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Current Trends in Biotechnology and PharmacyVol. 5 (1) 982-992 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)985After sterilization and cooling the medium, 3%(v/v) of inoculum was aseptically transferred tofermentation medium through peristaltic pump.The fermentation was run at agitation 300 rpm,aeration 2 l min -1 , temperature 30 °C, and pH 5.5automatically adjusted with 2 N HCl and 2 NNaOH. Antifoam (Silicone oil), 3.0 ml was alsoadded to the fermentation medium to avoid theexcessive foaming. Sterile syringes were used totake samples at regular time intervals duringfermentation for estimation of residual sugars,biomass and ethanol.Analytical methodsCMCase, FPase, xylanase and β-glucosidase activities were assayed as the methodmentioned in Saha et al. (11). One unit (U) ofeach enzyme activity is defined as the amount ofenzyme, which produces 1 µmol reducing sugaras glucose or xylose in the reaction mixture perminute under the above-specified conditions. β-glucosidase activity was determined by the releaseof p-nitrophenol from p-nitrophenyl-β-Dglucoside.Total reducing sugars (TRS) wereestimated by dinitrosalicylic acid method of Miller(12). Ethanol was estimated by gaschromatography (GC) (Agilent 4890D, USA) asdescribed by Chandel et al. (8). For yeast cellmass determination, biomass was harvested bycentrifugation and washed with 0.9 % (w/v) NaClin duplicates using 5 ml samples, dried at 105 °Cfor 2 h.Results and DiscussionChemical compositionGS used in this investigation contained35.7% cellulose and 18.7% hemicellulose, whichconstitutes total carbohydrate amount of 54.4%on dry solid (DS) basis (Table 1) (13).Table 1. Composition of Groundnut shell cell wallon % dry weight basis. [16]Component% dry weightVolatile solids 68.7Ash content 5.9Organic carbon 48.3Total nitrogen 0.8Cellulose 35.7Hemicellulose 18.7Lignin 30.2Parametric optimizations for FPaseproduction from a novel isolate, Aspergillussp. RCAL-5 under shake flask conditionsParametric studies were conducted to knowthe best parameter ranges influencing themaximum enzyme production from Aspergillussp. RCAL-5 under one parameter at-a-timestrategy.Optimization of incubation timeIncubation time was optimized by measuringthe maximum FPase production from the day 1to 7 at pH 4.5, 150 rpm, NaOH pretreated wheatbran as carbon source (10 g l -1 ), yeast extract asnitrogen source (2.5 g l -1 ) at 28 o C. Figure 2presents the results of incubation period study oncellulase production from Aspergillus sp. RCAL-5. The maximum FPase production (0.75 FPUml -1 ) was observed after 4 days of incubationsubsequently showing a regular down fall. In astudy by Kocher et al. (14) maximum cellulaseproduction (FPase 0.09 and CMCase 0.12 IU ml -1) was observed after 8 days by T. harzianum8230 using rice straw as carbon source.Chandra Sekhar et al
Current Trends in Biotechnology and PharmacyVol. 5 (1) 982-992 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)986Fig. 2. Effect of incubation time on exoglucanaseproduction by Aspergillus sp. RCAL-5. (pH 4.5, 150rpm, wheat bran (10 g l -1 ), yeast extract (2.5 g l -1 ), temp.28 o C.)Effect of carbon sourcesEffect of various NaOH pretreated carbonsources (dried distillery grains(DDG), wheat bran,bengal gram, groundnut shell) was studied formaximal FPase production under the followingconditions (pH 4.5, temp. 28 o C, agitation 150 rpm,yeast extract as nitrogen source (2.5 g l -1 ) andincubation time (4 days). Each substrate waspretreated with dilute sodium hydroxide solution(1 N NaOH, 110 o C, 15 min). A maximum of0.86 FPU ml -1 activity was found using pretreatedwheat bran (10 g l -1 ) as a carbon source (Figure3).Fig. 3. Effect of carbon source on exoglucanaseproduction by Aspergillus sp. RCAL-5 under followingconditions. (pH 4.5, temp. 28 o C, Agitation 150 rpm,yeast extract (2.5 g l -1 ) and incubation time (4 days)).Effect of nitrogen sourcesEffect of various nitrogen sources (yeastextract, peptone, ammonium nitrate, sodiumnitrate, casein, potassium nitrate, ammoniumchloride) was studied for maximal FPaseproduction at conditions (pH 4.5, temp. 28 o C,agitation 150 rpm, wheat bran 10 g l -1 , Incubationtime (4 days). Among all the nitrogen sourcestested, yeast extract (2.5 g l -1 ) showed themaximum FPase production (0.85 FPU ml -1 ).Figure 4 shows the effect of different nitrogensources on cellulase production from Aspergillussp. RCAL-5. Umikalsom et al. (15) showed themaximum FPase production (1.4 U ml -1 ) withpeptone (6 g l -1 ) as nitrogen source from theChaetomium globosum strain 414.Effect of pHThe effect of pH for cellulase productionby Aspergillus sp. RCAL-5 was investigatedwithin the range of 3.5–6.5. The pH was adjustedeither with 1N NaOH or 1N HCl. It was grownfor 7 days under submerged culture conditions(temp. 28 °C, agitation 150 rpm, wheat bran 10 gl -1 , yeast extract 2.5 g l -1 , incubation time (4 days).Figure 5 shows that maximum FPase (0.79 FPUml -1 ) production was obtained at pH 4.5 usingNaOH pretreated wheat bran as sole carbonsource. In general, lower pH is favored for highercellulase titer production from fungi when grownon different lignocellulosic substrates.Effect of temperatureThe effect of temperature on the cellulaseproduction was examined at the range of 24-36oC. The results were given in Figure 6. Theoptimum temperature for maximum FPase (0.84FPU ml -1 ) production was found at 28 o C(agitation 150 rpm, wheat bran 10 g l -1 , yeastextract 2.5 g l - , pH 4.5, incubation time (4 days).These results are in well accordance with thestudy of Narasimha et al. (16) who reportedFermentation of Enzymatically Saccharified Groundnut
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