J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6

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Journal of Radiation Research and Applied
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One-factor-at-a-time (OFAT) optimization of

xylanase production from Trichoderma viride-IR05
in solid-state fermentation

Muhammad Irfan*, Muhammad Nadeem, Quratulain Syed

Food and Biotechnology Research Center (FBRC), Pakistan Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Ferozpure Road, Lahore 54600, Pakistan

article info abstract

Article history: The present study dealt with the production of enzyme xylanase by solid substrate
Received 19 March 2014 fermentation using Trichoderma viride-IR05. Different substrates such as wheat bran, rice
Received in revised form polish, rice husk, soybean meal, sunflower meal, sugarcane bagasse or corn cobs were
25 April 2014 evaluated for enzyme production. Of all the substrates evaluated, sugarcane bagasse was
Accepted 27 April 2014 found to be best for enzyme synthesis. The substrate, sugarcane bagasse pretreated bio-
Available online 20 May 2014 logically, 2% H2SO4, 2.5% KOH or 3%H2O2. However 2.5% KOH gave maximum yield of
enzyme as evidenced by the SEM analysis of the pretreated substrate. The cultural con-
Keywords: ditions were optimized for the production of xylanase in 250 ml Erlenmeyer flask such as
Xylanase incubation period (seven days), substrate concentration (10 g), liquid to solid ratio (11:10),
Bagasse initial pH of diluent (4.5), incubation temperature (30
C) with inoculum size of 10%. Further
Trichoderma viride-IR05 supplementation of xylose, NaNO3 or tryptone and tween-80 as additional carbon source,
Solid state fermentation nitrogen and surfactant improved (72.4  1.42 U/g) the titer of xylanase by T. viride-IR05,
respectively.
Copyright 2014, The Egyptian Society of Radiation Sciences and Applications. Production
and hosting by Elsevier B.V. All rights reserved.

1993). Biodegradation of xylan is a complex process that re-

1. Introduction
Xylanases (endo-1, 4-b-D-xylan xylanohydrolase; EC 3.2.1.8) is
a group of enzymes that catalyze the hydrolysis of xylan, the
major constituent of hemicellulose, which is second to cellu-
lose in abundance in plant cell wall (Coughlan & Hazelwood,
quires the coordination of several xylanolytic enzymes that
hydrolyze xylan and arabinoxylan polymers. This enzyme
group includes endo-b1, 4-xylanase (1, 4-b-D-xylan xylanohy-
drolase, EC 3.2.1.8), which attack main chain of xylans, b-D-
xylosidase (1, 4-b-xylan xylanohydrolase, EC 3.2.1.37), which
hydrolyze xylo-oligosaccharides into D-xylose and a variety of

* Corresponding author.
E-mail addresses: mirfanashraf@yahoo.com, irfan.biotechnologist@gmail.com (M. Irfan).
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Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.jrras.2014.04.004
1687-8507/Copyright 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. All rights
reserved.
318 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
debranching enzymes i.e. a-L-arabinofuranosidases, a-glucu-
ronidases and acetyl esterases (Collins, Gerday, & Feller, 2005).
Many of the xylanase producing microorganisms express
multiple isoforms that have been ascribed to a variety of
reasons i.e. heterogeneity and complexity of xylan structure.
Xylanases are produced by a variety of microorganism such as
bacteria (Battan, Sharma, & Dhiman, 2006; Gilbert &
Hazelwood, 1999; Sunna & Antranikian, 1997), fungi
(Kuhadd, Manchanda, & Singh, 1998; Sunna & Antranikian,
1997), actinomycetes (Ball & McCarthy, 1989) and yeast
(Harmova, Beily, & Varzanka, 1984; Liu, Zhu, Lu, Kong, & Ma,
1998) which are cultivated in solid and submerged fermenta-
tions. Fungi are the most common sources of xylanases which
can produce thermophilic enzyme ranges from 40
C to 60
C
(Latif, Asgher, Saleem, Akram, & Legge, 2006).
