Scholarly article on topic 'Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation'

Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Muhammad Irfan, Umar Asghar, Muhammad Nadeem, Rubina Nelofer, Quratulain Syed

Abstract In this study an attempt was made to optimize the cultural and nutritional conditions for xylanase production by Bacillus species in submerge fermentation process. Whole fermentation process was carried out in 250 ml Erlenmeyer flask with agitation speed of 140 rpm. Bacillus subtilis exhibit maximum xylanase production at initial medium pH of 8, substrate concentration of 2% with inoculum size of 2% at 35 °C for 48 h of fermentation period. Further supplementation of sucrose, (NH4)2SO4 and peptone as a carbon and nitrogen sources favored enzyme production. The other strain Bacillus megaterium showed its peak xylanase production at initial medium pH of 8, inoculum size of 1.5% with substrate concentration of 1.5% at incubation temperature of 40 °C for 72 h of fermentation period. The best carbon and nitrogen sources are xylose, KNO3 and malt extract. Both strains can also utilize molasses at 0.5% concentration for xylanase production can grow in medium containing 0.2% NaCl (B. subtilis BS04) and 0.8% NaCl (B. megaterium BM07) respectively. The optimum temperature of xylanase was 50 °C and pH was 5 and 5.5 by B. subtilis BS04 and B. megaterium BM07 respectively.

Academic research paper on topic "Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation"

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Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation

Q4 Muhammad Irfan a'b'*, Umar Asghar b, Muhammad Nadeem b, Rubina Nelofer b, Quratulain Syed b

Q1 a Department of Zoology, University of the Punjab, New Campus Lahore, 54590, Pakistan

b Food & 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 10 September 2015 Received in revised form 12 October 2015 Accepted 24 October 2015 Available online xxx

Keywords: Xylanase

Bacillus sp. agricultural waste Submerged fermentation

In this study an attempt was made to optimize the cultural and nutritional conditions for xylanase production by Bacillus species in submerge fermentation process. Whole fermentation process was carried out in 250 ml Erlenmeyer flask with agitation speed of 140 rpm. Bacillus subtilis exhibit maximum xylanase production at initial medium pH of 8, substrate concentration of 2% with inoculum size of 2% at 35 °C for 48 h of fermentation period. Further supplementation of sucrose, (NH4)2SO4 and peptone as a carbon and nitrogen sources favored enzyme production. The other strain Bacillus megaterium showed its peak xylanase production at initial medium pH of 8, inoculum size of 1.5% with substrate concentration of 1.5% at incubation temperature of 40 °C for 72 h of fermentation period. The best carbon and nitrogen sources are xylose, KNO3 and malt extract. Both strains can also utilize molasses at 0.5% concentration for xylanase production can grow in medium containing 0.2% NaCl (B. subtilis BS04) and 0.8% NaCl (B. megaterium BM07) respectively. The optimum temperature of xylanase was 50 °C and pH was 5 and 5.5 by B. subtilis BS04 and B. megaterium BM07 respectively.

Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Xylanase (E.C 3.2.1.8) is the enzyme which degrades p-1, 4 xylan by cleaving p-1, 4 glycosidic linkages thus forming usable products such as xylose, xylobiose like xylo-oligosaccharides (Bernier, Desrochers, Jurasek, & Paice, 1983; Chakrit, Khin, & Khanok, 2006). In annual plants and hardwoods, xylan is the most abundant non-cellulosic poly-saccharide which accounts for 20-35% of the total dry weight

in biomass (Bernier et al., 1983; Elegir, Szakacs, & Jeffries, 1994). Xylanases can be produced by bacteria and fungi in both liquid culture and solid culture. The production of mi-crobial xylanases is preferred over plant and animal sources, because of their availability, structural stability and easy genetic manipulation (Bilgrami & Pandy, 1992). Mostly bacteria especially from the genus Bacillus are world widely used for the production of extracellular hemicellulases (Coughlan & Hazlewood, 1993; Srinivasan & Meenakshi, 1999).

