3 Biotech (2017) 7:158

DOI 10.1007/s13205-017-0755-0

ORIGINAL ARTICLE

Enzymatic saccharification of pretreated rice straw by cellulases

from Aspergillus niger BK01
Neeraj Kumar Aggarwal1 • Varsha Goyal1 • Anita Saini1 • Anita Yadav2 •

Ranjan Gupta3

Received: 28 November 2016 / Accepted: 30 January 2017 / Published online: 16 June 2017
Ó Springer-Verlag Berlin Heidelberg 2017

Abstract Alkali-assisted acid pretreated rice straw was Introduction

saccharified using cellulase from Aspergillus niger BK01.
The cellulase production by the fungus was enhanced by Lignocellulosic residues are low-cost renewable resources
parametric optimization using solid-state fermentation luxuriantly available in nature (Anwar et al. 2014; Nanda
conditions. Maximum cellulase production (12.0 U/gds of et al. 2014). Rice straw is one of the abundant lignocellu-
carboxymethyl cellulase, CMCase) was achieved in 96 h, losic crop residues of the world (Kim and Dale 2004;
using 6.0% substrate concentration, 7.5% inoculum con- Rahnama et al. 2014; Singh et al. 2016). The annual global
centration, 1:2 solid to liquid ratio, at pH 5.5, and tem- production of rice is about 526 million metric tons (Kim
perature 28 °C, by supplementation of the fermentation and Dale 2004). Estimates have shown generation of 1.35
medium with 0.1% carboxymethylcellulose and 0.1% tons of rice straw annually for every ton of harvested grain
ammonium nitrate. Characterization of crude cellulases (Kadam et al. 2000). Characteristics of the rice straw, such
showed that highest CMCase activity was observed at pH as low bulk density, high mineral, and silica contents, limit
4.8 and temperature 40 °C. The CMCase was stable from its applications (Jain et al. 2015). Its utilization as animal
pH 4.8–5.5 and at a temperature range of 35–50 °C. The fodder is also unattractive because of its low digestibility,
pretreated biomass was subjected to hydrolysis with the low protein content, high lignin, and silica contents (Kau-
fungal cellulases. The saccharification optimization studies sar et al. 2010). Therefore, a large part of the rice straw is
showed that 2% (v/v) enzyme concentration and hydrolysis left unused as a waste. Its disposal is also a problem due to
time of 2.5 h were optimum for maximum yield, i.e, its bulkiness, slow degradation in the nature, and harboring
23.78% sugars and 35.96% saccharification value. of diseases. It has been seen that burning rice straw in open
fields is a common practice all over the world, which leads
Keywords Aspergillus niger
Cellulases
Optimization
to air pollution (Gadde et al. 2009; Emtenan et al. 2012;
Pretreated
Rice straw
Saccharification Singh et al. 2016). An alternative to this is using rice straw
as a feedstock for production of cellulosic ethanol (Park
et al. 2011; Jain et al. 2015).
The cellulosic component of the lignocelluloses is an
attractive source of fermentable sugar, the glucose, which
can be obtained by enzymatic hydrolysis of the cellulose in
& Neeraj Kumar Aggarwal a process known as saccharification (Salehi et al. 2012).
[email protected]
However, the native cellulose is buried in a matrix of
1
Department of Microbiology, Kurukshetra University, hemicellulose and lignin posing physical barrier to its
Kurukshetra, Haryana 136119, India accessibility. Lignin present as a cover makes the entire
2
Department of Biotechnology, Kurukshetra University, structure recalcitrant (Khare et al. 2015). Therefore,
Kurukshetra, Haryana, India hydrolysis is mediated through a crucial step of pretreat-
3
Department of Biochemistry, Kurukshetra University, ment, which opens up the structure of lignocellulose
Kurukshetra, Haryana, India complex (Kumar et al. 2009). Recently, the enzymatic