Xylanases can be produced by submerged fermentation
and solid state fermentation processes. Mostly solid state
fermentation was employed for enzymes production due to its
numerous advantages such as high volumetric productivity,
relatively higher concentration of the products, less effluent
generation, requirement for simple fermentation equipment,
lower capital investment and lower operating cost (Holker &
Jurgen, 2005). This process was very good in developing
countries because it uses agro-industrial wastes as substrate
source which are very cheaper and easily available. The most
common substrates used in solid state fermentations are
sugar cane bagass, wheat bran, rice bran, saw dust, corncobs,
banana waste, tea waste etc (Pandey, Selvakumar, Soccol, &
Nigam, 1999). The major factors that affect microbial synthe-
sis of enzymes in an SSF system include; selection of a suitable
substrate and microorganism, pre-treatment of the substrate,
particle size of the substrate, water content and water activity
of substrate, relative humidity, type and size of the inoculum,
control of temperature of fermenting matter/removal of
metabolic heat, period of cultivation, maintenance of unifor-
mity in the environment of SSF system and gaseous atmo-
sphere, i.e., oxygen consumption rate and carbon dioxide
evolution rate (Pandey, 2003).
Xylanases have potential applications in various fields.
Some of the important applications are as fallows. Xylanases
are used as bleaching agent in the pulp and paper industry.
Mostly they are used to hydrolyzed the xylan component
from wood which facilitate in removal of lignin (Viikari,
Kantelinen, Buchert, & Puls, 1994). It also helps in bright-
ening of the pulp to avoid the chlorine free bleaching oper-
ations (Paice, Jurasek, Ho, Bourbonnais, & Archibald, 1989). In
bakeries the xylanase act on the gluten fraction of the dough
and help in the even redistribution of the water content of the
bread (Wong & Saddler, 1992). Xylanases also have potential
application in animal feed industry. They are used for the
hydrolysis of non-starchy polysaccharides such as arabi-
noxylan in monogastric diets (Walsh, Power, & Headon,
1993). Xylanases also play a key role in the maceration of
vegetable matter (Beck & Scoot, 1974), protoplastation of
plant cells, clarification of juices and wine (Biely, 1985)
liquefaction of coffee mucilage for making liquid coffee, re-
covery of oil from subterranian mines, extraction of flavors
and pigments, plant oils and starch (McCleary, 1986) and to
improve the efficiency of agricultural silage production
(Wong & Saddler, 1992).
2. Materials and methods
2.1. Chemicals/biochemicals
All the chemicals/biochemicals used in present study were of
analytical grade and purchased from Sigma (USA), Merck
(Germany), Fluka (Switzerland) and Acros (Belgium). Agricul-
tural residues such as bagasse, corn cobs, soybean meal, rice
husk, rice bran, wheat bran etc. were purchased from the local
market of Lahore city.
2.2. Isolation and identification of Trichoderma viride-
IR05
T. viride-IR05 was obtained from Microbiology Laboratory,
Food and Biotechnology Research Center (FBRC), Pakistan
Council of Scientific and Industrial Research (PCSIR) labora-
tories complex Ferozpur Road, Lahore, Pakistan. The culture
was maintained on slants containing potato-dextrose-agar
(PDA, Oxoid) stored at 4
C in a cold cabinet.
2.3. Pretreatment of substrate
2.3.1. Chemical treatment of substrate
The selected substrate (50 g) were soaked in different con-
centration of 2.5%KOH, 2%H2SO4, or 3%H2O2 solution at the
ratio of 1:10 (solid: liquid) for 2 h at room temperature as
described previously (Irfan et al., 2011). After that the samples
were heated at 127
C for 60 min at 20 lb psi. Then samples
were filtered and solid residues were washed up to neutrality.