* Corresponding author. Department of Zoology, University of the Punjab, New Campus Lahore, 54590, Pakistan. Q2

Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.10.008

1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Mostly the production of xylanases has been studied in submerged liquid culture but there are few reports concerning the xylanase production in solid state fermentation using lignocellulosic wastes (Couri, Terzi, Pinto, Freitas, & Costa, 2000; de Souza, de Souza, & Peralta, 2001; Kalogeris, Christakopoulos, Kekos, & MacRis, 1998). Submerged fermentation process is mostly preferable because of more nutrients availability, sufficient oxygen supply and less time required for the fermentation than other fermentation techniques (Gomes, Gomes, & Stiener, 1994; Gouda, 2000; Hoq, Hempel, & Deckwer, 1994; Veluz, Taksuo, Hiroshi, & Yusaku, 1999). Today, xylanases production is highly important due to its industrial exploitation like, pulp and paper industry, baking industry, clarification of juices and also in liquification of fruits and vegetables (Gupta & Kar, 2009). In this study we reported here the xylanase production from Bacillus subtilis BS04 and Bacillus megaterium BM07 in submerged fermentation. To our knowledge this is the first report on xylanase production by B. megaterium in submerged fermentation.

2. Materials and methods

2.1. Procurement of substrates

Sugarcane bagasse was procured from local market of Lahore, city and used as substrate for xylanase enzyme production in submerged fermentation.

Bacterial strain

Two bacterial strains B. subtilis BS04 and B. megaterium BM07 were locally isolated from soil. The strain was maintained on nutrient agar (Oxoid) slants and store at 4 °C.

2.3. Cultivation of bacterial cells

Twenty five milliliter of nutrient broth (Oxoid) was sterilized in each two 250 ml Erlenmeyer flask at 121 ° C for 15 min. After sterilization, the media was inoculated with a loopful of 24 h old strains of B. subtilis BS04 and B. megaterium BM07 incubated at 37 °C for 24 h with the agitation speed of 140 rpm. Vegetative cells were used as a source of inoculum throughout the study.

2.4. Fermentation technique

Twenty five milliliter of fermentation media (g/l: Sucrose 20, K2HPO4 0.5, NaCl 0.2, MgSO4.7H2O 0.16, Yeast extract 0.5.) with 2% substrate (sugarcane bagasse) were sterilized in each 250 ml Erlenmeyer flask at 121 °C for 15 min. After sterilization, the media was inoculated with 2% solution containing vegetative cells of 24 h old B. subtilis BS04 and B. megaterium BM07 in each flask and incubated at 37 °C for 48 h of fermentation period with the agitation speed of 140 rpm.

Preparation of enzyme

After the termination of fermentation period, the fermented broth was filtered through muslin cloth and finally by

centrifugation at 4 °C, 8000 x g for 10 min to remove the bacterial cells and unwanted particles. The clear filtrate obtained after centrifugation was used as a source of crude enzyme.

2.6. Assay of xylanase enzyme

Xylanase enzyme in the culture filtrate was estimated as reported earlier (Irfan, Nadeem, Syed, & Baig, 2012). 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, pH5.0) was incubated for 15 min at 50 °C. After incubation the reaction was stopped by the addition of 1.75 ml of 3, 5 dinitrosalicylic 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 enzyme activity was defined as the amount of enzyme required to produce 1 mmole reducing sugar as a xylose equivalent per minute under standard assay conditions. Units were calculated by using following formulae.

Xylanase activity (IU) =

Reducing sugars (mg/ml) x 1000 Incubation time (15 min) x 150

2.7. Optimization of cultural and nutritional conditions for xylanase production

Different cultural conditions like time course of fermentation (24-120 h), initial medium pH (4-10), incubation temperature (25-50 0C), inoculum size (0.5-3%), substrate concentration (0.5-3.0%) and various nutritional conditions such as additional carbon sources (glucose, sucrose, fructose, CMC, arab-inose & xylose), nitrogen sources (KNO3, NaNO3, (NH4)2SO4,NH4Cl, ammonium citrate, peptone, yeast extract, tryptone, Malt extract & urea) molasses supplementation (0.5-3.0%) and NaCl concentration (0.2-1.0%) were optimized for enhanced production of xylanase by two tested strains of B. subtilis BS04 and B. megaterium BM07 in submerged fermentation process.

