Food Sci. Biotechnol.
20(5): 1289-1298 (2011)
DOI 10.1007/s10068-011-0178-3
RESEARCH ARTICLE
Effect of Substrate and Fermentation Conditions on Pectinase and
Cellulase Production by Aspergillus niger NCIM 548 in Submerged
(SmF) and Solid State Fermentation (SSF)
Sanjay Kumar, H. K. Sharma, and B. C. Sarkar
Received: 25 March 2011 / Revised: 22 July 2011 / Accepted: 22 July 2011 / Published Online: 31 October 2011
© KoSFoST and Springer 2011
Abstract The present study deals with the optimization
of substrate and fermentation conditions for the production
of both pectinase and cellulase by Aspergillus niger NCIM
548 under same fermentation conditions in submerged
fermentation (SmF) and solid state fementation (SSF)
using a central composite face centered design of response
surface methodology (RSM). As per statistical design, the
optimum conditions for maximum production of pectinase
(1.64 U/mL in SmF and 179.83 U/g in SSF) and cellulase
(0.36 U/mL in SmF and 10.81 U/g in SSF) were, time 126
h, pH 4.6, and carbon source concentration 65 g/L in SmF
and were time 156 h, pH 4.80, and moisture content 65%
in SSF. The response surface modeling was applied
effectively to optimize the production of both pectinase and
cellulase by A. niger under same fermentation conditions to
make the process cost-effective in both submerged and
solid state fermentation using agro industrial wastes as
substrate.
Keywords: pectinase, cellulase, submerged fermentation,
solid state fermentation, Aspergillus niger, response surface
methodology
Introduction
Cellulases and pectinases have broad industrial and
commercial applications. Potential applications of cellulases
are in food, animal feed, textile, fuel, chemical industries,
Sanjay Kumar ( ), H. K. Sharma, B. C. Sarkar
Department of Food Engineering and Technology, Sant Longowal
Institute of Engineering and Technology, Distt. Sangrur 148106 (Pb.),
India
Tel: +91-9803-858320; Fax: +91-1672-253130
E-mail: sanjaysaharan12@gmail.com
paper and pulp industry, waste management, medical/
pharmaceutical industry, protoplast production, genetic
engineering, and pollution treatment and of pectinases are
in juice processing (extraction and clarification), vegetable
oil extraction, processing of alcoholic beverages, and a
variety of application in food industries.
Pectinases or pectinolytic enzymes are a heterogeneous
group of related enzymes that hydrolyze the pectic substances,
present mostly in plants. Pectinolytic enzymes are widely
distributed in higher plants and microorganisms. Almost all
the commercial preparations of pectinases are produced
from fungal sources. Aspergillus niger is the most
commonly used fungal species for industrial production of
pectinolytic enzymes (1). Cellulase refers to a group of
hydrolytic enzymes (cellulases) capable of hydrolyzing
cellulose to glucose. Cellulolytic enzymes are produced by
a large number of microorganisms including fungi and
bacteria (2). In microorganisms the enzymes are either cell-
bound or extra cellular. The ability to produce extra cellular
cellulolytic enzymes is widespread in fungi and these
enzyme systems have been most extensively studied (2).
Solid-state and submerged fermentation holds tremendous
potential for the production of enzymes. Agro-industrial
residues or wastes are generally considered as suitable
substrates for the production of enzymes especially
cellulases and pectinases in solid state fermentation (SSF)
and submerged fermentation (SmF) process (3). As
compared to SSF, SmF has been extensively employed for
the production of enzymes and to understand physiological
aspects of the synthesis of enzymes (4).
Due to the potential and wide applications of pectinases
and cellulases, it is necessary to study on several aspects
related to their production. The idea of using cheaper raw
materials for pectinase and cellulase production is an
important parameter in useful technological development.
