(PCSIR) Laboratories Complex Ferozpure Road Lahore, Pakistan Pakistan.

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1 !"#$#%&'(&! )*!' #' *' Muhammad Irfan 1*, Usman Irfan 2, Zunaria Razzaq 3, Quratulain Syed 1 and Muhammad Nadeem 1 1 Food & Biotechnology Research Center, Pakistan Council of Scientific & Industrial Research (PCSIR) Laboratories Complex Ferozpure Road Lahore, Pakistan Department of Microbiology, University of veterinary and Animal Science Lahore Pakistan. 3 Insititute of Biotechnology & Biochemistry, University of veterinary and Animal Science Lahore Pakistan. 1* (Corresponding Author: mirfanashraf@yahoo.com) #! The present study deals with the production of carboxymethy cellulase enzyme in submerged fermentation from by using wheat straw and rice straw as substrates. Different process parameters like substrate concentration, inoculum size, initial medium ph, carbon sources and mixing of substrates were optimized for maximum production of cellulase. Of all these parameters investigated maximum level of enzyme production was achieved at initial ph in the range of 5 with 3%(v/v) inoculum size using 25ml of media with agitation speed of 120pm. Wheat straw with 1.5% concentration was fond better in enzyme production. Further addition of glucose as a carbon source and mixing of wheat straw and rice straw in 1:1 ratio effectively improved the enzyme production. +&"*Agricultural wastes,, CMCase, Submerged fermentation *! Microbial enzymes can be produced by using either submerged fermentation or solid-state fermentation techniques 20. Today the production of cellulose degrading enzymes has been extensively studied in submerged fermentation as compared to solid state fermentation by using different microorganism 15. Among the various microorganisms fungus have a greater ability to produce cellulose degrading enzymes 13. For the production of cellulose degrading enzyme microorganism must be grown on cellulosic material which have inducer effect on enzyme synthesis 14.The major cellulose degrading classes of fungus are Ascomycetes ( and ), Basideomycetes ( ) and Deuteromycetes ( ). Among these species is the most common fungi which is present in human s environment because it is capable of utilizing every type of substrate due to their greater variety of enzyme production 15. The cellulose degrading enzymes of these funguses are of great interest due to its biotechnological application in conversion of lignocellulosic material into valueable products such as glucose, ethanol and single cell products 19. Mostly they are used in textile, food, pulp and paper, animal feed, fuel, chemical, waste management, pharmaceutical, protoplast production and in genetic engineering 10. In this article we reported the utilization of two agricultural wastes like wheat straw and rice straw for carboxymethy cellulase production from in submerged fermentation by optimizing various process parameters.

2 *(*,!!-' Sugar cane bagasse procured from Shakar Gunj Sugar mills (Pvt.) Limited, Jhang Road, Faisalabad, Pakistan, and wheat straw was purchased from local market was used as a source of lignocellulosic biomass. The biomass was washed and dried to remove the unwanted particles and then milled into powdered form (2mm) with hammer beater mill..))) The slants of five days old cultures were wetted by adding 10 ml of sterilized distilled water. The spores were scratched by sterile wire loop to break clumps and obtain homogeneous spore suspension. One ml of this spore suspension containing 1 x 10 8 spores, counted by haemocytometer was used as inoculum. /&'.*! was grown on medium as described by Mandles and Weber 17. The media contained (per liter of distilled water): Urea 0.3 g, (NH 4 ) 2 SO g, KH 2 PO g, CaCl g, MgSO 4-7H 2 O 0.3 g, protease peptone 1.0 g, FeSO 4-7H 2 O 5.0 mg, MnSO 4-7H 2 O 1.6 mg, ZnSO 4-7H 2 O 1.4 mg, CoCl mg. The ph of media was adjusted to 5.0 ± 0.2. Then, 25 ml of the liquid medium was placed in 250 ml Erlenmeyer flask and sterilized at 121 C for 15 min. After sterilization, the media was allowed to cooled and inoculated with 0.5 ml of spore suspension of containing approximately 10 6 spores per milliliter. The inoculated flasks were incubated at 30±1 o C for 96 hrs with the agitation speed of 120rpm. After termination of fermentation period the culture filtrate was centrifuged at 8000 rpm for 10 min at 4 C to remove unwanted particles and spores. The supernatants obtained after centrifugation were used as the crude extracellular enzyme source. /'$$ 500 µl of the enzyme sample along with 500 µl of 1% (w/v) CMC in 50 mm acetate buffer ph 5 was incubated, in a water bath at 50 o C, for 30 min. After incubation 1.5 ml of DNS was added and boiled for 5 minutes and absorbance was taken spectrophotometrically at 550nm. The reducing ends liberated were then measured with DNS 18. One unit of CMCase activity was defined as the amount of enzyme that required to release one micromole of glucose per minute under assay conditions. 0)'.*!.' Various production parameters such as initial medium ph (3-7), inoculum size (1-4% v/v), substrate concentration (0.5-2%), depth of medium (15ml, 25ml and 50ml), different carbon sources (cellulose, glucose and maltose) mixing of wheat straw and rice straw in different proportion under static and agitated conditions were optimized by in submerged fermentation using wheat straw and rice straw as substrate.!& All the data obtained were analyzed statistically by using computer based programme Graph pad Instate. 1**! Carboxymethyl cellulases are very important enzymes which are widely used in various industrial processes. Researchers had very much focused on this enzyme production due to its industrial importance. In this study different production parameters were optimized for maximum production of carboxymethyl cellulase by in submerged fermentation using wheat straw and rice straw as a substrate. /!#$!$$)*! Different concentrations of wheat straw and rice straw ranging from 0.5-2% were evaluated for maximum CMCase enzyme production from in submerged fermentation.

