Response surface optimization for enhanced production of cellulases with improved functional...

ORIGINAL PAPER

Response surface optimization for enhanced productionof cellulases with improved functional characteristicsby newly isolated Aspergillus niger HN-2

Harinder Singh Oberoi • Rekha Rawat •

Bhupinder Singh Chadha

Received: 21 July 2013 / Accepted: 17 October 2013 / Published online: 25 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Fungi isolated from partially decayed

wood log samples showing characteristic diversity

for spore colour, colony morphology and arrangement

of spores were assessed for cellulolytic enzyme

production. Isolates showing a cellulolytic index of

C2.0 were assayed for filter paper (FP) cellulase and b-

glucosidase (BGL) production. Molecular character-

ization confirmed the identity of the selected cellulo-

lytic isolate as a strain of Aspergillus niger (A. niger

HN-2). Addition of 2 % (w/v) urea enhanced FP and

BGL activity by about 20 and 60 %, respectively.

Validation studies conducted at parameters (29 �C, pH

5.4, moisture content 72 % and 66 h) optimized

through response surface methodology in a solid-state

static tray fermentation resulted in FP, BGL, cellobio-

hydrolase I (CBHI), endoglucanase (EG), xylanase

activity and protein content of 25.3 FPU/g ds, 750 IU/

g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and

0.9 mg/ml, respectively. In comparison, A. niger N402

which is a model organism for growth and develop-

ment studies, produced significantly lower FP, BGL,

CBHI, EG, xylanase activity and protein content of

10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds,

500 IU/g ds and 0.75 mg/ml, respectively under the

same process conditions as were used for A. niger HN-

2. Process optimization led to nearly 1.8- and 2.2-fold

increase in FP and BGL activity, respectively showing

promise for cellulase production by A. niger HN-2 at a

higher scale of operation. Zymogram analysis revealed

two isoforms each for EG and cellobiohydrolase and

three isoforms for BGL. Crude cellulase complex

produced by A. niger HN-2 exhibited thermostability

under acidic conditions showing potential for use in

biofuel industry.

Keywords Aspergillus niger HN-2 � b-

Glucosidase � Filter paper cellulase � Nitrogen

sources � Solid-state fermentation �Thermostability � Zymogram

Introduction

Cellulases have attracted worldwide attention because

of their diverse applications in various industries, such

as, textile and laundry, pulp and paper, animal feed

and fruit juice processing (Bhat 2000; Zaldivar et al.

2001). The recent thrust on bioconversion of agricul-

tural and industrial wastes to value-added products has

led to an extensive research on cellulolytic enzymes.

Cellulases are indispensable in the bioconversion of

lignocellulosic biomass to simple sugars, such as

glucose, xylose and arabinose, which can be fer-

mented to ethanol for its use as a biofuel. Today, these

H. S. Oberoi (&) � R. Rawat

Central Institute of Post-Harvest Engineering and

Technology, P.O. PAU, Ludhiana 141 004, India

e-mail: [email protected]

B. S. Chadha

Department of Microbiology, Guru Nanak Dev

University, Amritsar, India

123

Antonie van Leeuwenhoek (2014) 105:119–134

DOI 10.1007/s10482-013-0060-9

enzymes account for *20 % of the world enzyme

market, and are mostly produced through fermentation

by fungi belonging to the genera Trichoderma and

Aspergillus (Juwaied et al. 2011). Fungi can be

cultivated in a relatively short time by established

fermentation methods for regular production of the

desired enzymes. More than 14,000 fungal species

have been found to be active in degrading cellulose.

The use of improved microbial strains, media compo-

sition and process controls significantly contribute to

cellulase production (Szengyel et al. 2000).

Cellulase is a multi-enzyme system composed of

several enzymes with numerous isozymes, which act

synergistically and catalyze the hydrolysis of cellulose

and cello-oligosaccharide derivatives (Chinedu et al.

2008). The enzymatic process for the depolymeriza-

tion of cellulose involves three types of enzymes:

Endoglucanase (EG or CX), hydrolyzes internal b-1,4

glucan chains of cellulose randomly, primarily within

amorphous regions, but displays low hydrolytic

activity toward crystalline cellulose. Exoglucanase

i.e., exo-acting cellobiohydrolases (CBH), removes

cellobiose from the non-reducing end of cello-oligo-

saccharide and also from the crystalline, amorphous

and acid- or alkali-treated cellulose; Cellobiase or b-

glucosidase (BGL) hydrolyzes cellobiose to yield two

molecules of glucose which completes the depoly-

merization of cellulose. In addition to the cellulolytic

enzymes, there are also a number of auxiliary enzymes

which attack hemicellulose, such as glucuronidase,

acetylesterase, feruloyl esterase, xylanase, b-xylosi-

dase, galactomannanase and glucomannanase.

Solid-state fermentation (SSF) holds tremendous

potential in production of commercially important

enzymes. It is a process wherein an insoluble substrate

is fermented with sufficient moisture, but without free

water. The inherent advantages of SSF processes for

production of secondary metabolites have been well

documented (Holker et al. 2004). In SSF, cellulosic

substrate acts as the carbon source and might also help

induce cellulase production. The contribution of the

enzyme cost to bioethanol production cost is quite

significant (Klein-Marcuschamer et al. 2012) and one

of the ways to reduce this cost is to use the crop residues

and by-products available at either no or a very low cost

as substrates in a SSF process. In addition, the

utilization of agricultural residues for enzyme produc-

tion might not only help in establishing a commercial

use for such residues, but also mitigate the

environmental pollution problems caused by their poor

disposal in developing countries, like India. However,

most of the agricultural residues are poor sources of

nitrogen whose main function is the synthesis of plastic

and catalytic proteins, which significantly affect the

microbial physiology (Zabel and Morrell 1992).

Nitrogen sources present in different forms play an

important role in the growth of the microorganisms and

also in induction of enzymes. Thus, it is important to

evaluate different nitrogen sources for enhanced

cellulolytic enzyme production.

