ORIGINAL PAPER

Production of xylanase under solid-state fermentationby Aspergillus tubingensis JP-1 and its application

Jagruti J. Pandya • Akshaya Gupte

Received: 30 July 2011 / Accepted: 12 November 2011 / Published online: 24 January 2012

� Springer-Verlag 2012

Abstract The production of extracellular xylanase by a

locally isolated strain of Aspergillus tubingensis JP-1 was

studied under solid-state fermentation. Among the various

agro residues used wheat straw was found to be the best for

high yield of xylanase with poor cellulase production. The

influence of various parameters such as initial pH, mois-

ture, moistening agents, nitrogen sources, additives, sur-

factants and pretreatment of substrates were investigated.

The production of the xylanase reached a peak in 8 days

using untreated wheat straw with modified MS medium,

pH 6.0 at 1:5 moisture level at 30 �C. Under optimized

conditions yield as high as 6,887 ± 16 U/g of untreated

wheat straw was achieved. Crude xylanase was used for

enzymatic saccharification of agro-residues like wheat

straw, rice bran, wheat bran, sugarcane bagasse and

industrial paper pulp. Dilute alkali (1 N NaOH) and acid

(1 N H2SO4) pretreatment were found to be beneficial for

the efficient enzymatic hydrolysis of wheat straw. Dilute

alkali and acid-pretreated wheat straw yielded 688 and

543 mg/g reducing sugar, respectively. Yield of 726 mg/g

reducing sugar was obtained from paper pulp after 48 h of

incubation.

Keywords Aspergillus tubingensis JP-1 � Xylanase

production � SSF � Wheat straw � Saccharification

Introduction

The most abundant renewable biomass available on earth is

lignocellulose, which contains three major groups of

polymers, cellulose, hemicellulose and lignin [1]. Xylan is

a major hemicellulosic constituent of hard wood and soft

wood and is the most abundant renewable polysaccharide

after cellulose. It is a potential resource for producing

many valuable products [2]. It is a heterogeneous poly-

saccharide composed of b-1,4 linked xylose chains with

branches containing arabinose and 4-O-methyl glucuronic

acid. Complete hydrolysis of xylan involves the synergistic

action of an array of main- and side-chain cleaving

enzymes, among which xylanases [1,4 b-D-xylan xylano-

hydrolase (EC 3.2.1.8)] play a key role [3]. The other

enzymes, like b-D-xylosidase (EC 3.2.1.37), a-L-arabino-

furanosidase (EC 3.2.1.55), a-glucuronidase (EC 3.2.1.) and

acetyl xylan esterase (EC 3.1.1.6) are also considered for

carrying out other key reactions [4]. Xylan-degrading

enzymes have attracted much attention because of their

application in industrial processes such as modification of

cereal-based food stuffs, improving digestibility of animal

feed stocks [5], bioconversion of lignocellulosic material

and agro waste to fermentable products [6] and prebleach-

ing of paper pulps [7]. Cellulase-free xylanases have

received great attention in the development of an environ-

mental friendly technology in the paper and pulp industries

[8]. Benefits of the usage of xylanases in this industrial

sector have been demonstrated for (1) reduction of chlorine

base chemicals and H2O2, (2) increase in tear and burst

strengths of the paper produced and (3) Reduction in

chemical oxygen demand in the effluent discharge [9].

A variety of micro-organisms including bacteria, yeast

and filamentous fungi have been reported to produce

xylanolytic enzymes [10]. Filamentous fungi have

J. J. Pandya � A. Gupte (&)

Department of Microbiology, N. V. Patel College of Pure

and Applied Sciences, Vallabh Vidyanagar 388120,

Gujarat, India

e-mail: akshaya_gupte@hotmail.com

123

Bioprocess Biosyst Eng (2012) 35:769–779

DOI 10.1007/s00449-011-0657-1

demonstrated a great capability to secrete a wide range of

xylanases, with the genera Aspergillus and Trichoderma

being the most extensively studied and reviewed among the

xylanase producing fungi. There have also been some

reports of the xylanase production using different strains of

Aspergillus tubingensis [11, 12]. Xylanases are produced

either by solid-state fermentation (SSF) or submerged

fermentation. The use of SSF as a method of production for

xylanase could offer some apparent economic and engi-

neering advantages over the classical submerged fermen-

tation. These include high concentration of the product and

simple fermentation equipment as well as low requirement

for aeration and agitation during enzyme production. The

solid substrates used not only supply the nutrients to

the microbial cultures, but also serve as an anchorage to the

cell. Generally, carbon source has been very cost effective

in the production of xylanase and cost can be reduced usin

the agro waste that are abundant and considered as the best

substrates for SSF processes [13].

