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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 and bulk chemicals, including ethanol. For these purposes, how-
ever, the polysaccharides in the biomass must rst be hydrolyzed.
Plant cell walls are a source of lignocellulosic materials Enzymatic hydrolysis is advantageous over chemical conversion,
(biomass), the structure of which is represented primarily by the as substrate is not lost due to chemical modications and moder-
physico-chemical interaction of cellulose (a linear glucose poly- ate and non-corrosive physicalchemical operating conditions can
mer) with hemicellulose (a highly branched heteropolymer of be used. During enzymatic hydrolysis, the use of cellulases, hemi-
xylose, mannose, galactose, arabinose, glucose and various types cellulases and auxiliary enzymes, such as ferulic acid esterases, is
of uronic acids) and lignin (a very high molecular weight, cross- necessary to break the lignocellulosic composite structure of the
linked aromatic molecule). Depending on the type of sugar that biomass [4].
predominates, hemicelluloses, which bridge cellulose and lignin, Efcient cellulose hydrolysis requires the cooperative action
are referred to as xylans, mannans or galactans [1]. of endoglucanases (E.C. 3.2.1.4), which hydrolyze the cellulose
Photosynthesis produces 200 billion tons of biomass per year, polymer internally, exposing reducing and non-reducing ends,
from which 34% is used currently by man for food or non-food and exoglucanases or cellobiohydrolases (E.C. 3.2.1.91), which act
purposes [2,3]. Worldwide attention has focused on the major on the reducing and non-reducing ends, releasing cellobiose and
biotechnological uses of the carbohydrates in biomass as biomass cellooligosaccharides. The cellulose hydrolysis process is nal-
syrups that have sugars with ve or six carbons (derived from xylan ized through the action of -glucosidase (E.C. 3.2.1.21), which
and glucan) can be used as carbon sources in industrial fermen- cleaves cellobiose, liberating two molecules of glucosethe end
tations that produce antibiotics, industrial enzymes, amino-acids 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
Corresponding author at: Biochemistry Department, Chemistry Institute,
esterase (E.C. 3.1.1.6), feruloyl esterases (E.C. 3.2.1.73) p-coumaroyl
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.
esterases (E.C. 3.2.1.73) and -glucuronidase (E.C. 3.2.1.139). Fer-
E-mail address: ledagott@gmail.com (L.M.F. Gottschalk). 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 73