Different parameters such as pH, temperature,

cultivation time and moisture content can critically

influence enzyme production. Optimization of multiple

parameters is time consuming, hence response surface

methodology (RSM) can be used to evaluate the

significance of several factors, especially when inter-

actions exist among factors and are complex to

determine. Previous studies in the area of enzyme

production and bioprocessing which used RSM-opti-

mized parameters reported high product yield and

productivity (Brijwani et al. 2010; Oberoi et al. 2012). It

is strongly felt that the use of cheap, readily available

substrates coupled with the use of optimized process is

likely to have a significant impact on cellulase produc-

tion cost which in turn can influence the bioethanol

production cost from lignocellulosic biomass. Since,

microbial strains require different process conditions

for production of enzymes in optimal concentrations, it

becomes imperative to optimize the enzyme production

parameters for different isolates. Present study was

thus, aimed to screen various fungal isolates from the

partially decayed wooden log sample; conduct pre-

liminary studies to screen the most efficient cellulolytic

strain; evaluate the ideal nitrogen source for cellulase

production by the screened strain; ascertain the cellu-

lase production capability of the screened isolate using

optimized parameters by RSM; validate the optimized

parameters in a static tray SSF process; conduct the

zymogram analysis and evaluate the thermostability of

crude cellulase consortium at different pH values.

Materials and methods

Materials

Rice (Oryza sativa) straw of variety Pusa-44 was

procured from the Agronomy research fields of Punjab

120 Antonie van Leeuwenhoek (2014) 105:119–134

123

Agricultural University (PAU), Ludhiana, India. Rice

straw (RS) was cut, dried and size reduced to about

1 mm prior to its use for enzyme production. Wheat

bran (WB) was procured from a local wheat flour mill.

All the analytical grade chemicals and media ingre-

dients were procured from Hi-Media Pvt Ltd (Mum-

bai, India). The substrates used for enzyme assays

were purchased from Sigma-Aldrich (St. Louis, MO,

USA).

Isolation and screening of cellulolytic fungi

Isolation of fungi was done from the partially rotten

wood log samples. The rotten wood log sample was

procured from the agricultural fields of PAU, Ludhi-

ana, India. The protocol used for inoculum preparation

from the rotten wood log sample was the same as

described by Sandhu et al. (2012). On the basis of the

spore colour, arrangement of spores and structure of

conidiophores, distinct colonies were picked and

streaked separately on Rose-Bengal Chloramphenicol

(RBC) agar medium for obtaining pure cultures.

Cellulase production by the isolates was observed

with the use of Gram’s iodine using plate assay method

(Kasana et al. 2008). All those isolates which showed

characteristic zones were examined visually and

microscopically for colony and hyphal characteristics.

Cellulolytic index (CI) was determined and expressed

by the ratio between the diameter of the degradation

halo and the diameter of the colony (Teather and Wood

1982). Isolates showing CI of 2.0 or greater than 2.0

were further characterized for their cellulolytic ability

by filter paper (FP) cellulase and BGL assays.

SSF for cellulase production

Ten grams RS and WB in the ratio 4:1 were used as

substrate for cellulase production in 250-ml Erlen-

meyer flasks (Brijwani et al. 2010; Dhillon et al. 2011;

Oberoi et al. 2012). Out of the twelve isolates showing

morphological diversity through visual and micro-

scopic examination, only four isolates showed a CI of

C2.0. Spores from these four isolates were collected

using sterile distilled water from the 4 days old RBC

culture plates. Moisture content of 70 % (w/w) was

adjusted with the Mandel Weber (MW) medium

solution. The flasks were autoclave-sterilized for

15 min, cooled and inoculated using 1 ml inoculum

having concentration of 1 9 107 spores/ml in different

flasks. Initial pH of 5.0 was adjusted with the use of

5 N HCl and 5 N NaOH. The flasks were incubated at

30 �C in an incubator for 5 days and assayed for

cellulase production. A set of three flasks were

analyzed at a regular interval of 24-h until 120 h.

The crude enzyme was harvested by addition of

100 ml citrate buffer (50 mM, pH 5) followed by

shaking of the contents of the flasks at 150 rpm at

25 �C for 30 min to have the enzyme extract in the

solution. The contents were subsequently filtered and

centrifuged at 4 �C, 10,0009g for 15 min. Superna-

tant was assayed for FP and BGL activity. FP activity

is a relative measure of the overall cellulose hydro-

lyzing capacity of microbial cellulase preparations

(Urbanszki et al. 2000). Presence of BGL in the

cellulase mixture is necessary for conversion of

cellobiose to glucose. The isolate showing maximum

FP and BGL activity was identified through molecular

characterization and subsequently used for enzyme

production experiments.

Enzyme assays

Filter paper cellulase activity was reported in FP units

per gram dry substrate (FPU/g ds) using IUPAC

procedures recommended by Ghose (1987) and BGL

activity was determined using 5 mM, 4-nitrophenyl b-

D-glucopyranoside (pNPG). EG activity was deter-

mined according to IUPAC method using 2 % (w/v)

carboxymethyl cellulose (CMC), while xylanase

activity was determined using 2 % (w/v) birchwood

xylan as substrate. Endoglucanase I (EGI) & cello-

biohydrolase I (CBHI) were measured using 4-nitro-

phenyl-b-D-lactopyranoside with or without 10 mM

cellobiose and 100 mM glucose. The CBHI activity

was thus calculated by the subtraction method of

Bailey and Tahtiharju (2003). One unit of enzyme was

defined as the amount of enzyme required to release

1 lmol of product (glucose equivalents for FP and

CMC, p-nitrophenol for BGL and CBHI and xylose

for xylanase) from the appropriate substrates per

minute under the standard assay conditions. Total

protein content was determined by the protein dye

binding method (Bradford 1976).

Molecular characterization of the screened isolate

Genomic DNA was extracted from the selected

cellulolytic fungal isolate using fungal genomic

Antonie van Leeuwenhoek (2014) 105:119–134 121

123

DNA isolation kit (Chromous Biotech Pvt Ltd,

Bangalore, India). For molecular characterization of

the fungal strain, the D1, D2 and D3 domains of the

26 s rRNA gene were amplified using the primer set

LROR (50-ACCCGCTGAACTTAAGC-30) and LR7

(50-TACTACCACCAAGATCT-30). The PCR proto-

col applied for amplification of the 26 s rRNA gene

followed in this study has been described previously

(Sandhu et al. 2012). The 901 bp amplicon, thus

obtained was sequenced with ABI 3130 genetic

analyzer (Applied Biosystems, Foster City, CA,

USA). The sequence was then compared with the

sequences in nucleotide database (NCBI) using the

BLAST algorithm. Multiple sequence alignment was

done with CLUSTAL W. The neighbour-joining

phylogenetic analysis was carried out with MEGA

software programme (Tamura et al. 2007).