2.8. Effect of pH on activity of xylanase

The optimum pH for the enzyme was determined by incubating crude enzyme with substrate (1% xylan) prepared in appropriate buffers; 0.05 M citrate buffer (pH 3.0-6.0), 0.05 M sodium phosphate buffer (pH 6.0-8.0), 0.05 M Tris-HCl (pH 8.0-9.0) and 0.05 M glycine-NaOH (pH 9.0-11.0). Enzyme and substrate was incubated for 30 min at 50 0C. After incubation the reaction was stopped by the addition of DNS reagent and absorbance was measured at 550 nm.

2.9. Effect of temperature on activity of xylanase

The effect of temperature on activity of Xylanase was determined by incubating crude enzyme mixture in 1% xylan in 0.05 M citrate buffer, pH 4.8. at temperatures between 40 and 90 0C with regular interval of 5 0C. Enzyme activity was

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assayed by DNS method at different temperatures as described above.

2.10. Statistical analysis

The data obtained after experimentation was statistically evaluated using ANOVA at significance level of p < 0.05 by using computer based program SPSS.

Results and discussion

3.1. Time course study for xylanase production

Different experiments were conducted to study the optimum period for maximum xylanase production in submerged fermentation process. Results (Fig. 1) described that fermentation period of 48 h was optimum for xylanase production by B. subtilis BS04 while B. megaterium BM07 exhibited maximum production after 72 h of fermentation period. Further increase of fermentation period beyond this resulted decline in enzyme production which might be due to the production of toxic metabolites during microbial growth which inhibits the enzyme synthesis. Gupta and Kar (2009) studied on xylanase production by Bacillus sp. and reported that maximum xyla-nase production was observed in 48 h and 72 h using wheat bran and corn cob as a substrate respectively. Mrudula and Shyam (2012) reported 48 h of fermentation time for maximum production of protease from B. megaterium MTTC 2444. Murugan, Arnold, Pongiya, and Narayanan (2011) reported 96 h of fermentation period was optimum for xylanase production by Arthrobacter sp. MTCC 6915 in SSF using saw dust as a substrate. In an other study some strains of Bacillus showed maximum xylanase production after 24 h using digested bran and 48 h of fermentation using saw dust as a substrate respectively (Simphiwe, Ademola, Olaniran, & Pillay, 2011). Heck, Plinho, Marco, and Ayub (2002) isolated B. subtilis from soil which exhibit highest xylanase activity after 72 h of cultivation in SSF. Bacillus licheniformis MTCC 9415 can produce maximum xylanase after 72 h of fermentation period in solid state fermentation (Gupta & Kar, 2008). Sepahy, Ghazi,

I60] l5" J? 40

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I 20 H

and Sepahy (2011) reported fermentation period of 48 h by Bacillus mojavensis AG137 in submerge fermentation using oat bran as substrate.

3.2. Effect of initial medium pH for xylanase production

Most of the bacteria are dependent on pH and produce xyla-nase under high pH (Battan, Sharma, Dhiman, & Kuhad, 2007; Poorna & Prema, 2006). Many enzymatic processes and transport of various components across the cell membrane are strongly affected by the pH of medium (Kapoor, Nair, & Kuhad, 2008). Different pH ranges (4—10) were tested for maximum xylanase production by B. subtilis BS04 and B. megaterium BM07 in submerged fermentation. Results (Fig. 2) revealed that both strains of Bacillus sp. exhibited its peak activity at pH 8.0. Lower pH i.e. 4 and higher pH i.e. 10 of the medium retards xylanase secretion. These results indicated that both bacterial strains can tolerate in alkaline conditions. Similar findings were also reported by Sepahy et al. (2011) showing optimum pH of 8.0 for xylanase production by B. mojavensis AG137 in submerge fermentation. Simphiwe et al. (2011) reported that eight different strains of Bacillus sp. showed maximum xylanase production at pH 8.0. Bacillus pumilus showed maximum xylanase production at pH 7.0 (Monisha, Uma, & Murthy, 2009).

