Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2012, 2 (5):717-726
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ISSN : 2231 –3168

CODEN (USA) : JMBRB4

Alkali pretreatment of rice straw and enhanced cellulase production by a

locally isolated fungus Aspergillus fumigatus NITDGPKA3
Nibedita Sarkara and Kaustav Aikata*
a
Department of Biotechnology, National Institute of Technology, Durgapur, West Bengal, India
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ABSTRACT

Studies were conducted on the alkali pretreatment of rice straw by varying NaOH concentration from 0.1 M to 2.5
M. Cellulose content was maximum (62.19%) for 0.5 M NaOH. Lignin content showed a significant decrease on
increase in NaOH concentration up to 0.5M NaOH. Significant morphological changes were detected in the rice
straw structure by scanning electron microscopy after pretreatment. A locally isolated cellulolytic fungus
Aspergillus fumigatus NITDGPKA3 was used for cellulase (CMCase and FPase) and xylanase production under
submerged fermentation of rice straw pretreated with various NaOH concentrations. Maximum enzyme activites
were obtained for 0.5 M NaOH. Culture conditions (incubation temperature, initial pH and rotational speed) for
cellulase and xylanase production under submerged fermentation of thus pretreated rice straw by Aspergillus
fumigatus NITDGPKA3 were statistically optimized by response surface methodology based on central composite
design. The optimum conditions were found to be incubation temperature 30oC, rotational speed 120 rpm and initial
pH 4.17 (CMCase and FPase) and 4.5 (xylanase). A significantly higher titre of xylanase was obtained compared to
the available literature.

Key words: Alkali pretreatment , Cellulase , Rice straw , Submerged fermentation

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INTRODUCTION

Limited storage of petroleum based fuels has increased the demand of alternative liquid fuel from renewable
sources. In that aspect, lignocellulosic biomass is the only renewable carbon source available in large quantities and
thus can be a solution to the problems of energy. Agricultural residues generate million tones of agricultural wastes
each year such as wheat straw, corn straw, sugarcane baggase and rice straw. Rice straw is one of the most abundant
agricultural waste. According to FAO points 600-900 million tones of rice straw is produced each year globally [1].
Asia has over 90% of worldwide rice straw production. Most of the biomass remains unused, destroyed simply by
burning and increases the air pollution which consequently affects public health [2]. Therefore, rice straw is believed
to have potential as the preferential feedstock for fuel ethanol production in Asia [3] . Rice straw contains 25-45%
cellulose, 20-30% hemicellulose and 10-15% lignin [4].

The major polysaccharide cellulose is associated with hemicelluloses and surrounded by lignin. Tightly bound lignin
of the heteropolymer impedes enzymatic hydrolysis of underneath hemicellulose and crystalline cellulose [5].
Several pretreatment methods such as alkali hydrolysis, acid hydrolysis, steaming, liquid explosion are used to
remove lignin and hemicellulose to make cellulose available for the microorganisms to act upon [6]. The
pretreatment of lignocelluloses increases the accessibility of the surface area of cellulose for enzymatic conversion
[7].

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Cellulose can be hydrolyzed by acid or enzymatic treatments, for a high yield of soluble products of low molecular
weight, such as hexoses and pentoses [8]. Cellulases are the enzymes primarily implicated in the saccharification
process [9]. Cellulase is also used for the production of single cell protein and chemicals from renewable cellulosic
resources. Due to its potential application in deinking of mixed office waste (MOW), magazines and newspapers,
denim stone washing cellulase have gained significant commercial importance [10,11,12]. In addition,
saccharification of lignocellulosics

to fermentable sugars may be enhanced by the enzyme cocktails of cellulase and hemicellulase [14]. Large scale
bioethanol production is impeded by cost of the cellulase enzyme and substrate. Thus use of microbial cellulase and
cheap biomass as substrate can help to reduce the cost of bioethanol production. It is also necessary to improve the
yields of the enzymes to make the process economically attractive [13]. Cellulases are produced by a variety of
microorganisms including bacteria, actinomycetes and fungi where fungi are known to secrete cellulases in large
amounts [14]. Cellulase is a complex of enzymes that work synergistically to attack native cellulose. It is a cocktail
of the enzymes exoglucanase, endoglucanase, and β- glucosidase. At first endoglucanase (EC 3.2.1.4) acts
randomly on soluble and insoluble cellulose chains; then exoglucanase (cellobiohydrolase EC 3.2.1.91) acts to
liberate cellobiose from the reducing and non-reducing ends of cellulose chains and finally, β -glucosidase (EC
3.2.1.21) liberates glucose from cellobiose [15].

‘One factor at a time’ technique is a general optimization method . It is determined by varying one factor while
keeping the other factors at a constant level. This type of method is simple but time consuming and laborious.
Recently statistical designs for optimization has been successfully employed in enzyme production [16,9].

Response surface methodology (RSM) is a powerful strategic experimental tool that efficiently determine the
optimum conditions by considering both the effect of primary factors and their mutual interactions in a multivariate
system [17]. Among all nonlinear optimization techniques RSM is the most common method for process variable
optimization [18, 19, 20]. RSM is an effective statistical technique for optimizing a complex processes. It reduces
the number of experimental trials required to evaluate multiple parameters and their interactions. In the present
study RSM was employed to identify the optimum conditions for cellulase production from rice straw by a locally
isolated cellulolytic fungus Aspergillus fumigatus NITDGPKA3 under submerged fermentation .

MATERIALS AND METHODS

Microorganism
A locally isolated cellulolytic fungus Aspergillus fumigatus NITDGPKA3 was used for cellulase production. The
culture was maintained on Czapek modified medium (CMM) agar slants (0.2% NaNO3, 0.05% KCl, 0.05% MgSO4,
0.001% FeSO4, 0.1% K2HPO4, 0.2% peptone and 2% agar) at 4oC [23].

Pretreatment of Raw Material

Locally procured rice straw was washed, air dried, size fractioned to 0.5mm and stored at room temperature for
further use. The main components of untreated rice straw were determined to be 40.09 % cellulose, 26.8 %
hemicellulose, 18.9 % lignin and 10.76% ash. The rice straw was pretreated with NaOH of different concentrations
(0.1 – 2.5 M) at 121oC and 15 psi pressure for 1 hr at the ratio of 1:10 to substrate and NaOH solution. The
pretreated rice straw was washed with tap water until the pH of the filtrate reached 7.The washed straw was dried at
60oC overnight to constant weight and stored at room temperature for further use.

Estimation of cellulose, hemicellulose and lignin [21]

Cellulose estimation
For cellulose estimation, acetic/nitric reagent (150 ml of 80% acetic acid and 15 ml of concentrated nitric acid) was
added to 0.5 g sample and kept in a water bath at 100oC for 30 minutes. The mixture was cooled and centrifuged for
20 minutes. The pellet was washed with distilled water, mixed with 10 ml of 67% H2SO4 and allowed to stand for1
hour at room temperature. The solution (1 ml) was diluted to 100 ml and to 1 ml of this diluted solution was added
10 ml of freshly prepared anthrone reagent (0.2 % anthrone in concentrated H2SO4). The mixture was heated in a
boiling water bath for 10 minutes, cooled and the colour was measured at 630 nm. Cellulose powder (Hi Media,
India) was used as a standard.

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Hemicellulose estimation
In a refluxing flask, 1 g powdered sample was mixed with 10 ml cold neutral detergent solution. The neutral
detergent solution was prepared as follows.

Disodium ethylenediamine tetraacetate ( 18.61 g) and sodium borate decahydrate (6.81 g) were dissolved in about
200 ml of distilled water by heating and to this, 100 ml solution containing sodium lauryl sulphate (30 g) and ethoxy
ethanol (10ml) was added. A solution (100 ml) of 4.5% Na2HPO4 was then added to the mixture. The final volume
was made up to 1 litre with distilled water and pH adjusted to 7.

Decahydronapthalene (2 ml) and sodium sulphite (0.5 g) was added to the mixture of rice straw sample and cold
neutral detergent solution, which was then heated to boiling and refluxed for 1 hour. The contents were filtered
through sintered glass crucible (G-2) and washed with hot water. The contents were finally washed with acetone
twice and the residue transferred to a crucible. The sample was dried at 100oC for 8 hour, cooled in a desiccator and
weighed. The residue was designated as neutral detergent fiber (NDF). To calculate hemicellulose content, the
amount of acid detergent fiber (ADF) was subtracted from the amount of neutral detergent fiber (NDF). The acid
detergent fiber (ADF) was prepared as follows.

Powdered sample (1 g) was mixed with 100 ml acid detergent solution (2% cetyl trimethyl ammonium bromide in 1
N sulphuric acid). The sample was heated to boil in 5-10 minutes and refluxed for 1 hour after the onset of boiling.
The contents were filtered through a preweighed sintered glass crucible (G-2) by suction and washed with hot water
twice. The contents were washed with acetone until the filtrate was colorless, dried at 100oC for overnight and
weighed after cooling in a desiccator. The ADF content was expressed as a percentage i.e., W/S × 100 where W was
the weight of the fiber and S was the weight of the sample.

Lignin estimation
For lignin estimation the acid detergent fiber (ADF) was mixed with 25 ml of 72% H2SO4 and 1 g asbestos. The
mixture was kept for 3 hours at room temperature with intermittent stirring. The mixture was then diluted, filtered
with preweighed Whatman No. 1 filter paper, dried at 100oC and weighed after cooling in a desiccator. Then the
filter paper was transferred to a preweighed silica crucible and kept in a muffle furnace at 550oC for 3 hours. The
crucible was cooled in a desiccator and weighed to calculate ash content. Asbestos was used as blank. The acid
detergent lignin (% ADL) was determined using

{Weight 72% H 2SO 4 (Test - Asbestos blank) - Ash ( Test - Asbestos blank)}× 100
% ADL = Weight of the Sample

Inoculum
Pure culture of Aspergillus fumigatus NITDGPKA3 was subcultured on Czapek modified agar medium and
incubated at 30oC. Fully sporulated plates were obtained after 6 days. The sporulated plates were flooded using 20
ml of distilled water to harvest the spores and obtain the resulting spore suspension, which was used as inoculum in
subsequent experiments.

Cellulase production
Rice straw samples pretreated with different alkali concentrations (0.1M- 2.5 M) were used for enzyme production
to determine the maximum FPase, CMCase and xylanase activities. Enzyme production was carried out in 250 ml
Erlenmeyer flasks containing 0.5 gm of alkali pretreated rice straw in 50 ml basal medium (Czapek modified
medium). Tween 80 (5g/l) was added to the medium for the statistical optimization studies. The pH of the medium
was adjusted to 5. The flasks were autoclaved at 121oC (15psi) for 15 minutes and thereafter cooled to room
temperature and inoculated with 5% (v/v) of the inoculum containing (106 spores/ml) and incubated for 5 days at
30oC, 120 rpm. After incubation the culture broths were centrifuged at 8000 rpm at 4oC for 15 minutes and the
supernatants stored at 4oC for enzyme assay.

Enzyme assay
Enzyme activities were assayed by estimating total reducing sugar released. Enzyme activity was measured by the
methods of International Union of Pure and Applied Chemistry (IUPAC) Commission on Biotechnology [22].
CMCase and xylanase activities were determined using 2 % (w/v) carboxymethyl cellulose ( Himedia, India) and

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2% (w/v) oat spelt xylan ( Himedia, India) solution prepared in 0.05 M, pH 4.8 Na-citrate buffer respectively. The
reaction mixture, containing suitably diluted enzyme solution ( 0.5 ml) and 0.5 ml of substrate solution was
incubated at 50oC for 30 minutes and 10 minutes for CMCase and xylanase respectively . To determine FPase
activity 0.5 ml of enzyme solution was incubated with 1 ml of 0.05 M , pH 4.8 sodium citrate buffer containing 1cm
X 6cm (=50 mg) Whatman filter paper strip at 50oC for 60 minutes.

In all the cases, after incubation, the released reducing sugar was estimated by the DNS method with some
modifications [23]. 3,5- dinitrosalycylic acid (1 ml) was added to the reaction mixture and incubated for 5 minutes
in a vigorously boiling water bath. Na-K tartarate solution (1 ml) was then added to the mixtures and cooled rapidly.
The reducing sugar was estimated from the absorbance measured at 540 nm using glucose and xylose as standard.
Enzymatic activities were defined in International Units (IU). One unit of enzymatic activity is defined as the
amount of enzyme that releases 1 µmol reducing sugars/ml/minute.

Statistical optimization
Optimization of enzyme production was done using the response surface methodology with 0.5M NaOH pretreated
rice straw. For statistical optimization the basal medium and enzyme production procedure used were the same as
described above where initial pH, temperature and rotational speed were set according to experimental design. A
two-level fractional factorial design with six centre points was employed in order to determine the optimal process
conditions for the production of FPase, CMCase and xylanase. Three independent process variable including
incubation temperature (A), pH (B), rotational speed (C) were chosen for statistical optimization by RSM using
central composite design (CCD) and varied over five levels (Table 1).

Table. 1. Experimental range and levels of independent process variables

Factor Name Range of variables

-α -1 0 +1 +α
A Temperature 20 25 30 35 38
B pH 2 3 4.5 6 7
C Rotational speed 100 120 150 180 200

.The FPase, CMCase and xylanase activities were taken as the response. Both models constructed as a response
function were a second order polynomial regression model.

Y = b0 + ∑ bi Ai + ∑ bii Ai2 + ∑ bij Ai A j Eq.1

For three variable parameters, the model equation is given below ( Eq. 2)

Y = b0 + b1 A + b2 B + b3C + b11 A2 + b22 B 2 + b33C 2 + b12 AB + b13 AC + b23 BC Eq. 2

where Y is the predicted response ( dependent variable); b0 is a constant; b1, b2 and b3 are the linear coefficients; b12,
b13, b23 are the cross product coefficients and b11, b22, b33 are quadratic coefficients. A, B and C are the coded forms
of selected variables i.e. incubation temperature, pH and rotational speed respectively. A total of 20 experiments
including 6 centre points were generated by design of experiment using the statistical software 7.0.0 (Stat Ease,
USA).

Statistical significance of respective model equations was checked by ANOVA ( Analysis of Variance).3D model
graphs were obtained to analyze the effects of variables and their interactions.

RESULTS AND DISCUSSION

Alkali pretreatment of rice straw

The effect of different NaOH concentrations (0.1M – 2.5M) on chemical composition of rice straw such as cellulose,
hemicellulose and lignin was analyzed. Cellulose, hemicellulose and lignin contents are shown in Table 2 where
maximum cellulose content ( 62.19%) was obtained from 0.5 M NaOH pretreated rice straw. Further increase in
alkali concentration did not show any further increase in cellulose yield. Hemicellulose content was seen to decrease

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drastically with the increase at alkali concentration from 0.1M to 2.5M. The lignin content was seen to decrease at
alkali concentration from 0.1M to 0.5M and was consistent after 0.5M NaOH pretreatment.

Table. 2. Effect of alkali pretreatment on cellulose, hemicellulose, lignin and ash content of rice straw

NaOH Concentration (M) Cellulose (%) Hemicellulose (%) Lignin (%) Ash (%)
0.1 41.8 ± 0.01 25.6 ± 0.01 17.9 ± 0.02 11.24 ± 0.02
0.25 42.6 ± 0.02 24.1 ± 0.01 16.1 ± 0.01 12.12 ± 0.03
0.5 62.19 ± 0.02 14.5 ± 0.02 8.4 ± 0.01 14.54 ± 0.03
1 60.4 ± 0.01 10.21 ± 0.04 8.3 ± 0.01 16.75 ± 0.02
1.5 59.3 ± 0.02 8.34 ± 0.02 8.2 ± 0.02 17.54 ± 0.01
2 60.6 ± 0.01 7.78 ± 0.01 8.1 ± 0.01 17.78 ± 0.02
2.5 61.8 ± 0.01 6.92 ± 0.02 8.1 ± 0.03 18.23 + 0.02

The morphological changes of alkali pretreated rice straw with different NaOH concentration ( 0.1M – 2.5M ) were
examined by scanning electron microscopy (SEM) using S-530,SEM, Hitachi, Japan. The SEM micrographs of raw
and alkali pretreated rice straw samples in Fig 1(a -h ) provide the longitudinal section .Compared with the SEM
micrograph of raw straw a significant morphological change was observed in the pretreated rice straw. The untreated
rice straw showed rigid and highly ordered fibrils. The rigidity and order of the fibers were gradually lost and
microfibrils were fully exposed with the increase of alkali concentration. Thus external surface area and porosity
increased.

Figure.1. SEM micrographs of rice straw before and after pretreatment with NaOH at differenr concentration. The photographs have
been taken at 1000 magnification. A untreated. B 0.1% NaOH. C 0.25% NaOH. D 0.5% NaOH. E 1% NaOH. F 1.5% NaOH. G 2%
NaOH. H 2.5% NaOH.