Effect of nitrogen sources on cellulase production

Different nitrogen sources, such as ammonium sul-

phate, potassium nitrate, urea, beef extract and pep-

tone were evaluated for cellulase production by the

screened strain. The above nitrogen sources were

added separately at a concentration of 0.2 % (w/w) to

the basal medium composed of the substrate and 2 g/l

KH2PO4, 0.3 g/l MgSO4�7H2O, 0.3 g/l CaCl2�2H2O

and 0.1 % Tween 80 (w/w). Initial pH of the medium

was adjusted to 5.0 using 5 N HCl or 5 N NaOH.

Flasks containing 10 g substrate impregnated with

basal medium to a moisture content of 70 % (w/w) and

the nitrogen sources mentioned previously were

autoclave-sterilized for 15 min. The flasks were

cooled and inoculated with 10 % (w/v) inoculum

having spore concentration of 1 9 107 spores/ml,

incubated at 30 �C and a set of three flasks were

removed at a regular interval of 24-h until 120 h and

analyzed for FP and BGL activity.

Statistical optimization of parameters through

RSM

All the statistical optimization experiments were per-

formed in 250 ml Erlenmeyer flasks. A four-factor

central composite design (CCD), consisting of 27

experimental runs was employed. The experimental

design consisted of a 24 factorial design, eight axial

points and three replications at central points. The

design space consisted of four independent variables:

temperature (X1, �C), initial pH (X2), incubation time

(X3, h) and moisture content (X4, %). Response

variables were FP activity (Y1, FPU/g ds) and BGL

activity (Y2, IU/g ds). The range selected for optimi-

zation was: temperature (25–35 �C); pH (4.0–7.0);

incubation time (36–96 h) and moisture content (50–

80 %). The range for the four independent variables

was selected on the basis of our preliminary studies and

previously published work (Brijwani et al. 2010). Spore

and substrate concentration mentioned previously were

used during the optimization process. On the basis of

the results obtained from experiments on screening of

nitrogen sources discussed elsewhere in this paper; urea

was added to the basal medium at a concentration of

0.2 % (w/w) during the optimization process. Actual

values and corresponding values for the four indepen-

dent variables, X1, X2, X3 and X4 are mentioned in

Table 1. Experimental data from the CCD was ana-

lyzed using RSM algorithm Design-Expert 7.1 (Sta-

tease Inc., MN, USA) and fitted according to Eq. (1) as a

second-order polynomial equation including main

effects and interaction effects for each variable:

Yn ¼ b0 þX4

i¼1

biXi þX4

i¼1

biiX2i þ

X X4

i\j¼1

biXiXj

ð1Þ

where Yn is the predicted response, b0 is the constant

coefficient, bi is the linear coefficient, bii is the

Table 1 Independent variables and their coded levels chosen for optimization through CCD

Independent variables Symbol Coded levels

-2(-a) -1 0 1 2(a)

Temperature (�C) X1 20.00 25.00 30 35 40.00

pH X2 2.50 4.00 5.50 7.00 8.50

Incubation time (h) X3 6.00 36 66 96 126.00

Moisture content (%) X4 35.00 50 65 80 95.00


123

quadratic coefficient, and bij is the interaction

coefficient.

Analysis of variance (ANOVA) and surface plots

were generated using Design-Expert 7.1 software.

Optimized values of four independent variables were

obtained using the numerical optimization package

and model graphs suggested by the software.

Solid-state static tray fermentation for cellulase

production

In our previous studies, we had successfully validated

the results of the preliminary/optimization experi-

ments in the solid-state static tray fermentation,

though the preliminary/optimization experiments

were conducted in flasks (Brijwani et al. 2010; Dhillon

et al. 2011). It is noteworthy to mention here that the

tray SSF method is generally employed for commer-

cial production of enzymes. Therefore, the validation

experiments were conducted in plastic trays having

dimensions: 40 cm (length), 25 cm (breadth) and

8 cm (height) as per the procedure of Dhillon et al.

(2011). Although, the optimization experiments were

conducted in flasks, validation studies were conducted

in trays as 555 g of RS and WB (90 % dry matter)

were mixed in the ratio 4:1 in glass beakers and

supplemented with 640 ml basal medium solution

containing urea (0.2 % w/v) to achieve a moisture

content of 72 % (w/w). Initial pH for the fermentation

process was adjusted to 5.4. The contents were

thoroughly mixed and autoclave-sterilized for

15 min. The material after sterilization was spread in

trays to achieve a depth of about 2 cm and this process

was carried out under sterile air in the laminar air flow.

The trays were covered with a wet muslin cloth and

incubated in a fumigated environment chamber main-

tained at 29 �C, 90–95 % RH for 66 h. The optimized

process parameters were obtained through the analysis

of the statistically designed process described else-

where in this paper. The trays were removed after 66 h

and the crude filtrate extract was analyzed for different

enzyme activities. All the experiments were conducted

in triplicate and the mean and SD values were

determined with the MS Excel software. In order to

appraise the potential of A. niger HN-2 for cellulase

enzyme production for commercial exploitation in

future, the production capability of this isolate was

compared with the N402 strain which is generally

considered as a model A. niger strain for growth and

development studies, under the same optimum condi-

tions, that were used for A. niger HN-2. The A. niger

N402 (MTCC 271) was procured from the Microbial

Type Culture Collection and Gene Bank (MTCC),

Institute of Microbial Technology (IMTECH), Chan-

digarh, India.