1290
The synthesis of pectolytic and cellulolytic enzymes by
microorganisms has been reported to be highly influenced
by the components of the growth medium. Most
extracellularly induced enzymes are known to be
synthesized in higher quantities when inducers are present
in the cultivation medium. The production of pectolytic
and cellulolytic enzymes using different sources and the
effect of physical parameters such as temperature, aeration
rate, and type of fermentation have been investigated and
reported in literature. Various agro-industrial wastes such
as apple pomace (5), lemon peel (6), sugarcane bagasse
(7,8), sugar cane bagasse and pectin (7), and wheat bran
and dextrose (9) have been explored for the microbial
production of pectinase. The raw materials used to induce
cellulase production are saw dust (10) and bagasse, corn
corbs, computer cards, sawdust (11), etc using different
strains of A. niger.
Response surface methodology (RSM) is one of the
most practical optimization methods. This method enables
us to identify the effects of individual variables and to
efficiently seek the optimum conditions for a multivariable
system. With this methodology, the effect of interaction of
various parameters can be understood, generally resulting
in high production yields and simultaneously limiting the
number of experiments. This methodology is also widely
used for optimization studies in several biotechnological
and industrial processes. There have been several studies
published recently concerning the pectinase or cellulase
production individually from different cheaper raw
materials under submerged and solid state fermentation (9-
12). However, the production of both pectinase and
cellulase by same organism under same fermentation
conditions are required to be investigated. Therefore the
present study deals with the optimization of fermentation
conditions using different carbon-sources with respect to
optimum yield of both pectinase and cellulase by A. niger
NCIM 548 and comparative evaluation under submerged
and solid state conditions.
Materials and Methods
Materials The substrates (carbon-sources) such as wheat
bran, corn bran, and kinnow peel were obtained from local
market of Longowal, Sangrur, India. Both wheat bran and
corn bran were separated by 425-µm (40 mesh) sieve. The
kinnow peels were chopped into small pieces, milled into
smaller particles and then separated by 425-µm (40 mesh)
sieve. The flow-through material was used for submerged
and solid state fermentation as described below:
Microorganism and maintenance The experimental
organism Aspergillus niger NCIM 548 was procured from
Kumar et al.
National Chemical Laboratory (NCL), Pune, India. The
culture was maintained on potato dextrose agar (PDA)
(Himedia Laboratories, Mumbai, India) slant at 4oC and
sub-cultured on fresh sterile PDA slant and incubated for
72-120 h.
Inoculum preparation The inoculums were prepared by
growing the microorganism on PDA slants for 72 h at
30oC. The spores were harvested by addition of 10 mL of
isotonic NaCl solution.
Preliminary experiments for the selection of carbon
sources Preliminary experiments were performed for the
selection of carbon (C)-source for the production of both
pectinase and cellulase in single fermentation step. Wheat
bran, corn bran, rice bran, rice straw, and kinnow peel were
used individually and in combination for the production of
these enzymes by A. niger NCIM 548 under same
temperature (30oC), agitation speed (170 rpm), and other
media components in g/L [(NH4)2SO4-1.0, MgSO4-5.0,
FeSO4. 7H2O-0.005, and KH2PO4-5.0]. The samples from
each combination were assayed for enzyme activity. Wheat
bran, corn bran, and kinnow peel in combination with a
fixed ratio of 2:1:2 respectively gave the maximum
production of both pectinase and cellulase and it was
selected as C-source for optimizing the production of
cellulase and pectinase.
Experimental design and statistical analysis for enzyme
production RSM consists of a group of empirical
techniques devoted to the evaluation of relation existing
between a cluster of controlled experimental factors and the
measured responses, according to one or more selected
variables under investigation are necessary for achieving a
more realistic model. Three variables were selected to find
the optimized condition for the production of pectinase and
cellulase using central composite face centered design. The
independent variables studied were time (X1, h), pH (X2),
and concentration of C-source (X3, g/L) for submerged
fermentation (SmF) and time (X1, h), pH (X2), and
moisture content (X3, %) for solid state fermentation
(SSF), while response variables were pectinase yield and
cellulase yield for both models. The range and the levels of
the experiment variables used in the coded and uncoded
form for both SmF and SSF in this study are given in
Table 1.
The central value (zero level) chosen for experiment
design were as time 144 (h), pH 5, and concentration of
carbon source 60 (g/L) for SmF and time 144 (h), pH 5,
and moisture content 60 (%) for SSF, in developing the
regression equation, the test factors were coded according
to the equation.