arabinose side chains of hemicellulose [7]. Enzyme blends that 2. Materials and methods
present cellulases, xylanases and also FAEs improve the degrada-
tion of plant cell walls [8,9] due to increased accessibility. 2.1. Pretreated sugarcane bagasse
Enzymes for the degradation of the polysaccharide part of
biomass have been produced, mostly by fungi belonging to the Pretreated sugarcane bagasse was kindly provided by the sugar
genus Trichoderma [10]. Even though Trichoderma reesei has been and ethanol industry, Usina Vale do Rosrio (Santa Elisa Vale Con-
considered the best microorganism to carry out cellulose degrada- glomerate), located in Morro Agudo, So Paulo State, Brazil. This
tion, several works have claimed that this fungus is inefcient for plant processes 250 tons of bagasse per day, during the harvest
breaking down lignocellulosic substrates into glucose molecules, season, to be used as cattle feed. The bagasse is pretreated with
due to its low -glucosidase titre (0.1 IU/mL) [11,12]. To over- steam at 200 C for 7 min (average pressure of 15 atm), followed by
come the Trichoderma enzyme pool deciency studies have been gradual decompression. In our laboratory, the bagasse was washed
carried out on the supplementation of the Trichoderma enzymes with warm distilled water to remove residual soluble sugars and
by Aspergillus enzymes that typically present high levels of - dried overnight at 60 C. The carbohydrate and lignin content of
glucosidase. As such, Trichoderma viride and Aspergillus ustus the unpretreated and pretreated bagasse samples were determined
enzymes were separately produced, blended and used for the sac- using the NREL procedure [21].
charication of sugarcane bagasse [13]. Moreover Trichoderma and
Aspergillus enzymes were produced by mixed fungal cultures of T.
2.2. Fungal propagation and enzyme production
reesei and Aspergillus niger in solid substrate fermentation [14] and
in a stirred tank bioreactor [15]. Submerged shake asks co-culture
Stock cultures of T. reesei RUT-C30 (ATCC 56765) and A. awamori
of T. reesei and Aspergillus phoenicis are also reported [12].
2B.361 U2/1 [22,23] were propagated on potato dextrose agar
Sugars syrups, which are derived from the hydrolysis of biomass,
(PDA) plates at 30 C for 7 days. To prepare the seed culture, the
can be used for the production of ethanol. This is currently a large-
conidia from sporulating cultures were suspended in 5 mL of sterile
scale option that can be used by the transportation sector to ght
water, and 1 mL of the suspension (106 spores) was transferred to a
climate change through the decrease of greenhouse gas (GHG)
500 mL Erlenmeyer ask containing 100 mL of the growth medium,
emissions. Ethanol is currently produced through the conversion of
which was incubated in an orbital shaker for up to 3 days at 30 C
carbohydrates from dedicated crops such as corn (USA), sugarcane
and 200 rpm. A total volume of 30 mL of the mycelium suspen-
(Brazil) and sugar beet (Europe). However, the use of land for these
sion (obtained from the seed cultures) was used to initiate cell
fuels competes with the food supply and environmental preserva-
growth in 1 L asks, containing 300 mL of the growth medium. The
tion. As such, efforts have been made towards the development and
growth media composition used for both, the seed culture and for
implementation of advanced process technologies to produce cel-
enzyme production by T. reesei (A) and A. awamori (B) were as fol-
lulosic ethanol from low value agricultural co-products or wastes
lows: medium A (g/L): 30 lactose, 6 yeast extract, 0.3 urea, and
such as corn stover, wheat straw, sugarcane bagasse and straw, or
0.6% (v/v) corn steep liquor; plus salts (g/L): 1.4 (NH4 )2 SO4 , 2.0
wood wastes [16]. The potential use of these residues worldwide
KH2 PO4 , 0.3 CaCl2 and 0.3 MgSO4 7H2 O; and trace elements (mg/L):
is indeed signicant; in Brazil the agroindustry of corn, sugarcane,
5 FeSO4 7H2 O, 20 CoCl2 , 1.6 MnSO4 and 1.4 ZnSO4 [24]. Medium
rice, cassava, wheat, citrus, coconut and grass collectively generate
B (g/L): 30 wheat bran, 6 yeast extract, 1.2 NaNO3 , 3 KH2 PO4 ,
597 million tons of residues per year [17].
6 K2 HPO4 , 0.2 MgSO4 7H2 O and 0.05 CaCl2 2H2 O. The asks were
Brazilian sugar and alcohol industries generate 195 million tons
incubated for up to 7 days, and the production of enzymes was
of sugarcane bagasse per year [17], and these have been burnt in
monitored by periodical sampling. The cells were separated by l-
mills inefciently for energy cogeneration and as a way to reduce
tration using glass lter membranes, and the culture supernatants
the bagasse disposal problem. Despite this, a 12% surplus remains
were concentrated by ultraltration (Stirred Cells, Amicon) using
unexploited and could be used as a raw material for the produc-
a 10-kDa cut-off membrane. T. reesei and A. Awamori supernatants
tion of lignocellulosic ethanol, thereby increasing fuel production
were concentrated 2- and 3-fold, respectively.
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]. 2.3. Enzyme activity assays
To bioconvert lignocellulosic biomass into sugars customized,
effective and economical pretreatments need to be developed such The lter paper (FPU), endo-1-4--glucanase (CMCase) and
that a high yield of fermentable sugars can be obtained in the sub- -glucosidase (BGU) activities were determined using standard
sequent enzymatic hydrolysis step; pretreatment conditions must IUPAC procedures and expressed in international units (IU) [25].
also be well balanced to avoid the formation of inhibitors of the Reducing sugars were estimated according to Miller [26], using
hydrolysis and fermentation processes of the biomass [20]. More- glucose as standard. Glucose concentration was measured using
over, low cost, stable and efcient enzymes are also of paramount a Biochemistry Analyzer YSI 2700 Select. Xylanase activity was
importance, as the high cost of enzymes restricts their use in large- determined by mixing 50 l of the enzyme preparation to 100 l
scale applications for the conversion of lignocellulosic materials. of soluble oat spelts xylan (1%, w/v) in 100 mM sodium acetate
The present work studied the effectiveness of enzyme prepara- buffer, pH 5.0 at 50 C for 30 min. Reducing sugars were deter-
tions produced by either T. reesei or Aspergillus awamori cultures, mined according to Miller [26], using xylose as the standard. One
or that of different enzyme blends from both fungi, to hydrolyze unit of xylanase activity was dened by the formation of 1 mol
steam-treated sugarcane bagasse, which is produced industrially of reducing sugar per minute. Ferulic acid esterase (FAE) activity
in Brazil to be used as cattle feed. The enzymes were concentrated was assayed by measuring the release of ferulic acid in a reaction
using ultraltration and blended to obtain preparations with dif- mixture containing 10 l of the enzyme preparation, 20 l of 1%
ferent levels of exo- and endoglucanases, -glucosidase, xylanase ethyl ferulate in dimethylsulphoxide (DMSO), 100 l of 1 M acetate
and ferulic acid esterase. The hydrolytic efciency of the enzyme buffer (pH 5.0), and 870 l of water. After incubation at 50 C for
preparations was quantied using HPLC measurements of glu- 20 min, the reaction was terminated by boiling the mixture for
cose, cellobiose and xylose released from the bagasse. It was also 5 min, followed by the quantication of ferulic acid by HPLC. One
investigated the synergism between the T. reesei and A. awamori unit of FAE corresponded to the formation of 1 mol of ferulic acid
enzymes. 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 Table 1


