full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 982-992 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)984Inoculum preparation and cellulaseproduction from Aspergillus sp. RCAL-5The inoculum for cellulase production fromAspergillus sp. RCAL-5 was prepared accordingto the method of Chandel et al. (10) after somemodifications. The medium ingredients were keptsame except carbon source. NaOH pretreatedwheat bran (1 N, 110 o C, 15 min) was used as asole carbon source (10 g l -1 ). Cellulase wasproduced by Aspergillus sp. RCAL-5 using thegrowth medium consisted of (g l -1 ): alkalipretreated wheat bran 10, peptone 3, yeast extract3, KH 2PO 42, MgSO 41, CaCl 21 and olive oil 2.Erlenmeyer flasks (1 l) containing of 250 mlgrowth medium was inoculated with a sporessuspension (10 4 spores ml -1 ) and incubated at 28oC for 4 days at 150 rpm. Parametric optimizationstudies were done using one parameter at-a-timeapproach to know the most influential growthparameters for the maximum cellulase productionfrom isolate, Aspergillus sp. RCAL-5 under shakeflask conditions. The parameters involved were- effect of incubation time (1-7 days), carbonsource (dried distillery grains, wheat bran, bengalgram, groundnut shell), nitrogen source (yeastextract, peptone, ammonium nitrate, sodiumnitrate, casein, potassium nitrate, ammoniumchloride), pH (3.5–6.5), temperature (24-36 o C)and agitation (50-250 rpm). The obtained culturefiltrate was recovered by centrifugation (10,000rpm for 10 min at 4 o C) and assayed for FPase(Filter paperase), CMCase (Carboxy methyalcellulase), β-glucosidase and xylanase. Therecovered culture filtrates were subsequentlylyophilized (Scanvac, Coolsafe, Denmark) and theenzyme powder was used for saccharificationexperiments.Enzymatic hydrolysisEnzymatic hydrolysis of 532.5 g (dry wt.)of NaOH pretreated GS was carried out in 10 Lround bottom glass vessel containing 8 l of citratebuffer (pH 5.0, 50 mM, 50 °C) at 100 rpm. Thecellulosic substrate was soaked in the citratebuffer for 2 h before adding the enzymaticsolution. Sodium azide was added at aconcentration of 0.005 % to restrict any microbialgrowth during the course of enzymatic hydrolysis.The substrate soaked in citrate buffer wassupplemented with enzyme cocktail fromAspergillus sp. RCAL-5: cellulase 30 FPU g -1 ,β-glucosidase 55 U g -1 and xylanase 150 U g -1 ofthe dry substrate. Samples were withdrawn atvarious intervals, centrifuged and supernatantanalyzed for total reducing sugars released.The saccharification efficiency wascalculated as:Hydrolysis (%) =Reducing sugar concentration obtained x 0.98x100Amount of NaOH pretreated substrate takenInoculum preparation and ethanolfermentationInoculum was prepared by harvesting theyeast cells grown for 24 h at 30 °C in the culturemedium containing 10.0 g l -1 each of xylose andglucose with the medium defined by Nigam (9)at 150 rpm.The fermentation medium consisted of 7 lgroundnut shell enzymatic hydrolysatesupplemented with the defined media ingredientsdescribed by Nigam (9). Fermentation of GSenzymatic hydrolysate (49 g l -1 total reducingsugar) was carried out in 10 l capacity glassfermenter (Sartorius B Plus, Bangalore, India)with a working volume of 7 l. The GS enzymatichydrolysate (7 l volume) was taken andsupplemented with the other medium ingredientsas described by Nigam (9) to form a completemedium prior to sterilization at 120 °C for 20 min.Fermentation of Enzymatically Saccharified Groundnut