2.3.2. Biological treatment of substrate
Fifty grams of substrate was taken in 1 L conical flask and
moistened with 60 ml of Vogels medium and autoclaved at
121
C for 15 min. After autoclaving, the contents of the flask
were allowed to cool at room temperature. After cooling the
flask was inoculated with 10 ml spore suspension of T. viride-
IR05 and incubated at 30
C for seven days. The contents of the
flask were mixed each day during incubation. After seven days
of incubation the substrate was washed, dried and used as a
biologically treated sample source for enzyme production.
2.4. Scanning electron microscopy of substrate
Samples of untreated and treated sugarcane bagasse were
oven-dried at 50
C for 1 h and thick layers were supported in
the sample holder fixed on a carbon ribbon. This assembly
was maintained in vacuum-desiccators until the analysis. The
SEM type S-3700 microscope (Hitachi) was used for observing
the bagasse fibers in both treated and untreated samples.
2.5. Inoculum preparation
In present study, conidial inoculum was used. The spore
suspension was prepared by adding 10 ml of sterile distilled
water in to a 7 days old slant culture aseptically. Conidial
clumps were broken using inoculation needle. The tube was
shaken to make homogeneous mixture of conidial
suspension.
 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6 319

2.6. Fermentation technique
The production of xylanase was carried out using SSF in
250 ml Erlenmeyer flask. Ten ml of diluents (Vogels media)
was transferred into the flask containing 10 g of bagasse and
mixed well. The flasks were cotton plugged and sterilized
them in an autoclave at 121
C for 15 min at 15 lbs/in2. After
cooling the flasks at room temperature, inoculated them with
1.0 ml of fungal conidial suspension under aseptic condition.
The flasks were kept at 30  1
C for seven days in the incu-
bator. All experiments were run parallel in duplicate.
2.7. Extraction of enzyme
lauryl sulfate) were optimized for enhanced production of
xylanase by T. viride-IR05 in solid state fermentation process.
2.10. Protein determination
Total protein content was determined by the method as
described by Lowery, Rosebrough, Farr, and Randall (1951).
2.11. Statistical analysis
Treatment effects were compared by the protected least sig-
nificant difference method after using computer software
SPSS.

After seven days of fermentation, 50 ml of extractants

(distilled water, 0.1% glycerol, 0.1% NaCl, 0.1% tween-80 and
citrate buffer pH 5) was added in to the each flask containing 3. Results and discussion
fermented mash and rotated them on rotary shaker at
150 rpm for 2 h at 30  1
C for maximum enzyme extraction. 3.1. Time course study
Then filtered slurry through muslin cloth followed by centri-
fugation at 8000 rpm at 4
C for 10 min to separate fungal Xylanase production was checked by incubating the inocu-
spores and small particles. The clear supernatant was used as lated flasks for various time periods and its was noted that
a crude xylanase source. enzyme production was gradually increased with increase in
fermentation period and maximum production was achieved
after seven days of fermentation period as shown in Fig. 1. As
2.8. Estimation of xylanase activity
the fermentation period was increased decrease in enzyme
production was observed. Okafor, Emezue, Okochi,
Xylanase activity was assayed as described earlier (Irfan,
Onyegeme-Okerenta, and Nwodo-Chinedu (2007) isolated a
Nadeem, Syed, & Baig, 2010). Reaction mixture containing
strain of Penicillium chrysogenum PCL501 from wood wastes and
0.5 ml of appropriately diluted culture filtrate with 0.5 ml of 1%
reported that highest xylanase activity of 6.47 units mL1 was
birchwood xylan (Sigma) solution prepared in citrate buffer
obtained with wheat bran after 96 h of fermentation period
(0.05 M, pH 5.0) for 15 min at 50
C. After incubation the re-
and lowest activity of 0.79 U/ml after 120 h. Abdel-Satera and
action was stopped by the addition of 1.75 ml of 3,5-dini-
El-Said (2001) obtained maximum production of xylanase
trosalicylic acid and heated for 10 min in boiling water bath.
