Indian Journal of Multidisciplinary Research 02 ISSN - 0973 - 2225

Ind. J. Multi. Res. 2008. Vol. 4 (4) : 507 - 516

OPTIMIZATION OF XYLANASE PRODUCTION UNDER SOLID STATE

FERMENTATION BY ISOLATED ASPERGILLUS FUMIGATUS (MTCC 9372)
NAGAMALLESWARARAO BEERAKA*, PRASANTH KUMAR KATIKALA*,
VARAPRASAD BOBBARALA** AND PRABHAKAR TADIMALLA*.
*Pharmaceutical Biotechnology Division, A.U.College of Pharmaceutical Sciences,
Andhra University, Visakhapatnam-530 003.,
**For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam,
A. P-530017, India., * Corresponding Author: E-mail : prof.tprabhakar@gmail.com
(Received 28 July 2008, Accepted 6 October 2008)

ABSTRACT

Recent days xylose based products are gaining more importance in the market.
Xylanase is a hydrolytic enzyme which act on xylan and release the xylose. In industries
xylanse is used to produce the pentose sugars from the agro-industrial waste materials.
Aspergillus sp. were found to be potential producers for this enzyme at industrial scale. In
the present study isolated Aspergillus fumigatus is used to produce the xylanase under solid
state fermentation. Screenings of various agro-industrial materials were done and wheat
bran was found to be the best source for the enzyme yield. Various processes as well as
nutritional parameters were optimized for enhanced xylanase production.
Key words:- Solid state fermentation, Xylanase production, Optimization Process
parameters, A. fumigates.

INTRODUCTION
Xylanases play a key role in xylan hydrolysing to xylooligosaccharides. Microbial
xylanases mainly include xylanase or endoxylanase (1, 4-ß-D-xylan xylanohydrolase, E.C.
3.2.1.8) and ß-xylosidase (1, 4-ß-xylan xylohydrolase, E.C. 3.2.1.37) (Ghosh 1993).
Endoxylanase (EC 3.2.1.8) primarily cleaves ß-1, 4-linked xylan back bone and ß-xylosidase
(EC 3.2.1.37) hydrolyses xylo-oligomers. From a commercial viewpoint, xylanases are an
important group of carbohydrolases and have a worldwide market of around $200 million
each year. Xylanases have been widely applied in food, animal feed, bioconversion, textile,
and in paper and pulp industries (Suvarnalakshmi et al 2008). However, high cost and low
yields of xylanase have been the main problems for its industrial production. Therefore,
there is a great need to develop a new fermentation medium with inexpensive substrates that
provides a high xylanase yield.
Among existing technologies in the fermentation industry, solid-state fermentation
(SSF) shows many advantages over fermentation with submerged culture, such as lower
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 517
cost and much higher reactor volume (Prakasham et al. 2005). The application of SSF
process has a considerable economical potential in the food, feed, pharmaceutical, and
agricultural industries. There are a great number of literatures reported to use the SSF
process for producing enzymes with industrial importance, such as protease, cellulase,
polygalacturonase, xylanase, pectinase, amylase, and glucoamylase. Carbon and nitrogen
sources together with fermentation time have been reported to play significant roles in
the determination of the final morphology of the culture. Therefore, it is significant to
optimize these conditions for low-cost enzyme production. Therefore, the aim of this
study was to select the suitable solid substrate for xylanase production by using the
isolated Aspergillus fumigatus and optimize the nutrient medium for high yield of
xylanase in SSF.

MATERIALS AND METHODS

Preparation of solid substrates:

Different agro-industrial waste materials (Sugar cane bagasse, wheat bran, corn
cobs, green gram husk, read gram husk, Peanut meal and rice bran) were evaluated as
substrate for Xylanase enzyme production. Five grams each of the substrate was taken
separately in 250 ml Erlenmeyer flasks and moisturized with 5 ml of distilled water. These
flasks were sterilized and inoculated with 2 ml of spore solution. The flasks were mixed
thoroughly and incubated at 30oC in an incubator for different time intervals.

Effect of particle size

To understand the role of particle size on xylanase production, the experiments were
carried out with different size of substrate particles ranging from 0.3 to 2.8 mm. These were
further sub-divided into 4 groups i.e., group 1 (particle size 0.3 to 1.0 mm), group 2 (particle
size 1.0 to 1.4 mm), group 3 (particle size 1.4 to 2.0 mm) and group 4 (particle size 2.0 to 2.8
mm). To get these group particles, solid material subjected to sieving by using the USA
standard testing sieves.