The purpose of the present study was to investigate the

potential xylanase-producing fungi from soil and to iden-

tify the strain that secretes the maximum amount of

enzyme. The nutritional requirements for xylanase pro-

duction by isolated strain Aspergillus tubingensis JP-1

under SSF have been examined. The optimization of

medium composition (pH, temperature, carbon source,

nitrogen source, moisture level, surfactants, and additives)

was investigated by one factorial methodology.

Materials and methods

Isolation and identification of xylanolytic

microorganisms

The soil samples for the isolation of xylanolytic micro-

organisms were collected from different areas of agri-

cultural field (Anand, Gujarat, India). One gram of soil

was suspended in 10 ml sterile distilled water, vortexed

and a 100-ll aliquot of the clear supernatant was plated

on MEA xylan agar plate (malt extract 2%, birch wood

xylan 0.5%, agar-agar 2.0%). Positive xylanolytic iso-

lates were detected based on the clear zone of hydrolysis

on the MEA xylan agar plate. Further confirmation of

potential xylanase producers was carried out using the

defined xylan agar (DX) media (Birch wood xylan 1.0%,

KH2PO4 0.1%, (NH4)2SO4 0.2%, NaCl 0.1%, agar-agar

2.0%) [14]. The isolates obtained were further screened

for xylanase activities by cultivating in DX broth for

12 days under shaking condition. The selected isolate

was identified by Bangalore GeNei (Bangalore, India)

using partial 18S rRNA, ITS1, 5.8S rRNA, ITS2 and

partial 28S rRNA gene sequencing. Potential isolate was

subcultured and maintained at 4 �C on potato dextrose

(PDA) agar slant.

Xylanase production under SSF

Erlenmeyer flasks (250 ml) containing 5 g of wheat straw

and 25 ml of Mandels and Sternburg’s (MS) medium [15]

(g/l): peptone, 1.0; (NH4)2SO4, 1.4; KH2PO4, 2.0; urea,

0.3; CaCl2, 0.3; MgSO4�7H2O, 0.3 and trace elements (mg/

l): FeSO4�7H2O, 5.0; MnSO4�H2O, 1.6; ZnSO4�7H2O, 1.4;

CoCl2, 2.0; Tween 80, 0.1% (v/v) pH 6.0) were autoclaved

at 121 �C for 30 min, cooled, inoculated with 2 9 106

spores/ml and incubated at 30 �C for 10 days. The flasks

were gently tapped intermittently to mix the content. At the

desired intervals, the flasks were removed and the contents

extracted with 50 mM sodium citrate buffer (pH 5.0).

Enzyme extraction

The enzymes from wheat straw were extracted with

50 mM sodium citrate buffer (pH 5.0) and squeezed

through muslin cloth. The enzyme extract obtained was

centrifuged at 10,000 rpm at 4 �C for 15–20 min. The clear

supernatant obtained was used as the enzyme sample for

assay.

Enzyme assay

Xylanolytic activity of the cell free supernatant was deter-

mined according to Bailey et al. [16]. One unit of xylanase

is defined as the amount of enzyme that liberates 1 lmol

reducing sugar as xylose/ml/min under the assay condition.

b-Xylosidase activity was determined according to Judith

and Nei [17]. One unit of b-xylosidase is defined as the

amount of enzyme required to liberate 1 lmol P-nitrophe-

nol/ml/min under the assay condition. Filter paper cellulase

activity was measured according to the methods described

by Ghose [18]. One unit of cellulase (FPA) is defined as the

amount of enzyme that liberates 1 lmol reducing sugar

glucose/ml/min under the assay condition. Protease activity

was measured according to the methods described by

Gessesse et al. [19]. One unit of protease activity is defined

as the amount of enzyme that liberates 1 lmol tyrosine/ml/

min under the assay condition. Soluble protein content was

determined according to Lowry method [20] using bovine

serum albumin as the standard.

Xylanase production on various lignocellulosic

substrates

The fungal isolate Aspergillus tubingensis JP-1 was grown

in 250-ml Erlenmeyer flasks containing 5 g each of various

lignocellulosic substrates (wheat straw, wheat bran, rice

770 Bioprocess Biosyst Eng (2012) 35:769–779

123

bran, sugarcane bagasse) and moistened with MS medium.

The enzyme was extracted and assayed.

Effect of particle size of wheat straw

To study the effect of particle size on xylanase production,

the experiments were performed with different size of

substrate particle ranging from 0.07 to 1.7 mm. These were

further divided into three subgroups, i.e. (0. 07–0.3,

0.33–0.5 and 0.7–1.7 mm).