from Trichoderma harzianum after 8 days of fermentation
After cooling the reducing sugars liberated were measured by
period. Goyal, Kalra, and Sareen (2008) achieved maximum
spectrophotometrically at 550 nm and expressed as xylose
enzyme production for 14e17 days of fermentation period
equivalent. Xylose was taken as standard. One unit of activity
using strain of T. viride. Increased fermentation time and
was defined as the amount of enzyme, which liberates
decreased enzyme synthesis might be due to the depletion of
reducing sugar (equivalent to xylose) from 1.0% Birch wood
macro- and micronutrients in the fermentation medium with
xylan under standard assay conditions.
the passage of time, which altered the fungal physiology
resulting in the inactivation of secretary machinery of the
2.9. Optimization of cultural and nutritional conditions enzymes (Nochure, Roberts, & Demain, 1993).
for xylanase production

Various cultural conditions like time course of fermentation

(1e10days), initial medium pH (4e8), incubation temperature
(20e50
C), inoculum size (5e30%), substrate concentration
(5e30 g/500 ml flask) and various nutritional conditions such
as screening of substrates (wheat bran, rice polish, rice husk,
soybean meal, sunflower meal, sugarcane baggase and corn
cobs) substrate pretreatment (H2SO4, KOH, H2O2 and biological
treatment), diluent selection (Vogels, Zepicks, citrate buffer
pH 4, phosphate buffer pH 5, tab water and distilled water),
diluent to substrate ratio (5:10, 7:10, 9:10, 11:10, 13:10 and
15:10), additional carbon sources (glucose, xylose, starch,
Days
maltose, cellulose, galactose, sucrose & arabinose), nitrogen
sources (NH4NO3, NaNO3, (NH4)2SO4,NH4Cl, (NH4)2H2PO4, Fig. 1 e Time course of xylanase production in solid state
Ammonium citrate, Peptone, yeast extract, tryptone, casein, fermentation by Trichoderma viride-IR05. Y-error bars
skim milk, lablamco powder and urea) and surfactants represent the SD among duplicates which differs
(tween-80, triton X-100, sodium dodecyl sulfate and sodium significantly at P 0.05.
320 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
3.2. Selection of substrate
Different agricultural wastes such as wheat bran, rice polish,
rice husk, soybean meal, sunflower meal, sugarcane bagasse
and corn cobs were evaluated for xylanase production by T.
viride-IR05 in solid state fermentation. Results (Fig. 2) indi-
cated that maximum xylanase yield of 56.6  1.21 U/g was
obtained by sugarcane bagasse which was followed by corn
cobs (50.0  0.97 U/g) and wheat bran (29.3  0.84 U/g),
respectively. High protein content (0.9  0.23 mg/ml) was
found in case of sugarcane bagasse while lowest protein
secretion was found in sunflower meal (0.36  0.21 mg/ml) and
rice polish (0.65  0.31 mg/ml), respectively. Some researchers
obtained maximum yield of xylanase enzyme production
using sugarcane bagasse (Rezende, Barbosa, Vasconcelos, &
Sakuarda, 2002) and corn cobs (Damaso, Carolina, &
Andrade, 2002) as a substrate in solid and submerged
fermentation, respectively. Qinnghe, Xiaoyu, Tiangui, Cheng,
and Qiugang (2004) optimized the cultural conditions for
xylanase production by Pleurotus ostreatus SYJ042 in shake
flask cultures using 2.5% corn cob 2.5% wheat bran as carbon
source. Wheat bran is most widely used substrate for enzyme
production like xylanases due to its nutritional constituents
(Okafor et al., 2007; Querido, Coelho, Araujo, & Chaves-Alves,
2006; Simoes & Tauk-Tornisielo, 2005). Maize straw was the
best inducer followed by jowar straw for xylanase production
among all the tested lignocellulosic substrates (Goyal et al.,
2008). Corn cob and coba husk, have high tendency to pro-
duce xylanase which is used to develop low-cost media for the
mass-production of xylanase (Fang, Chang, & Lan, 2008).
