b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
Available online at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Using Amazon forest fungi and agricultural residues as
a strategy to produce cellulolytic enzymes
Priscila da Silva Delabona a,b, Rosangela D.P. Buzon Pirota a,b, Carla Aloia Codima a,b,
Célia Regina Tremacoldi c, André Rodrigues d, Cristiane Sanchez Farinas b,*
a
Universidade Federal de São Carlos, 13565-905 São Carlos, São Paulo, Brazil
Embrapa Instrumentação, Rua XV de Novembro 1452, 13560-970 São Carlos, São Paulo, Brazil
c
Embrapa Amazônia Oriental, Trav. s/n. Caixa Postal, 48 Belém, Pará, Brazil
d
Universidade Estadual de Santa Cruz, 45662-900 Ilhéus, Bahia, Brazil
b
article info
abstract
Article history:
The successful strategy to produce cellulolytic enzymes includes both microorganism
Received 21 June 2011
selection and improved fermentation process conditions. This work describes the isolation,
Received in revised form
screening and selection of biomass-degrading fungi species from the Amazon forest and
6 December 2011
analyzes the enzymatic complex produced by a selected strain of Aspergillus fumigatus
Accepted 8 December 2011
cultivated using different agro-industrial residues (wheat bran, sugarcane bagasse,
Available online 24 December 2011
soybean bran, and orange peel) as substrate in solid state fermentation (SSF). The profile of
endoglucanase (CMCase), FPase, b-glucosidase and xylanase enzymatic activities obtained
Keywords:
during 120 h of cultivation is presented. Enzyme activities up 160.1 IU g1 for CMCase,
Enzymes
5.0 FPU g1 for FPAse, 105.82 IU g1 for b-glucosidase and 1055.62 IU g1 for xylanase were
Amazon forest
achieved. The enzymatic extract with higher CMCase activity was used to run a zymogram
Cellulases
analysis that showed 3 bands of endoglucanase activity. Characterization studies of this
Agro-industial residues
extract showed that the CMCase was most active at either 65 C or pH 3e3.5, indicating that
Solid state fermentation
this microorganism produces a thermophilic and acid endoglucanase. These data
Bioethanol
demonstrate that the fungal isolates from the Amazon forest are a potential source of
cellulases and xylanases, providing support to further studies related to the use of these
microorganisms to obtain the enzymes needed for biomass conversion.
ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Cellulosic ethanol has attracted attention as a potential
alternative renewable transportation fuel. One of the most
promising routes for the conversion of cellulosic materials
into ethanol is the enzymatic hydrolysis followed by
fermentation [1,2]. The biomass-degrading enzymes needed
in this process, the cellulases, are multienzyme systems
composed of several enzymes, which act in synergy [3,4].
Cellulases are the third largest industrial enzyme product
worldwide, by dollar volume, but will certainly become the
largest volume industrial enzyme upon its use for converting
biomass into biofuels [5]. However, the major bottleneck for
a broader application of industrial cellulases is their high
cost. The successful strategy to produce cellulases can be
achieved through microorganism selection and improved
fermentation process conditions. These include screening for
new enzyme activities and developing pre-treatments that
alter the cellulose lattice structure, increasing enzyme
accessibility.
* Corresponding author. Tel.: þ55 16 2107 2908; fax: þ55 16 2107 2902.
E-mail address: [email protected] (C.S. Farinas).
0961-9534/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.12.006
244
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
It is therefore necessary to search for microorganisms
that have a high rate of cellulase production. Prospecting for
cellulose-producing fungi is one of the possible strategies to
obtain the necessary enzyme complex to hydrolyze plant
biomass [6,7]. Fungal strains that produce cellulases include
Aspergillus, Fusarium, Penicillium and Trichoderma genera. One
of the most reported microorganisms for cellulase production in the literature is the Rut C30 strain mutant of the
filamentous fungus Trichoderma reesei [8,9]. However, it is
known that the cultivation of T. reesei has some limitations
including product inhibition of cellulase activity and lower
cellobiase activities compared to other cellulolytic strains
[10]. To reduce these limitations many studies have used
genetic engineering methods. However, the avoidance of
genetic modified organisms (GMOs) for large scale industrial
use may facilitate site approval and avoid capital costs
associated with their containment. In this context, Brazil’s
high biodiversity level stands out as a potential environment to be explored in the search for microorganisms that
have the desired characteristics for such application. The
Amazon biome occupies nearly half of the Brazilian territory
and is the world’s largest reserve of biological diversity,
presenting special soil and climate characteristics for
microorganism growth [11].