Optimization of process parameters

To maximize the production of xylanase, various physico-chemical and nutritional

parameters were optimized. Various parameters such as pH (3.0-9.0), temperature
(25-35oC), initial moisture content of the substrate (20 -140% v/w), inoculum size
(0.5-3ml) were studied. To study the effect of nutrients on enzyme production the
selected carbon and nitrogen sources were added individually at 0.2% level to the solid
medium before autoclaving. Results reported in this study were averages of triplicate
findings. CHN analysis was performed using CHN analyzer (Elementar, Model: Vario
E L, Germany).
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 518
 RESULTS AND DISCUSSION

Screening of different agro industrial materials for xylanase production:

2250

2000 24hrs
48hrs
72hrs
1750 96hrs
120hrs
144hrs
1500 168hrs
Xylanase activ ity (U gds )
-1

1250

1000

750

500

250

0
Sugar cane bagasse Wheatbran Corn cobs Greengram husk Redgram husk Peanut meal Rice ban

Fig 1: Effect of different agro-industrial waste materials on xylanase production

by Aspergillus sp. at different time intervals.
In recent years, considerable research has been carried out using agricultural wastes,
which are renewable and abundantly available to produce value-added products. The role of
different agro-industrial waste materials impact on xylanase production by isolated Aspergillus
sp. was evaluated. The xylanase enzyme production data presented in Fig 1 revealed that all
selected agro-industrial materials support the growth of this isolated fungal strain and production
of xylanase enzyme. The enzyme production pattern however, varied with the incubation
time. Among selected different materials the maximum enzyme activity was noticed with
the wheat bran (2016 U/gds), maximum enzyme production was noticed at 5th day of incubation
and further increase in fermentation time resulted in reduction of xylanase activity. These
values are much higher than the xylanase produced by Aspergillus fumigatus SBS58 where
as it is able to produce 608 IU gds-1 (Suprabha et al 2008). Lenartovicz et al (2002) reported
that wheat bran is a best substrate for xylanase production by Aspergillus fumigatus in
submerged fermentation than synthetic xylan. Similar variation of enzyme yield was also
found in all fermentation experiments (Fig 1). Maximum xylanase production varied from
259-2016 U/ml depending on the selected agro-material (Fig 1) depending on the type of
agro industrial waste materials. The variation on the production is attributed to variation in
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 519
the CHN composition of the materials; table 1 gives the CHN composition of the selected
ago-industrial materials. Among all selected materials the minimum enzyme production was
noticed with green gram husk and red gram husk. Such agro industrial material dependent
variation in xylanase production was also noticed in Aspergillus sp RSP-6 (Suvarna et al
2008), Penicillium canescens (Bakri 2003) and Thermoascus aurantiacus (Kalogeris 1998).
Table 1: CHN analysis of the selected agro-industrial waste material.
S. No Name Carbon Nitrogen Hydrogen
1 Sugar cane bagasse 40.84 1.6 5.11
2 Wheatbran 41.23 1.3 6.38
3 Corn cobs 41.32 1.68 3.97
4 Greengram husk 38.94 1.68 4.28
5 Redgram husk 96.57 1.67 4.78
6 Peanut meal 38.67 1.94 4.53
7 Rice ban 46.38 0.95 5.31

Effect of mixed substrates:

In recent years many authors were reported that mixed substrates are enhance the
productivity rather than single substrates (Sathish et al 2008). In order to observe the mixed
substrates affect on the xylanase production the best three substrates (Fig 1) were chosen
2250

2000

1750
Xylanase activity (U gds )
-1

1500

1250

1000

750

500

250

0
SCB WB CC SCB+WB SCB+CC WB+CC

Fig 2: Effect of mixed substrates on xylanase production

IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 520
and they were mixed in the 1: 1 ratios in different combinations. It was observed that the
substrates at individual levels yield the more product than their combinations with other
materials (Fig 2). The reduction of enzyme production due to release of other metabolic
enzymes by this isolated Aspergillus sp. However, A.fischeri and Cephalosporium sp.
Have been reported to produce high levels of xylanase on wheat bran compared to rice
straw and bagasse (Bansod et al 1999, Chandra and Chandra 1995) it is evident that the
preference for lignocellulosic substrate depends on the strain. Malarvizhi et al, (2003) observed
30-fold enhancement of xylanase production in solid state fermentation than liquid culture
when wheat bran was used as the substrate for a culture of Ganoderma lucidum. Hence,
further enzyme improvement studies with this isolated fungal strain were aimed with wheat
bran as a solid support material.