3.3. Effect of substrate concentration
Suitable substrate level for xylanase production was also
checked by changing the amount of selected substrate (sug-
arcane bagasse) in 500 ml Erlyenmer flask from 5 to 30 g. Of all
these tested concentrations of substrate 10 g in 500 ml flask
showed optimum enzyme production (64.2  1.24 U/g). As the
concentration of substrate was increased above this concen-
tration, decreased in enzyme production and protein secre-
tion were observed as shown in Fig. 3. Our findings were in
accordance with Haq, Javed, and Saleem (2006) who also re-
ported that 10% substrate level was best for CMCase
Fig. 2 e Selection of substrate for xylanase production by T.
viride-IR05 in SSF. The different letters show significant
difference (P < 0.05).
Fig. 3 e Effect of different substrate concentrations on
xylanase production by T. viride-IR05 in SSF. The different
letters show significant difference (P < 0.05).
production by using T. viride. Xia and Cen (1999) reported that
30% substrate was best for cellulase accumulation. Reis,
Costa, and Peralta (2003) obtained maximum xylanase activ-
ity (130  16 IU/ml) with 5% sugarcane bagasse as a carbon
source in submerged fermentation using Aspergillus nidulans.
Substrate concentration of 14% w/v bagasse produced
maximum xylanase activity of 27.6 U/ml using strain of T.
harzianum Rifai (Rezende et al., 2002). High concentration of
carbon sources inhibits the enzyme synthesis (Naidu & Panda,
1998).
3.4. Selection of pretreatment condition
Five different conditions of substrate were used to check the
maximum xylanase production. The substrate used were raw
sugarcane bagasse, biologically treated bagasse, 2% H2SO4
treated bagasse, 2.5% KOH treated bagasse and 3%H2O2
treated bagasse was investigated. Maximum xylanase activity
of 72.4  1.42 U/g was observed with 2.5% KOH treated bagasse
with protein secretion of 0.88  0.11 mg/ml. Lowest enzyme
activity of 26.4  0.91 U/g was observed in 3%H2O2 treated
bagasse which was 50% low yield as compared to untreated
bagasse. Acid (2% H2SO4) treated bagasse improved enzyme
production with yield of 71.0  1.02 U/g which was higher as
compared to untreated bagasse as shown in Fig. 4a. The sub-
strate was further analyzed by advanced techniques such as
scanning electron microscopy (Fig. 4b) indicating alteration in
structure which lead to fully attacked by the microorganism
which ultimately increased enzyme synthesis. Alkali was also
used for the pretreatment of lignocellulosic biomasses and its
action depends upon the lignin content present in the
biomass (Fan, Gharpuray, & Lee, 1987; McMillan, 1994). The
xylanase production could be further improved by using alkali
treated straw as carbon source (Goyal et al., 2008).
3.5. Selection of diluent
Fig. 5 represented the effect of different diluents for xylanase
production. Vogels media, Czepeks media, Citrate buffer pH
4, Phosphate buffer pH 5, Tap water or distilled water were
used as diluent in solid state fermentation. Vogels media
found suitable diluent for xylanase production with enzyme
yield of 66.7  1.94 U/g and protein secretion of 0.82  0.18 mg/
ml. Distilled water and citrate buffer pH 4 also showed best
 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
Fig. 4 e Effect of different pretreatments of substrate on xylanase production by T. viride-IR05 in SSF. SEM of untreated and
treated bagasse. Arrows indicate the effect of chemical (2.5% KOH) causing pores in the substrate. The different letters show
significant difference (P < 0.05).
activity of 61.3  1.20 U/g and 57.3  1.65 U/g, respectively.