In addition to SEM micrographs and determination of cellulose, hemicellulose , lignin and ash contents, all the straw
samples were used for cellulase production .The FPase, CMCase and xylanase activities obtained from each sample
are shown in Fig. 2. It was observed that FPase, CMCase and xylanase activities gradually increased with increase in
NaOH concentration where 0.5M NaOH pretreated rice straw resulted in maximum enzyme production. An
improvement of enzyme activity may be due to the increase of available cellulose surface and decrease of lignin
with the gradual increase of NaOH concentration.

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Figure. 2 Enzyme activities obtained from pretreated rice straw samples at different NaOH concentration. Symbols: FPase (▲); CMCase
(♦); xylanase (■)

Response surface approach by central composite design

The effect of three independent variables on cellulase and xylanase production was investigated and Central
Composite Design (CCD) was used for optimization of production where 20 experimental runs were carried out
with different combinations of the three factors. Experimental design and actual response along with the predicted
response are given in Table 3. The predicted response was calculated by the regression equation. With the design
conditions the CMCase, FPase and xylanase activities markedly varied in range of 1.475 – 13.9 IU/ml , 0.395 –
2.435 IU/ml and 28 – 249.11 IU/ml respectively.

Table. 3. Experimental and predicted values of FPase, CMCase and xylanase obtained in experimental set up of response surface
methodology

Serial No Temperature pH Rotational speed (rpm) FPase CMCase Xylanase

(oC) Actual / Predicted Actual / Predicted Actual / Predicted
(IU/ml) (IU/ml) (IU/ml)
1 25 3 120 1.375 (1.386) 7.865 (7.812) 158.100 (159.270)
2 35 3 120 1.811 (1.896) 6.752 (6.826) 140.000 (141.020)
3 25 6 120 1.182 (1.196) 7.591 (7.574) 148.900 (148.190)
4 35 6 120 1.095 (1.095) 8.239 (8.161) 157.110 (158.020)
5 25 3 180 1.112 (1.159) 7.544 (7.629) 150.899 (150.470)
6 35 3 180 1.325 (1.359) 4.555 (4.578) 90.830 ( 92.020)
7 25 6 180 1.520 (1.474) 7.861 (7.791) 155.980 (155.440)
8 35 6 180 1.025 (1.064) 6.254 (6.313) 125.760 (125.070)
9 20 4.5 150 0.395 (0.400) 3.467 (3.502) 67.120 ( 67.660)
10 38 4.5 150 0.561 (0.483) 1.476 (1.431) 28.000 ( 26.780)
11 30 2 150 2.001 (1.911) 10.032 (9.959) 197.200 (195.680)
12 30 7 150 1.485 (1.503) 11.151 (11.218) 213.310 (214.160)
13 30 4.5 100 2.181 (2.130) 10.132 (10.183) 208.000 (206.810)
14 30 4.5 200 1.935 (1.913) 8.530 ( 8.476) 171.191 (171.700)
15 30 4.5 150 2.373 (2.397) 13.752 (13.658) 245.220 (245.170)
16 30 4.5 150 2.425 (2.397) 13.911 (13.658) 248.300 (245.170)
17 30 4.5 150 2.405 (2.397) 13.652 (13.658) 243.150 (245.170)
18 30 4.5 150 2.319 (2.397) 13.722 (13.658) 244.360 (245.170)
19 30 4.5 150 2.432 (2.397) 13.525 (13.658) 240.780 (245.170)
20 30 4.5 150 2.435 (2.397) 13.455 (13.658) 249.110 (245.170)

A comparison of predicted values with the experimental values of FPase, CMCase and xylanase activities has been
shown in Fig.3(a-c) respectively. ANOVA was employed to ascertain the significant variable and their interaction
effect on FPase, CMCase and xylanase production. The ANOVA of the quardratic regression model are summarized

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in Table 4. The computed R2 values for endoglucanase, FPase and xylanase were obtained between 95.3% and
99.9% which determines a good fitness of the model.

Figure.3. Plots of predicted vs actual responses. A FPase. B CMCase. C Xylanase.

FPase
FPase represents the total cellulolyticactivity. FPase activity (2.435 IU/ml) was obtained at the combination of
incubation temperature 30oC, initial pH 4.5 and rotational speed 150 rpm. Moreover, all the middle level runs gave
higher yields compared to other combinations. Polynomial equation for FPase activity (Y) as a function of
temperature (A), initial pH (B) and rotational speed (C) has given below.

YFPase = 2.40 + 0.025 * A − 0.12 * B − 0.065 * C − 0.15 * A * B − 0.077 * A * C + 0.13 * B * C

− 0.69 * A 2 − 0.24 * B 2 − 0.13 * C 2 Eq. 3a

A high Model F-value (194.21) along with high multiple correlation coefficient (R2) value of 0.9943 and an
insignificant lack of fit indicates a good effect of the parameters on FPase production. From Table.4 it is observed
that the FPase production was significantly affected by temperature and rotational speed (p < 0.0001) in linear as
well as in squared terms (p < 0.0001). Squared terms of initial pH also had good effect on FPase production (
p<0.0001). The interaction between temperature and pH as well as initial pH and rotational speed was significant at
p < 0.0001 and p<0.0004 level respectively. CV values obtained from the model was 4.06. The "Pred R-
Squared"(0.9648) is in reasonable agreement with the "Adj R-Squared"(0.9892). It indicates a good corelation
between the observed and predicted values.

Table. 4. Analysis of variance for the response surface quadratic model

Source F value Prob > F

FPase CMCase Xylanase FPase CMCase Xylanase
Model 194.2065 1573.774 1413.67 < 0.0001 < 0.0001 < 0.0001
A- Temperature 1.821216 278.323 325.7634 0.2069 < 0.0001 < 0.0001
B- pH 43.94319 102.8132 66.54751 < 0.0001 < 0.0001 < 0.0001
C- Rotational speed 12.43943 189.1116 240.2979 0.0055 < 0.0001 < 0.0001
AB 40.62608 66.51023 63.67056 < 0.0001 < 0.0001 < 0.0001
AC 10.49225 114.5999 130.4972 0.0089 < 0.0001 < 0.0001
BC 27.84377 4.309912 20.80353 0.0004 0.0646 0.0010
A2 1505.25 12122.26 11400.22 < 0.0001 < 0.0001 < 0.0001
B2 187.6506 912.0597 471.4539 < 0.0001 < 0.0001 < 0.0001
C2 55.66657 1813.496 909.5977 < 0.0001 < 0.0001 < 0.0001
Lack of Fit 2.92 0.36 0.26 0.1320 0.8581 0.9199

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The three dimensional plots ( Fig. 4a and 4b) represent the interaction between temerature and initial pH as well as
rotational speed and initial pH on FPase production. Fig 4a showed that FPase activity was increased with the
increase of temperature and was optimum at 30oC. Any increase or decrease in temperature lowered FPase
production and initial pH did not show considerable effect on enzyme activity. In Fig. 4b the enzyme activity
gradually increased with increase in initial pH as well as rotaional speed to a certain level and then decreased, which
indicates that initial pH and rotational speed were critical to obtain maximum response. The pH and rotational speed
for optimum response were 4.5 and 150 rpm respectively.

Figure. 4. Three dimensional plots of FPase (response). In each plot, the influence of two variables is shown while the third is set at the
central level. A Temperature (A) vs initial pH (B) and their interaction effect with rotational speed (C) set at center level. B Initial pH
(B) vs rotational speed (C) and their interaction effect with temperature (A) set at center level.

CMCase
From Table.3 it is observed that a maximum CMCase (13.9 IU/ml) was produced under the culture conditions
similar to FPase. The experimental results were analyzed by regression analysis that reproduced the following
equation in terms of linear, quardratic and interaction effect on temperature (A), initial pH (B) and rotatinal speed
(C) on CMCase production.

YCMCase = 13.66 − 0.62 * A + 0.37 * B − 0.51* C + 0.39 * A * B − 0.52 * A * C + 0.10 * B * C

− 3.96 * A 2 − 1.09 * B 2 − 1.53 * C 2 Eq.3b

The Model F value (1573.774) with low probability value ( p < 0.0001) indicated that the model was statistically
significant The model significance was also implied by high R2 (0.9993) value and an insignificant lack of fit
determines that the model was efficient to explain the effect of three parameters on CMCase production. All of the
linear and quadratic terms were significant . Interaction coefficients between temperature and initial pH as well as
temperature and rotational speed were significant at p< 0.0001 where interaction of initial pH and rotational speed
was significant at p < 0.5 ( Table 4). CV values obtained from the model was 1.49. Like FPase the "Pred R-
Squared"(0.9977) is also in reasonable agreement with the "Adj R-Squared"(0.9987). Interaction effects of
temperature and initial pH as well as rotational speed and initial pH on CMCase production are depicted in Fig. 5a
and 5b respectively. The maximum response in two plots was obtained under the same conditions as for FPase.

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Figure. 5. Three dimensional plots of CMCase (response). In each plot, the influence of two variables is shown while the third is set at
the central level. A Temperature (A) vs initial pH (B) and their interaction effect with rotational speed (C) set at center level. B Initial
pH (B) vs rotational speed (C) and their interaction effect with temperature (A) set at center level.

Xylanase
Significance of the coefficients of linear, quadratic and interaction terms were determined by the following equation.
YXylanase = 245.17 − 12.15* A + 5.49* B − 10.44* C + 7.02* A * B − 10.05* A * C + 4.01* B * C
−69.99* A2 − 14.23* B 2 − 19.77 * C 2 Eq.3c

Y is the dependent variable response of xylanase production (IU/ml). The results by ANOVA showed that the model
F value was 1413.67 with a probability value p < 0.0001 (Table 4). The coefficient of determination R2 (0.9992) and
an insignificant lack of fit ensured satisfactory adjustment of the quadratic model to the experimental data. The
model represented a reasonable agreement between the "Pred R-Squared"(0.9979) and"Adj R-Squared" (0.9985)
value. All the linear as well as quadratic terms significantly affected xylanase production ( p < 0.0001). Interaction
effect of temperature and initial pH as well rotational speed and temperature was more significant ( p < 0.0001)
compared to rotational speed and initial pH ( p < 0.005).

Figure. 6. Three dimensional plots of xylanase (response). In each plot, the influence of two variables is shown while the third is set at
the central level. A Temperature (A) vs initial pH (B) and their interaction effect with rotational speed (C) set at center level. B Initial
pH (B) vs rotational speed (C) and their interaction effect with temperature (A) set at center level.

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The interaction effect of temperature and initial pH as well as rotational speed and initial pH on xylanase production
has been shown in Fig. 6a and 6b respectively. Maximum xylanase activity in both plots was obtained under the
similar conditions of cellulase production.

Optimization of process parameters

The optimum values of FPase, CMCase and xylanase by the adjusted model was predicted by numerical
optimization step in CCD. The goals for variables of temperature and initial pH were set as ‘‘in range”. Rotational
speed was set as “ minimize” for low energy consumption. The goal for the response ( FPase, CMCase and
xylanase) was set as ‘‘maximize” because the maximum value of yield is the aim. The software Design Expert.7.0.0
produced a number of solutions and ranked according to their desirability. The solution with the highest desirability
was selected and its predicted value of the response was 2.38 IU/ml, 12.55 IU/ml and 235.88 IU/ml for FPase,
CMCase and xylanase respectively. The optimized conditions for optimum FPase and CMCase were temperature
30oC, initial pH 4.17 and rotational speed 120 rpm. Optimized conditions for optimum xylanase production were
same except initial pH (4.5). Experimentally optimized value (FPase 2.43 IU/ml, CMCase 12.62 IU/ml and xylanase
235.84 IU/ml) with conditions of selected solution showed good agreement with the predicted value. It was ensured
that the developed model was enough significant for predicting the experimental results. The optimized yield
obtained in this study is a reasonable production of cellulase enzyme under submerged condition and further
improvements in product yield will be achieved with the fine tuning of other parameters.

CONCLUSION

It can be concluded that the pretreated rice straw with 0.5M NaOH resulted in maximum FPase, CMCase and
xylanase activities. SEM micrographs exhibited significant morphological changes in alkali pretreated rice straw
compared to untreated rice straw. Locally isolated Aspergillus fumigatus NITDGPKA3 is a potential microorganism
for cellulase production under submerged condition. Statistical optimization of cellulase production from alkali
pretreated rice straw has been carried out using response surface methodology. The culture conditions were
optimized for optimal FPase, CMCase as well as xylanase production.

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