Regarding fermentation processing conditions, the current
enzyme production technologies are conducted either in
a liquid phase, known as submerged fermentation (SF), or
using solid substrates, termed solid state fermentation (SSF).
Approximately 90% of all industrial enzymes are produced by
SF, often using genetically modified microorganisms [12].
However, most of these enzymes could be produced by SSF
using wild-type microorganisms. Though there are reports on
cellulase production through SSF, the large scale commercial
processes are still using SF [13,14]. However, growth of filamentous fungi by SSF is considered advantageous since the
solid medium simulates its natural habitat [15]. This benefit is
extended to the production of enzymes, yielding greater
productivity when compared to SF [12]. Another advantage of
SSF is the use of agro-industrial residues as a solid substrate,
acting both as carbon and energy source.
The choice of these lignocellulosic materials should be
based on their abundance and cost, as well as their physicalchemical characteristics. In Brazil, sugarcane is one of the
largest agricultural monocultures, supplying an enormous
amount of residues. Because of its low ash content, bagasse
offers numerous advantages, in comparison to other crop
residues such as rice straw and wheat straw, for use in
bioconversion processes [16] however, it has a low nutrient
value. Soybean and wheat bran are good sources of nitrogen
and have been used in mixtures with sugarcane bagasse for
the production of cellulases [17]. Citrus peels are the principal
by-product of the citrus processing industry and account for
about 50% of fresh fruit weight [18], which also represents an
abundant residue in Brazil.
This work describes the isolation, screening and selection
of biomass-degrading fungi species from the Amazon forest
and analyzes the enzymatic complex produced by a selected
strain of Aspergillus fumigatus cultivated using different agroindustrial residues (wheat bran, sugarcane bagasse, soybean
bran, and orange peel) as substrate in SSF.
2.
Materials and methods
2.1.
Fungi isolation and identification
The fungal strains used in this study were isolated by
screening 100 soil and decomposed wood samples from the
natural Amazon forest reserve of Embrapa Eastern Amazon
(Brazilian Company for Agricultural Research), located in
Belém, Pará - Brazil. Approximately 5 g of soil or pieces of
decomposed wood (length of 10e20 mm) were transferred to
tubes containing 5 mL of a nutrient medium [19]. After 72 h of
incubation at 37 C, the samples were transferred to potato
dextrose agar and to a media with 30 g L1 of malt-agar.
Purified fungi were transferred to plates that contained
(g L1): 2.0 NaNO3, 1.0 K2HPO4, 0.5 MgSO4, 0.5 KCl, 2.0 Avicel, 0.2
peptone, 17 agar and 0.25 chloramphenicol. Fungal identification was carried out using morphological markers according to classical taxonomical keys [20e22]. Selected strains are
maintained at Embrapa culture collection (São Carlos, SP,
Brazil).
2.2.
Initial screening
Initial screening for cellulose degrading strains were carried
out in Petri plates using a synthetic agar medium composed of
(g L1) 3.0 NaNO3, 0.5 MgSO4, 0.5 KCl, 0.01 FeSO4.7H2O, 1.0
KH2PO4 and 30.0 agar supplemented with 5.0 g L1 of crystalline cellulose (Avicel) (Sigma, St. Louis, USA) as the only
source of carbon. Fungal isolates were inoculated and plates
were incubated at 37 C during 96 h. Selection was based on
fungal growth under this condition.
2.3.
Agro-industrial residues
The sugarcane bagasse used as solid substrate was kindly
provided by Edra Eco Sistemas (Ipeúna, SP, Brazil). It was used
without any pre-treatment and a particle size between 1 and
2 mm was selected. Exploded sugarcane bagasse was kindly
provided by a local sugarcane mill (Usina Nardini, Vista Alegre
do Alto, SP, Brazil). Soybean and wheat bran where purchased
from a local store. Orange peel was kindly provided by a local
orange processing industry (Citrosuco, Matão, SP, Brazil).
These three materials were thoroughly washed with water,
dried at room temperature, milled with a knife mill and classified to particle size between 1 and 2 mm. All processed
material was stored in sealed plastic bags and maintained
refrigerated at 4 C until experimentation.
2.4.