Vogels media is the most widely used medium for the culti-
vation of fungi for production of xylanases by Trichoderma sp.
and Aspergillus sp. in fermentation processes (Simoes & Tauk-
Tornisielo, 2005; Simoes, Tauk-Tornisielo, & Tapia, 2009). Nair,
Sindhu, and Shashidhar (2008) isolated 70 fungal strains from
soils collected from different parts of southern Kerala, India
and Czapeks agar medium was used for screening of xylanase
production. Meshrama, Kulkarni, Jayaraman, Kulkarni, and
Fig. 5 e Selection of different diluents for xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
321
Lele, (2008) produced xylanase from Penicilium janthinellum
NCIM 1169 in submerged fermentation using MandelseWeber
medium, sugarcane bagasse as a carbon source.
3.6. Effect of diluent to substrate ratio on xylanase
production
Every microorganism has its own water activity for their
growth in solid state fermentation. Different experiments
were performed by changing the amount of diluent and
keeping solid ratio constant. Results in Fig. 6 indicated that by
increasing liquid to solid ratio, enzyme production was
enhanced. Highest enzyme production (64.3  1.57 U/g) was
observed in ratio of 11:10 (liquid: solid) and by further
increasing the amount of liquid there was decrease in
enzyme production. In SSF the optimal moisture content
depends on the requirement of microorganism, type of the
substrate and the types of end products (Kalogeris Iniotaki,
Topakas, Christakopoulos, Kekos, & Macris, 2003). Pang,
Darah, Poppe, Szakacs, and Ibrahim (2006) reported that
moisture content of 80% was optimum for xylanase produc-
tion by Trichoderma sp. in solid state fermentation using
sugarcane bagasse as substrate. Gao, Weng, and Zhu (2008)
reported the moisture level of 80% was best for enzyme
production. When the moisture level was too increased the
media become clumped and there is poor aeration and poor
322 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
Fig. 6 e Effect of diluent to substrate ratio for xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
growth so the enzyme production will decrease (Alam,
Mohammad, & Mahmat, 2005). Muniswaran and Charyulu
(1995) observed that high moisture level increases the free
excess liquid in the medium which ultimately decrease in
growth and enzyme production.
3.7. Effect of different inoculum size
Results in the Fig. 7 indicated the effect of different inoculum
size on xylanase production by T. viride-IR05 in solid state
fermentation using sugarcane bagasse as substrate. Results
indicated that maximum xylanase production was observed
with10% inoculum size yielding enzyme activity of
(59.7  1.8 U/g) with protein secretion of 0.83  0.2 mg/ml.
Inoculum size beyond this level declined the enzyme pro-
duction. Inoculum size controls and shortens the lag phase,
smaller inoculum size increased the lag phase whereas the
larger inoculum size increases the moisture content which
ultimately decreased the growth and enzyme production
(Sharma, Tiwari, & Behere, 1996). The pretreated wheat straw
had maximum enzyme production with 10% of inoculum size
which was in good agreement with our findings (Fadel, 2000).
Omojasola and Jilani (2009) worked on cellulase production
and reported that maximum glucose production was observed
with 8% inoculum size.
3.8. Effect of initial pH
To check the optimum initial medium pH for xylanase pro-
duction, experiments were carried out at different pH of the
medium ranging from 4 to 8. pH of the medium was adjusted
with 0.1 N NaOH/HCl before sterilization. From the experi-
ments it was observed that maximum enzyme production
(67.1  1.6 U/g) and protein secretion (0.87  0.11 mg/ml) as
shown in Fig. 8. Bakri, Jawhar, and Arabi (2008) produced
xylanase from newly isolated Cochliobolus sativus Cs5 strain in
submerged fermentation and reported that initial medium pH
of 4.5e5.0 was optimum for xylanase production. Different
investigations on xylanase production reported that initial
medium pH of 4.5 (Fadel, 2001), 6.0 (Qinnghe et al., 2004) and
Fig. 7 e Effect of different inoculum size on xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
6.5 (Carmona, Fialho, & Buchgnani, 2005) were best for xyla-
nase production by different fungi in fermentation process.
These reports indicating that most of the fungus exhibit acidic
environment for their growth.