3 Results (Fig. 1) showed that enzyme production was increased by increasing the concentration of substrates and by further increasing the substrate concentration enzyme production was decreased. Maximum enzyme production was observed at 1% and 1.5% substrate concentration using rice straw and wheat straw respectively. Further increase or decrease in substrate concentration beyond this decreased the enzyme production. Acharya et al. 1 found maximum level of CMCase activity (0.1813U/ml) with substrate (saw dust) concentration of 9.6%. Irfan et al 8 achieved maximum CMCase activity with 2% pretreated substrate in submerged fermentation. Ojumu et al. 19 gained maximum enzyme production with 3% saw dust, corn cobs and sugarcane bagasse. Figure 1. Effect of different substrate concentration on CMCase production by in submerged fermentation. Error bars indicate the SD among triplicates. /!'*')2$$)*! Figure 2 showed the effect of different initial medium ph on CMCase production by in submerged fermentation. Results showed that by increasing the ph of the medium enzyme production was increased and maximum at ph 4.0 (0.456 ± 0.11 IU) in case of rice straw and ph 5.0 (0.577 ± 0.13 IU) in wheat straw. As the ph of the medium further increased up to 7.0 the enzyme production was decreased. These findings indicated that was able to produce cellulose enzyme in ph range of 4-5. Juwaied et al. 9 recorded the optimum ph of 4.5 for best production of CMCase by using sugarcane waste as substrate. Jaafaru and Fagade 9 reported maximum enzyme production with ph of 5.0 using strain of YL128. Acharya et al. 1 observed maximum CMCase activity of IU/ml at ph 4.0 using saw dust as substrate. In another study 3, maximum enzyme activity was found at ph 4.5 and 7.5 using strains of.

4 Figure 2. Effect of different initial medium ph on CMCase production by in submerged fermentation. Error bars indicate the SD among triplicates. 3'4$$)*! Proper utilization of volume of fermentation flask affects the enzyme synthesis by microbes. In this experiment, 250ml capacity of flask is used with three different volumes of medium such as 15ml, 25ml and 50ml to test the enzyme production. Results (Fig.3) revealed that among all the three tested volumes, 25ml volume of medium in 250ml capacity flask gave maximum (0.3 ± 0.08 IU) enzyme production as compared to rest of the volume used which was 15ml and 50ml. Malik et al. 16 also reported similar findings for the production of cellulase by. Figure 3. Effect of different volume of medium in 250ml flask for CMCase production by in submerged fermentation. Error bars indicate the SD among triplicates. /!!*$$)*! This experiment was conducted to check weather agitation affects the enzyme production or not. Results in the figure 4 indicated that agitation conditions favored better enzyme yield as compared to static conditions. Our findings were in agreement with Acharya et al. 1 who also

5 obtained maximum levels of enzyme with agitation speed of 120rpm for 96h. Ojumu et al. 19 also obtained maximum enzyme yield of IU/ml at 35 o C with agitation speed of 200 rpm. Figure 4. Effect of static and agitation conditions on CMCase production by for 94h of fermentation. /!!'$$)*! Different experiments were conducted to check the optimum level of cellulose by in submerged fermentation. Inoculum size of 1-4% was tested (Fig. 5), maximum enzyme production was found at 3% v/v yielding enzyme activity of ± 0.09 IU using wheat straw as a substrate. Decline enzyme production was observed by increasing the inoculum s concentration of 4% v/v. By using 10 discs of 8 mm size of maximum cellulose activity of IU/mL was found 1.! "#" Figure 5. Effect of different inoculum size on CMCase production by in submerged fermentation. Error bars indicate the SD among triplicates.