Enzyme production
Solid state fermentation was used for fungal cultivation and
evaluation of the enzymatic complex produced using different
carbon sources. For preparation of fungal inoculums for
fermentation, 2 mL of sterile distilled water containing
1.0 g L1 of Tween 80 was introduced into the sporulated
slants of each fungus and the spores were dislodged into the
liquid by gentle pipetting. Fungi were cultured in cottonplugged 250 mL Erlenmeyer flasks containing 5 g of
substrate moistened with mineral salt medium [19] in order to
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
achieve 60% moisture. Each flask was sterilized by autoclaving
at 121 C for 20 min before inoculation with spores at 107 mL1,
measured by the Neubauer chamber count method. For the
second screening step, ten selected strains from the first
screening on avicel were cultivated using only wheat bran as
substrate. The selected fungus strain based on endoglucanase
activity was then cultivated in different agro-industrial residues. Lignocellulosic substrates (wheat bran, exploded
sugarcane bagasse, sugarcane bagasse, sugarcane bagasse
and wheat bran (1:1), soybean, orange peel and orange peel
and wheat bran (1:1)) were prepared as described above. The
flasks were incubated at 37 C for 5 days. After incubation
period, 50 mL of citrate buffer (pH 5.0, 50 mmol L1), prepared
as described by Gomori [23], was added to each flask. The
suspensions were stirred at 2 Hz in an orbital shaker incubator
(Solab, Brazil) at 35 C for 30 min. The solids were separated by
centrifugation at 10,000 g at 4 C for 15 min and the enzyme
activity of the supernatant was assayed.
2.5.
Enzyme assays
Cellulase activity on filter (FPase), endoglucanase (CMCase),
xylanase and b-glucosidase activities were determined. All
enzymes were analyzed according to the standard procedure
recommended by the Commission on Biotechnology, IUPAC
[24] with some modifications. Filter paper activity was assayed
by incubating the suitable diluted enzyme (0.1 mL) with 0.9 mL
citrate buffer (50 mmol L1, pH 5.0) containing filter paper
Whatman no. 1 (50 mg, 1 6 cm). The reaction mixture was
incubated at 50 C for 60 min. Endoglucanase activity was
carried out by using 0.1 mL of suitably diluted enzyme and
0.9 mL of 40 g L1 carboxymethylcellulose (CMC) (Sigma, St.
Louis, EUA) solution in citrate buffer (50 mmol L1, pH 5.0). This
mixture was incubated at 50 C for 15 min. Xylanase activity
was determined under similar conditions as described above,
except that 1% xylan birchwood (Sigma, St. Louis, EUA) solution
was used as substrate. Finally, the b-glucosidase activity was
determined by using cellobiose (Sigma, St. Louis, EUA) as
substrate and quantifying the sugars released by using an
enzimatic kit for glucose measurement (Laborlab, São Paulo,
Brazil). All the enzyme assays were performed in duplicate.
One unit of FPase (expressed as filter paper units - FPU) and
endoglucanase activity corresponds to 1 mmol of glucose
released per minute. One unit of xylanase activity corresponds
to 1 mmol of xylose released per minute. The quantification of
the reducing groups was performed using the dinitrosalicylic
acid (DNS) method [25]. Results were expressed as activity units
per mass of initial dry solid substrate (IU g1 or FPU g1).
2.6.
Partial crude enzyme characterization: influence of
pH, temperature and thermal stability
The temperature profile for CMCase activity (supernatant corresponding to the optimal production condition on SSF) was
determined by assaying the activity at different reaction
temperatures (30, 35, 40, 45, 50, 55, 60, 65 and 70 C) in
50 mmol L1 sodium citrate buffer (pH 5.0). Accordingly,
CMCase activity was assayed in different reaction buffers
(50 mmol L1 glycine-HCl for pH 3.0; 50 mmol L1 sodium
citrate for pH 3.5e6.0; 50 mmol L1 citrate phosphate for pH
245
6.5e7.0; 50 mmol L1 phosphate for pH 7.5e8.0; 50 mmol L1
TriseHCl for pH 8.5e10.0) at 50 C in order to determine the pH
profile. For thermal stability determination, the crude supernatant obtained in the optimal production condition (CMCase
initial activity of 190 IU g1) was incubated at 60 C for 1 h in the
absence of substrate. The residual CMCase activity was
measured at different time periods. At the end of incubations
the test tubes containing the enzyme were immediately cooled
down by placing them on ice and then kept at 4 C overnight.