Pectinase and Cellulase by Aspergillus niger 1291

Table 1. Experimental range and levels of the independent variables in SmF and SSF
Range and level
Variable
-1 0 1
Time (X1, h) 48 144 240
Submerged fermentation (SmF) pH (X2) 3 5 7
Conc. of C-source (X3, g/L) 40 60 80
Time (X1, h) 48 144 240
Solid state fermentation (SSF) pH (X2) 3 5 7
Moisture content (X3, %) 40 60 80

Table 2. Central composite face centered design with experimental values for the responses in SmF and SSF
SmF SSF
Uncoded variable Response Uncoded variable Response
St. order1) Time Conc.of Pectinase yield Cellulase yield Time Moist. cont.2) Pectinase yield Cellulase yield
pH pH
(h) C-source (g/L) (U/mL) (U/mL) (h) (%) (U/g) (U/g)
X1 X2 X3 Exp3) Exp3) X1 X2 X3 Exp3) Exp3)
1 48 3 40 1.33 0.14 48 3 40 111.61 5.53
2 240 3 40 1.19 0.25 240 3 40 113.38 7.77
3 48 7 40 1.18 0.10 48 7 40 125.37 5.86
4 240 7 40 1.17 0.24 240 7 40 108.11 7.74
5 48 3 80 1.52 0.28 48 3 80 156.46 6.12
6 240 3 80 1.18 0.27 240 3 80 129.73 9.59
7 48 7 80 1.33 0.25 48 7 80 140.31 7.04
8 240 7 80 1.19 0.25 240 7 80 107.28 10.62
9 48 5 60 1.63 0.30 48 5 60 170.71 7.10
10 240 5 60 1.49 0.34 240 5 60 147.84 10.43
11 144 3 60 1.55 0.32 144 3 60 168.48 10.02
12 144 7 60 1.50 0.31 144 7 60 153.37 9.92
13 144 5 40 1.46 0.26 144 5 40 138.14 8.72
14 144 5 80 1.51 0.33 144 5 80 158.01 10.16
15 144 5 60 1.61 0.37 144 5 60 185.93 10.77
16 144 5 60 1.60 0.38 144 5 60 182.04 10.99
17 144 5 60 1.63 0.35 144 5 60 191.04 10.88
18 144 5 60 1.62 0.39 144 5 60 176.78 10.44
19 144 5 60 1.63 0.37 144 5 60 184.06 10.22
20 144 5 60 1.69 0.39 144 5 60 185.50 11.02
1)
Standard run-non randomized; 2) Moisture content; 3)Experimental value

Xi=(X'i –Xxi)/∆Xi (1) regression coefficients for intercept, linear, quadratic, and
th interaction terms, respectively. Xi and Xj are the coded
where, Xi is the coded value of the i independent variable,
value of the ith and jth independent variables. The variable
X’i is the natural value of the ith independent variable, Xxi
Xi Xj represents the first order interaction between Xi and
is the natural value of the ith independent variable at the
Xj for (i<j).
center point, and ∆Xi is the step change value or difference
between maximum and minimum value.
Selection of relevant variables and experimental ranges
The following quadratic response function for n variables
The initial step was the selection of relevant factors in
with interaction terms was considered for the mathematical
enzyme production and the experimental ranges for the
relationship between independent and dependent variables.
independent variables. Three independent variables were
n n n
selected for each model. Two variables, time and pH were
Y=β0+ ∑ βi Xi + ∑ βii Xi2+ ∑ ∑ βij XiXj (2)
selected for both SmF and SSF models where as the third
i=1 i=1 i<j = 1
factor was concentration of C-source for SmF and moisture
where, Y is the measured response β0, βi, βii, and βij are the content for SSF.