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saccharification of the cellulose is gaining interest world- hemicellulose, and lignin contents was done using standard
wide, especially due to the potential of glucose for its biochemical analysis methods (Goerging and van Soest
conversion into bioethanol fuel, which can offer a potent 1975).
alternative to the exhaustible fossil fuel energy sources.
Cellulases are the key enzymes in enzymatic sacchari- Optimization of cellulase production by solid-state
fication of the cellulosic biomass (Sukumaran et al. 2009). fermentation (SSF)
The complete cellulase system is comprised of endoglu-
canases, exoglucanases, and b-glucosidases enzymes, The solid-state fermentation was carried out using pre-
which act synergistically for complete hydrolysis of cel- treated rice straw. Mandel and Sternburg’s medium (1976)
lulose to sugars (Sadhu and Maiti 2013). A wide variety of (pH 5.0) containing KH2PO4, 0.2%; Urea, 0.03%;
microorganisms, including bacteria, fungi, and actino- MgSO4
7H2O, 0.03%, CaCl2, 0.03%; Peptone, 0.075%,
mycetes, are known to produce cellulases (Wilson 2011). Yeast extract, 0.025%, and trace element solution
Most of the commercial cellulases production focuses on (FeSO4
7H2O, 5 mg/ml; MnSO4.4H2O, 1.6 mg/ml;
fungi. Aspergillus niger is among potent cellulase pro- ZnSO4
7H2O, 1.4 mg/ml and CoCl2
6H2O, 20 mg/ml) was
ducers (Mrudula and Murugammal 2011). Solid-state fer- used as moistening agent. The solid-to-liquid ratio was
mentation is known to be an efficient technique for the maintained as 1:1.5. The incubation was done at 25 °C for
production of hydrolytic enzymes (Sukumaran et al. 2009), 96 h. Cellulase production by the fungus was enhanced by
in which fungi are cultivated in conditions simulating optimizing parameters of substrate concentration
natural environments. In this study, enhanced cellulases (4.0–9.0% w/v), inoculum concentration (6.0–9.0% v/v),
production has been achieved from A. niger by parametric incubation period (24–120 h), pH (4.0–7.0), temperature
optimization under solid-state conditions and the cellulases (20–40 °C), moisture level (1:1–1:5 biomass to moistening
obtained have been used for the saccharification of pre- agent ratio), supplementation with carbon (0.1% w/v mal-
treated rice straw. tose, sucrose, carboxymethyl cellulose, cellulose powder,
lactose), and nitrogen sources (0.1% w/v ammonium
nitrate, ammonium sulphate, ammonium chloride, beef,
Methodology tryptone, urea, and potassium nitrate).

Microorganism, maintenance, and inoculum Characterization of cellulases

preparation
The enzyme produced from A. niger BK01 was extracted
The microorganism used was Aspergillus niger BK01, using tenfolds (w/v) of 0.1 M citrate buffer (pH 4.8). The
which was isolated from rice field soil (Goyal et al. 2014a). contents were mixed thoroughly followed with separation
The culture was maintained on potato dextrose agar (PDA) of liquid, which was subjected to centrifugation at 4 °C at
medium slants and preserved under refrigeration at 4 °C. 10,000 rpm for 20 min. Finally, the crude enzyme was
For inoculum development, spores of A. niger BK01 obtained by filtering the supernatant through Whatman
were inoculated in 30 ml of Potato Dextrose broth (pH 5.0) filter paper no. 1. The crude enzyme was characterized by
contained in Erlenmeyer flasks followed with incubation at studying the effect of pH and temperature on CMCase
28 °C for 72 h under stationary conditions. Finally, the activity as well as the stability of the enzyme. To study the
spores of activated culture were harvested using sterilized effect of pH, the enzyme was incubated with buffers of
water containing 0.1% Tween 80 (Smith et al. 1996). different pH values (3.0–10.0), i.e., 0.1 M citrate buffer
(pH 3.0–6.0), 10 mM sodium phosphate buffer (pH
Pretreatment of rice straw 6.0–8.0), 0.05 M Tris–HCl (pH 8.0–9.0), and 0.05 M gly-
cine-NaOH (pH 9.0–10.0). The effect of temperature was
Rice straw, variety Basmati, was procured from the local studied by carrying out reactions at different temperatures
fields of Haryana state in India. The biomass was thor- ranging from 20–60 °C.
oughly washed, then chopped, and dried at 60 °C till
constant weight followed with grinding to the particle size Optimization of saccharification of rice straw
of 0.5 mm. Subsequently, it was subjected to two-stage
pretreatment: first with 0.5 M KOH at room temperature The crude enzyme produced by A. niger BK01, under
for 4 h, and then with 0.1 N H2SO4 at room temperature optimized conditions of SSF, was used for saccharification
for 1 h (bath ratio 1:10) (Goyal et al. 2014b). Subsequently, of the pretreated rice straw. To optimize the saccharifica-
the biomass was washed using water till neutral pH. The tion conditions, the effect of different concentrations of
compositional analysis of the biomass for cellulose, enzyme (loaded @ 1.0, 1.5, 2.0 and 2.5% v/v) and