Zymogram analysis of cellulase enzyme

For visualization of EG isoforms, samples were

applied to 10 % (w/v). Native-PAGE gel containing

0.2 % (w/v) CMC. Following electrophoresis, gel was

washed with 0.1 M sodium citrate buffer (pH 5.0) and

incubated at 50 �C for 30 min. The gel was then

stained with congo-red dye (0.1 %, w/v) for 15 min

and visualization of the clear zones against the red

background was facilitated by washing the gel with

1 M NaCl. Zymogram analysis of the BGL and CBH

was carried out by incubating the native gel (10 %,

w/v) after electrophoresis in 0.1 M sodium citrate

buffer (pH 5.0) containing 10 mM 4-methylumbel-

liferyl-b-D-glucoside and 4-methylumbelliferyl-b-D-

lactopyranoside, respectively. The gel was incubated

at 50 �C for 15 min and the active bands were

visualized under UV light using gel documentation

system (Syngene-GBox, UK).

Temperature and pH stability

The temperature profiles of the crude cellulase were

assessed for FP and BGL activity and were obtained

between 30 and 80 �C. The optimal pH was deter-

mined by measuring the FP and BGL activity between

pH 3.0 and 10.0, using 50 mM acetate (pH 3.0–5.0),

phosphate (pH 6.0–8.0) and glycine NaOH (pH

9.0–10.0) buffers at 50 �C on the basis of the results

obtained from enzyme characterization studies men-

tioned elsewhere in this paper. For the determination

of temperature and pH stability, the enzyme aliquots

were incubated at different temperatures and pH range

for 0–360 min and assayed for FP activity. Samples

were drawn at 60 min interval and the residual activity

was determined using the standard assay method

described previously in this paper. All the experiments

were conducted in triplicate and mean and SD values

were calculated using the MS Excel software. The

LSD values for treatment means were calculated using

the JMP software (SAS Inc., MN, USA).


123

Results

Screening of cellulolytic fungal isolates

Six out of the twelve fungal isolates were found to be

cellulolytic on the basis of sharp and distinct zone

around the colonies on the CMC agar plates. However,

only four out of the six isolates which showed a CI of

2.0 or above 2.0 were identified on the basis of the

spore colour, colour of the mycelium, colony diameter

and vesicle shape. On the basis of the colony

characteristics and microscopic examination for the

arrangement of spores and vesicle shape, the four

isolates were identified as strains of A. fumigatus,

A. flavus, A. oryzae and A. niger (Table 2).

Except, A. fumigatus isolate which showed max-

imum FP production at 72 h, FP activity for the

remaining isolates reached the plateau at 96 h

(Fig. 1a). The LSD (p \ 0.5) values clearly indicate

that there was a significance increase in the FP

activity until 96 h, though the difference was not

remarkable for FP activity between 72 and 96 h for

the isolates, excepting A. fumigatus. All the four

isolates showed a similar trend for BGL production

(Fig. 1b). Increase in incubation time beyond 72 h

for A. fumigatus and 96 h for the remaining three

isolates led to a fall in the enzyme production which

is corroborated by the LSD values (Fig. 1b). Pre-

sence of cellulase and BGL in optimum concentra-

tions is necessary for hydrolysis of lignocellulosic

biomass. On the basis of the FP and BGL activity

obtained using the four Aspergillus isolates (Fig. 1a,

b), it was decided to use A. niger for cellulase

production experiments. In order to corroborate the

identity of the most efficient Aspergillus isolate,

molecular characterization of this isolate was done

through sequencing of the 26 s rDNA region.

Identification and characterization of the screened

fungal strain

Sequencing and analysis of the 26 s rDNA region of

the screened fungal strain revealed that this region had

the highest identity with A. niger. Phylogenetic

relationships were drawn using the alignment and

cladistic analysis of homologous nucleotide sequences

of known microorganisms (Fig. 2). The evolutionary

distances were computed using the maximum com-

posite likelihood method. The analysis involved 11

nucleotide sequences, out of which one was the

Table 2 Morphological features and CI of the four cellulolytic isolates belonging to the genus Aspergillus

Isolate Colour of the

mycelium

Spore

colour

Colony

diameter (mm)

Ring

diameter (mm)

CIa Vesicle shape

A. fumigatus White Grey 40 84 2.10 Subclavate

A. oryzae White Green 26 75 2.88 Inflated globose

A. niger White Black 28 79 2.82 Globose to sub globose

A. flavus White Greenish yellow 22 60 2.73 Inflated globose

a Cellulolytic index (CI) = ratio between the ring diameter (mm)/colony diameter (mm)

(a)

(b)

0

2

4

6

8

10

12

14

16

FP

cel

lula

se (

FP

U/g

-ds)

Time (h)

A. fumigatus A. niger A. flavus A. oryzae

0

100

200

300

400

500

600

24 48 72 96 120

24 48 72 96 120

ββ-g

luco

sid

ase

(IU

/g-d

s)

Time (h)

A. fumigatus A. niger A. flavus A. oryzae

Fig. 1 Effect of incubation time on a FP cellulase production

and b BGL production by the four screened Aspergillus isolates.

The values for LSD (p \ 0.05) for incubation period were 0.60

and 9.6 for FP cellulase and BGL, respectively


123

sequence of the isolated Aspergillus strain and the

remaining ten were taken from the National Centre for

Biotechnology Information (NCBI). The evolutionary

analyses were conducted using MEGA 5 software and

the phylogenetic tree was prepared (Fig. 2). The

isolated fungal strain belonged to the same branch as

A. niger (NCBI Accession No. HM 008328) with

99 % homology in the 26 s rDNA region. On the basis

of the morphology and the comparison of 26 s rRNA

gene sequence, the isolated fungal strain was con-

firmed as a strain of A. niger and is henceforth referred

to as A. niger HN-2. The 26 s rRNA gene sequences

for A. niger HN-2 were submitted to GenBank under

Accession No. HQ 825091. The isolate A. niger HN-2

has been deposited with the Microbial Type Culture

Collection and Gene Bank (MTCC), Institute of

Microbial Technology (IMTECH), Chandigarh, India

with the Accession No. MTCC11098.