1292 Kumar et al.

The experimental ranges for the independent variables Optimization and validation of the model Design
were selected as time range, 48-240 h; pH, 3-7; concentration Expert version 6.0.10 (Trial version; STAT-EASE Inc.,
of C-source, 40-80 g/L for SmF and time, 48-240 h; pH, 3- Minneapolis, MN, USA) software was used for regression
7; moisture content, 40-80% for SSF with respect to the and graphical analysis of the data obtained. The optimum
reported literature (10,12-14). values of the selected variables were obtained by solving
The central composite face centered design was used to the regression equation and also by analyzing the response
design a series of experiments to provide data to determine surface contour plots. The validity and adequacy of the
the relationship between the responses (i.e. pectinase yield predictive models was done by experimental analysis at
and cellulase yield) and the 3 process parameters, with the suggested optimum conditions by the design expert.
specific ranges for SmF and SSF (Table 2).
Results and Discussion
Production medium For SmF a total of 20 shake flasks
media (100 mL in 250-mL Erlenmeyer), including the Central composite designs and response surface analysis
repetitions performed at center points, were prepared as per SmF: The actual yields of pectinase and cellulase obtained
design, given in Table 2. The medium which was in the experiments are given in Table 2, whereas the
optimized for cellulase and pectinase production contained regression coefficients and significance levels of the terms
in g/L: (NH4)2SO4-1.0, MgSO4-5.0, FeSO4. 7H2O-0.005, are given in Table 3. It is evident from Table 3 that the
and KH2PO4-5.0 (8). Medium was autoclaved for 15 min model used in the present study gave a satisfactory fit
at 120oC. The fermentation was carried out at 30oC (p<0.001). The desired the second-order polynomial models
temperature and at 170 rpm agitation speed in SmF as per for pectinase and cellulase yield can be expressed as:
the reported literature (10,12).
Pectinase yield=1.64−0.077X1 −0.04X2 +0.04X3
For SSF also, 20 flasks of media were prepared as per
+0.041X1X2 −0.041X1X3 −0.001X2X3 −0.087X12
design given in Table 2. In SSF 10 g of the same C-source
−0.123X22 −0.163X32 (3)
as above was used with same other ingredients. Medium
was autoclaved for 15 min at 120oC. The fermentation Cellulase yield=0.37+0.028X1 −0.012X2 +0.039X3
temperature was kept 30oC as in SmF. The fermentation + 0.006X1X2 −0.033X1X3 +0.001X2X3 −0.040X12
was then carried out as per the designed conditions for the −0.045X22 −0.065X32 (4)
production of cellulase and pectinase under SmF and SSF
(Table 2). Table 3. Regression coefficients of predicted quadratic
polynomial models for the responses for SmF and SSF
Analysis of response variables SmF SSF
1)
Pectinase and cellulase activity: The pectinase activity Coefficient Pectinase Cellulase Pectinase Cellulase
was determined using pectin (Sisco Research Laboratories yield yield yield yield
Pvt., Ltd., Mumbai, India) as substrate. The reaction Intercept 1.640* 0.369* 180.71* 10.60*
mixture containing equal amount of substrate (2%) Linear
prepared in citrate buffer (0.05 m, pH 4.4) and suitably X1 -0.077*** 0.028** -9.810** 1.450***
diluted enzyme was incubated at 50oC for 30 min in water X2 -0.040** -0.011** -4.520* 0.220*
bath. The release of reducing sugars was measured by X3 0.040** 0.039** 9.520** 0.790***
DNS method (15) using galactouronic acid (Sigma- Quadratic
Aldrich, St. Louis, MO, USA) as standard. The enzyme X12 -0.088** -0.040** -16.150** -1.670***
(pectinase) activity was defined as the amount of enzyme X22 -0.123*** -0.045** -14.500** -0.460**
required to release 1 µmol equivalent of galactouronic X32 -0.163*** -0.065*** -27.350*** -0.990**
acid/min under assay conditions. The activity was
Crossproduct
expressed as U/mL. X1X2 0.041** 0.006** -3.170 -0.033
Cellulase activity assay was also carried out according to X1X3 -0.041* -0.033** -5.530** 0.370**
the methods by Ghose (16), using carboxymethyl cellulose X2X3 -0.001* 0.001* -5.890** 0.210
(CMC) (Central Drug House (P) Ltd., New Delhi, India) as
R2 0.9877 0.9804 0.9733 0.9834
substrate. The reducing sugar was measured by the DNS
Adj. R2 0.9767 0.9638 0.9493 0.9684
method using glucose (Qualigens Fine Chemicals,
CV 1.94 5.17 4.26 3.70
Mumbai, India) as standard (15). In this study, 1 unit (U) of F-value 89.32 55.65 40.49 65.69
enzyme (cellulase) activity was defined as the amount of 1) 2
R , coefficient of multiple determination; Adj R2, adjusted R2; CV,
enzyme that liberates 1 µmol of glucose equivalent/min coefficient of variance
2)
under the specified conditions. ***p<0.001, **p<0.05, and *p<0.10
Pectinase and Cellulase by Aspergillus niger
where, X1, X2, and X3 are the coded factors of time, pH,
and concentration of C-source, respectively.
