010) 7278 75

Table 2
The levels of FPA, CMCase, -glucosidase and xylanase in the A. awamori and T. reesei concentrated enzyme preparations and in the enzyme blends.

Enzyme blends Activities

Filter paper (FPU/L) Carboxymethyl cellulase (IU/L) -Glucosidase (IU/L) Xylanase (IU/L)

T. reesei 1700 20,000 340 12,600

A. awamori 420 4900 45,600 79,100
25/75 T. reesei/A. awamori 1550 16,820 32,790 67,500
40/60 T. reesei/A. awamori 1900 22,160 27,760 54,960
50/50 T. reesei/A. awamori 2120 23,550 22,790 52,160
60/40 T. reesei/A. awamori 2340 25,490 19,380 42,490
75/25 T. reesei/A. awamori 2390 31,300 12,090 31,970

Fig. 1. Synergistic enhancement of the FPA, CMCase, -glucosidase and xylanase
activities using mixtures of the concentrated T. reesei and A. awamori preparations
at ratios of 25:75, 40:60, 50:50, 60:40 and 75:50.
Fig. 2. The concentration of glucose (lled symbols) and cellobiose (open sym-
bols) in the hydrolysate during enzymatic hydrolysis of the steam-treated sugarcane
bagasse using enzymes produced by T. reesei ( and ) or by A. awamori ( and ).
The substrate and enzyme concentrations were 20 g/L and 10 FPU/g, respectively.

3.4. The hydrolysis of sugarcane bagasse using T. reesei, A.

preparations and in the blends. In all the T. reeseiA. awamori
enzyme blends, it was observed a synergistic enhancement of FPA
(exoglucanase) activity within the range of 83103%, and of CMCase
(endoglucanase) within the range of 73110% (Fig. 1). T. reesei
RUT-C30 secretes two cellobiohydrolase (CBH), ve endo-1-4--
glucanases (EG), a number of endo-1-4--xylanases and at least
one -xylosidase [29] whereas Aspergillus species are well known
to produce -glucosidase, -xylanases and ferulic acid esterase [6].
The A. awamori strain used in this study, besides producing very
high levels of the aforementioned enzyme, also produced impor-
tant levels of endoglucanase and exoglucanase. Although the FPA
and CMCase synergistic activity enhancement was likely related
to the A. awamori -glucosidase supplement, which responded, in
the blends, for FPU:BGU ratios within 1:20 to 1:5, the A. awamori
cellulases might also have been involved in the observed syner-
gism. Further chemical and biochemical characterization of the A.
awamori cellulases could help to explain how the different enzy-
matic proteins showing FPA and CMCase activities, produced by
both fungi, would cooperate advantageously for cellulose hydroly-
sis [30,31]. The synergy results are of economical importance as a
lower enzymatic protein load can respond for the required enzyme
activity load. No synergism was observed for the activities of -
glucosidase and xylanase.
awamori or T. reesei:A. awamori enzyme blends
Table 3 presents the cellulases, xylanase, -glucosidase and fer-
ulic acid esterase activity levels per gram of bagasse for all of the
enzyme preparations that were used in the hydrolysis experiments.
The preparations presented the same FPA load (10 IU/g) and differ-
ent loads of endoglucanases (from 111 to 170 IU/g), -glucosidase
(from 2 to 1086 IU/g), xylanases (from 74 to 1883 IU/g) and fer-
ulic acid esterase (from 0 to 3.8 IU/g). Fig. 2 shows the hydrolysis
time course when either A. awamori or T. reesei enzymes were used.
Hydrolysis using the A. awamori enzymes resulted in the forma-
tion of 3.5 g/L glucose within 72 h, corresponding to 37% cellulose
hydrolysis. As expected, it was not observed the accumulation of
cellobiose, due to the high -glucosidase load (1086 IU/g bagasse).
Hydrolysis using the T. reesei enzyme resulted in the formation of
6.3 g/L glucose, corresponding to 65% cellulose hydrolysis. It was
observed the accumulation of up to 1.0 g/L cellobiose in the ini-
tial stages of hydrolysis due to the poor -glucosidase load (2 IU/g
bagasse). These results fully agree to previous studies that reported
the requirement for supplementing T. reesei cellulases with -
glucosidase to overcome its low -glucosidase titres [32], This
initial phase of cellobiose accumulation was followed by a gradual
decrease in the concentration of cellobiose over 72 h. Although pre-

Table 3
The level of cellulases, xylanase, -glucosidase and ferulic acid esterase activities per gram of biomass for all enzyme preparations that were used for the hydrolysis of the
pretreated sugar cane bagasse. All of the hydrolysis experiments presented 10 FPU/g bagasse.