Effect of particle size on xylanase production

In solid-state fermentation process, the availability of surface area play a vital role
for microbial attachment, mass transfer of various nutrients and substrates and subsequent
growth of microbial strain and product production. The availability of surface area in turn
depends on the particle size of the substrate/support matrix. The experimental data revealed
that xylanase production was affected by the particle size. Maximum enzyme production
(2418 U/gds) was noticed with 1.4-0.71mm particle size wheat bran material (Fig 3). Altering
the substrate particle size in either side of this resulted in reduction of xylanase production.
2600

2400

2200
Xylanase activity (U gds )
-1

2000

1800

1600

1400

1200

1000

2.8-1.4 1.4-0.71 0.71-0.3 0.3-0.21 <0.21

Particle size (mm)

Fig 3: Effect of particle size on xylanase production by Aspergillus sp.

IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 521
The observed reduction of xylanase production with altered particle size could be attributed
to intra-particulate associated aeration, available surface area for microbial attachment and
substrate mass transfer and subsequent growth and enzyme production. These results are in
accordance with the literature data on particle size-mediated influence on microbial enzyme
production in Bacillus sp. (Prakasam et al 2006), B. subtilis (Krishna and Chandrasekaran,
1996) and in Rhizopus oligosporus (Ikasari et al., 1999).

Effect of amount of substrate on xylanase production

To understand the influence of concentration of the particle size on enzyme production,

further experiments were performed with variation of solid material supplementation in the
medium. Variation of solid material quantity did affect the enzyme production pattern and

2500

2250

2000
Xylanase activity (U gds )
-1

1750

1500

1250

1000

750

500

250

0
3 5 7 10
Weight of material (gm)

Fig 4: Influence of substrate concentration on xylanase production by Aspergillus sp.

maximum (2497 U/gds) being observed with 5 gm of material compared to 3, 7 and 10 gm of
material (Fig 4). This may be attributed to the fact that higher quantity of solid material (7&
10 gm) supplementation may provide better support for attachment of microbial strain but
nutrient mass transfer may be affected during fermentation as reported in various solid state
fermentations (Prakasham et al 2006). However, in case of 3gms supplemented environment,
the solid material may not be sufficient enough to provide support for microbial attachment.

Effect of pH on xylanase production

Enzymes production by microbial strains strongly depends on the extra-cellular pH

because culture pH strongly influences many enzymatic processes and transport of various
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 522
components across the cell membranes, which in turn support the cell growth and product
production (Prakasham et al 2005). Hence, the influence of pH of the medium on xylanase
production by this fungal strain was studied. Optimum enzyme production of 2834 U/gds
was noticed at pH 5.0 (Fig 5). Further analysis of the enzyme production data denoted that
xylanase enzyme production did not vary much in the acidic pH range of 4 - 7.0 and drastically
reduced at acidic pH and no enzyme production was noticed in pH 2.0 (Fig 4) indicating the
growth potential of isolated fungal strain at different pH environments. The xylanase production
is varied in pH is observed with the variation of the fungal species Christopher et al (2005)
reported that Thermomyces lanuginosus is produce the higher xylanase at pH 7 indicating
that the tolerance to acidic pH is varied from fungal strains.

3000

2500
Xylanase activity (U gds )
-1

2000

1500

1000

500

1 2 3 4 5 6 7 8 9 10
pH
Fig 5: Effect of pH on xylanase production by Aspergillus sp

Influence of moisture content

Among the several factors that are important for microbial growth and enzyme
production under solidstate fermentation using a particular substrate, moisture level (content)/
water activity is one of the most critical factors (Pandey et al. 1999; Prakasam et al 2006).
Because, solid-state fermentation processes are different from submerged fermentation
culturing, since microbial growth and product formation occurs at or near the surface of the
solid substrate particle having low moisture contents (Pandey et al. 1999). Thus, it is crucial
to provide optimized water level that controls the water activity (aw) of the fermenting
substrate for achieving maximum product. In present study, maximum enzyme production
was observed with 100% moisture content, (3165 U/gds). Linearity between moisture content
and enzyme production was observed up to 100%, and thereafter, further increase in moisture
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 523
level in the fermentation medium resulted in reduction of xylanase production (Fig 6). The
percent reduction in enzyme production from either side of the optimum moisture level varied.
The role of moisture level in SSF media and its effect on microbial growth and biosynthesis
of xylanase have been attributed to the impact of moisture on the physical properties of the
solid substrate (Virupakshi et al 2005). Christopher et al. reported that 83% of moisture
content is favorable for xylanase production by Thermomyces lanuginosus. The moisture
content beyond the optimum level inhibits enzyme production because the higher moisture
levels decrease the porosity due to gummy texture of the substrate, alters the rice bran
particle structure, leads to poor oxygen transfer and decreases the diffusion. The lower
moisture level, than optimum leads to the poor solubility of the nutrient of the solid substrate,
improper swelling and a higher water tension (Prakasham et al 2005).
Moisture content (% v/w)
40 60 80 100 120 140 160
3500