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incubation time (0.5–3.0 h) was studied. The reaction was Table 1 Effect of pretreatment on biomass composition of rice straw
carried out at 40 °C using alkali-assisted acid pretreated Lignocellulosic content Untreated biomass Pretreated biomass
rice straw at 10% (w/v) concentration and the amount of
reducing sugars released was determined. Finally, the Cellulose (% w/w) 38.40 ± 1.40 59.50 ± 1.54 (54.94):
percent saccharification was calculated using formula: Hemicellulose (% w/w) 24.0 ± 1.50 8.26 ± 0.23 (65.58);
(Reducing sugars produced 9 0.9 9 dilutions/amount of Lignin (% w/w) 19.0 ± 1.0 5.17 ± 0.10 (72.78);
the cellulose) 9 100 (Begum and Alimon 2011). Figures in parenthesis indicate % change in the value due to alka-
li ? acid pretreatment as compared to the values for untreated sample
Enzyme assay

Carboxymethyl cellulase (CMCase/endoglucanase) activity 10

CMCase Activity (U/gds)

was assayed by the DNS (3, 5-dinitrosalicylic acid) method 8
(Miller 1959). The reaction mixture consisted of 900 ll of
substrate (CMC in 0.1 M citrate buffer, pH 4.8) and 100 ll 6
of crude enzyme and was incubated at 35 °C for 60 min. 4
The reaction was terminated by adding 3 ml of 3, 5-dini-
trosalicylic acid reagent. The tubes were incubated for 2
15 min in a boiling water bath for the color development 0
and the contents were cooled rapidly. The activity of the 4 5 6 7 8 9
reaction mixture was measured against a reagent blank at Substrate Concentration (% w/v)

540 nm. The concentration of the glucose released by the Fig. 1 Effect of substrate concentration on CMCase production by A.
enzyme was determined by comparing against a standard niger BK01 under SSF (fermentation conditions: pH 5.0; 6.0% v/v
curve plotted similarly using known concentrations of inoculum size; temperature 25 °C; incubation time 96 h; solid to
glucose. One enzyme unit (IU) is defined as the amount of liquid ratio 1:1.5)
enzyme required to hydrolyze 1 lg of substrate per min
under the assay conditions. The amount of the enzyme a decline in the activity. This can be attributed to the fact that
production was expressed as units per gram dry substrate high substrate concentration results in lower enzyme yields
(U/gds). due to the inhibitory effect of the byproducts released in large
quantities (Ramos et al. 1993). Different levels of the sub-
strate are required depending on the type of the substrate and
Results and discussion the microbial species. In a study by Gori and Malana (2010),
4% wheat straw was found optimum for maximum CMCase
Pretreatment of rice straw production by Aspergillus sp. Sherief et al. (2010) reported
5% rice straw as the best substrate concentration under SSF
Alkali-assisted acid pretreatment resulted in the change in conditions. In another study, 3% substrate concentration was
the biomass composition, i.e., increase in the cellulose found suitable for maximum CMCase production by Tri-
content as a result of the decrease in lignin and hemicel- choderma viride (Ahmed et al. 2010) and Trichoderma
lulose contents during alkali and acid pretreatments, harzianum (Iqbal et al. 2010) under SSF using wheat straw.
respectively (Goyal et al. 2014a, b); (Table 1).
Effect of inoculum size
Optimization of cultural conditions for cellulase
production under solid-state fermentation Fungal sporulation and metabolic activities are greatly
conditions influenced by the size of the inoculum (Domingues et al.
2000). On studying the effect of different inoculum levels
Effect of substrate concentration (6.0–9.0%, v/v), maximum CMCase activity of
8.84 ± 0.07 U/gds was recorded at 7.5% inoculum level
Optimum substrate concentration is an essential requirement (Fig. 2). The results highlight the importance of the
of the SSF to ensure the appropriate growth of microorgan- inoculum density in SSF. Lower inoculum size requires
isms. On studying the effect of the substrate concentration longer time for fungal multiplication and substrate uti-
(4.0–9.0%, w/v), it was found that the CMcase production by lization, whereas higher inoculum size increases the spore
A. niger BK01 increased maximum to 8.52 ± 0.04 U/gds density as well as the water content in the medium causing
when the concentration was raised from 4 to 6% (Fig. 1). hindrance in oxygen penetration resulting in the inhibited
However, an increase in concentration beyond 6% resulted in fungal growth and enzyme production (Vu et al. 2011).

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10 10
CMCase Activity (U/gds)

CMCase Activity (U/gds)

9
8 8
7
6 6
5
4 4
3
2
2
0 1
6 6.5 7 7.5 8 8.5 9 0
Inoculum size (% v/v) 24 48 72 96 120
Incubation time (h)
Fig. 2 Effect of inoculum size on CMCase production by A. niger
BK01 under SSF (fermentation conditions: pH 5.0; 6.0% w/v Fig. 3 Effect of incubation time on CMCase production by A. niger
substrate concentration; temperature 25 °C; incubation time 96 h; BK01 under SSF (fermentation conditions: pH 5.0; 6.0% w/v
solid to liquid ratio 1:1.5) substrate concentration; 7.5% v/v inoculum size; temperature
25 °C; solid to liquid ratio 1:1.5)

Fadel (2001) reported maximum cellulase activity by A.

12
niger with 10% inoculum size using wheat straw as a

CMCase Activity (U/gds)

substrate. Omojasola and Jilani (2009) reported 8% 10
inoculum size suitable for maximum cellulase production 8
by A. niger. Murad and Azzaz (2013) have reported 7% 6
inoculum size optimum for maximum cellulase production
4
by Aspergillus flavus using rice straw.
2

Effect of incubation time 0

4 4.5 5 5.5 6 6.5 7
pH
CMCase production by A. niger BK01 reached maximum
levels after 96 h of incubation yielding 9.06 ± 0.06 U/gds Fig. 4 Effect of pH on CMCase production by A. niger BK01
of CMCase. Thereafter, the enzyme production started (fermentation conditions: 6.0% w/v substrate concentration; 7.5% v/v
inoculum size; temperature 25 °C; solid to liquid ratio 1:1.5;
decreasing significantly (Fig. 3). This could be due to the incubation time 96 h)
loss of moisture, denaturation of the enzyme as a result of
variation in pH during fermentation, or the accumulative
effect of cellobiose inhibitory to the CMCase enzyme A pH value lower or higher than the optimum affects the
(Melo et al. 2007; Singh et al. 2009). The optimal incu- metabolic activities of the organism. It also influences
bation time varies with the type and composition of the stability of the enzyme and may lead to the protein
fermentation medium, initial pH, and different fungal denaturation (Kalra and Sandhu 1986). In different fungal
species employed for enzyme production. Similar to our species, the optimum pH for CMCase production has been
observations, Milala et al. (2005) and Ilyas et al. (2011) found to vary from 3.0 to 6.0 (Rodriguez et al. 2005;
also reported maximum CMCase production by A. niger in Niranjane et al. 2007). Different workers have reported an
96 h. In other studies, the optimal incubation period for optimum pH of 5.5 for maximum CMCase production by
maximum CMCase production was documented to be 3, 5, A. niger AT-3 (Dutt and Kumar 2014), Aspergillus fumi-
and 10 days in Aspergillus sp. SU14-M15 (Vu et al. 2011), gatus (Sherief et al. 2010), T. viride (Ahmed et al. 2010),
Trichoderma reesei (Fatma et al. 2010), and Rhizopus and T. reesei RUT-C30 (Haq et al. 2001; Xiong et al.
stolonifer (Pothiraj et al. 2006), respectively, using differ- 2004). Fadel (2001) found pH 4.5 optimal for maximum
ent substrates. The reports have indicated that the optimum CMCase synthesis by A. niger under SSF. Acharya et al.
time for the synthesis of cellulolytic enzymes during SSF (2008) documented that optimum pH for cellulase pro-
of lignocellulosic residues lies in the range of 3–8 days duction by A. niger, using saw dust substrate, was between
(Jecu 2000; Panagiotou et al. 2003; Narasimha et al. 2006). 4.0 and 4.5. Sohail et al. (2009) have found more acidic
pH, i.e., 4.0 optimal for cellulases production by A. niger
Effect of pH MS82.

On studying the effect of initial pH, the maximum pro- Effect of incubation temperature
duction of CMCase, i.e., 9.54 ± 0.06 U/gds, was observed
at pH 5.5 (Fig. 4). Optimal pH is an important parameter Temperature strongly affects the SSF process. Optimiza-
for the microbial growth as well as the enzyme production. tion of temperature is essential, because it significantly

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12 12

CMCase activity (U/gds)

CMCase Activity (U/gds)

10 10
8
8
6
6
4
4
2
2 0
1:1 1:2 1:3 1:4 1:5
0
Solid to liquid ratio
20 25 28 30 35 40
Temperature (°C)
Fig. 6 Effect of moisture level on CMCase production by A. niger
BK01 under SSF (fermentation conditions: 6.0% w/v substrate
Fig. 5 Effect of incubation temperature on CMCase production by A.
concentration; 7.5% v/v inoculum size; incubation time 96 h; pH
niger BK01 (fermentation conditions: 6.0% w/v substrate concentra-
5.5; temperature 28 °C)
tion; 7.5% v/v inoculum size; solid to liquid ratio 1:1.5; incubation
time 96 h; pH 5.5)
optimal water levels in the solid substrate appear to be
40–60% (by mass) under solid-state fermentation condi-
influences the metabolic activities of an organism. A tions. Fatma et al. (2010) demonstrated a ratio of 1:3
temperature lower or higher than the optimum may lead to (substrate: moistening agent) optimal for maximum
the decreased transport across cell envelope or enzyme CMCase production by T. reesei. In another study by Vu
denaturation, respectively (Dutt and Kumar 2014). It also et al. (2011), maximum CMCase production by Aspergillus
plays a vital role in production of the end-products (Ahmed sp. SU14 was observed using 50% moisture content,
et al. 2009). Even slight changes in the temperature can whereas 70% moisture content was found suitable for
affect the enzyme production. In the present work, maxi- maximum cellulase production by A. niger (Ilyas et al.
mum production of CMCase by A. niger BK01 was 2011).
achieved at 28 °C resulting in 9.86 ± 0.05 U/gds enzyme
activity (Fig. 5). Similarly, 28 °C temperature was found Effect of carbon sources
suitable for maximum CMCase production by T. reesei
(Singhania et al. 2006) and A. niger (Acharya et al. 2008). The SSF production medium was supplemented with dif-
In other studies, an optimum temperature of around ferent carbon sources (0.1% w/v), from which CMC
30 ± 2 °C has been reported for CMCase production by A. showed the stimulatory effect for maximum CMCase
niger (Ilyas et al. 2011; Mrudula and Murugammal 2011). (11.75 ± 0.05 U/gds) production by the fungus A. niger
Dutt and Kumar (2014) have found 35 °C temperature BK01 (Fig. 7). In a study by Irfan et al. (2012), an increase
optimum for highest levels of cellulases synthesis by A. in CMCase production was recorded in T. viride on addi-
niger AT-3. tion of CMC (0.5%) in the fermentation medium as a
carbon source. On the other hand, Vu et al. (2011) men-
Effect of moisture level on CMCase production tioned that CMCase was expressed maximum when
Aspergillus sp. SU14-M15 was grown in the presence of
To determine the effect of moisture level, the substrate was (1%) rice starch and corn starch under solid-state fermen-
moistened by Mandel and Sternburg’s medium in different tation. Irfan et al. (2011) documented glucose as the best
solid-to-liquid ratios ranging from 1:1 to 1:5. A ratio of 1:2 additional carbon source while producing CMCase from
was found to be best for producing highest levels of Aspergillus sp. It is evident from various research studies
CMCase (10.98 ± 0.07 U/gds) by A. niger BK01 (Fig. 6). that the cellulolytic systems in different fungi are induced
Moisture is the most significant factor in the solid-state to different levels in the presence of diverse sources of
fermentation. The efficiency of the mass transfer in the carbon (Amore et al. 2013). The presence of an easily
solid-phase particles depends on the substrate characteris- utilizable form of carbon, supportive for the growth of the
tics and the appropriate moisture (Liu and Yang 2007). fungus, may not be inductive for high cellulase production
Very high moisture content results in decreased substrate by the same fungal species (Tong and Rajendra 1992). A
porosity and reduced oxygen penetration (Vu et al. 2010). study by Nazir et al. (2010) has also shown differential
On the other hand, excessively low moisture levels lead to expression of endoglucanases and b-glucosidases isoforms
poor microbial growth and poor accessibility to nutrients by A. terreus in the presence of different carbon sources
(Vu et al. 2010). Narasimha et al. (2006) reported that and culture conditions.

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Lactose peptone was recorded as the best nitrogen source (Acharya

Carbon Source (0.1 % w/v)

et al. 2008). Like carbon sources, the nitrogen sources also

Cellulose Powder
cause differential expression of cellulolytic genes in dif-
Galactose ferent microbial species to different levels.
Carboxymethyl cellulose

Sucrose Characterization of cellulases

Maltose
Effect of pH on activity and stability of enzyme
0 2 4 6 8 10 12 14
CMCase activity (U/gds)
The optimum pH level for the enzyme activity was deter-
Fig. 7 Effect of carbon source on CMCase production by A. niger mined by incubating crude enzyme from A. niger BK01
BK01 (fermentation conditions: 6.0% w/v substrate concentration; with CMC at different pH levels. Highest CMCase activity
7.5% v/v inoculum size; incubation time 96 h; pH 5.5; temperature of the enzyme was observed at pH 4.8. However, the
28 °C; solid to liquid ratio 1:2)
optimum pH range recorded for CMCase activity ([80%)
was 4.8–6.0. The enzyme showed[80% stability in the pH
Effect of nitrogen sources
range of 4.8–5.5 (Fig. 9). From the results, it was con-
cluded that the CMCase from the fungal isolate needed an
The presence of additional nitrogen sources along with the
acidic environment to be active. Increasing or decreasing
nitrogenous compounds present in the substrate could
pH beyond optimum range resulted in a significant decline
promote enhanced growth and consequent enzyme pro-
in the enzyme activity. Any change in the pH is known to
duction. The effect of various nitrogen sources (0.1% w/v)
cause changes in the enzyme’s active site resulting in a
was, therefore, studied on CMCase production by A. niger
change in the enzyme activity. Akiba et al. (1995) reported
BK01. The results depicted highest levels of CMCase
pH 6.0–7.0 optimum for CMCase from A. niger. Saha
(12.0 ± 0.07 U/gds) production by the fungus in the
(2004) reported pH range 4.0–7.0 optimum for activity of
presence of ammonium nitrate (Fig. 8). Nitrogen is one of
CMCase from M. circinelloides. Cellulases produced from
the major elements of cellular proteins. The stimulation of
Chrysosporium lucknowense and A. fumigatus were found
cellulase activity by the ammonium salts might be due to
stable at pH 5.0 (Gusakov et al. 2005; Sherief et al. 2010).
their direct entry in the protein synthesis (Mandels 1975).
In a study by Gokhale et al. (1991), A. niger NCIM 1207
Effect of temperature on activity and stability of enzyme
showed enhanced cellulase production in the presence of
ammonium sulphate, ammonium dihydrogen orthophos-
Activity and stability of the crude cellulase from A. niger
phate, and corn-steep liquor. Singhania et al. (2006) also
BK01 were also tested at different temperatures. It was
observed that ammonium nitrate increased the CMCase
found that activity of the CMCase increased rapidly when
production by T. reesei NRRL 11460 during SSF. Vyas
the temperate was raised from 20 to 30 °C. The enzyme
et al. (2005) found ammonium sulphate suitable for max-
showed [80% activity in the range of 30–50 °C and
imum CMCase production by Aspergillus terrus using
highest activity was recorded at 40 °C. However, an
pretreated groundnut shells. In another study on CMCase
increase in temperature beyond 50 °C resulted in a sharp
production by A. niger under solid-state fermentation, 0.1%
decline in the activity. This could be due to the reason that
increasing temperature beyond the optimum value causes a
Nitrogen Source (0.1 % w/v)

Potassium nitrate
Urea
Beef 120 Stability Activity
CMCase activity (%)

Tryptone 100
Ammonium nitrate 80
Ammonium Phosphate 60
Ammonium Chloride 40
0 5 10 15 20
CMCase activity (U/gds) 0
3 4 4.8 5 5.5 6 7 8
Fig. 8 Effect of nitrogen sources on CMCase production by A. niger pH
BK01 (fermentation conditions: 6.0% w/v substrate concentration;
7.5% v/v inoculum size; incubation time 96 h; pH 5.5; temperature Fig. 9 Effect of different pH on activity and stability of crude
28 °C; solid to liquid ratio 1:2; 0.1% w/v CMC) CMCase from A. niger BK01

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120 Stability Activity 40 1% 1.50% 2% 2.50%

CMCase activity (%)