3.3. Effect of incubation temperature for xylanase production

Various incubation temperatures (25—50 0C) were tested for maximum production of xylanase enzyme. Fig. 3 illustrates that, at 25 0C bacterial strains did not grow well thus resulting in decreased enzyme production. B. subtilis BS04 showed highest enzyme production (51.60 ± 0.53 IU/ml) at 35 0C while B. megaterium BM07 produced maximum titer of xylanase yield (41.66 ± 0.58 IU/ml) at 40 0C. Increased in incubation temperature up to 50 0C significantly reduced in enzyme production. Sepahy et al. (2011) reported optimum temperature of 370 C for xylanase production by B. mojavensis AG137 in submerge fermentation. Different strains of Bacillus sp. gave maximum yield of xylanase production at incubation temperature of

□ B. subtilis B B.megaterium d d

24 48 72 96 120

Time period (h)

Fig. 1 - Time course study of xylanase production by Bacillus sp. in submerged fermentation at 370C. Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

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El B. subtilis ■ B.iregaterium

4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 Initial medium pH

Fig. 2 - Effect of initial medium pH for xylanase production in submerged fermentation of 48 h. Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

45 0C and 55 0C (Simphiwe et al., 2011). Monisha et al. (2009) reported that incubation at 37 0 C gave maximum xylanase production by B. pumilus.

3.4. Effect of substrate concentration on xylanase production

Fig. 4 describes the effect of different substrate (corn cobs) concentrations (0.5-3.0%) on xylanase production by Bacillus sp. in submerged fermentation. Results indicated that B. sub-tilis BS04 showed maximum enzyme (47.33 ± 1.17 IU/ml) production with 2% substrate concentration while B. megaterium BM07 showed its peak production (40.00 ± 1.00 IU/ml) at 1.5% substrate concentration. Further increase in substrate concentration did not significantly affect the enzyme production. Saleem, Akhtar, and Jamil (2002) isolated a new strain of B. subtilis which can produce best xylanase using 0.5% bagasse as a substrate in submerge fermentation. Li, Lin, Meng, Lu, and Gu (2006) worked on endoxylanase production by Aspergillus awamori ZH-26 under submerged fermentation reporting optimum substrate (wheat bran) concentration of 4.93% was best for endoxylanase production. Immanuel, Dhanusha, Prema,

and Palavesam (2006) also reported that maximum endoglu-canase production was maximum with 1.5% substrate (coir powder) concentration using Cellulomonas sp., Bacillus sp. and Micrococcus sp.

3.5. Effect of inoculum size on xylanase production

Different inoculum levels (0.5-3.0%) were tested for enhanced production of xylanase enzyme by Bacillus sp. in submerged fermentation. When low inoculum level i.e. 0.5% was used, enzyme production was minimum but as the inoculum level increased the enzyme production was also increased. Maximum yield of xylanase enzyme was observed at 2% with Bacillus subtiltis BS04 and 1.5% inoculum level with B. megaterium BM07 (Fig. 5). Increased level of inoculum mostly reduced xylanase production in industrial fermentation process (Battan et al., 2007). This may be due to the depletion of nutrients from the fermentation medium which resulted decline in enzyme synthesis. Sepahy et al. (2011) reported inoculum size of 2% was best for xylanase production by B. mojavensis AG137 in submerged fermentation.

B.megaterium

30 35 40 45 50

Temperature (°C)

Fig. 3 - Effect of incubation temperature for xylanase production by Bacillus sp. with initial medium pH of 8 for 48 h of fermentation period. Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

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B.megaterium

Substrate Cone. (%)

Fig. 4 - Effect of substrate concentration on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8,48 h period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

□ B. subtilis ■ B.megaterium

Inoculum Size (%)

Fig. 5 - Effect of inoculum size on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

3.6. Effect of additional carbon sources on xylanase production

Xylanase production was further enhanced by supplementing the fermentation medium with suitable additional carbon source. Table 1 depicts that sucrose and xylose at concentration of 0.5% were the best xylanase inducer by B. subtilis BS04 and B. megaterium BM07 in submerged fermentation respectively. Any other carbon sources like fructose, glucose, CMC

Table 1 - Effect of supplementation of additional carbon sources on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h period).

Sr. No.