After this period the samples were taken to test the enzyme
activity under standards pH and temperature conditions.
2.7.
Zymogram
Crude enzyme preparations (70 mg of protein, measured
according to the Bradford method [26]) were fractionated by
native polyacrylamide gel electrophoresis (PAGE) using 10%
acrylamide gel [27]. For developing the CMCase, the PAGE gel
was incubated for 15 min in 0.05 mol L1 sodium citrate buffer
(pH 5.0) and then overlaid with polyacrylamide gel containing
CMC (0.5%, w/v) for 30 min at 50 C. The overlaid gel was
removed and stained with 0.2% Congo Red solution (Sigma, St.
Louis, EUA). Bands corresponding to CMCase appeared as
clear zones against a dark background after destaining with
1M NaCl followed by treatment with 10% (v/v) acetic acid
solution.
3.
Results and discussion
3.1.
Fungal isolation and screening studies
Screening studies were carried out in two steps. The first
selection was based on the ability of fungal isolates to grow in
a medium containing only crystalline cellulose (Avicel) as
a carbon source. Selected strains from the first step were then
grown in wheat bran under SSF and a second selection criterion was based on the highest CMCase activity produced after
5 days of incubation.
Of all 110 fungal strains isolated from the Amazon Forest,
46 isolates were able to grow in a nutrient medium containing Avicel as the only carbon source. Of this total, 10 fungal
strains that showed a more expressive growth on Avicel
plates were selected for cellulase production evaluation
under SSF on wheat bran (Fig. 1). All 10 fungal strains evaluated were able to produce significant titers of endoglucanase. Higher endoglucanase activities were obtained by the
species A. fumigatus P40M2 (43.65 IU g1), Aspergillus niger
P47C3 (39.15 IU g1) and Trichoderma harzianum P34P9
(36.16 IU g1). Based on these results, the A. fumigatus P40M2
strain was selected for further studies on the influence of the
carbon source on enzyme production by using different agroindustrial residues as substrates of SSF.
3.2.
Influence of biomass on enzyme production by A.
fumigatus strain P40M2
In order to evaluate the effect of the nature of the carbon source
on enzyme production, the selected strain A. fumigatus P40M2
was cultivated by SSF using different agro-industrial residues
246
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
Fig. 1 e Production of cellulase under SSF using wheat bran
for 10 selected fungal strains growing with avicel to select
the best producer. Fungal strains are denoted on the Xaxis. Results were expressed as activity units per gram of
initial dry solid substrate (IU gL1).
as substrates (wheat bran, exploded sugarcane bagasse,
sugarcane bagasse, sugarcane bagasse and wheat bran (1:1),
soybean bran, orange peel and orange peel and wheat bran
(1:1)). The effect of the carbon source on the enzymatic
production was evaluated in terms of cellulase and xylanase
activities. Cellulase is a complex of enzymes that contains: (1)
endoglucanases (EG) which cleave internal bonds in the
amorphous regions of the cellulose chains, (2) cellobiohydrolases (CBH) which act on the ends of the chains liberating
cellobiose moieties, and (3) b-glucosidases which hydrolyze
cellobiose to glucose [28]. Xylan is the principal type of hemicelluloses and represents a vast resource that can be used to
produce fermentable sugars and fuels. The complete cleavage
of xylan is carried out by the synergistic action of b-xylanase
and its accessory enzymes, including b-xylosidase, a-arabinofuranosidase, a-glucuronidase and acetyl xylan esterase [29].
The enzyme production profile of endoglucanase, FPase, bglucosidase and xylanase activities obtained during 120 h of
fermentation were carried out and are presented as follows.
3.2.1.
Endoglucanase production profile
Fig. 2 shows the endoglucanase production profile by A.
fumigatus P40M2 cultivated under SSF using different carbon
sources. It can be observed that the nature of the carbon
source in the culture medium had a significant influence on
endoglucanase production. Soybean bran was found to result
in maximal endoglucanase production (160.1 IU g1 of
substrate), followed by wheat bran (122.8 IU g1) and a mixture
of sugarcane bagasse and wheat bran (1:1) (112.6 IU g1). The
highest production for all three substrates was achieved after
72 h of incubation. After this period, there was a decrease in
endoglucanase activity, which was also observed when using
wheat bran and a mixture of sugarcane bagasse and wheat
bran (1:1) as substrates.
Many Aspergillus species are described in the literature as
good producers of endoglucanase. Soni et al. [30] reported
Fig. 2 e Effect of different carbon sources on the production
of CMCase by A. fumigatus under solid state fermentation
(SSF).
a maximal production of endoglucanase (98.5 IU g1 of
substrate) in rice straw when comparing different sources of
carbon during cultivation of A. fumigatus under SSF. Wheat
bran has also been reported to induce high levels of endoglucanase in Aspergillus sp. Thygesen et al. [31] observed
endoglucanase activity of 66.6 IU g1 in SSF using wheat bran
as substrate for the fungus A. niger.
Sugarcane bagasse resulted in lower levels of endoglucanase
production (16.71 IU g1) after 96 h of fermentation, when
compared to the other carbon sources. Soni et al. [30] reported
endoglucanase production of 29.2 IU g1 when using bagasse as
substrate during A. fumigatus growth. Grigorevski-Lima et al. [32]
obtained endoglucanase activity up to 21.06 IU g1 in solid state
using sugarcane bagasse as substrate. Gupte and Madamwar [33]
found an endoglucanase activity of 14.55 IU g1 when using
sugarcane bagasse, in comparison to other substrates during
cocultivation of Aspergillus ellipticus and A. fumigatus.
In the present work, endoglucanase activity was 18.42 IU g1
when orange peel was used as substrate, and this value was
increased to 32.92 IU g1 when orange peel was mixed with
wheat bran (1:1 ratio). It was verified that the use of wheat bran in
combination with other substrates was very effective in
increasing enzyme production. Further productivity improvements could be achieved by optimizing the SSF relevant
parameters, such as temperature, moisture content, aeration,
supplementation of the medium and pre-treatment of the
substrate. Mamma, Kourtoglou and Christakopoulos [18] were
able to increase endoglucanase activity from 9.2 to 60.5 IU g1 in
solid state using orange peel as substrate and fungus A. niger after
optimizing the initial moisture content of the solid medium.
Even though it is difficult to compare enzyme production values
given the different cultivation conditions and microorganisms
employed in each study, the values reported here using fungal
isolates from the Amazon forest were able to demonstrate that
the selected strain of A. fumigatus can efficiently produce endoglucanases when growing in agro-industrial residues.
3.2.2.
FPAse production profile
Fig. 3 shows the effect of carbon source on the FPase
production profile by A. fumigatus. Higher productions of
FPase were obtained when wheat bran as well as a mixture of
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
Fig. 3 e Effect of different carbon sources on the production
of FPase by A. fumigatus under solid state fermentation
(SSF).
sugarcane bagasse and wheat bran (1:1) were used as solid
substrates, reaching values of up to 5.0 FPU g1. This result is
rather interesting when compared to the other substrates
evaluated, since soybean bran resulted in FPAse activity
values that were less than half of those obtained with wheat
bran (2.4 FPU g1). The other substrates did not result in
significant FPase activities. The maximum activity of FPase
occurred after 96 h incubation.
Grigorevski-Lima et al. [32] also had maximum FPase
activity (5.35 FPU g1) when using wheat bran as substrate,
followed by sugarcane bagasse (4.4 IU/g). Soni et al. [30] obtained FPase activities of 2.11 and 3.37 FPU g1 when using
wheat bran and rice straw, respectively, as solid substrates and
a strain of A. fumigatus. Although wheat bran is considered as
an inducer of FPase for fermentation with Aspergillus sp. [34],
the addition of wheat bran to orange peel did not induce FPase
activity significantly. The observed difference in FPase activities can be attributed to the structural heterogeneity of the
cellulosic substrates, as well as to the differences in culture
conditions during fermentation [35]. Sukumaran et al. [36] obtained 4.55 FPU g1 when growing A. niger MTCC 7956 in wheat
bran as substrate and Gupte and Madamwar [33] obtained
3.75 FPU g1 during cocultivation of A. ellipticus and A. fumigatus
using sugarcane bagasse as substrate and 2.1 FPU g1 when A.
ellipticus was used alone. The values reported here using fungal
isolates from the Amazon forest were in the same order of
magnitude of the values reported in the literature, some of the
values achieved only after SSF process optimization.
3.2.3.
247
There was an increase in b-glucosidase activity when
sugarcane bagasse was supplemented with wheat bran, since
when sugarcane bagasse was used as the sole substrate, no
significant enzyme production was observed (Fig. 4). The
literature suggests that b-glucosidase induction requires
specific operational conditions and wheat bran, mainly due to
its mineral composition, favors the induction of b-glucosidase
in the fermentation medium [37]. The fungus T. reesei, one of
the most studied microorganisms for the production of
cellulases, produces a relatively low amount of b-glucosidase,
which is a disadvantage in terms of biomass saccharification
process [38]. Brijwani et al. [39] reported that the deficit of
b-glucosidase was overcome when T. reesei was co-cultured
with Aspergillus oryzae, allowing a high b-glucosidase activity
after 96 h incubation (10.71 IU g1) by using soybean hulls and
wheat bran. Kang et al. [40] related a maximum b-glucosidase
activity of 100.0 IU g1 by using rice straw as substrate by A.
niger KK2 and Gao et al. [41] related 119 IU g1 using corn stover
as substrate by Aspergillus terreus M11. Regarding b-glucosidase activity, the values reported here were mostly in the
same order of magnitude of the values reported in the
literature.
3.2.4.
Xylanase production profile
Xylanase enzymes are responsible for xylan hydrolysis, which
is the main polysaccharide component of hemicelluloses.
Xylans account for 20e35% of the total dry weight of hardwoods and herbaceous plants and represent a vast resource
that can be used for the production of fermentable sugars and
fuels [42]. In SSF cultures, wheat bran and rice are used as
xylanases inducers. Alternative substrates for enzyme
production have also been reported, such as sugarcane
bagasse, rice husks and wood pulp [43].
Concerning xylanase activity, higher yields were also obtained using wheat bran as substrate (1055.62 IU g1), followed
by sugarcane bagasse and wheat bran (1:1) (821.52 IU g1),
soybean (484.21 IU g1) and orange peel and wheat bran (1:1)
(437 IU g1) (Fig. 5). All of the other substrates did not result in
significant activities. Soni et al. [30] obtained xylanase activity
of 1722 IU g1 by using wheat bran as solid substrate and
a maximum activity of 2782 IU g1 by using wheat straw as
b-glucosidase production profile
Wheat bran also resulted in maximal production of b-glucosidase (105.82 IU g1), followed by a mixture of sugarcane
bagasse and wheat bran (1:1) (38.04 IU g1) (Fig. 4). The highest
activity occurred after 96 h of incubation. It is difficult to
compare the results described in the literature, since the
conditions to measure b-glucosidase activity and enzyme
production were different. Soni et al. [30] obtained b-glucosidase activity of 99.4 IU g1 by using wheat bran as solid
substrate and a maximum activity of 250.9 IU g1 by using
wheat straw as solid substrate.
Fig. 4 e Effect of different carbon sources on the production
of b-glucosidase by A. fumigatus under solid state
fermentation (SSF).
248
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
Fig. 5 e Effect of different carbon sources on the production
of xylanase by A. fumigatus under solid state fermentation
(SSF).
Fig. 7 e Effect of pH on endoglucanase activity produced by
A. fumigatus P40M2 grown under solid state fermentation
in soybean. Temperature was kept constant at 50 C. Data
points are the average of duplicate experiments.
CMCases obtained from A. fumigatus P40M2 grown in soybean
under SSF conditions presented maximal activity at 65 C
(Fig. 6). The activity decreased when incubated at 70 C, but
still retained about 60% after one hour incubation. Our results
strongly suggest that cellulases being produced seem to be
thermophilic, which is considered ideal for many biotechnological processes. Grigorevski-Lima et al. [32] related
a maximum activity of the enzyme by the fungus A. fumigatus
at 65 C and when incubated at 80 C the enzyme still
remained 30% of its activity. Saqib et al. [46] also obtained an
optimum temperature of 65 C under SSF by the fungus A.
fumigatus. The pH profiles (Fig. 7) showed more than 100%
activity (187.0 IU g1) in the acidic pH (3.0), according to Gao
et al. [41] this remarkable characteristic was also detected in
a thermoacidophilic fungus, A. terreus M11. Grigorevski-Lima
et al. [32] obtained a maximum activity of the enzyme
CMCase obtained by A. fumigatus at pH 2.0. When CMCase was
incubated at alkaline pH the activity was very low, decreasing
about 25%. This biochemical characteristic is very interesting
for processes that require acid conditions. Besides, acid
Fig. 6 e Effect of temperature on endoglucanase activity
produced by A. fumigatus P40M2 grown under solid state
fermentation in soybean. The pH was kept constant at 5.0.