The response surface curves were plotted to explain the
interaction of the variables and to determine the optimum
level of each variable for maximum response. The response
surface curves for pectinase and cellulase are shown in Fig.
1. Each figure demonstrates the effect of 2 factors while the
3rd factor was fixed at middle level.
Figure 1A is the response surface curve for variation in
the yields of pectinase, as a function of time (X1) and pH
(X2), keeping the concentration of C-source (X3) at middle
level 60 g/L. The interactive effect of the variables on the
production of pectinase was significant (p<0.05). It was
evident that while cultivation time was at low level, the
effect of pH on the response was negligible. When pH in
medium was at a higher level, pectinase production
steadily increased with increasing cultivation time. The
activity of pectinase was 1.66 U/mL when cultivation time
was 96 h and pH 4.50 and it declined beyond these limits.
The optimized fermentation period for the production of
polygalactouronase was observed after 94 h of fermentation
using A. niger (17). Patil and Dayanand (14) observed the
increased level of pectinase production at pH 5.0 in
submerged and solid-state conditions by A. niger.
Figure 1B shows the effects of cultivation time (X1) and
concentration of C-source (X3) on pectinase production,
while the pH (X2) is fixed at its middle level 5. It was
evident that pectinase production increased with increase in
cultivation time and C-source concentration up to 96 h of
cultivation time and 63.8 g/L of concentration of C-source.
The maximum production of pectinase under these
conditions was 1.66 U/mL. With further increase in the
concentration of C-source the production decreased slowly
with increasing time indicating that excessive increase in
the fermentation time would not increase the yield of
pectinase any more. These facts are important in shortening
fermentation periods during the potential industry
application by keeping appropriate substrate concentration
in the medium. Yugandhar et al. (18) observed that
pectinase production by A. niger NCIM 548 was
maximum at 3.71%(w/v) tapioca starch concentration.
Palaniyappan et al. (12) found that enzyme activity was
maximum (5.17 U/mL) for 1% wheat flour as substrate for
A. niger. The different results for concentration of C-source
may be due to different nutrient composition, concentration,
different nature of the substrate (particle size and
consistency), and the process physiological conditions.
The effect of varying fermentation period (X1) and pH
(X2) on cellulase production, while concentration of C-
source (X3) fixed at central point (60 g/L), is shown in Fig.
1C. It was evident that as the fermentation time and pH
increased, the cellulase production increased and maximized
at pH 4.6 after 170 h of fermentation. The production of
1293
cellulase decreased beyond these limits of the pH and time.
The production of the cellulase was slightly more sensitive
to the change in pH than time. Kang et al. (19) reported
that cellulase activity of A. niger KK2 increased with time
and was maximum at the 5-6 days of fermentation. The
variation in period of fermentation for the production of
enzymes may be due to the different nature of the medium,
fermenting organism, concentration of nutrients, and the
process physiological conditions. Gokhale et al. (20)
reported that the optimum pH for cellulase production by
A. niger NCIM 1207 was 3.0-5.5. The cellulase activity
has a broad pH range between 3.0 and 9.0. Akiba et al.
(21) found the optimal pH in between 6.0 and 7.0 for
cellulase from A. niger. Such different results may appear
because of differences within the same genus.