Carbon source

Xylanase activity (IU/ml)

56 B. subtilis B. megaterium

57 1 Control 19.96 ± 1.55a 16.63 ± 1.18a

58 2 Xylose 48.30 ± 0.61e 43.06 ± 1.10e

59 3 Fructose 38.80 ± 1.25d 26.60 ± 1.21b

60 4 Sucrose 54.80 ± 0.52f 39.27 ± 0.94d

61 5 Glucose 36.93 ± 1.10d 34.90 ± 1.15c

62 6 CMC 26.23 ± 1.08c 18.16 ± 1.04a

63 7 Arabinose 23.20 ± 0.72b 25.67 ± 1.53b

Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

(carboxymethyl cellulose) and arabinose gave less enzyme production. Azeri, Tamer, and Oskay (2010) isolated different strains of Bacillus sp. and all exhibit maximum xylanase production by using birchwood xylan as a carbon source. Saleem et al. (2002) reported that supplementation of sucrose to the fermentation medium significantly enhance the xylanase production by B. subtilis. In 96 h of fermentation, wheat bran was best carbon source for xylanase production by Strepto-myces sp. (Sharma & Bajaj, 2005).

3.7. Supplementation of nitrogen sources

Among all the tested inorganic and organic nitrogen sources, tryptone and (NH4)2SO4 are best for B. subtilis BS04 while KNO3 and malt extract for B. megaterium BM07 for xylanase synthesis in submerged fermentation as shown in Table 2. Therefore, these nitrogen sources were selected as optimum for respective bacterial strains. Earlier studies reported that organic nitrogen sources have been found to stimulate xylanase production in Bacillus species (Battan et al., 2007). Yeast extract + tryptone and yeast extract + NH4NO3 have stimulatory effect on xylanase production from B. mojavensis AG137 in submerge fermentation (Sepahy et al., 2011). Sharma and Bajaj (2005) isolated different species of Streptomyces sp. and reported that best xylanase production was observed with

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Table 2 — Effect of different nitrogen 48 h period). sources on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8,

Sr.# Inorganic nitrogen sources Xylanase activity (IU/ml) Organic nitrogen sources Xylanase activity (IU/ml)

B. subtilis B. megaterium B. subtilis B. megaterium

1 Control 3.07 ± 0.058a 3.56 ± 0.15a Control 11.70 ± 0.30a 10.10 ± 0.10b

2 KNO3 21.33 ± 0.15d 34.5 ± 0.25f Peptone 48.93 ± 0.90e 16.50 ± 0.20d

3 NaNO3 17.63 ± 0.15c 18.66 ± 0.25c Yeast extract 39.90 ± 0.85d 12.43 ± 0.23c

4 (NH4)2SO4 38.03 ± 0.06e 21.33 ± 0.15d Tryptone 55.66 ± 0.41f 21.30 ± 0.30e

5 NH4G 20.27 ± 0.15d 24.03 ± 0.06e Malt extract 23.40 ± 0.72c 43.66 ± 0.06f

6 Ammonium citrate 14.11 ± 1.72b 12.56 ± 0.15b Urea 20.13 ± 1.02b 8.50 ± 0.30a

Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

soybean meal and yeast extract as nitrogen sources in the medium.

3.8. Effect of different concentrations of molasses on xylanase production

Effect of different concentrations of molasses supplementation was also evaluated for xylanase production by tested bacterial strains. Results (Fig. 6) revealed that supplementation of 0.5% molasses concentration favored enzyme production by both bacterial strains i.e. B. subtilis BS04 and B. megaterium BM07. Increased supplementation of molasses caused decreased enzyme production which might be due to increased sugars level and many other nutrients present in molasses which resulted in catabolite repression. Shabeb, Younis, Hezayen, and Nour-Eldien (2010) achieved best cellulase production with 10% molasses containing medium. Rafi, Asghar, Yaqube, and Ghour (1998) obtained highest yield of xylanase production in a medium containing 2% cane molasses. Supplementation of 0.125% molasses concentration enhances the CMCase yield (Gori & Malana, 2010).

3.9. Effect of different concentration of NaCl on xylanase production

Different salt concentrations ranging from 0.2 to 1.0% were tested for xylanase production. B. subtilis BS04 showed maximum xylanase production (48.1 ± 1.00 IU/ml) at 0.2% NaCl concentration and further increased concentration resulted decline in enzyme production up to 12.20 ± 0.35 IU/ml at 1%

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NaCl as shown in Fig. 7. The other strain B. megaterium BM07 exhibited maximum activity (45.93 ± 0.11 IU/ml) with 0.8% NaCl concentration showing its halophilic character. Sajitha, Vasanthabharathi, Lakshminarayanan, and Jayalakshmi (2011) isolated a strain of B. megaterium which have good growth in medium containing 0.3% NaCl and gave maximum yield of amylase. Shaheen, Shah, Hameed, and Hasan (2008) worked on protease production by B. subtilis reporting that 1.5% NaCl concentration was best for maximum production of protease in submerged fermentation.