3.9. Effect of incubation temperature
Incubation temperature is also a critical factor in the growth of
fungus. Different experiments were performed on various
incubation temperatures ranging from 20 to 50
C. Results of
the study indicated that maximum enzyme production was
noted at 30
C yielding enzyme activity of 64.3  1.3 U/g as
shown in Fig. 9. When the fungus was grown at 35
C enzyme
yield of 60.1  1.6 U/g was obtained. As the incubation tem-
perature was further increased decrease in enzyme produc-
tion was also observed. Abdel-Satera and El-Said (2001)
screened xylan degrading filamentous fungi and reported that
T. harzianum produced maximum xylanase production at in-
cubation temperature of 35
C. Goyal et al. (2008) also reported
the incubation temperature of 25
C was best for xylanase
production by T. viride. Fusarium oxysporum in shake flask
cultures also produces maximum xylanase yield at incubation
temperature of 30
C (Kuhadd et al., 1998). These variations in
different incubation temperatures were due to the different
nature of microorganism and its environmental conditions.
Fig. 8 e Effect of initial pH of diluent on xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
Fig. 9 e Effect of incubation temperature on xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
3.10. Supplementation of nitrogen and additional carbon
sources
Supplementation of different carbon sources to the medium
was also investigated by changing in medium using glucose,
xylose, starch, maltose, cellulose, galactose, sucrose or arab-
inose. Highest yield of xylanase was found in case of xylose
(59.7  0.94 U/g) with protein content of 0.94  0.25 mg/ml as
compared to control. Low enzyme yield was recorded when
medium was supplemented with arabinose as shown in
Fig. 10. Isil and Nilufer (2005) studied some physiological
conditions affecting the xylanase production from T. harzia-
num 1073 D3. Their study indicated that xylose was found best
carbon source for xylanase production. Maximum production
Fig. 10 e Supplementation of nitrogen and additional carbon sources on xylanase production by T. viride-IR05 in solid state
fermentation. The different letters show significant difference (P < 0.05).
323
Fig. 11 e Effect of different surfactants on xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
of xylanase was observed in case of T. harzianum using
maltose and starch as carbon source.
Effect of different nitrogen (inorganic and organic) sources
was also checked for maximum xylanase production. NaNO3
and tryptone proved to be best for maximum xylanase pro-
duction by T. viride with activity of 62.4  1.44 U/g and
67.07  1.36 U/g with protein secretion of 0.85  0.23 mg/ml
and 0.96  0.33 mg/ml, respectively. Supplementation of me-
dium with any other nitrogen source do not favored best
enzyme production (Fig. 10). Goyal et al. (2008) achieved
maximum xylanase production by supplementing the me-
dium with sodium nitrate as nitrogen source with 5% maize
straw as a substrate as a carbon source. Qinnghe et al. (2004)
reported that supplementation of peptone to the
324 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
Fig. 12 e Effect of tween-80 concentrations on xylanase
production by T. viride-IR05 in SSF. The different letters
show significant difference (P < 0.05).
fermentation medium enhanced the xylanase production by
P. ostreatus. Kalogeris et al. (2003) stated that addition of 0.04 g
of ammonium sulfate per gram of substrate favored the better
enzyme production. Kuhad, Manchanda, & Singh (1998) re-
ported that wheat bran and peptone were found best for
highest xylanase production among various tested agricul-
tural residues and inorganic/organic nitrogen sources. Xylan
and NaNO3 were best carbon and nitrogen sources for
maximum xylanase production C. sativus Cs5 strain in sub-
merged fermentation (Bakri et al., 2008).