Effect of nitrogen sources on cellulase production

by A. niger HN-2

Addition of urea to the basal medium increased the FP

activity by nearly 20 %, when compared with the

activity obtained using MW medium (Fig. 3a). The

inorganic sources of nitrogen, such as ammonium

sulphate and urea significantly enhanced enzyme

production, compared to the organic nitrogenous

sources. Similar trend was observed in the case of

BGL production with the use of inorganic nitrogen

sources (Fig. 3b). The LSD (p \ 0.5) values clearly

indicate a significance increase in the FP and BGL

activity until 96 and 72 h, respectively after which a

fall in activity was seen (Fig. 3). An increase of about

33, 50 and 60 % in BGL production was observed with

the use of basal nutrient solution supplemented with

potassium nitrate, ammonium sulphate and urea,

respectively as compared to the activity obtained

using MW medium (Fig. 3b). In addition, the maxi-

mum BGL production was observed at 72 h after

incorporation of the nitrogen sources in the production

medium, leading to higher enzyme productivity. EG

and xylanase activity of 73.43 and 2,141.3 IU/g ds,

respectively were observed when urea was used as a

nitrogen source. On the basis of these results, it was

decided to use urea as nitrogen source along with the

basal nutrient solution during process optimization

through RSM.

Statistical optimization of cellulase production

by A. niger HN-2

The results presented in Table 3 showed a wide

variation from 0.40 to 27.2 FPU/g ds which reflects

the importance of process parameter optimization.

Highest FP cellulase activity (27.2 FPU/g ds) and

BGL activity (790.4 IU/g ds) were observed at 25 �C,

pH of 4.0, 80 % (w/w) moisture and incubation time of

96 h (Table 3). The data in Table 3 indicates that

increase in incubation time and higher moisture

Fig. 2 Phylogenetic

dendrogram for A. niger

HN-2 and related strains

with their accession

numbers based on the 26 s

rRNA gene sequence


123

content at a given temperature had a positive effect on

the FP and BGL activity. Results of this study

indicated that the cellulolytic enzyme production

capability of A. niger HN-2 was not drastically

affected when the initial pH varied from 4 to 7.

However, at extreme pH of 2.5 and 8.5, the cellulase

production capability by A. niger HN-2 was highly

compromised (Table 3).

The overall quadratic model was significant with an

R2 value of 0.87 and adjusted R2 value of 0.80 for FP

and R2 value of 0.86 and adjusted R2 value of 0.78 for

BGL activity. Lack of fit was not significant in both the

cases, indicating no reason to doubt the fitness of the

model. Statistical analysis indicated that the three

independent variables (initial moisture content, tem-

perature and incubation time) were statistically sig-

nificant for cellulase and BGL production at 5 %

significance level. The interaction between tempera-

ture and incubation time and squares of all the four

parameters were also significant at a 5 % significance

level for cellulase and BGL production.

The final response function to predict FP activity

after eliminating the non-significant terms at 5 %

significance level was

Y ¼ �270:81þ 8:8755X1 þ 22:6375X2 þ 0:9233X3

þ 1:9601X4 � 0:01329X1X3 � 0:1383X21

� 2:0986X22 � 0:0028X2

3 � 0:0134X24:

ð2ÞThe final response function to predict BGL activity

after eliminating the non-significant terms 5 % sig-

nificance level was

(a)

(b)

02468

101214161820

FP

cel

lula

se (

FP

U/g

-ds)

Time (h)

Ammonium sulphate Beef ExtractPeptone Potassium nitrateUrea

0

100

200

300

400

500

600

700

24 48 72 96 120

24 48 72 96 120

ββ-g

luco

sid

ase

(IU

/g-d

s)

Time (h)

Ammonium sulphate Beef ExtractPeptone Potassium nitrateUrea

Fig. 3 Effect of different nitrogen sources on a FP cellulase

production and b BGL production by A. niger HN-2. The values

for LSD (p \ 0.05) for incubation period were 1.40 and 35 for

FP cellulase and BGL, respectively

Table 3 Effect of different process parameters on FP activity

(FPU/g ds) and BGL activity (IU/g ds) during the optimization

experiment

X1

(temp

�C)

X2

(pH)

X3

(incubation

time, h)

X4

(moisture

content,

%)

FPU/g ds IU/g ds

30 5.5 66 35 0.40 20.10

40 5.5 66 65 7.80 110.90

25 7 36 50 5.00 104.80

25 7 96 80 19.20 471.80

30 2.5 66 65 3.00 80.40

30 5.5 66 65 22.80 755.00

25 7 36 80 80.00 200.80

25 7 96 50 13.50 229.70

30 5.5 66 95 21.70 617.00

30 5.5 66 65 25.60 677.60

30 5.5 66 65 26.50 750.50

35 4 96 80 13.30 214.00

25 4 36 50 7.60 189.40

35 7 96 50 9.10 208.80

30 5.5 6 65 0.40 11.60

30 5.5 126 65 25.60 658.10

25 4 36 80 12.40 208.40

35 7 36 80 9.00 254.10

35 7 96 80 15.00 254.40

35 4 96 50 10.50 289.70

25 4 96 50 19.20 459.60

20 5.5 66 65 10.80 254.70

35 4 36 50 7.50 165.00

35 4 36 80 10.00 254.60

35 7 36 50 7.50 144.40

25 4 96 80 27.20 790.40

30 8.5 66 65 5.50 95.60


123

Y ¼ �9288:80763þ 328:94742X1 þ 725:34861X2

þ 30:74643X3 þ 60:32403X4 � 0:45804X1X3

� 5:13937X21 � 67:63750X2

2 � 0:10052X23

� 0:42021X24:

ð3ÞThe lower p value and insignificant lack of fit

suggests the good fit of the quadratic model. A higher

coefficient of regression (R2) suggests that there was a

good agreement between the predicted and estimated

values. The results for two responses show similarity for

the significant factors and also for the significant

interaction, because production of cellulolytic enzymes

is drastically affected by process parameters, such as

moisture content, incubation time and temperature. In

order to determine the optimum levels for the variables

showing significant interaction for maximizing FP and

BGL production, three dimensional graphs were plotted

to understand the relationship between the response and

experimental levels for each variable. The response

surface plots were constructed by varying the two

independent variables at fixed concentrations of the

remaining two parameters. Three dimensional graphs

of temperature and incubation time (Fig. 4a, b) at

constant pH and moisture content showed significant

effect for interactions between temperature and incu-

bation time on enzyme production. As temperature and

incubation time were changed from their optimum

values, FP and BGL activity decreased significantly.