Enzyme Activities

Filter paper (FPU/g) Carboxymethyl cellulase (IU/g) -Glucosidase (IU/g) Xylanase (IU/g) Ferulic acid esterase (IU/g)

T. reesei 10 118 2 74 0.0

A. awamori 10 117 1086 1883 3.8
T. reesei: A. awamori 25:75 10 170 437 843 1.6
T. reesei: A. awamori 50:50 10 111 205 489 0.7
T. reesei: A. awamori 75:25 10 122 50 249 0.2
76 L.M.F. Gottschalk et al. / Biochemical Engineering Journal 51 (2010) 7278

Fig. 4. The concentration of xylose in the hydrolysate during enzymatic hydrolysis

Fig. 3. The concentration of glucose in the hydrolysate during enzymatic hydrolysis
of the steam-treated sugarcane bagasse using the enzymes produced by T. reesei
(), A. awamori (), and in the enzyme blends of the T. reesei and A. awamori cel-
lulases at ratios of 25:75 (), 50:50 () and 75:50 (). The substrate and enzyme
concentrations were 20 g/L and 10 FPU/g, respectively.
vious studies reported that cellobiose accumulation causes severe
feedback inhibition of endoglucanase and exoglucanase [12,32], the
decrease in cellobiose concentration, over the hydrolysis experi-
ment, did not improve the bagasse hydrolysis rate.
Interestingly, nevertheless the T. reesei or A. awamori experi-
ments presented the same FPU load (10 FPU/g bagasse) the T. reesei
cellulases showed to be much more efcient than the A. awamori
cellulases.
The enzymatic saccharication of alkali pretreated sugarcane
bagasse by a mixture of cellulases produced by T. viride and A. ustus,
has been reported [13]. Nevertheless the observed hydrolysis yield,
of 90%, was higher than that observed in the present study, of 80%,
the differences in the sugarcane pretreatment conditions hinder the
comparison of the enzyme blends effectiveness nevertheless both
works used comparative saccharication conditions regarding the
biomass enzyme load and incubation temperature. However, as A.
ustus is an animal and plant pathogen [33,34] its application may
not be feasible.
Hydrolysis experiments using the blends of the T. reesei and A.
awamori enzymes at ratios of 25:75, 50:50 and 75:25 resulted in
comparable glucose concentrations. These lay within the range of
6.57.2 g/L, corresponding, after 48 h, to cellulose hydrolysis yields
of 6774%, respectively. The yields increased to 80% after 72 h of
incubation (Fig. 3 and Table 4). It was observed, in all cases, a fast
of the steam-treated sugarcane bagasse using the enzymes produced by T. reesei
(), A. awamori (), and in the enzyme blends of the T. reesei and A. awamori cel-
lulases at ratios of 25:75 (), 50:50 () and 75:50 (). The substrate and enzyme
concentrations were 20 g/L and 10 FPU/g, respectively.
hydrolysis rate within the rst 6 h of the reaction (the average rate
was 0.65 g glucose/L h), corresponding to 41% cellulose hydroly-
sis. This rst phase was followed by a slow phase (rate of 0.074 g
glucose/L h) that lasted up to 72 h. No cellobiose accumulation
was observed when the T. reesei:A. awamori enzyme blends were
used. These data, collectively, indicated that all blends presented
the minimum necessary load for all enzyme activities. Further
work will be carried out increasing the T. reesei:A. awamori ratio,
above 75:25, aiming to reach the minimum proportion for the A.
awamori enzymes pool supplementation to the T. reesei enzymes
pool.
The initial rates for xylan hydrolysis and the xylose nal con-
centrations were higher and equivalent for the T. reesei:A. awamori
blends of 25:75 and 50:50 and also for the individual use of the A.
awamori preparation. Xylan hydrolysis may have been favoured by
FAE activity that cleaves diferulic bridges between the xylan chains,
besides releasing lignin structures from the xylan. It was observed a
nal xylose concentration of 1.6 g/L, corresponding to 91% hydroly-
sis of the bagasse residual xylan within 72 h (Fig. 4 and Table 5). The
xylan hydrolysis rate was approximately 0.2 g of xylose/L h for the
rst 6 h, followed by a slow phase (at a rate of 0.009 g of xylose/L h)
that lasted for up to 72 h. These results indicate that most of the
hemicellulose was degraded within the rst 6 h. The lower xylan
hydrolysis yields observed with the individual use of the T. ree-
sei enzymes or with the T. reesei:A. awamori blend of 75:25 could
relate to xylanases loads lower than 249 IU/g bagasse and or to the
poor effectiveness of the T. reesei xylanases, besides the low ferulic

Table 4
Glucose hydrolysis yields (expressed as the percentage of the theoretical yields) after 6, 24, 48 and 72 h of sugarcane bagasse hydrolysis.

Time (h) Glucose hydrolysis yield (%)

T. reesei A. awamori 25/75 T. reesei/A. awamori 50/50 T. reesei/A. awamori 75/25 T. reesei/A. awamori

6 17.7 0.3 7.8 0.1 33.4 0.3 37.3 0.1 40.7 3.1
24 40.0 0.4 20.1 0.2 59.7 0.5 59.1 0.2 54.1 0.3
48 52.6 0.9 30.7 0.2 67.3 1.8 74.0 0.6 69.2 1.5
72 65.9 1.1 36.6 0.1 80.1 2.6 81.1 1.2 80.8 2.2

Table 5
Xylose hydrolysis yields (expressed as the percentage of the theoretical yields) after 6, 24, 48 and 72 h of sugarcane bagasse hydrolysis.

