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One-factor-at-a-time (OFAT) optimization ofxylanase production from Trichoderma viride-IR05in 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

a r t i c l e i n f o

Article history:

Received 19 March 2014

Received in revised form

25 April 2014

Accepted 27 April 2014

Available online 20 May 2014

Keywords:

Xylanase

Bagasse

Trichoderma viride-IR05

Solid state fermentation

* Corresponding author.E-mail addresses: mirfanashraf@yahoo.c

Peer review under responsibility of The Egy

Production and hosting by El

http://dx.doi.org/10.1016/j.jrras.2014.04.0041687-8507/Copyrightª 2014, The Egyptian Socreserved.

a b s t r a c t

The present study dealt with the production of enzyme xylanase by solid substrate

fermentation using Trichoderma viride-IR05. Different substrates such as wheat bran, rice

polish, rice husk, soybean meal, sunflower meal, sugarcane bagasse or corn cobs were

evaluated for enzyme production. Of all the substrates evaluated, sugarcane bagasse was

found to be best for enzyme synthesis. The substrate, sugarcane bagasse pretreated bio-

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-

ditions were optimized for the production of xylanase in 250 ml Erlenmeyer flask such as

incubation period (seven days), substrate concentration (10 g), liquid to solid ratio (11:10),

initial pH of diluent (4.5), incubation temperature (30 �C) with inoculum size of 10%. Further

supplementation of xylose, NaNO3 or tryptone and tween-80 as additional carbon source,

nitrogen and surfactant improved (72.4 � 1.42 U/g) the titer of xylanase by T. viride-IR05,

respectively.

Copyright ª 2014, The Egyptian Society of Radiation Sciences and Applications. Production

and hosting by Elsevier B.V. All rights reserved.

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,

om, irfan.biotechnologistptian Society of Radiation

sevier

iety of Radiation Sciences

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

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

@gmail.com (M. Irfan).Sciences and Applications

and Applications. Production and hosting by Elsevier B.V. All rights

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 and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6318

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 themost 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

fermentationwas 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 thewater 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 substrateThe 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 substrateFifty grams of substrate was taken in 1 L conical flask and

moistened with 60 ml of Vogel’s 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

flaskweremixed 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

wasmaintained 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.

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2.6. Fermentation technique

The production of xylanase was carried out using SSF in

250 ml Erlenmeyer flask. Ten ml of diluents (Vogel’s 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

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

fermented mash and rotated them on rotary shaker at

150 rpm for 2 h at 30 � 1 �C for maximum enzyme extraction.

Then filtered slurry through muslin cloth followed by centri-

fugation at 8000 rpm at 4 �C for 10 min to separate fungal

spores and small particles. The clear supernatant was used as

a crude xylanase source.

2.8. Estimation of xylanase activity

Xylanase activity was assayed as described earlier (Irfan,

Nadeem, Syed, & Baig, 2010). Reaction mixture containing

0.5ml of appropriately diluted culture filtratewith 0.5ml of 1%

birchwood xylan (Sigma) solution prepared in citrate buffer

(0.05 M, pH 5.0) for 15 min at 50 �C. After incubation the re-

action was stopped by the addition of 1.75 ml of 3,5-dini-

trosalicylic acid and heated for 10 min in boiling water bath.

After cooling the reducing sugars liberated were measured by

spectrophotometrically at 550 nm and expressed as xylose

equivalent. Xylose was taken as standard. One unit of activity

was defined as the amount of enzyme, which liberates

reducing sugar (equivalent to xylose) from 1.0% Birch wood

xylan under standard assay conditions.

Days

Fig. 1 e Time course of xylanase production in solid state

fermentation by Trichoderma viride-IR05. Y-error bars

represent the SD among duplicates which differs

significantly at P £ 0.05.

2.9. Optimization of cultural and nutritional conditionsfor 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 (Vogel’s, Zepick’s, 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,

maltose, cellulose, galactose, sucrose & arabinose), nitrogen

sources (NH4NO3, NaNO3, (NH4)2SO4,NH4Cl, (NH4)2H2PO4,

Ammonium citrate, Peptone, yeast extract, tryptone, casein,

skim milk, lablamco powder and urea) and surfactants

(tween-80, triton X-100, sodium dodecyl sulfate and sodium

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.

3. Results and discussion

3.1. Time course study

Xylanase production was checked by incubating the inocu-

lated flasks for various time periods and its was noted that

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

the fermentation period was increased decrease in enzyme

production was observed. Okafor, Emezue, Okochi,

Onyegeme-Okerenta, and Nwodo-Chinedu (2007) isolated a

strain of Penicillium chrysogenum PCL501 fromwoodwastes and

reported that highest xylanase activity of 6.47 units mL�1 was

obtained with wheat bran after 96 h of fermentation period

and lowest activity of 0.79 U/ml after 120 h. Abdel-Satera and

El-Said (2001) obtained maximum production of xylanase

from Trichoderma harzianum after 8 days of fermentation

period. Goyal, Kalra, and Sareen (2008) achieved maximum

enzyme production for 14e17 days of fermentation period

using strain of T. viride. Increased fermentation time and

decreased enzyme synthesis might be due to the depletion of

macro- and micronutrients in the fermentation mediumwith

the passage of time, which altered the fungal physiology

resulting in the inactivation of secretary machinery of the

enzymes (Nochure, Roberts, & Demain, 1993).

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).

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 and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6320

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

secretionwas found in sunflowermeal (0.36� 0.21mg/ml) and

rice polish (0.65� 0.31mg/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 cobþ 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-costmedia 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).

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 observedwith 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. Vogel’s media, Czepek’s media, Citrate buffer pH

4, Phosphate buffer pH 5, Tap water or distilled water were

used as diluent in solid state fermentation. Vogel’s media

found suitable diluent for xylanase production with enzyme

yield of 66.7� 1.94 U/g and protein secretion of 0.82� 0.18mg/

ml. Distilled water and citrate buffer pH 4 also showed best

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).

J o u rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6 321

activity of 61.3 � 1.20 U/g and 57.3 � 1.65 U/g, respectively.

Vogel’s 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 Czapek’s agarmediumwas 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).

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 xylanaseproduction

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

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).

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).

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 and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6322

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 thatmaximumglucose productionwas 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

6.5 (Carmona, Fialho, & Buchgnani, 2005) were best for xyla-

nase production by different fungi in fermentation process.

These reports indicating thatmost 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 producesmaximumxylanase 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. 9 e Effect of incubation temperature on xylanase

production by T. viride-IR05 in SSF. The different letters

show significant difference (P < 0.05).

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).

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3.10. Supplementation of nitrogen and additional carbonsources

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 sou

fermentation. The different letters show significant difference (

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

rces on xylanase production by T. viride-IR05 in solid state

P < 0.05).

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).

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 and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6324

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 synthesiswas significantly stimulated by the addition

of wheat bran and tween-80 to the medium.

3.12. Effect of various concentration of tween-80 onxylanase 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

maximumsynthesis 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 froma solidmaterial 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

order0.1%tween-80>citratebufferpH5>distilledwater>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 rn a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 3 1 7e3 2 6 325

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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