3000
Xylanase activity (U gds )
-1

2500

2000

1500

1000

500

0
1 2 3 4 5 6 7 8 9
Moisture content (v/w)

Fig 6: Influence of moisture content on xylanase production by Aspergillus S-6

strain under solid state fermentation.
Effect of inoculum size on enzyme production:

In order to observe the effect of inoculum size on xylanase production 0.5-3 ml of

spore solution was added to the solid medium. A parabolic nature of the production was
observed along with the increment of inoculm concentration in this present investigation (Fig
7). The maximum xylanase production was observed at 2 ml inoculum concentration. Above
and below this concentration decreased productivity was observed. Similar experiments were
performed by the virupakshi et al 2005 & Archana and Satyanarayana by Bacillus sp. JB-99
& B. licheniformis where they observed that 10% & 15 % inoculum is optimum. It indicates
that the inoculm concentration is varied by the different organisms.
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 524
 3500

3000

2500

Xylanase activity (U gds )

-1

2000

1500

1000

500

0.5 1.0 1.5 2.0 2.5 3.0

Inoculum concentration (ml)

Fig 7: Effect of inoculum concentration on xylanase production.

Effect of temperature on xylanase production

Temperature plays a vital role in the microorganism growth and product formation
in order to search the optimum temperature the spores inoculated solid medium was incubated
at different temperatures ranging from 26-34oC with 2oC variation. It is observed that at
30oC is favorable for the maximum xylanse production (Fig 8) by this isolated Aspergillus
sp. Above 30oC is unfavorable for the enzyme production.Similar reports are available on
xylanase production by Bacillus sp. JB-99 (Virupaksha et al 2005) and B. licheniformis
(Archana and Satyanarayana 1997).

3500

3000
Xylanase activity (U gds )
-1

2500

2000

1500

1000

500

26 28 30 32 34
o
Temperature ( C)

Fig 8: Influence of incubation temperature on xylanase production

IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 525
Effect of carbon source on enzyme production:

Carbon source is one of the essential constituents of the microbial fermentation

medium which has major role overall cellular growth and metabolism. Different carbon
sources (glucose, xylose, fructose, maltose, mannose, galactose, arabinose, lactose and
glycerol) impact on xylanase production was studied and compared with wheat bran as
carbon source (control). Glucose being conventional carbon source though improved the
xylanase titer values over control but it was only 5% (Fig 9). Similar results were noticed
with Aspergillus foetidus (Shah and Madamwar, 2005). It was noticed that the enzyme
production was improved by the glucose and other carbohydrates is not able to increase the
production. Such data reveal that the enzyme production in this fungal strain is constitutive in
nature and regulated by only glucose as a carbon source. These results were in accordance
with reported xylanase production in presence of different sugars (Virupakshi et al., 2005;
Shah and Madamwar, 2005; de Souza et al 2001). Further the concentration of glucose
could be optimized by using the statistical approach.

3500

3000
Xylanase activity (U gds )
-1

2500

2000

1500

1000

500

0
Glucose Xylose Fructose Maltose Mannose Galactose Arabinose Lactose Glycerol Control

Fig 9: Influence of different carbon sources on xylanase production by Aspergillus sp.

Role of nitrogen sources on enzyme production:

The impact of inorganic (ammonium sulphate, ammonium nitrate, ammonium chloride,

sodium nitrate, potassium nitrate and urea) and complex nitrogen (yeast extract, peptone,
beef extract, typtone, soya bean meal, corn steep liquor and pea nut meal) sources was
evaluated on xylanase production by Aspergillus sp. by supplementing the 0.2 % of each
selected nitrogen source individually in the fermentation medium before autoclaving. Among
IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008 526
 4000

3500

Xylanase activity (U gds )

3000
-1

2500

2000

1500

1000

500

0
Y.E Pe B.E Tr So CSL Ca U A.S A.N A.C S.N P.N P.MControl

Fig 10: Effect of additional nitrogen sources on xylanase production by

Aspergillus sp.
all nitrogen sources studied, yeast extract supported the maximum enzyme production (4026
U/gds) followed by beef extract (3765 U/gds) and pea nut meal (3746 U/gds) (Fig 10).
Though all selected inorganic nitrogen sources supported the enzyme production but not as
efficient as yeast extract. These results are similar to the Virupakshi et al. 2005. George
et al. (2001) reported that (NH4)2SO4 was a highly effective inorganic nitrogen source in
the media for the xylanase production by Thermomonospora sp. However, no significant
effect was observed for (NH4)2SO4 in the medium as the nitrogen source. These indicated
that the ability of utilization of nitrogen sources for xylanase production by fungus varied
each other.
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