Time (h) Xylose hydrolysis yield (%)

T. reesei A. awamori 25/75 T. reesei/A. awamori 50/50 T. reesei/A. awamori 75/25 T. reesei/A. awamori

6 43.1 0.4 65.1 0.3 61.2 0.3 73.2 2.8 53.2 3.1
24 55.7 1.4 73.4 1.8 76.8 0.2 78.9 0.4 62.3 1.1
48 63.8 0.5 78.2 0.5 81.1 1.6 85.9 0.7 71.3 1.2
72 70.1 0.6 87.4 4.0 91.3 3.5 91.0 0.5 82.0 0.9
 L.M.F. Gottschalk et al. / Biochemical Engineering Journal 51 (2010) 7278 77

Table 6
Glucose hydrolysis yields (expressed as the percentage of the theoretical yields) after 24, 48 and 72 h of hydrolysis, using different lter paper activity enzyme loadings (5,
10, 15 and 20 FPU/g) and a bagasse concentration of either 20 or 200 g/L.

Enzyme load (FPU/g bagasse)/bagasse concentration (g/L) Glucose hydrolysis (%)

24 h 48 h 72 h

5/20 38.1 0.4 52.1 0.2 50.9 1.1

10/20 54.1 0.3 69.2 1.5 80.8 2.2
15/20 57.3 1.0 72.1 1.2 74.3 2.4
20/20 61.0 1.3 74.3 1.5 78.3 3.3
10/200 43.1 1.5 60.2 0.4 68.4 1.1

In hydrolysis experiments presenting 200 g bagasse/L it was

observed a signicant decrease of the hydrolysis yields that was
of 68%, in 72 h, in comparison to the observed 81% yield for 20 g
bagasse/L (Table 6). Similar results have been observed for other
lignocellulosic materials [32,35] and may be related to end product
feedback inhibition of cellulases by high glucose concentration.

4. Final remarks

This study showed the efciency of blends of enzyme produced

Fig. 5. The concentration of reducing sugars in the hydrolysate during enzymatic
hydrolysis of the steam-treated sugarcane bagasse using the enzymes produced by
T. reesei (), A. awamori (), and in the enzyme blends of the T. reesei and A. awamori
cellulases at ratios of 25:75 (), 50:50 () and 75:50 (). The substrate and enzyme
concentrations were 20 g/L and 10 FPU/g, respectively.
acid esterase activity of 0.2 IU/g bagasse in the T. reesei:A. awamori
blend.
The time course for the reducing sugars concentration resulting
from the bagasse (Fig. 5) was always higher than that of the corre-
sponding glucose concentrations and corresponded to the sum of
glucose, xylose and cellobiose concentrations, indicating that there
was no accumulation of oligosaccharides.
It was evaluated the hydrolytic effectiveness of the T. reesei:A.
awamori 75:25 blend with FPA loads ranging from 5 to 20 FPU/g and
a xed -glucosidase/FPA ratio of 5. As shown in Fig. 6, the glucose
concentration increased markedly by the use of the cellulase load of
10 FPU/g bagasse compared to 5 FPU/g whereas from 10 to 20 FPU/g,
the glucose concentration levelled off. It was observed 81% cellulose
hydrolysis within 72 h, conrming the data presented in Table 4.
by the fungi T. reesei RUT-C30 and A. awamori 2B.361 U2/1 for the
hydrolysis of steam-pretreated sugarcane bagasse, which reached
81% cellulose hydrolysis and 91% xylan hydrolysis in reaction mix-
tures presenting 20 g bagasse/L, 10 FPU/g bagasse, incubated at
50 C for 72 h. It was also observed 68% cellulose hydrolysis, in reac-
tion mixtures presenting 200 g bagasse/L. The use of the T. reesei:A.
awamori enzyme blends at the ratios of 75:25; 50:50 and 25:75
resulted in equivalent data for hydrolysis rates and yields. It was
observed a 2-fold synergistic enhancement of the lter paper (FPA)
and endoglucanase (CMCase) activities in all the T. reesei:A. awamori
enzyme blends. The enzymes load, per gram of sugarcane bagasse,
of the T. reesei:A. awamori 75:25 blend were as follows: 10 FPU; 120
CMCase; 50 BGU; 250 xylanase; 0.2 FAE. Enzymes from the T. reesei
RUT-C30 were more effective for the cellulose bagasse hydrolysis
whereas A. awamori 2B.361 U2/1 enzymes were more effective for
the xylan bagasse hydrolysis.
Acknowledgments
This work was supported by the Research and Projects Financing
(FINEP), the Brazilian Research Council (CNPq) and by the Brazilian
Foundation for Graduate Students (CAPES). Usina Vale do Rosrio
is gratefully acknowledged for supplying the steam-pretreated
bagasse. The authors are thankful to Dr. Marlia Martins Nishikawa
from the National Institute of Quality Control in Heath of Oswaldo
Cruz Foundation (FIOCRUZ) for the fungi strains preservation and
validation.

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