3.10. Effect of pH on activity

Different pH ranges from 4 to 9 was tested to check the optimum activity of xylanase by using various buffers. Results revealed (Fig. 8) that pH 5 and 5.5 was found optimum for maximum xylanase activity B. subtilis BS04 and B. megaterium BM07 by using 0.05 M citrate buffer respectively. Further increase in pH resulted decline in enzyme activity. The optimum pH of xylanase enzyme from different Bacillus sp. was reported 5 (Pratumteep, Sansernsuk, Nitisinprasert, & Apiraksakorn, 2010) 7 (Sanghi, Garg, Gupta, Mittal, & Kuhad, 2010), 8 (Kamble & Jadhav, 2012) and 9 (Annamalai, Thavasi, Jayalakashmi, & Blasubramanian, 2009).

3.11. Effect of temperature on activity

The activity of the xylanase enzyme was assayed at various temperatures ranging from 35 0C to 70 0 C to check the optimum temperature. Results (Fig. 9) indicated that enzyme

□ B.subtilis ■ B.meg&terium

"1-1-1-г

1 1.5 2 2.5 3

Molasses Cone. (%)

Fig. 6 - Effect of different concentrations of molasses on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

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□ B.subtiüs ■ B.megaterium d

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NaCl Cone. (%)

Fig. 7 - Effect of different concentration of NaCl on xylanase production by Bacillus sp. in submerged fermentation (initial pH 8, 48 h period). Values presented were the means of triplicates and different letters differ significantly at p < 0.05.

E 50 g 40 1 30

i 20 я

- B.subtiüs ■ B.megaterium

3 3.5 4 4.5 5 5.5

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Fig. 8 - Effect of pH on activity of xylanase produced by Bacillus sp. in submerged fermentation.

activity was increased with increase in temperature and highest activity was observed at 50 °C. After that, as the temperature was increased from 60 °C to 70 °C, there was a sharp decline in enzyme activity was observed. Xylanase enzyme from various Bacillus species has optimum temperature of 40-60 °C (Khandeparker, Verma, & Deobagkar, 2011; Kumar, Panday, & Naik, 2011; Pratumteep et al., 2010; Roy & Rowshanul, 2009; Sanghi et al., 2010; Sa'-Pereira, CostaFerreira, & Aires-Barros, 2002).

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4. Conclusion

The results of the present study revealed that B. megaterium BM07 can produce xylanase enzyme using sugarcane bagasse as a substrate in submerged fermentation which was not reported earlier. In this study bacterial strains were used for xylanase enzyme production which has advantage of short period of growth as compared to the fungi. Results of this

-♦— B.subtiüs -"—B.megaterium

-1-1-1-1-1-1-1-1-1

30 35 40 45 50 55 60 65 70 75 Temperature (°Q

Fig. 9 - Effect of temperature on activity of xylanase produced by Bacillus sp. in submerged fermentation.

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study indicated that nutrients and cultural properties played a pivotal role in enzyme production. The optimization of all the process parameters are being considered as pre-requisites to make the process of enzyme production cost effective at large scale.

REFERENCES

Annamalai, N., Thavasi, R., Jayalakshmi, S., &

Balasubramanian, T. (2009). Thermostable and alkaline tolerant xylanase production by Bacillus subtilis isolated from marine environment. Indian Journal of Biotechnology, 8, 291—297.

Azeri, C., Tamer, A. U., & Oskay, M. (2010). Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp. African Journal of Biotechnology, 9(1), 63—72.

Battan, B., Sharma, J., Dhiman, S. S., & Kuhad, R. C. (2007).

Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry. Enzyme and Microbial Technology, 41(6—7), 733—739.

Bernier, R., Desrochers, M., Jurasek, L., & Paice, M. G. (1983). Isolation and characterization of a xylanase from Bacillus subtilis. Applied and Environmental Microbiology, 46(2), 511—514.

Bilgrami, K. S., & Pandy, A. K. (1992). In E. S. K. Jain (Ed.), Industry and fermentation in introduction to biotechnology (pp. 149—165).