Figure 1D demonstrates that at low C-source
concentration, the cellulase production increased steadily
with increase in fermentation time, but at higher C-source
concentration (65 g/L) the production of cellulase increased
gradually to attain maxima after 170 h fermentation time.
With the further increase in time the production decreased
slowly with increasing concentration of C-source
indicating that excessive increase in the fermentation time
would not increase the yield of pectinase further. Acharya
et al. (10) observed that maximum cellulase activity
(0.1813 U/mL) was yielded by A. niger at 9.6% substrate
(saw dust) concentration.
SSF: Table 2 shows the 3 independent variables (actual
values) and dependent variables (pectinase and cellulase
yield) in the design matrix. Using the designed experimental
data, the polynomial proposed models for pectinase and
cellulase yield, under SSF conditions was regressed by
only considering the significant terms, are represented as:
Pectinase yield=180.71−9.81X1 −4.52X2 +9.52X3
−3.17X1X2−5.53X1X3 −5.89X2X3 −16.15X12 −14.50X22
−27.35X32 (5)
Cellulase yield=10.60+1.45X1+0.22X2+0.79X3
−0.033X1X2+0.37X1X3 +0.21X2X3 −1.67X12 −0.46X22
−0.99X32 (6)
where, X1, X2, and X3 are the coded factors of time, pH,
and moisture content, respectively.
Figure 2A shows a 3-dimensional plot for pectinase yield
as a function of time (X1) and pH (X2) at 60% moisture
content (X3) level. The interactive effect of time and pH
was not significant. It was evident that with increase in
cultivation time and pH, pectinase production increased
and reached to maxima after 116 h of cultivation time at
pH 4.8. However the yield declined with further increase in
the pH and cultivation time. The maximum pectinase
activity was reported by Baladhandayutham and
Thangavelu (22) after 96 h of fermentation using A. niger
1294
Fig. 1. Surface plot of pectinase and cellulase activity of A. niger NCIM 548 in SmF. Effect of time and pH (A) and C-source
concentration and time (B) on pectinase activity; effect of time and pH (C) and C-source concentration and time (D) on cellulase activity
under solid state fermentation. Patil and Dayanand (14)
observed the increased level of pectinase production at pH
5.0 in submerged and solid-state conditions by A. niger.
The effect of varying cultivation time (X1) and moisture
content (X3) on pectinase production, keeping pH (X2)
fixed at central point 5, is shown in Fig. 2B. The pectinase
production increased with increase in cultivation time and
moisture content; however the response was more sensitive
to the change in moisture content than time. The results of
this study are in accordance with the findings of Patil and
Dayanand (14) who observed that 65% moisture content of
the substrate was optimum for the maximum production of
pectinase by A. niger in solid-state condition. The low level
of moisture content leads to reduction of substrate swelling,
nutrient diffusion and solubility of solid substances. These
facts would result in insufficient nutrient supply for
microorganisms, which in turn causes the decrease of
microbial growth and enzyme production (23). However
higher level of moisture content (80%) reduces the activity
of both enzymes. The adverse effect of too high level
moisture content is attributed to the reduction of substrate
porosity, low heat and mass transfer through the culture
and decrease of air exchange, which in turn result in
decrease of fungi growth and product formation.
Kumar et al.
Figure 2C is the response surface curve for variation in
the yield of cellulase, as a function of time (X1) and pH
(X2), keeping the concentration of C-source (X3) at middle
level (60 g/L). The interactive effect of the variables on the
production of cellulase was not significant. While cultivation
time was at a low level, the effect of pH on the response
was negligible. The cellulase production increased with
increasing cultivation time and pH, but decreased slowly
beyond 186 h of time and 5.38 pH. The maximum
CMCase production by A. niger was after the 8th day of the
fermentation in SSF (24).
A 3-dimensional response surface plot (Fig. 2D) shows
the interaction effect of varying time (X1) and moisture
content (X3) on cellulase yield at fixed pH (X2), at its
centre point 5. It was observed that the cellulase activity
was increased with increasing time and moisture content
and maximized after 190 h of fermentation at 70%
moisture content; however the production of cellulase
decreased with further increase in moisture content and
time. Lee et al. (25) observed that the optimum moisture
content for the production of cellulase by A. niger using
sugarcane bagasse and palm kernel cake as substrate was
70%.
Pectinase and Cellulase by Aspergillus niger
Fig. 2. Surface plot of pectinase and cellulase activity of A. niger NCIM 548 in SSF. Effect of time and pH (A) and time and moisture
content (B) on pectinase activity; effect of time and pH (C) and time and moisture content (D) on cellulase activity
Fitting the models The experimental values for responses
(pectinase and cellulase yield) under different combination
of production conditions are given in Table 2 for both SmF
and SSF. The results showed that in SmF, pectinase and
cellulase yield ranged from 1.17 to 1.69 U/mL and from
0.096 to 0.39 U/mL respectivily and in SSF, pectinase and
cellulase yield ranged from 107.28 to 191.04 U/g and from
5.53 to 11.02 U/g, respectivily. In SmF the maximum yield
for both pectinase and cellulase was obtained when the
time, pH, and C-source concentration were 144 h, 5, and
60 g/L, respectively. The pectinase yield was minimum
after 240 h at pH 7 and 40 g/L C-source concentration,
while the cellulase yield was minimum after 48 h at pH 7
and 40 g/L C-source concentration (Table 2).
In SSF pectinase yield was minimum at 240 h of time,
at pH 7 and 80% of moisture content, where as in case of
cellulase the yield was minimum at 48 h of time, pH 3, and
40% of moisture content. The maximum yield of both
pectinase and cellulase was obtained when time, pH, and
concentration of C-source were 144 h, 5, and 60%
respectively.
The parameters of regression equations obtained by fitting
of yield pectinase and cellulase data for both SmF and SSF
are given in Table 3. The fitness and adequacy of the
1295
model was judged by the coefficient of determination (R2).
The R2 which can be defined as the ratio of the explained
variation to the total variation was a measure of the degree
of fit. The closer the R2 value to unity, the better the
empirical model fits the actual data. The coefficients of
determination, R2, in SmF were 0.9877 and 0.9804 for the
regressed models predicting the yield pectinase and
cellulase, respectively, and in case of SSF the R2 values
were 0.9733 and 0.9834 for the regressed models predicting
the yield pectinase and cellulase respectively, suggesting a
good fit for both SmF and SSF models. The predicted
models seemed to reasonably represent the observed
values. Thus, the responses were sufficiently explained by
the models.
The adjusted R2 was a corrected value for R2 after
elimination of the unnecessary model terms. If many non-
significant terms have been included in the model, the
adjusted R2 would be remarkably smaller than the R2. In
this study, the adjusted R2 for pectinase and cellulase were
0.9767 and 0.9638, respectively in SmF and 0.9493 and
0.9684, respectively in SSF which were very close to their
corresponding R2 value in both models. High values of
adjusted R2 also advocated significance of the models for
all responses. The coefficient of variation (CV) describes
1296 Kumar et al.

Table 4. Optimization of process variables with respect to cellulase and pectinase yield under SmF and SSF conditions
Optimum value (in the range) Optimum value (targeted)
Time (h) 126.22 126
Variables
pH 4.66 4.60
Submerged fermentation Conc. of C-source (g/L) 64.64 65
(SmF) Predicted value Experimental value
Responses Pectinase (U/mL) 1.66 1.64
Cellulase (U/mL) 0.37 0.36
Desirability 0.933
Optimum value (in the range) Optimum value (targeted)
Time (h) 156.40 156
Variables
pH 4.86 4.80
Solid state fermentation Moisture content (%) 65.01 65
(SSF) Predicted value Experimental value
Responses Pectinase (U/g) 180.26 179.83
Cellulase (U/g) 10.90 10.81
Desirability 0.92

the extent to which the data are dispersed. The coefficient
of variation is a measure of residual variation of the data
relative to the size of the mean; the small values of CV
give better reproducibility. The small CV values 1.94 and
5.17 of the responses pectinase yield and cellulase yield
respectively in SmF, and 4.26 and 3.70 respectively in SSF
(Table 3) revealed that the experimental results were
precise and reliable.
The significance of each coefficient was determined
using the F-test and p-value (Table 3). The F-value of
89.32 and 55.65 for pectinase and cellulase yield,
respectively in SmF and 40.49 and 65.69 respectively in
SSF implied that both the models were significant
(p<0.001). The corresponding variables would be more
significant if the absolute F value becomes greater and the
p-value becomes smaller. The F-value for all responses for
both models was greater than the tabulated F value
indicating the adequecy of the models to predict different
responses at different fermentation conditions in both
models.
Optimization of the models Design Expert software
was used to optimize the fermentation conditions like time,
pH, and concentration of C-source in SmF and time, pH,
and moisture content in SSF to maximize the production of
cellulase and pectinase in both models. The software uses
second order model to optimize the responses. Predicted
values of different responses on optimum conditions (in the
range constraint) for both models are given in Table 4.
When constraint in the range were selected then the
optimum conditions were found as time 126.22 h, pH 4.66,
and concentration of C-source 64.64 g/L in SmF and time
156.40 h, pH 4.86, and moisture content 65.01% in SSF.
But in practice, it is difficult to maintain the recommended
conditions during processing and some deviation is
expected. Therefore, optimum conditions were targeted as
time 126.00 h, pH 4.60, and concentration of C-source
65 g/L in SmF and time 156 h, pH 4.80, and moisture
content 65% in SSF. Predicted values of different responses
on targeted optimum conditions for both models are given
in Table 4. Response surface in function of 2 variables and
the value of the missing independent variable in plot was
kept at the centre point is shown in Fig. 1 and 2 for SmF
and SSF models, respectively.
Verification of predictive models Under the optimum
condition (target constraint), experiments were conducted
for checking the variation in production of pectinase and
cellulase in both SmF and SSF. The experimental values of
different responses under the optimum conditions of
different variables in SmF and SSF are given in Table 4,
which showed that the experimental results were very close
to the predicted one for both SmF and SSF models. This
implied that there was a high fit degree between the values
observed in experiments and the values predicted from the
regression model. Hence, the response surface modeling
could be applied effectively to optimize the conditions for
the maximum production of both pectinase and cellulase
under submerged as well as solid state conditions.
Comparison of SmF and SSF for pectinase and
cellulase production The production of both pectinase
and cellulase were higher in SSF as compared to SmF. The
pectinase and cellulase yield under optimized conditions in
SmF were 25.23 and 5.54 U/g, where as in SSF were
179.83 and 10.81 U/g for pectinase and cellulase,
Pectinase and Cellulase by Aspergillus niger
Table 5. Pectinase and cellulase production under SmF and
SSF conditions
Response SmF1) SSF
Pectinase yield (U/g) 25.23 179.830
Cellulase yield (U/g) 05.54 10.81
1)
Activities calculated from U/mL
respectively (Table 5). The data showed that the production
of pectinase and cellulase is 7.13 and 1.95 times higher
respectively in SSF than SmF as given in the Table 5. It
was reported (7) that SSF generally allows more
production of crude enzymes as compared to liquid state
fermentation. Acuna et al. (26) reported up to 50 times
higher production of exo-pectinase by A. niger CH4 in SSF
as compared to SmF. Maldonado and Strasser de Saad (27)
found that PGase production by A. niger was 6 times
higher in SSF than in SmF.
The higher production of enzymes in SSF may be due to
fact that the SSF takes place in the absence or near absence
of free water, thus being close to the natural environment
to which microorganisms are adapted (28). One important
biological factor in favour of SSF is the low catabolite
repression, which appeares to be limiting enzyme production
by A. niger in SmF (29). Viniegra-Gonzalez et al. (30)
demonstrated, using logistic and Luedekind-Piret equations,
that the higher productivity of invertase, pectinase, and
tannase in SSF was due to better growth of A. niger in SSF,
resulting in higher biomass production, and more efficient
biosynthesis of enzymes under conditions without catabolite
repression. Moreover, the breakdown of enzymes by
contaminating proteases was 8 times higher in SmF than in
SSF.
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