Data points are the average of duplicate experiments.
Fig. 8 e Residual activity expressed as a percentage of the
maximum endoglucanase activity produced by A.
fumigatus P40M2 grown under solid state fermentation in
soybean. Data points are the average of duplicate
experiments.
solid substrate. Senthilkumar et al. [44] related a maximum
activity of 1.024 IU g1 using wheat bran as substrate by
Aspergillus fischeri. When the fungus was cultivated on cane
bagasse and orange peel, the maximum activity was of 68.10
and 43.05 IU g1, respectively. Lemos et al. [45] obtained
a maximum activity of 2.50 IU g1 by Aspergillus awamori on
cane bagasse and Mamma, Kourtoglou and Christakopoulos
[18] related a maximum activity of 77.1 IU g1 by A. niger BTL
on orange peel as the sole carbon source. The values reported
here for xylanase activity were mostly superior to the values
reported in the literature.
3.3.
Effect of temperature, pH and thermostability
analysis
b i o m a s s a n d b i o e n e r g y 3 7 ( 2 0 1 2 ) 2 4 3 e2 5 0
Fig. 9 e Zymogram showing multiplicity of CMCase in (a)
soybean, (b) wheat bran, (c) cane bagasse and wheat bran
(1:1).
stability is also important in order to reduce microbial
contamination. Considering the optimal temperature, strain
P40M2 can be considered as producer of a thermoacidophilic
endoglucanase.
Thermostability of the enzyme refers to its resistance to
unfolding upon heating, i.e., to the thermal denaturation.
Various parameters are used to determine the thermostability
of enzymes, including the half life of the enzyme [47]. The A.
fumigatus P40M2 supernatant was able to retain 100% of the
residual activity at 60 C for 50 min and 91% after 1 h incubation (172.0 IU g1) (Fig. 8). Saqib et al. [46] evaluated the
production of endoglucanse in supernatant obtained by the
fungus A. fumigatus under SSF using rice bran as substrate and
found 50% of the original CMCase activity at 75 C after 1 h of
incubation. Grigorevski-Lima et al. [32] obtained similar
results, the supernatant obtained from A. fumigatus FBSPE was
able to retain 100% of the residual CMCase activity at 50 C for
2 h of incubation. These results were similar to the results
obtained in the literature and showed the potential of the
enzymes produced, since crude extracts were evaluated,
which were not subject to preliminary purification.
3.4.
Detection of endoglucanase activity by zymogram
staining
The extracts obtained by culturing A. fumigatus P40M2 on
wheat bran and soybean, showed the expression of three
forms of endoglucanases (Ia, Ib and Ic) and in the bagasse only
two EG isoforms on native PAGE (Fig. 9). Soni et al. [30] related
four isoforms in the presence of corn cob and in the bagasse
only two EG isoforms. Grigorevski-Lima et al. [32] showed the
expression of six EG isoforms in the presence of sugarcane
bagasse under submerged culture. The observed differential
expression of cellulases can be attributed to a structural
heterogeneity of the cellulosic substrates, as well as differences in culture conditions during cultivation [46]. Such
multiple forms of endoglucanases have also been reported in
different Aspergillus strains.
4.
Conclusion
The fungal isolates from the Amazon forest were efficient
producers of cellulase and xylanase. The use of agroindustrial residues as substrate for microbial growth also
249
showed to be a promising alternative, with good perspectives
for scaling up. The characterization of the crude enzyme has
shown A. fumigatus P40M2 endoglucanase to be active in the
acidic pH range 3e5, with maximal activity at either pH 3.0, or
65 C, hence a promising organism for the production of
acidophilic and thermophilic cellulases. It is important to
point out that cultivations were carried at a specific condition
and further productivity improvements can be achieved by
optimizing the SSF relevant parameters. These data demonstrate that the fungal isolates from the Amazon forest are
a potential source of cellulases and xylanases, providing
support to further studies related to the use of these microorganisms to obtain the enzymes needed for biomass
conversion.
Acknowledgments
The authors are grateful to Embrapa (Brazil) for the financial
support.
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