Effect of optimized parameters on cellulase

production

Design Expert 7.1 software analyzed the data and

suggested solutions on the basis of the selected

criteria. The data analysis also allowed us to study

the combined effect of independent variables on the

enzyme activity. The optimized variables were found

using a desirability objective function that assigns

relative importance to the responses. Since, no

significant difference in FP and BGL activity was

observed by increasing the incubation time from 66

to 96 h, the target for incubation time was fixed at

66 h, whereas the other three variables were kept in

range during numerical optimization. On the basis of

the solutions with higher desirability suggested by the

software and the results of model graphs, optimum

parameters for cellulase production were selected as

temperature: 29 �C, pH: 5.4, incubation time: 66 h

and moisture content: 72 % (w/w). At these opti-

mized conditions, the predicted responses for FP and

BGL activity from the model Eqs. 2 and 3 were

25.99 FPU/g ds and 752.68 IU/g ds, respectively.

The experimental FP and BGL activity obtained

using the suggested levels of the parameters were

25.3 FPU/g ds and 750 IU/g ds, respectively sug-

gesting a good agreement between the experimental

and predicted values. FP cellulase, BGL, CBHI, EG,

xylanase activity and protein content of 25.3 FPU/

g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds,

2890 IU/g ds and 0.9 mg/ml, respectively were

obtained during the validation process. Under similar

culture conditions, A. niger N402 produced nearly

60, 85, 80, 78, 82 and 12 % lower FP, BGL, CBHI,

EG, xylanase activity and protein, respectively as

compared to those produced by A. niger HN-2. These

results confirm the superiority of A. niger HN-2 over

A. niger N402.

(a)

(b)

25.00 27.50

30.00 32.50

35.00

36.00 51.00

66.00 81.00

96.00

0

7

14

21

28

FP

(F

PU

/gds

)

Temperature (deg C) Incubation time (h)

25.00 27.50

30.00 32.50

35.00

36.00 51.00

66.00 81.00

96.00

10

205

400

595

790

bet

a- g

luco

sida

se (

IU/g

ds)

Temperature (deg C) Incubation time (h)

Fig. 4 Response surface plots showing the effect of tempera-

ture and incubation time on a FP cellulase activity and b BGL

activity


123

Protein profile and zymogram analysis of cellulase

enzyme

Crude enzyme extract from A. niger HN-2 showed a

number of protein bands with differential intensities

indicating expression of different extracellular pro-

teins. The enzyme extract when analysed for activity

staining of different components of cellulase enzyme

complex viz., EG, CBH and BGL, showed variable

number of isoforms for each enzyme (Fig. 5). Zymo-

gram analysis for EG enzyme clearly indicated the

production of two isoforms corresponding to the

molecular size of about 55 and 42 kDa. On the other

hand, zymogram studies on CBH and BGL enzyme

revealed the presence of two and three isoforms,

respectively. The molecular weight of three isoforms

for BGL enzyme was estimated to be about 152, 130

and 51 kDa while it was 62 and 105 kDa for the two

isoforms of CBH enzyme.

Temperature, pH and stability of crude cellulase

consortium

Crude cellulases exhibited optimal FP and BGL

activity at 50 and 60 �C, respectively (Fig. 6a, b).

The pH profile showed more than 80 % FP and BGL

activity at a pH of 3.0–5.0 and 3.0–7.0, respectively

(Fig. 6b). However, decline in both FP and BGL

activity was seen at temperature beyond 70 �C and at

alkaline pH range. Crude enzyme exhibited relatively

more stability for BGL than FP activity at higher

temperature and alkaline pH range (Fig. 6a, b). The

stability of crude enzyme was studied for 360 min

within its near optimal pH range of 3.0–5.0 at

50–70 �C. At pH 3.0, the enzyme retained more than

80 % FP activity after 240 min at 50 and 60 �C,

whereas it retained only 65 % activity at 70 �C

(Fig. 7a). Crude enzyme was able to retain 100 %

FP activity after 240 min at pH of 4.0. At pH of 4.0, the

crude enzyme exhibited 83 and 70 % FP activity at 60

and 70 �C, respectively after 240 min (Fig. 7b). Crude

enzyme retained 80 % FP activity at pH of 5.0 until

180 min at 60 and 70 �C and until 240 min at 50 �C,

however, a sharp decline in enzyme activity was seen

beyond 240 min, irrespective of temperatures

(Fig. 7c).

Discussion

In order to economize the enzyme production process,

it is important to reduce the production time, so that

more number of batches could be completed in

relatively less time. In addition, the optimal time to

produce a desired concentration of enzymes also

depends upon the initial inoculum concentration,

moisture content of the medium, interaction of organ-

ism with substrate and incubation temperature. A fall

in enzyme activity after 72 or 96 h for different

isolates could possibly be due to the organism entering

Fig. 5 Molecular

characterization of cellulase

enzyme produced by A.

niger HN-2. Lane M

molecular weight markers,

Lane 1 SDS PAGE of crude

extract, Lane 2 Zymogram

analysis of EG, Lane 3 BGL,

Lane 4 CBH


123

stationary phase of growth; depletion of the nutrients

and production of other by-products in the fermenta-

tion medium or combination of all of the above

mentioned factors. Initially, fungi consume the readily

available sugars and produce hydrolytic enzymes, but

due to reduction in sugar concentration with time, the

fungi begin to use these hydrolytic enzymes for

production of sugars, leading to a decline in enzyme

activity (Botella et al. 2005).

Incorporation of urea in the fermentation medium

significantly enhanced the cellulase production by

A. niger HN-2. Maeda et al. (2010) reported a

significant increase in cellulase production by Peni-

cillium funiculosum by incorporation of urea and

ammonium sulphate in the fermentation medium.

Jatinder et al. (2006) reported that addition of urea to

RS used as carbon source proved beneficial during

optimization of medium components for cellulase

production by Melanocarpus sp. MTCC 3922 under

SSF. The results obtained through the present study

indicate that supplementing the fermentation medium

with the inorganic nitrogen sources resulted in higher

cellulase production, compared to the organic sources.

This may be attributed to the fact that complex

substances like amino acids and vitamins in organic

nitrogen sources could trigger the biomass production,

thus making it unnecessary for the fungus to produce

cellulase (Reddy and Reddy 1985). The other advan-

tages associated with urea as a nitrogen source are its

easy availability, low price and buffering ability.