Effect of the initial culture pH and temperature

on xylanase production

To evaluate the effect of initial culture pH on xylanase

production, the initial pH of MS medium was adjusted to

3.0–9.0. The effect of temperature on xylanase production

by Aspergillus tubingensis JP-1 was studied by incubating

the flasks at 25, 30, 35, 40 and 45 �C for 8 days.

Effect of different moistening agents on xylanase

production

Wheat straw was moistened with different mineral salt

solution, which were distilled water and tap water added

with Tween 80 (0.1% v/v), MS medium, modified MS

medium I—(g/l): (NH4)2SO4, 1.4; KH2PO4, 2.0; CaCl2, 0.3;

MgSO4�7H2O, 0.3; (mg/l): FeSO4�7H2O, 5.0; MnSO4�H2O,

1.6; ZnSO4�7H2O, 1.4; Tween 80 0.1% (v/v), pH 6.0, MS

medium II—(g/l): KH2PO4, 2.0; CaCl2, 0.3; MgSO4�7H2O,

0.3; (mg/l): FeSO4�7H2O, 5.0; MnSO4�H2O, 1.6; ZnSO4�7H2O, 1.4; Tween 80 0.1% (v/v), pH 6.0 and Toyoma’s

mineral solution—(g/l): (NH4)2SO4, 10.0; KH2PO4, 3.0;

urea, 0.3; CaCl2, 1.0; MgSO4�7H2O, 0.5; Tween 80 0.1% (v/v)

at pH 6.0.

Effect of moisture level on xylanase production

The influence of moisture level on the xylanase production

was evaluated by varying the ratio (w/v) of wheat straw to

MS medium (1:1, 1:2, 1:3, 1:4, 1:5, 1:6 and 1:7).

Effect of nitrogen sources on xylanase production

The effect of nitrogen sources on the enzyme production

was studied in the complex MS medium with different

concentrations of inorganic and organic nitrogen sources.

Effect of surfactant on xylanase production

To study the effect of different surfactants (Tween 20,

Tween 40, Tween 60, Tween 80, Triton X-100 and SDS)

[0.01–0.2% (v/v)] on enzyme production, they were

incorporated into the production medium.

Effect of additives on xylanase production

Additives such as glucose, maltose, lactose, xylose, birch

wood xylan and oat spelt xylan (0.5–4% w/w) were

incorporated with wheat straw.

Effect of pre treatment of substrate on xylanase

production

In order to study the influence of pretreatment of substrate

on xylanase production, wheat straw was soaked in 1 N

HCl, 1 N NaOH and 1% w/v Ca(OH)2 as 5% slurry and

then thoroughly washed until it attained neutral pH and was

later dried. Steam treatment was given at 121 �C for 2 h.

Time course study of xylanase production

Wheat straw (5 g) was moistened with 25 ml of unoptim-

ized and optimized MS medium (250 ml flasks), auto-

claved at 121 �C, inoculated with 2 9 106 spores/ml and

incubated at 30 �C for 16 days. The content of the flasks

were harvested after every 24 h and assayed.

Saccharification of different lignocellulosic substrates

with xylanase

Natural lignocellulosic materials such as wheat bran, rice

bran, wheat straw, sugarcane bagasse and industrial paper

pulp and wheat straw treated with dilute acid and alkali

were saccharified using crude enzyme. The reaction mix-

ture contained 5 g of substrate, 80 ml buffer and 20 ml

crude enzyme. The reaction was allowed to proceed for

48 h. Aliquots were taken at regular intervals, centrifuged

and supernatant assayed for total reducing sugar.

All the experiments were carried in triplicates and the

data represents the mean value of three replicates.