Chakrit, T., Khin, L. K., & Khanok, R. (2006). Purification of

xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column. Process Biochemistry, 41(12), 2441—2445.

Coughlan, M. P., & Hazlewood, G. P. (1993). ß-1,4-D-Xylan degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnology and Applied Biochemistry, 17,259—289.

Couri, S., Terzi, S. D. C., Pinto, G. A. S., Freitas, S. P., &

Costa, A. C. A. D. (2000). Hydrolytic enzyme production in solid-state fermentation by Aspergillus niger 3T5B8. Process Biochemistry, 36(3), 255—261.

de Souza, D. F., de Souza, C. G. M., & Peralta, R. M. (2001). Effect of easily metabolizable sugars in the production of xylanase by Aspergillus tamarii in solid-state fermentation. Process Biochemistry, 36(8—9), 835—838.

Elegir, G., Szakacs, M., & Jeffries, T. W. (1994). Purification, characterization and substrate specificities of multiple xylanases from Streptomyces sp. strain B-12-2. Applied and Environmental Microbiology, 60(7), 2609—2615.

Gomes, D. J., Gomes, J., & Stiener, W. (1994). Production of highly thermostable xylanase by a wild strain of thermophilic fungus Thermoascus aurantiacus and partial characterization of the enzyme. Journal of Biotechnology, 37, 11—22.

Gori, M. I., & Malana, M. A. (2010). Production of carboxymethyl cellulase from local isolate of Aspergillus species. Pakistan Journal of Life and Social Sciences, 8(1), 1—6.

Gouda, M. K. (2000). Purification and partial characterization of cellulose free xylanase produced in solid state and submerged fermentation by Aspergillus tamarii. Advances in Food Science, 22, 31—37.

Gupta, U., & Kar, R. (2008). Optimization and scale up of cellulase free endo xylanase production by solid state fermentation on corn cob and by immobilized cells of a thermotolerant bacterial isolate. Jordan Journal of Biological Science, 1(3), 129—134.

Gupta, U., & Kar, R. (2009). Xylanase production by a thermo-tolerant Bacillus species under solid-state and submerged fermentation. Brazilian Archives of Biology and Technology, 52(6), 1363—1371.

Heck, J. X., Plinho, F., Marco, H., & Ayub, A. Z. (2002). Cellulase and xylanase production by isolated amazon Bacillus strains using

soybean industrial residue based solid-state cultivation. Brazilian Journal of Microbiology, 33, 213-218.

Hoq, M. M., Hempel, C., & Deckwer, W. D. (1994). Cellulase free xylanase by Thermomyces lanuginosus RT9: effects of aeration, agitation and medium components on production. Journal of Biotechnology, 37, 49-58.

Immanuel, G., Dhanusha, R., Prema, P., & Palavesam, A. (2006). Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. International Journal of Environment Science and Technology, 3(1), 25-34.

Irfan, M., Nadeem, M., Syed, Q.., & Baig, S. (2012). Effect of medium composition on xylanase production by Bacillus subtilis using various agricultural wastes. American-Eurasian Journal of Agriculture & Environmental Science, 12(5), 561-565.

Kalogeris, E., Christakopoulos, P., Kekos, D., & MacRis, B. J. (1998). Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus, characterization of two isozymes. Journal of Biotechnology, 60(3), 155-163.

Kamble, R. D., & Jadhav, A. R. (2012). Isolation, purification, and characterization of xylanase produced by a new species of Bacillus in solid state fermentation. International Journal of Microbiology, 2012, 8. http://dx.doi.org/10.1155/2012/683193 (Article ID 683193).

Kapoor, M., Nair, L. M., & Kuhad, R. C. (2008). Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora. Biochemical Engineering Journal, 38(1), 88-97.

Khandeparker, R., Verma, P., & Deobagkar, D. (2011). A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40, gene cloning and sequencing. New Biotechnology, 28, 814-821.

Kumar, S. S., Panday, D. D., & Naik, G. R. (2011). Purification and molecular characterization of low molecular weight cellulase-free xylanase from thermoalkalophilic Bacillus spp. JB 99. World Journal of Science & Technology, 1, 09-16.

Li, Y., Lin, J., Meng, D., Lu, J., & Gu, G. (2006). Effect of pH, cultivation time and substrate concentration on the endoxylanase production by Aspergillus awamori ZH-26 under submerged fermentation using central composite rotary design. Food Technology and Biotechnology, 44(4), 473-477.