It is evident from the results of optimization

experiment that the initial moisture content, temper-

ature and incubation time had a significant effect on

cellulase production. Temperature is a cardinal factor

which affects the amount and rate of growth of an

organism and has profound influence on the

(a)

(b)

0

20

40

60

80

100

120

% R

elat

ive

acti

vity

Temperature (oC)

FP cellulasebeta-glucosidase

0

20

40

60

80

100

120

30 40 50 60 70 80

2 3 4 5 6 7 8 9

% R

elat

ive

acti

vity

pH

FP cellulase beta-glucosidase

Fig. 6 Activity profiles for FP cellulase and BGL at different

a temperatures and b pH range

(a)

(b)

(c)

0

20

40

60

80

100

120

% R

elat

ive

acti

vity

Time (min)

50 degC 60 deg C 70 degC

0

20

40

60

80

100

120

% R

elat

ive

acti

vity

Time (min)

50 degC 60 deg C 70 degC

0

20

40

60

80

100

120

60 120 180 240 300 360

60 120 180 240 300 360

60 120 180 240 300 360

% R

elat

ive

acti

vity

Time (min)

50 degC 60 deg C

Fig. 7 Stability of FP cellulase at different temperatures at

a pH 3.0 b pH 4.0 and c pH 5.0


123

production of the desired end product. At lower

temperature, the transport of substrate across the cells

is suppressed resulting in lower product yields. At

higher temperature, the maintenance energy require-

ment for cellular growth is high due to thermal

denaturation of enzymes of the metabolic pathway

resulting in minimum product formation (Aiba et al.

1973). Moisture content of the fermentation medium is

one of the major factors that determines the success of

the process. Since, fungi are known to thrive in moist

environments; an optimum moisture level has to be

maintained for their efficient growth and production of

secondary metabolites. Lower moisture levels reduce

nutrient diffusion, microbial growth, enzyme stability

and substrate swelling, while higher moisture level

leads to particle agglomeration, gas transfer limitation

and competition from bacteria (Gowthaman et al.

1993). Gwande and Kamat (1999) reported maximum

xylanase production by A. niger when the ratio of WB

to mineral solution was kept at 1:5 (83 % moisture,

w/w). The change in pH from the initial pH value had a

little effect on FP and BGL activity within the

experimental range. Robustness against changes in

pH from its initial value during enzyme production

would be beneficial in shielding any adverse effect on

enzyme activity due to variations in pH. The optimum

pH for fungal cellulases varies from species to species,

though in most cases the optimum pH ranges from 3.0

to 6.0 (Niranjane et al. 2007). Maximum BGL

production by A. terreus was achieved at a pH in the

range of 4.0–5.5 (Pushalkar et al. 1995).

Validation studies conducted with A. niger HN-2 in

static trays using optimized parameters showed an

increase in FP and BGL activity by nearly 1.8- and 2.2-

fold, respectively. Cellulase activity obtained through

validation experiments was in close agreement with

the results predicted by the model equations, indicat-

ing successful validation of the model. In a previous

study, Soni et al. (2010) reported that A. fumigatus

fresenius strain (AMA) showed FP and BGL activity

of 9.73 FPU/g ds and 470 IU/g ds, respectively after

optimization of enzyme production parameters using

Box-Behnken design. Further, the concentrated cul-

ture extracts hydrolyzed Solka floc SW 40 and

bagasse, resulting in 90 and 87 % saccharification,

respectively. Optimization of process parameters

using the two step approach employing Plackett–

Burman and Box-Behnken designs, led to a two-fold

increase in BGL production by Paecilomyces variotii

MG3 (Job et al. 2010).

Zymogram studies revealed the presence of two

isoforms each for EG and CBH and three isoforms for

BGL. Our results are similar to the findings of some of

the recent studies wherein, multiple isoforms of EG,

CBH and BGL from Aspergillus sp. have been

reported (He et al. 2013; Kaur et al. 2013). Multiplicity

of cellulase enzyme is well known in several fungi. In

some cases, presence of multiple isoforms has been

shown to represent the products of separate genes i.e.

more than one gene encoding for proteins with EG

activity (Warren 1996). According to Willick and

Seligy (1985), some multiplicity may also be the result

of partial proteolysis during culture or storage, inter-

action and aggregation of enzymes with each other and

differential glycosylation of a common polypeptide

chain. Multiplicity in cellulases could be essential,

considering the vast and diverse roles these enzymes

play in fungal metabolism and survival. Differential

expression of the various enzymes could help the fungi

to respond positively to the rapidly changing

environments.

Thermal stability studies on crude cellulase con-

sortium produced by A. niger HN-2 showed more than

50 % retention for FP activity after 4 h at 70 �C. Half-

lives of 8 h at 60 �C and 1 h at 70 �C for crude enzyme

from A. niger has been reported previously (Aguiar

2001). Cellulases (EG) from A. fumigatus had a half

life of 330 min at 50 �C, declining slowly to 193 min

at 70 �C (Saqib et al. 2010). It is evident from the

results obtained in the present study that the crude

cellulase complex produced by A. niger HN-2 exhib-

ited better thermostability characteristics than the

crude cellulases produced by some of the other

Aspergillus strains (Aguiar 2001; Javed et al. 2009;

Saqib et al. 2010). As mentioned previously, differ-

ential expression of various enzymes as found in the

case of cellulase enzyme complex by A. niger HN-2

could be a plausible reason for adaptation of this

enzyme to varying temperature and pH conditions.

Thermostable enzymes are classified into three

groups, defined by their range of temperature stability:

moderately thermostable (45–65 �C), thermostable

(65–85 �C) and extremely thermostable ([85 �C)

(Yano and Poulos 2003). Thus, cellulase produced

by A. niger HN-2 could be classified in the category of

moderately thermostable to thermostable enzymes.


123

The results obtained through this study are significant

as the crude cellulase complex exhibited thermosta-

bility at acidic pH range. The thermostability of

cellulases under acidic conditions is an important trait

for achieving simultaneous saccharification and fer-

mentation of lignocellulosic biomass to ethanol

(Senthilkumar and Gunasekaran 2005).

It is important to explore the enzymes which have

compatible pH optima with that of yeast so that

simultaneous saccharification and fermentation could

be efficiently employed for ethanol production from

lignocellulosic biomass. Advantages of simultaneous

saccharification and fermentation for ethanol produc-

tion have been well documented (Sandhu et al. 2012).