Results and discussion

Isolation and identification of xylanase producer

Based on the initial screening program, a total of 40

isolates were capable of exhibiting xylanolytic activities on

MEA xylan agar plate with the diameter of clear

zones ranging from 20 to 40 mm. Among the 40 isolates,

5 isolates were selected for further confirmation using the

Bioprocess Biosyst Eng (2012) 35:769–779 771

123

defined xylan agar (DX) media. The isolate JP-1, dem-

onstrated reproducible zones of hydrolysis of 30–45 mm

diameter and the isolate was selected as a potential

producer of xylanase. The isolates were further tested for

xylanase production by cultivating them in DX broth

medium. It was observed that JP-1 produced higher

xylanase activity (185.34 U/ml) on 8th day of cultiva-

tion. Compared with JP-1, lower xylanase production

was obtained with JP-2 (59.3 U/ml), JP-3 (48.7 U/ml),

JP-4 (51.3 U/ml) and JP-5 (43.5 U/ml), respectively. The

highest xylanase-producing strain (JP-1) was thus selec-

ted for our further studies. Identification of JP-1 was

based on structural morphology and nucleotide sequence

analysis of enzymatically amplified partial 18S rRNA,

ITS1, 5.8S rRNA, ITS2 and partial 28S rRNA gene

sequencing. BLAST similarity search analysis based on

ITS and 18S rRNA gene sequences revealed that the

isolates belong to the genus Aspergillus. The closest

phylogenetic neighbor was found to be Aspergillus tu-

bingensis EF 634380 (NRRL 35179) with 100%

homology. Phylogenetic relationship could be inferred

through the alignment and cladistic analysis of homolo-

gous nucleotide sequences of known fungi, and the

approximate phylogenetic position of the strain is shown

in Fig. 1. The gene sequence has been deposited in the

GenBank database of NCBI under accession number EU

867248 (1,147 bp). The strain Aspergillus sp. JP-1 was

thus identified as Aspergillus tubingensis and named as

Aspergillus tubingensis JP-1.

Solid-state fermentation using different lignocellulosic

substrates

Carbon source is one of the most important factors during

the growth and metabolic process of micro-organisms. The

choice of an appropriate carbon source and the cost of

substrate play a crucial role in the economics of xylanase

production. Various agro-waste residue viz., wheat straw,

wheat bran, rice bran and sugarcane bagasse were studied

using MS medium as the moistening agents in the propor-

tion of 1:5. Among the lignocellulosic materials tested as

carbon source, wheat straw was found to be most appro-

priate for the production of xylanase (1,478 ± 11 U/g).

When used as substrates, rice bran, wheat bran and sugar-

cane bagasse showed 66, 76, 98% decrease in the xylanase

yield, respectively (Table 1). Difference in xylanase pro-

duction is observed with wheat straw as carbon source. This

may be due to its hemicellulose nature, favorable degra-

dability and the presence of some nutrients [21]. The use of

wheat straw as a suitable substrate for xylanase production

has also been reported with Penicillium canescens [22] and

Paecilomyces themophila [10].

Effect of particle size of wheat straw on xylanase

production

In SSF process, the availability of surface area plays a vital

role for microbial attachment, mass transfer of various

nutrients/substrates, subsequent growth of microbial strain

Fig. 1 Phylogenetic

dendogram for Aspergillustubingensis JP-1 based on 18S

rRNA, 5.5S rRNA, and partial

28S rRNA gene sequencing.

Numbers following the names

of the strains are accession

numbers of published

sequences. The bootstrapped

unrooted tree was constructed

by neighbor-joining method

from the distance data generated

by multiple alignments of

nucleotide sequences

772 Bioprocess Biosyst Eng (2012) 35:769–779

123

and product formation. The availability of surface area in turn

depends on the particle size of the substrate. The results thus

obtained reveal that the xylanase production is affected by the

particle size. Maximum enzyme production of 1,478 ± 11 U/

g was obtained with the particle size of 0.7–1.7 mm of wheat

straw (Fig. 2). The particle size-mediated influence on

xylanase production has also been reported in Rhizopus

oligosporus [23] and in Bacillus sp. [24].

Effect of initial culture pH and growth temperature

on xylanase production

pH and temperature are important environmental parame-

ters that determine growth rate of microorganisms and

significantly affect the level of xylanase produced. The

enzyme production by microbial strains strongly depends

on the initial culture pH, as it influences many enzymatic

processes and transport of various components across the

cell membranes [25]. The influence of pH on xylanase

production was studied by adjusting the initial medium

(MS medium) pH from 3 to 9 (Fig. 3). The maximum

xylanase production was obtained at an initial pH 6.0,

corresponding to 1,478 ± 11 U/g. The enzyme production

reduced in acidic and in highly alkaline range. This may be

due to the unfavorable pH which may limit the growth and

production by reducing accessibility of the hemicellulosic

substrates [26].

To examine the effect of temperature on xylanase pro-

duction, growth of A. tubingensis JP-1 was studied in the

range of 25–45 �C. Maximum xylanase production

(1,478 ± 11 U/g) was obtained at 30 �C on the 8th day of

incubation. Further increase in temperature was detrimental

to fungus in turn reducing the xylanase production. Highest

xylanase titer in fungal system has been reported to happen

generally at temperatures that are optimum for growth of

culture in SSF [13, 27]. However, at low temperature

transport of substrate across the cell is decreased and low

yield of products are obtained.