The dry matter-based composition of the biomass components (%) of steam-treated
enzyme activity synergism
bagasse.

Enzyme blends were obtained by mixing the concentrated Biomass component Unpretreated bagasse Pretreated bagasse
supernatants of the two fungi cultures at the following T. reesei:A. Glucan 41.4 0.1 48.4 0.3
awamori ratios: 25:75, 40:60, 50:50, 60:40 and 75:25. The FPA, Xylan 22.5 0.2 10.3 0.1
CMCase, -glucosidase, xylanase and FAE activities were measured Galactan 1.3 0.05 0
Arabinan 1.3 0.04 0
in the individual T. reesei and A. awamori concentrated supernatants
Mannan 3.4 0.04 0
as well as in all enzyme blends. To investigate the existence of Lignin + ash 23.6 34
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 3.2. T. reesei and A. awamori enzyme production and activity
of the theoretical activity. proles

2.5. Sugarcane bagasse hydrolysis experiments In this study the T. reesei and A. awamori enzymes were produced
separately in spite of previous reports regarding enzyme produc-
Hydrolyses were carried out in 250 mL Erlenmeyer asks con- tion by mixed cultures of T. reeseiA. niger [14,15], and T. reeseiA.
taining 100 mL of the reaction mixture (20 g bagasse/L, suspended phoenicis [12] aiming mostly to complement the Trichoderma cel-
in 50 mM sodium citrate buffer, pH 4.8), which were incubated at lulase enzymes with the Aspergillus -glucosidase. However, as the
50 C and 200 rpm for 72 h in an orbital shaker. The experiments A. awamori strain used in this work was found to produce, besides
used either T. reesei or A. awamori enzyme concentrates, or blends high levels of -glucosidase and xylanase, cellulases and ferulic
of T. reesei and A. awamori enzyme at ratios of 25:75, 50:50 or 75:25. acid esterase, it was carried out, beforehand, a medium optimiza-
The hydrolysis experiments were designed to present, in all cases, tion study to maximize the A. awamori enzyme pool accumulation.
10 FPU/g of pretreated bagasse. All experiments were performed Culture conditions for cellulases accumulation by T. reesei, which
in triplicate, and the average values (standard deviations were less have been very much studied, were used in this work [27]. As a
than 4%) were reported. result maxima enzyme accumulation by A. awamori and T. reesei
The blend presenting T. reesei:A. awamori in the ratio 75:25 was achieved in growth media with different compositions, mostly
(10 FPU/g and 5 BGU/g) was used in hydrolysis experiments pre- regarding the main carbon source that was found to be wheat bran
senting 200 g bagasse/L, which were carried out in an agitated for A. awamori and lactose for T. reesei. Moreover, whereas lac-
reactor BIOSTAT BPLUS 1 L MO. Experiments were initiated tose induced T. reesei cellulases, as expected [27], it repressed A.
with 100 g bagasse/L (750 mL, 50 C and 100 rpm) followed, after awamori enzymes (unpublished data from our laboratory). It has
4 h, by a continuous fresh bagasse supply, up to 100 g, parallel been reported, though, the use of lactose fed batch fermentation
to the supply of the corresponding enzyme load. In all of the on mixed culture of T. reesei RUT-C30 and Aspergillus niger LMA
hydrolysis experiments, samples were withdrawn regularly and [15] that may have lessened the lactose repressive effect on the
centrifuged to separate lignin plus residual polysaccharides from Aspergillus culture.
the glucosexylose rich supernatant (i.e., the biomass syrup). In the T. reesei cultures, peak enzyme activities (IU/L): FPA
1200; CMCase 15,000; -glucosidase 150 and xylanase 10,000,
2.6. HPLC analysis were detected within 5 days incubation, whereas in the A. awamori
cultures, peak activities (IU/L): FPAse 150; CMCase 2500; -
Glucose, cellobiose and xylose were analyzed using high- glucosidase 18,800; xylanase 25,000 and FAE 160 were detected
performance liquid chromatography (HPLC) equipped with a within 7 days.
Lichorospher 100 NH2 5 m column at 35 C. The samples were The activity prole of the T. reesei enzymes preparation,
eluted using a mobile phase of acetonitrile:water (80:20) at a ow which were ultraltrate (IU/L): FPA 1700; CMCase 20,000; -
rate of 1.5 mL/min. A refraction index detector was used to mea- glucosidase 340; xylanase 12,600, showed activities of FPA and
sure the sugars. Ferulic acid was analyzed using HPLC equipped CMCase that were 4 times higher than those in A. awamori
with a Lichrocart-250-4.6 PurospherStar RP-18e (5 m) column (420 and 4900 IU/L, respectively) as it is shown in Table 2. The
at 25 C. Samples were eluted using a mobile phase of acetoni- activities of -glucosidase and xylanase were 134 and 6 times
trile:0.5% methanol in 0.01 M H3 PO4 (with an gradient of 96.5% lower than those in A. awamori (45,600 and 79,100 IU/L, respec-
acetonitrile at 0 min; 90% acetonitrile at 11 min; 80% acetonitrile tively). As such, the activity proles of T. reesei RUT-C30 and
at 22.5 min and 96.5% acetonitrile at 23.5 min), using a ow rate A. awamori 2B.361 U2/1 were quite complementary concerning
of 1.0 mL/min until 24.5 min. A diode array detector was used for the main enzymatic activities that are required to hydrolyze cel-
measurements at 320 nm. lulose and xylan. Moreover, and in accordance to a previous
work, which reports FAE as an Aspergillus exoenzyme [28], A.
3. Results and discussion awamori produced high levels of ferulic acid esterase (160 IU/L),
an enzymatic activity that was not detected in the Trichoderma
3.1. Effect of steam treatment on sugarcane bagasse supernatant. As FAE cleaves the linkages between xylan and lignin,
it creates more sites for the hydrolysis of glucan and xylan,
The normalized composition of unpretreated and pretreated enhancing the effectiveness of the cellulase and xylanase enzyme
bagasse (Table 1) indicates that the steam treatment alters the ini- blends.
tial composition of the biomass such that the xylan content was
decreased by 54% and the glucan and lignin content was increased 3.3. The activity prole of the T. reesei and A. awamori blends and
by 17% and 54%, respectively. Furthermore, the pretreatment have the synergistic activity of the enzyme blends
either fully removed the galactan, arabinan and mannan content in
the bagasse or have decreased these components to undetectable Table 2 presents the levels of FPA, CMCase, -glucosidase and
levels. 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. 2. The concentration of glucose (lled symbols) and cellobiose (open sym-
bols) in the hydrolysate during enzymatic hydrolysis of the steam-treated sugarcane
Fig. 1. Synergistic enhancement of the FPA, CMCase, -glucosidase and xylanase bagasse using enzymes produced by T. reesei ( and ) or by A. awamori ( and ).
activities using mixtures of the concentrated T. reesei and A. awamori preparations The substrate and enzyme concentrations were 20 g/L and 10 FPU/g, respectively.
at ratios of 25:75, 40:60, 50:50, 60:40 and 75:50.

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


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


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


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