35

Saccharification (%)
100
30
80
25
60
20
40
15
20
10
0 5
20 30 35 40 45 50 60
0
Temperature (°C)
0.5 1 1.5 2 2.5
Time (h)
Fig. 10 Effect of different temperatures on activity and stability of
crude CMCase from A. niger BK01 Fig. 11 Effect of A. niger BK01 crude cellulase enzyme concentra-
tion on saccharification of pretreated rice straw
decrease in the catalytic rate of the enzyme as a result of its
denaturation. The stability ([80%) of the crude enzyme Maximum 23.78% sugars were released after 2.5 h incu-
was achieved in the range of 35–50 °C (Fig. 10). El-Azab bation period with cellulase loading of 2% (Table 2). The
(2007) reported that 45–55 °C temperature is optimum highest saccharification value recorded under optimized
range for CMCase activity. Optimum temperatures repor- conditions was 35.96% (Fig. 11; Table 3).
ted by other workers were 55 °C for that from T. viride Many workers have used microbial enzymes for the
(Sharma et al. 1991) and 40 °C for that from A. fumigatus hydrolysis of lignocellulosic materials. The saccharification
(Sherief et al. 2010). On the other hand, the crude cellu- of cotton, filter paper, and newspaper using T. viride culture
lases of M. circinelloides showed an optimum temperature filtrate resulted in a saccharification rate of 9.9, 59.4, and
of 55 °C (Saha 2004). 41.8%, respectively (Mandels et al. 1974). The rate of sac-
charification was 3.5, 1.5, and 3.0% during hydrolysis of the
Saccharification optimization saw dust, filter paper, and newspaper, respectively, using
Sporotrichum thermophile culture filtrate (El-Naghy et al.
The crude cellulase enzyme produced from A. niger BK01 1991). Ja’afaru and Fagade (2007) achieved 5.0% sacchari-
using pretreated rice straw, through SSF under optimized fication rate for treated corn cob using A. niger crude
conditions, was used for saccharification of the alkali-as- enzyme. Wati et al. (2007) reported the hydrolysis of alkali-
sisted acid pretreated rice straw. The different enzyme treated paddy straw with a commercial preparation of cel-
preparations were loaded @ 1, 1.5, 2, 2.5% (v/v) concen- lulase resulting in the release of 65% total reducing sugars.
trations and hydrolysis was carried out for different time Fatma et al. (2010) reported enzymatic saccharification of
intervals at 40 °C and pH 4.8. The results showed maxi- alkali-treated rice straw with cellulases of T. ressei and
mum hydrolysis of the biomass occurred in 2.5 h. observed maximum glucose yield of 1.07% after 16 h of
Increasing the enzyme loadings from 1 to 2% enhanced the incubation. Another work by Kumar and Pushpa (2012)
rate of saccharification. Further increase in the enzyme showed the release of 73.30 mg/g of reducing sugars after
concentration released lesser amounts of the sugars. treatment of rice straw by T. reesei.

Table 2 Effect of cellulase concentration on the total reducing sugars released from pretreated rice straw at different time intervals with fungal
crude cellulase enzyme from A. niger BK01
Time (h) Total reducing sugars (% w/w) at different enzyme concentration (% v/v)
1 1.5 2 2.5

0.5 3.68 ± 0.22 5.87 ± 0.17 6.43 ± 0.06 5.21 ± 0.10

1.0 7.15 ± 0.31 9.43 ± 0.25 11.55 ± 0.29 8.92 ± 0.17
1.5 10.86 ± 0.32 14.72 ± 0.21 17.35 ± 0.18 13.86 ± 0.23
2.0 12.72 ± 0.11 18.24 ± 0.16 20.56 ± 0.15 17.78 ± 0.11
2.5 16.43 ± 0.02 21.15 ± 0.04 23.78 ± 0.13 20.57 ± 0.19
Alkali-assisted acidic pretreated rice straw was used at 10% conc. and reaction was carried out at 40 °C

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Table 3 Saccharification study of pretreated rice straw using crude cellulase enzyme from A. niger BK01
Enzyme Pretreated rice straw Reducing sugar (g/g of Reducing sugar (g/g of Saccharification
concentration substrate) cellulose) value

Crude cellulase enzyme from A. niger 10 g (5.95 g) 0.2378 0.3996 35.96

isolate BK01
Figures in parenthesis indicate the cellulose present in the substrate
Conditions: substrate concentration 10% w/v, Enzyme loading 2% v/v, 40 °C, pH 4.8, time 2.5 h

Conclusion Ahmed I, Zia MA, Iqbal HMN (2010) Bioprocessing of proximally

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the Haryana State Council of Science and Technology (HSCST),
dase enzymes of Aspergillus niger grown under solid state
Haryana, India, for providing the financial aid for this research work.
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Fatma H, El-Zaher Abd, Fadel M (2010) Production of bioethanol via
Compliance with ethical standards
enzymatic saccharification of rice straw by cellulase produced by
Trichoderma reesei under solid state fermentation. N Y Sci J
Conflict of interest The authors declare that there is no conflict of
3:72–78
interest regarding publication of this paper.
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