Crude enzyme stability at 50 �C, especially in the

acidic range is an important characteristic which could

be exploited for simultaneous saccharification and

fermentation.

It is clear from the results of this study that though

RS and WB are good sources of carbon and nutrients,

addition of a nitrogen source and supplementing the

fermentation medium with additional nutrients is

essential for augmenting cellulase production. How-

ever, maintenance of high humidity at a higher scale of

operation in SSF with fungi is still a challenge. Most of

the previous studies on cellulase production have not

analyzed the CBH activity which has an important role

to play in hydrolysis of cellulose. CBHI is the major

component of the cellulase system required for

degradation of crystalline cellulose and constitutes

about 60 % of the protein secreted by Trichoderma

reesei (Uusitalo et al. 1991). It is thus, important to

have all the components of cellulase in a single

enzyme mixture, thereby obviating the need for

addition of cellulase components separately. An

enzyme consortium having all the cellulase and

xylanase components might result in efficient hydro-

lysis resulting in both hexose and pentose sugars,

which could be subsequently fermented to ethanol,

which in turn is likely to have a substantial effect on

bioethanol production cost.

Gielkens et al. (1999) characterized the two CBH-

encoding genes from A. niger (cbhA and cbhB) and

reported that the overall structure of cbhB was similar

to those of fungal CBH of family 7, while cbhA lacked

the cellulose binding domain (CBD) and the linker

peptide. It is possible that cellulases with CBDs are

required in the early stages of cellulose degradation,

when most of the substrate is insoluble and at laterTa

ble

4S

um

mar

yo

fso

me

of

the

rece

ntl

yp

ub

lish

edre

sult

so

nce

llu

lase

pro

du

ctio

nu

sin

gd

iffe

ren

tsu

bst

rate

san

dm

icro

bia

lst

rain

sth

rou

gh

SS

F

Mic

rob

ial

stra

inS

ub

stra

teF

P(F

PU

/

gd

s)

EG

(IU

/

gd

s)

b-G

luco

sid

ase

(IU

/

gd

s)

Xy

lan

ase

(IU

/

gd

s)

CB

H(I

U/

gd

s)

Ref

eren

ces

Asp

erg

illu

so

ryza

eS

oy

bea

nh

ull

4.5

04

7.1

01

3.3

03

90

–B

rijw

ani

and

Vad

lan

i

(20

11

)

Asp

erg

illu

sn

iger

US

MA

I

1

Su

gar

can

eb

agas

se:

pal

mk

ern

el

cak

e(1

:1)

3.4

0–

––

–L

eeet

al.

(20

11)

Asp

erg

illu

sfu

mig

atu

sR

ice

stra

w:

wh

eat

bra

n(7

:3)

0.9

31

4.7

18

.51

42

.70

0.6

8S

her

ief

etal

.(2

01

0)

Asp

erg

illu

sfu

mig

atu

s

fres

eniu

s

Ric

est

raw

:w

hea

tb

ran

(1:3

)2

.11

40

.92

43

.70

44

4–

So

ni

etal

.(2

01

0)

Asp

erg

illu

sn

iger

MT

CC

79

56

Wh

eat

bra

n4

.55

13

5.4

42

1.3

9–

–S

uk

um

aran

etal

.(2

00

9)

Asp

erg

illu

sn

iger

NR

RL

Ap

ple

po

mac

e1

33

17

26

01

,41

2–

Dh

illo

net

al.

(20

12)

Asp

erg

illu

sn

iger

NS

2W

hea

tb

ran

17

.03

10

.03

3.0

––

Ban

sal

etal

.(2

01

2)

Asp

erg

illu

sn

iger

HQ

-1–

42

.43

05

.11

58

.5–

–Z

han

get

al.

(20

12

)

Asp

erg

illu

sn

iger

HN

-2R

ice

stra

w:

Wh

eat

bra

n(4

:1)

25

.30

19

07

50

2,8

90

13

.2P

rese

nt

stu

dy


123

stages during solubilization of substrate, enzymes

without CBDs are preferred. This suggests that the A.

niger isolates can be suitable candidates for produc-

tion of the cellulase complex overcoming the limita-

tions that have been known for T. reesei for efficient

hydrolysis of lignocellulosic biomass. Most of the

previous studies conducted on the model A. niger

strain, such as N402 (ATCC 6497, MTCC 271),

extensively used for growth and development studies

have focused on strain improvement through muta-

genesis, gene cloning and expression for enhancing

cellulase and xylanase production by the recombinant

strains. Coupled with the results reported in Table 4 on

cellulolytic potential of different Aspergillus isolates

and the comparison of A. niger HN-2 with the

extensively studied model A. niger N402, it could be

inferred that the in overall terms, the cellulase activity

reported in the present study was significantly higher

than the activity reported for different cellulase

components in recently published literature. There-

fore, this study holds promise for cellulase production

using RS as a major substrate which is abundantly

available in countries like India at almost no cost. We

now plan to further manipulate the culture conditions

using metabolic foot printing methods for selective

expression of the desired isoforms and use this crude

enzyme preparation for the hydrolysis of the pre-

treated lignocellulosic biomass to evaluate its hydro-

lytic efficiency and also assess the commercial

feasibility of the crude cellulase preparation.

Conclusions

The use of agricultural residues and by-products,

optimized process parameters and use of A. niger HN-2

are likely to have a significant impact on cellulase

production as is evident from the results obtained in

this study. Experimental methods to enhance the

enzyme production are imperative for success in

biofuel industry, which uses enzymatic and microbial

fermentation platforms. Substantial increase in cellu-

lase production through statistical process optimiza-

tion using the newly isolated A. niger strain holds

promise for scale-up studies. The results obtained

through this study are significant as thermostability

under acidic conditions is required for effective ethanol

production from lignocellulosic biomass. This study

has therefore, set a platform for conducting cellulase

production studies through SSF at a higher scale of

operation using the optimized parameters and a newly

isolated A. niger HN-2 strain.

Acknowledgments Authors thankfully acknowledge the

financial assistance received under the NAIP Project (418301-

3) funded by World Bank from the Indian Council of Agricultural

Research, New Delhi, India for conducting this study.

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