6 /!*$#!$$)*! Carbon source is very essential component for microbial growth and product formation. Sometime it enhances the product formation as well as growth of the microorganism. In this experiment fermentation media was supplemented with glucose, maltose and cellulose with concentration of 0.25%. Addition of glucose to the medium enhanced the enzyme production as compared to control using rice straw and as substrate. When wheat straw was used as substrate and supplemented with carbon sources, no comparable results were found as shown in figure 6. Jaafaru and Fagade 9 studied cellulase synthesis by and reported that addition of sugarcane bagasse as sole carbon source gave maximum yield (10.11 U/ml). Some workers 6,7,11 reported that sugarcane bagasse and wheat bran produced maximum cellulase enzyme synthesis. Gautam et al. 5 optimize the medium constituents for cellulose production by in submerged fermentation. According to their results sucrose at the concentration of 1.0% was the most effective carbon source. Figure 6. Effect of different carbon sources for CMCase production by in submerged fermentation at 30 o C. Error bars indicate the SD among triplicates. % #$$)*! Rice straw and wheat straw were mixed in different proportion to check the optimum enzyme production by in submerged fermentation. Results (Fig. 7) showed that mixing of both substrates in equal amount (1:1) improved enzyme (0.56 ± 0.06 IU) production as compared to individual substrates. Kavitha and Nagaranjan 11 obtained maximum endoglucanase production from in liquid cultures using combination of 4:1 using rice bran and wheat bran as a substrates. Benkun et al. 2 achieved highest yield of CMCase production by combination of 4:1 ratio of rice bran and wheat bran in solid state fermentation using strains of.

7 !!! $% Figure 7. Effect of different combinations of rice straw to wheat straw for CMCase production by in submerged fermentation. Error bars indicate the SD among triplicates. 1! 1. Acharya PB, Acharya DK, Modi HA. Optimization for cellulase production by using saw dust as substrate., 2008; 7 (22): Benkun Q, Yaoa R, Yua Y, Chena Y. Influence of different ratios of rice straw to wheat bran on production of cellulolytic enzymes by zy-01 in solid state fermentation.!",2007; 6 (9): Coral G, Arikan B, Unaldi MN, Guvenmes H. Some properties of crude carboxymethyl cellulase of Z10 wild-type Strain. #, 2002; 26: Fan LT, Gharpuray MM, Lee YH. Cellulose hydrolysis. Berlin, Germany.SpringerVerlag,1987; 3, pp Gautam SP, Bundela PS, Pandey AK, Awasthi MK. Sarsaiya S. Optimization of the medium for the production of cellulase by the using submerged fermentation. $, 2010; 1:4. 6. Haq I, Javed MM, Khan TS. An innovative approach for hyper production of cellulolytic and hemicellulolytic enzymes by consortium of MSK-7 and MSK-10., 2006; 5: Immanuel, G., Dhanusha, R., Prema, P. and Palavesam, A. (2006). Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. $, 2006;3, Irfan M, Syed QA, Gulsher M, Abbas S, Nadeem M, Baig S. Pretreatment of corncobs for the production of hydrolytic enzymes from -IR01. Int. J Agr. Vet. Med. Sci. 2010; 4(3):81-87.

8 9. Jaafaru MI, Fagade OE. Optimization studies on cellulase enzyme production by an isolated strain of YL128. %&', 2010; 4(24): Juwaied AA, Al-amiery AAH, Abdumuniem Z, Anaam U. Optimization of cellulase production by and using sugar cane waste. (! ',2011; 2(2): Kang SW, Park YS, Lee JS, Hong SI, Kim SW. Production of cellulase and hemicellulase by KK2 for lignocellulosic biomass. 2004; 91: Kavitha S, Nagarajan P. Fermentative production of Endoglucanse - kinetics and Modeling. $ 2011; 3(3): Kim KC, Yoo SS, Oh YA, Kim SJ. Isolation and characteristics of FJ1 producing cellulases and xylanase, %& 2003; 13: Lee SM, Koo YM. Pilot-scale production of cellulase using rut C-30 in fed-batch mode.%& 2001; 11: Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. Microbial cellulose utilization: Fundamentals and biotechnology. %&%'2002;66: Malik SK, Mukhtar H, Farooqi AA,Haq I: optimization of process parameters for the Biosynthesis of cellulases by #) 2010; 42(6): Mandels M, Weber J. The production of cellulases. Adv. Chem. Ser, 1965;95: Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugars. 1959;31: Ojumu TV, Solomon BO, Betiku E, Layokun SK, Amigun B. Cellulase production by Linn isolate NSPR 101 fermented in sawdust, bagasse and corncob. 2003;2(6): Ramamurthy V, Kothari RM. Comparison of fungal protease production by submerged and surface cultivation. 1993; 27,: