Scholarly article on topic 'One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation'

One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation Academic research paper on "Chemical engineering"

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Abstract of research paper on Chemical engineering, author of scientific article — Muhammad Irfan, Muhammad Nadeem, Quratulain Syed

Abstract 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 biologically, 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 conditions 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.

Academic research paper on topic "One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation"

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One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma uiride-IR05 in solid-state fermentation

Q3 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: Received 19 March 2014 Received in revised form 25 April 2014 Accepted 27 April 2014 Available online xxx

Keywords: Xylanase Bagasse

Trichoderma viride-IR05 Solid state fermentation

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 biologically, 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 conditions 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 © 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 cellulose in abundance in plant cell wall (Coughlan & Hazelwood,

1993). Biodegradation of xylan is a complex process that requires the coordination of several xylanolytic enzymes that hydrolyze xylan and arabinoxylan polymers. This enzyme group includes endo-ß1, 4-xylanase (1, 4-ß-D-xylan xylanohydrolase, EC 3.2.1.8), which attack main chain of xylans, ß-D-xylosidase (1, 4-ß-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). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications

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.

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debranching enzymes i.e. a-L-arabinofuranosidases, «-glucuronidases 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 fermentations. 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 developin; 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 synthesis 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 uniformity in the environment of SSF system and gaseous atmosphere, 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 brightening of the pulp to avoid the chlorine free bleaching operations (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, recovery 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). Agricultural 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. IR05

Isolation and identification of Trichoderma viride-

T. viride-IR05 was obtained from Microbiology Laboratory, Food and Biotechnology Research Center (FBRC), Pakistan Council of Scientific and Industrial Research (PCSIR) laboratories 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.

g 2.3. Pretreatment of substrate

2.3.1. Chemical treatment of substrate The selected substrate (50 g) were soaked in different concentration 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 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 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.

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 incubator. 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 0.5 ml of appropriately diluted culture filtrate with 0.5 ml 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 reaction 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.

2.9. Optimization of cultural and nutritional conditions for xylanase production

Various cultural conditions like time course of fermentation (1-10days), initial medium pH (4-8), incubation temperature (20-50 °C), inoculum size (5-30%), substrate concentration (5-30 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 significant difference method after using computer software SPSS.

Results and discussion

3.1. Time course study

Xylanase production was checked by incubating the inoculated 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 from wood wastes 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 14-17 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 medium with the passage of time, which altered the fungal physiology resulting in the inactivation of secretary machinery of the enzymes (Nochure, Roberts, & Demain, 1993).

5 6 Days

Fig. 1 - Time course of xylanase production in solid state fermentation by Trichoderma uiride-IR05. Y-error bars represent the SD among duplicates which differs significantly at P £ 0.05.

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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) indicated 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 produce 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 (sugarcane 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 concentration, decreased in enzyme production and protein secretion were observed as shown in Fig. 3. Our findings were in accordance with Haq, Javed, and Saleem (2006) who also reported that 10% substrate level was best for CMCase

Fig. 2 - Selection of substrate for xylanase production by T. viride-IR05 in SSF. The different letters show significant difference (P < 0.05).

10 15 20 25

Substrate Cone. (g/500ml flask)

Fig. 3 - Effect of different substrate concentrations on xylanase production by T. uiride-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 activity (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. 4. The substrate was further analyzed by advanced techniques such as scanning electron microscopy (Fig. 4) 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).

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 ± 0.18 mg/ ml. Distilled water and citrate buffer pH 4 also showed best

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Untreated Biologicaly Acid Treated KOH treated H202 treated treated

Substrate Conditions

Untreated Bagasse

2.5% KOH treated Bagasse

Fig. 4 - 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. Vogel's media is the most widely used medium for the cultivation 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 agar medium was used for screening of xylanase production. Meshrama, Kulkarni, Jayaraman, Kulkarni, and

50 90 j

51 80 -

52 3 70 -

53 » 60 -

54 « 50 -

55 n tt 40 -

56 i 30 -

57 fl > 20 -

58 X 10 -

59 0 -

Citrate buffer 4

Phosphate Tap Water buffer 5

Distilled water

Diluents

Fig. 5 - Selection of different diluents for xylanase production by T. uiride-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 Mandels-Weber 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 production 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

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5:10 7:10 9:10 11:10 13:10 Liquid : Solid ratio (ml : g)

Fig. 6 - 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 production. 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 production, 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 experiments 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 4.5-5.0 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

Inoculum size (%)

Fig. 7 - Effect of different inoculum size on xylanase production by T. uiride-IR05 in SSF. The different letters show significant difference (P < 0.05).

6.5 (Carmona, Fialho, & Buchgnani, 2005) were best for xylanase 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 temperature was further increased decrease in enzyme production was also observed. Abdel-Satera and El-Said (2001) qi screened xylan degrading filamentous fungi and reported that T. harzianum produced maximum xylanase production at incubation 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.

5.5 6 6.5 Initial medium pH

Fig. 8 - Effect of initial pH of diluent on xylanase production by T. viride-IR05 in SSF. The different letters show significant difference (P < 0.05).

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25 30 35 40 45 Incubation Temperature (°C)

Fig. 9 - 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

Control

Tween-80 Triton X-100

Fig. 11 - 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 production 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 medium with any other nitrogen source do not favored best enzyme production (Fig. 10). Goyal et al. (2008) achieved maximum xylanase production by supplementing the medium 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

Inorganic Nitrogen Source

Organic Nitrogen sources

Fig. 10 - 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).

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0.4 0.5 0.6 0.7 Cone, of Tween-80 (%)

Fig. 12 - 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) reported that wheat bran and peptone were found best for highest xylanase production among various tested agricultural residues and inorganic/organic nitrogen sources. Xylan and NaNO3 were best carbon and nitrogen sources for maximum xylanase production C. sativus Cs5 strain in submerged 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 production 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

Distilled water 0.1% Glycerol 0.1% NaCI 0.1% Tween-80 Citrate buffer pH

Leaching Agents

Fig. 13 - 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. 0.1-1.0% 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 concentration 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% concentration 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 fermented mash. Results (Fig. 13) indicated that maximum extraction was observed in 0.1% tween-80 (63.4 ± 2.11 U/g) followed 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 processes. Rezende et al. (2002) used two extraction methods for enzyme recovery: (A) Tween 80, 0.1% (v/v), in physiological saline, and (B) 50 mM sodium acetate buffer, pH 5.0, under agitation (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.

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.

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Acknowledgment

Q2 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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