Monisha, R., Uma, M. V., & Murthy, V. K. (2009). Partial

purification and characterization of Bacillus pumilus xylanase from soil source. Kathmandu University Journal of Science Engineering and Technology, 5(II), 137-148.

Mrudula, S., & Shyam, N. (2012). Immobilization of Bacillus

megaterium MTCC 2444 by Ca-alginate entrapment method for enhanced alkaline protease production. Brazilian Archives of Biology and Technology, 55(1), 135-144.

Murugan, S., Arnold, D., Pongiya, U. D., & Narayanan, P. M. (2011). Production of xylanase from Arthrobacter sp. MTCC 6915 using saw dust as substrate under solid state fermentation. Enzyme Research, 2011, 7. http://dx.doi.org/10.4061/2011/696942 (Article ID 696942).

Poorna, C. A., & Prema, P. (2006). Production and partial

characterization of endoxylanase by Bacillus pumilus using agro industrial residues. Biochemical Engineering Journal, 33(2), 106-112.

Pratumteep, A., Sansernsuk, J., Nitisinprasert, S., &

Apiraksakorn, J. (2010). Production, characterization and hydrolysation products of xylanase from Bacillus subtilis GN156. KKU Research Journal, 15, 343-350.

Rafi, S., Asghar, M., Yaqube, M., & Ghour, M. A. (1998). Production of xylanase from corn stover by Arachniotus sp. Pakistan Journal of Biological Sciences, 1(4), 380-382.

Roy, N., & Rowshanul, H. M. (2009). Isolation and characterization of xylanase producing strain of Bacillus cereus from soil. Iranian Journal of Microbiology, 1, 49-53.

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Sa'-Pereira, P., Costa-Ferreira, M., & Aires-Barros, M. R. (2002). Enzymatic properties of a neutral endo-1,3(4)-ß-xylanase Xyl II from Bacillus subtilis. Journal of Biotechnology, 94, 265—275.

Sajitha, N., Vasanthabharathi, V., Lakshminarayanan, R., & Jayalakshmi, S. (2011). Amylase from an estuarine Bacillus megaterium. Current Research Journal of Biological Sciences, 3(2), 110—115.

Saleem, M., Akhtar, M. S., & Jamil, S. (2002). Production of

xylanase on natural substrates by Bacillus subtilis. International Journal of Agricultural Biology, 4(2), 211—213.

Sanghi, A., Garg, N., Gupta, V. K., Mittal, A., & Kuhad, R. C. (2010). One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ASH. Brazilian Journal of Microbiology, 41, 467—476.

Sepahy, A. A., Ghazi, S., & Sepahy, M. A. (2011). Cost-effective production and optimization of alkaline xylanase by indigenous Bacillus mojavensis AG137 fermented on agricultural waste. Enzyme Research, 9. http://dx.doi.org/ 10.4061/2011/593624 (Article ID 593624).

Shabeb, M. S., Younis, A. M., Hezayen, F. F., & Nour-Eldien, M. A. (2010). Production of cellulase in low-cost medium by Bacillus subtilis KO strain. World Applied Science Journal, 8(1), 35—42.

Shaheen, M., Shah, A. A., Hameed, A., & Hasan, F. (2008).

Influence of culture conditions on production and activity of protease from Bacillus subtilis bs1. Pakistan Journal of Botany, 40(5), 2161—2169.

Sharma, B., & Bajaj, B. K. (2005). Production and partial characterization of alkali-tolerant xylanase from an alkalophilic Streptomyces sp. CD3. Journal of Scientific and Industrial Research, 64, 688—697.

Simphiwe, P., Ademola, B., Olaniran, O., & Pillay, B. (2011).

Sawdust and digestive bran as cheap alternate substrates for xylanase production. African Journal of Microbiology Research, 5(7), 742—752.

Srinivasan, M. C., & Meenakshi, V. R. (1999). Microbial xylanase for paper industry. Current Science, 77(1), 137—142.

Veluz, G., Taksuo, K., Hiroshi, M., & Yusaku, F. (1999). Screening Rhizopus sp. Journal of Faculty of Agriculture, 43, 419—423.

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