Effect of different moistening agents

Four different moistening salt solutions (MS medium,

modified MS medium I and II and Toyoma’s mineral salt

solution) along with distilled water and tap water with

0.1% v/v Tween 80 were used as moistening agents. The

highest enzyme production of 1,478 ± 11 U/g of substrate

was obtained when wheat straw was moistened with MS

medium. Lower xylanase production was obtained when

modified MS medium I (211 ± 12 U/g), MS medium II

(352 ± 10 U/g) and Toyoma’s mineral salt solution

(526 ± 10 U/g) were used. Moistening media like tap

water (78 ± 10 U/g) and distilled water (18 ± 2 U/g)

when used without supplementation of any nitrogen source

poorly supported the xylanase production. Similar results

Table 1 Xylanase production under Solid State Fermentation using different lignocellulosic substrates at 30 �C by Aspergillus tubingensis JP-1

Substrates Xylanase activity (U/g) Protease activity (U/g) Cellulase activity (FPA) (U/g) Protein (mg/g)

Wheat bran 364 ± 13 0.557 ± 0.09 0.024 ± 0.005 13.16 ± 0.11

Rice bran 509 ± 16 1.12 ± 0.11 0.037 ± 0.005 6.89 ± 0.09

Sugarcane bagasse 17 ± 3 0.678 ± 0.11 0.064 ± 0.002 3.11 ± 0.11

Wheat straw 1,478 – 11 0.608 ± 0.13 0.669 ± 0.007 10.99 ± 0.13

All the values in the table are the average of triplicate with ±standard deviation

Bold value in the table indicates the maximum xylanase activity

Fig. 2 Effect of particle size on xylanase production by Aspergillustubingensis JP-1 under SSF system (pH 6.0) at 30 �C on wheat straw Fig. 3 Effect of pH on xylanase production under SSF on wheat

straw at 30 �C using Aspergillus tubingensis JP-1

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123

have also been reported using variety of mineral salt

solutions [28, 29].

Effect of different moisture level

Initial moisture content is one of the key factors influencing

the xylanase production. The influence of moisture content

was studied in the ratio of 1:1 to 1:7 (w/v) of the wheat

straw to MS medium. Maximum xylanase production of

1,478 ± 11 U/g was obtained at 1:5 initial moisture con-

tent followed by 1:6 and 1:7 (Fig. 4). The lower enzyme

production at higher moisture level could be attributed to

the decreased porosity, alteration in particle structure or

lower oxygen transfer. Likewise, lower moisture contents

can lead to reduced diffusion of the nutrients in the sub-

strate, lower degree of swelling and higher water tension.

Similar effects of moisture content on xylanase production

have also been reported by other researchers [22, 30]. High

moisture enhanced fungal growth and subsequent early

initiation of the enzyme production when lignocellulosic

substrates were used as carbon sources [10, 31].

Effect of different nitrogen sources on xylanase

production

The mechanism that governs the formation of extracellular

enzyme can be influenced by the availability of the nitro-

gen source. The nitrogen source can significantly affect pH

of a medium during the course of fermentation and in turn

may substantially influence the activity of the enzyme.

Supplementation of nitrogen source is not always essential

in the SSF system, as it depends on the availability of

nitrogen in the substrate and requirement of the organisms.

Xylanases with minimal cellulase can be produced by the

low C:N source [32].

In the present study, combinations of different organic

and inorganic nitrogen sources were tested using wheat straw

as a substrate. It was observed that the maximum xylanase

production was found to occur in the presence of 0.1%

peptone, 0.28% (NH4)2SO4 and 0.03% urea (combination

IX), with high enzyme activity of 3,030 ± 14 U/g on the 8th

day of cultivation (Table 2). These results are in agreement

with those reported in literature where fungi produced higher

xylanase on similar nitrogen sources [13, 33].

Effect of surfactant on xylanase production

Addition of various surfactants to the culture medium

exerts a range of effects on enzyme secretion. Various

surfactants like Tween 80, Triton X-100, SDS or fatty acids

have been added to the medium for xylanase production

[34]. To study the effect of different surfactants, Tween 20,

Tween 40, Tween 60, Tween 80, Triton X-100 and SDS

were added to the medium in the concentration ranging

from 0.01 to 0.2% (v/v). Maximum xylanase production of

3,719 ± 4 U/g of substrate was obtained with Triton

X-100 (0.01% v/v) (Table 3), which could be due to its

favorable effect on cell permeability, thus affecting the

secretion of certain proteins [34, 35].

Effect of additives on xylanase production

The choice of an appropriate additive is of great impor-

tance for the successful production of xylanases. TheFig. 4 Effect of moisture level on xylanase production under SSF (at

30 �C, pH 6.0) on wheat straw using Aspergillus tubingensis JP-1

Table 2 Effect of nitrogen sources on the xylanase production under

SSF at 30 �C, pH 6.0 on wheat straw using Aspergillus tubingensisJP-1

Set no. Xylanase activity

(U/g)

Protein

(mg/g)

Specific activity

(U/mg)

I 505 ± 11 12.60 ± 0.13 40.07

II 1,344 ± 15 16.04 ± 0.16 83.79

III 1,818 ± 16 23.19 ± 0.23 78.39

IV 1,620 ± 16 27.75 ± 0.10 58.37

V 1,685 ± 11 23.72 ± 0.17 71.03

VI 1,478 ± 13 24.67 ± 0.12 59.91

VII 1,343 ± 16 25.06 ± 0.12 53.59

VIII 2,700 ± 20 17.11 ± 0.17 157.80

IX 3,030 – 14 18.20 – 0.15 166.48

X 1,642 ± 14 20.36 ± 0.18 80.64

XI control 1,478 ± 11 10.99 ± 0.13 134.48

All the values in the table are the average of triplicate with ±standard

deviation

I: 0.05% P, 0.14% AS, 0.03% urea; II: 0.15% P, 0.14% AS, 0.03% urea;

III: 0.2% P, 0.14% AS, 0.03% urea; IV: 0.1% P, 0.14% AS, 0.015%

urea; V: 0.1% P, 0.14% AS, 0.045% urea; VI: 0.1% P, 0.14% AS, 0.06%

urea; VII: 0.1% P, 0.07% AS, 0.03% urea; VIII: 0.1% P, 0.21% AS,

0.03% urea; IX: 0.1% P, 0.28% AS, 0.03% urea; X: 0.05% P, 0.05% YE,

0.14% AS, 0.03% urea; XI: 0.1% PP, 0.14% AS, 0.03% urea

P peptone, PP proteose peptone, AS ammonium sulfate, YE yeast

extract

Bold values in the table indicate the maximum xylanase activity

774 Bioprocess Biosyst Eng (2012) 35:769–779

123

additive not only serves as carbon and energy source, but

also provides the necessary inducing compounds for the

organisms, preferably for an extended period of time, since

then a prolonged production phase can result in an

increased overall productivity of the fermentation process.

Figure 5 shows the influence of various additives, indi-

cating the inducible nature of enzyme production by

Aspergillus tubingensis JP-1. The addition of xylose along

with wheat straw enhanced the xylanase production up to

(46.15%). Xylose has been described as an effective

inducer and carbon source for xylanase production in

several microorganisms [36–38]. The effect of xylose

concentration on xylanase production was further exam-

ined in the range of 0.1–1.0% (w/w) (Table 4). The opti-

mum xylose concentration was found to be 0.5% w/w

which suggested that sugar acts as an inducer rather than

carbon source. Xylanase production decreased with lower

and higher xylose concentrations suggesting that the pres-

ence of repression phenomena as a xylose concentration

exciding 0.5% concentration. Gessesse and Mamo [39]

reported that xylanase production is strongly repressed by

xylose concentration above 1%.Effect of pretreatment of substrates on xylanase

production

The lignocellulosic matrix is highly resistant to microbial

attack. The utilization of lignocellulosic substrates as a

potential low-cost carbohydrate source for monomeric

sugar has been impeded by the low efficiency with which

the hemicellulosic portion of these material is enzymati-

cally hydrolyze to xylose. Several types of pretreatments

have been shown to be effective in enhancing the enzy-

matic hydrolysis of lignocellulosic substrates. In order to

obtained high amounts of fermentable sugar and enzyme

production, various pretreatments have been used in the

present study. An ideal pretreatment would accomplish

reduction in crystallinity, lignin content, increase in surface

area, pore size, depolymerization and deacetylation of

hemicelluloses which enhance the availability of sub-

strates. In the present study wheat straw was pretreated

Table 3 Effect of surfactants

on xylanase production under

SSF (pH 6.0) on wheat straw at

30 �C using Aspergillustubingensis JP-1

All the values in the table are

the average of triplicate with

±standard deviation

Bold value in the table indicates

the maximum xylanase activity

Surfactant

concentration

% (v/v)

Xylanase activity (U/g)

Tween 20 Tween 40 Tween 60 Tween 80 Triton X-100 SDS

0.01 1,988 ± 14 2,269 ± 9 3,281 ± 7 2,164 ± 5 3,719 – 4 2,065 ± 5

0.02 2,389 ± 5 2,358 ± 6 3,291 ± 5 2,237 ± 7 3,590 ± 4 2,376 ± 6

0.025 2,350 ± 4 2,519 ± 5 3,359 ± 8 3,393 ± 5 3,521 ± 5 2,343 ± 5

0.05 2,357 ± 8 2,443 ± 6 3,341 ± 4 2,359 ± 4 3,530 ± 3 2,248 ± 8

0.075 2,333 ± 7 2,190 ± 9 3,083 ± 6 2,355 ± 11 3,488 ± 4 2,152 ± 5

0.1 2,273 ± 3 2,051 ± 9 3,172 ± 10 1,890 ± 5 3,309 ± 6 1,938 ± 10

0.15 1,949 ± 5 1,975 ± 6 3,108 ± 11 648 ± 9 3,282 ± 7 1,642 ± 11

0.2 1,706 ± 6 1,470 ± 6 2,315 ± 8 556 ± 15 3,130 ± 4 1,603 ± 3

Fig. 5 Effect of additives on xylanase production by Aspergillustubingensis JP-1 under SSF system at 30 �C, pH 6.0 on wheat straw.

BWX birch wood xylan, OSX oat spelt xylan

Table 4 Effect of xylose concentration on xylanase production under

SSF at 30 �C, pH 6.0 using Aspergillus tubingensis JP-1 with wheat

straw as a substrate

Concentration of

xylose % (w/w)

Xylanase

activity

(U/g)

Protein

(mg/g)

Specific

activity

(U/mg)

0.1 5,055 ± 21 22.54 ± 0.20 224.26

0.2 5,128 ± 23 23.58 ± 0.13 217.47

0.3 5,473 ± 16 23.88 ± 0.16 229.18

0.4 5,592 ± 18 25.51 ± 0.15 219.20

0.5 6,887 – 16 28.20 – 0.21 244.21

0.6 5,257 ± 16 25.50 ± 0.16 206.15

0.7 5,121 ± 19 23.72 ± 0.18 215.89

0.8 4,674 ± 19 23.72 ± 0.24 197.04

0.9 4,687 ± 25 30.14 ± 0.23 155.50

1.0 3,312 ± 20 23.05 ± 0.14 143.68

All the values in the table are the average of triplicate with ±standard

deviation

Bold values in the table indicate the maximum xylanase activity

Bioprocess Biosyst Eng (2012) 35:769–779 775

123

with mild acid (1 N HCl), mild alkali [1 N NaOH and 1%

w/v Ca(OH)2] as well as steam treatment (121 �C for 2 h).

None of the pretreatments used showed any advantages to

increase the production of xylanase by Aspergillus

tubingensis JP-1 (Table 5). Similar observations were also

reported by Shah and Madamwar [13] and Ferreira et al.

[30]. The application of mild alkali and steam treatment

can result in weight loss of the substrate as a result of

solubilization of lignin. These treatments are also respon-

sible for the delignification process. More easily available

substrate is readily depleted and causes drastic reduction in

enzyme production [40], whereas mild acid treatment does

not remove the lignin from the substrate but modifies the

lignin carbohydrate linkages. Thus lignin remains a barrier

for enzymatic attack, thereby affecting the overall fer-

mentation and production of the enzyme [41].

Time course study of xylanase production

Time course study of xylanase production by Aspergillus

tubingensis JP-1 was investigated before and after media

optimization in SSF system. Figure 6 shows the xylanase

production increased fivefold with the maximum activity of

6,887 ± 16 U/g of substrate on 8th day of cultivation.

Further incubation did not show any increment in the level

of xylanase production. The production of xylanases using

other substrates such as wheat bran, sugarcane baggase,

corn cobs, soybean hull and rice straw has also been

reported by other researchers [13, 28, 31]. Kheng and Omar

[37] reported xylanase production of 35 U/g of palm kernel

cake (PKC) using Aspergillus niger USM AI 1 under SSF

system. Lower xylanase production of 15–17 U/g of wheat

bran was obtained by Bacillus licheniformis [42]. In the

present study, very poor cellulase (FPA) (0.674 ± 0.007 U/g)

production was observed along with higher xylanase

(6,887 ± 16 U/g) and b-xylosidase (9.19 ± 0.005 U/g).

Saccharification of lignocellulosic substrates

with xylanase

The structure of xylan is more complex than cellulose and

requires several different enzymes with different specifi-

cation for complete hydrolysis. The fermentable sugars

thus obtained have high market value. The utilization of

enzymatic hydrolysis to obtain sugar from agricultural

residues is of great interest in mordent biotechnology,

particularly for the production of xylooligosaccharides and

bioethanol. As the enzymatic reaction, by nature, is a more

specific process, in the present study, we attempted the

saccharification of various agro-residues like wheat straw,

wheat bran, rice bran, sugarcane bagasse and pulp using

crude xylanase produced by Aspergillus tubingensis JP-1

using wheat straw under SSF. Saccharification was carried

out using 2,000 U/g of substrate and the remaining sugar

was measured at different time intervals. Pretreatment of

lignocellulosic biomass is a crucial step before enzymatic

hydrolysis, as it enhances enzymatic saccharification [43].

As compared with untreated wheat straw, higher yield of

reducing sugars (688 and 544 mg/g) were obtained when

wheat straw was pretreated with 1 N NaOH and 1 N

H2SO4, respectively (Fig. 7). Other lignocellulosic sub-

strates such as rice straw, wheat bran and sugarcane

bagasse were found to be less accessible towards enzy-

matic saccharification as they released low amounts of

reducing sugar. The effect of xylanase treatment was more

intensive on paper pulp as it produced maximum reducing

sugar of 726 mg/g of substrate after 48 h of incubation.

Industrial application of xylanase is increasing in pre

bleaching of kraft pulp so as to minimize the use of toxic

chlorine containing chemicals in the bleaching step [44].

Maximum liberation of reducing sugars from paper pulp

samples suggests the feasibility of a biopulping process

using the crude xylanase produced from A. tubingensis

JP-1.

Table 5 Effect of pretreatment of substrate on the xylanase pro-

duction under SSF at 30 �C, pH 6.0 on wheat straw using Aspergillustubingensis JP-1

Pretreatment of

substrates

Xylanase

activity

(U/g)

Protease

activity

(U/g)

Cellulase

activity

(FPA) (U/g)

Steam (121 �C/2 h) 832 ± 16 0.519 ± 0.013 0.966 ± 0.008

1 N HCl 309 ± 14 0.658 ± 0.013 0.286 ± 0.008

1 N NaOH 163 ± 8 0.522 ± 0.016 0.685 ± 0.008

1% w/v Ca(OH)2 238 ± 14 0.679 ± 0.016 0.683 ± 0.012

Untreated 6,887 – 16 0.609 – 0.013 0.673 – 0.006

All the values in the table are the average of triplicate with ±standard

deviation

Bold values in the table indicate the maximum xylanase activity

Fig. 6 Time course study of xylanase production by Aspergillustubingensis JP-1 on unoptimized and optimized medium under SSF

system at 30 �C, pH 6.0 on wheat straw

776 Bioprocess Biosyst Eng (2012) 35:769–779

123

Alkali pretreatment of substrate reduces the lignin

content of agroresidues. Dilute sodium hydroxide causes

separation of the structural linkages between the lignin and

carbohydrate and disruption in the lignin structure leads to

increase in the internal surface area and pore size, as well

as decrease in the degree of polymerization and crystal-

linity. It may be due to saponification of intermolecular

ester bonds cross-linking the hemicelluloses and lignin.

The porosity of the lignocellulosic materials increases with

the removal of the cross links, leading to swelling and

enhances the accessibility to the enzymes [45, 46]. Kong

et al. [47] also reported that alkali removed acetyl groups

from hemicellulose, particularly from acetylated xylan, and

thereby reduced the steric hindrance of hydrolytic enzymes

and greatly enhanced carbohydrate digestibility. Chapla

et al. [48] also used dilute NaOH pretreatment for wheat

straw, rice straw and corncobs and maximum reducing

sugar obtained were found to be 151.6, 163.06 and

172.66 mg/g, respectively. Dilute sulfuric acid acts as a

swelling agent and reduces the degree of polymerization.

The dilute acid pretreatment can achieve high reaction

rates and significantly improve enzymatic hydrolysis.

Wheat straw pretreated with dilute H2SO4 produced

543 mg/g of reducing sugars. The main disadvantage of

this pretreatment method is the necessity of neutralization

of pH for the enzymatic hydrolysis, as well as different

chemical inhibitors might be produced during pretreatment

which reduce enzyme activity; therefore, neutralization is

necessary for the pretreated biomass before enzymatic

hydrolysis [46, 49]. The main advantage of this method is

the possibility to recover a major portion of the hemicel-

lulose sugars. This indicates that application of pretreatment

to different lignocellulosic substrates may be beneficial for

the production of sugar-rich hydrolysates by enzymatic

hydrolysis.

Conclusion

The results reported indicate that the isolate A. tubingensis

JP-1 was found to be an active producer of xylanase with

negligible level of cellulase and protease, using available

inexpensive agricultural waste like wheat straw under SSF.

Different fermentation parameters such as pH of the

medium, particle size, moisture ratio, moistening agents,

nitrogen sources, surfactants, additives and their concen-

trations regulate the fungal metabolism related to xylanase

production. Xylanase production increased fivefold upon

optimization of various fermentation parameters. The crude

cellulase poor xylanase so produced was found to be

suitable for saccharification of various lignocellulosic

substrates and paper pulp. The results thus demonstrate the

potential of xylanase for application in pulp and feed

industries and in production of sugar-rich hydrolysates for

the production of bioethanol.

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