3.11. Effect of different surfactants
Xylanase production was enhanced by the addition of various
enhancers such as tween-80, Triton X-100 and sodium
dodecyl sulfate (SDS). Results in Fig. 11 indicated that tween-
80 enhanced the enzyme production (66.2  1.66 U/g) as
compared to control (45.5  1.33 U/g). Triton X-100
(59.6  1.38 U/g) and SDS (48.2  1.13 U/g) also enhanced the
xylanase production up to some extent. Highest total protein
(0.96  0.14 mg/ml) secretion was found in case of tween-80
supplementation to the medium. Kuhad, Manchanda, and
Singh (1998) optimized cultural conditions for xylanase pro-
duction by a hyperxylanolytic mutant strain (NTG-19) of F.
oxysporum in shake flask cultures. They reported that enzyme
production was also enhanced by supplementation of tween-
80 and olive oil to the medium. Liu et al. (1998) stated that
Fig. 13 e Effect of different leaching agents on xylanase
activity. The different letters show significant difference
(P < 0.05).
enzyme synthesis was significantly stimulated by the addition
of wheat bran and tween-80 to the medium.
3.12. Effect of various concentration of tween-80 on
xylanase production
Further experiments were performed to test the suitable
concentration of tween-80 supplementation to the medium.
0.1e1.0% tween-80 concentrations were tested, among all
these tested concentration 0.2% found to be better for
maximum synthesis of xylanase from T.viride-IR05 under solid
state fermentation as shown in Fig. 12. Increased concentra-
tion of tween-80 beyond this resulted in decline in enzyme
synthesis. Total protein content of 0.91  0.21 mg/ml was also
noted at 0.2% tween-80 supplementation. Saleem, Akhtar, and
Jamil (2002) reported that supplementation of 0.2% concen-
tration of tween-80 had a positive effect on the production of
xylanase by Bacillus subtilis.
3.13. Effect of different leaching agents
Recovery of enzyme from a solid material is a critical process in
solid state fermentation. Different leaching agents such as
distilled water, 0.1% glycerol, 0.1% NaCl, 0.1% tween-80 and
citrate buffer pH 5 were tested to extract the enzyme from fer-
mented mash. Results (Fig. 13) indicated that maximum
extraction was observed in 0.1% tween-80 (63.4  2.11 U/g) fol-
lowed by citrate buffer pH 5 (60.1  1.76 U/g), distilled water
(59.2  1.22 U/g), 0.1% NaCl (52.3  1.51 U/g) and 0.1% glycerol
(48.4  1.38 U/g). Enzyme activity decreased in the following
order 0.1% tween-80 > citrate buffer pH 5 > distilled water > 0.1%
NaCl > 0.1% glycerol. Different workers (Biswas, Mishra, &
Nanda, 1988; Silveira, Melo, & Filho, 1997) used tween-80 for
the recovery of enzyme under solid state fermentation pro-
cesses. Rezende et al. (2002) used two extraction methods for
enzyme recovery: (A) Tween 80, 0.1% (v/v), in physiological sa-
line, and (B) 50 mM sodium acetate buffer, pH 5.0, under agita-
tion (180 rpm) for 15, 30 and 60 min. Both extraction methods
recovered an average of 15U/ml of xylanase activity after single
extraction. Chandra, Reddy, and Choi (2008) reported that a
single wash with 20 ml distilled water gave maximum enzyme
yield. Haq, Mukhtar, and Daudi (2003) stated that the chemical
composition of the buffer might show inhibitory effect on the
enzyme activity. Aikat and Bhattacharyya (2000) also reported
highest enzyme yield when potassium phosphate buffer pH 8.0
was used as an extractant, which showed comparatively less
activity than distilled water extraction.
4. Conclusion
This strain (T. viride-IR05) had the potential to utilize ligno-
cellulosic waste, such as sugarcane bagasse, as a carbon
source to produce valuable enzymes, thus reducing enzyme
production cost. Pretreatment of the substrate plays a pivotal
role in enzyme production due to the increased accessibility of
nutrients to the fungus hindered by thick hard layer of lignin.
Optimization of process parameters is a pre-requisite to
enhance the yield, which is very helpful in large-scale
production.
 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7 e3 2 6
Acknowledgment
The authors would like to thank the Ministry of Science and
Technology (MoST), Islamabad, Pakistan for the financial
support of this work through the project Production of Bio-
energy from Plant Biomass.
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