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Current Trends in Biotechnology and PharmacyVol. 5 (1) 982-992 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)983pentose sugars fermenting yeast, Pichia stipitishas shown promise for industrial application forethanol production due to its ability to fermentwider range of sugars rapidly with a high ethanolyield and apparently produces no xylitol (8).In this communication, enzymatic hydrolysisof NaOH delignified GS was carried out usingcellulases derived from Aspergillus sp. RCAL-5 for the recovery of fermentable carbohydrates.The fermentation of GS enzymatic hydrolysatewas undertaken for ethanol production from P.stipitis NCIM3498 under batch conditions using10 l capacity fermenter.Materials and MethodsRaw MaterialGS were collected from Chagalamarri,Kurnool Dt. Andhra Pradesh. The material wasfurther processed in a laboratory disintegrator toattain a particular size between 4-10 mm followedby washing with tap water until the washings wereclear and colorless and then gently dried at 40 °Cfor overnight. This material was used throughoutall the experiments.Delignification750 g of dry GS pulverized material wassuspended in 1 N NaOH solution (1:10 ratio) andautoclaved at 121 °C for 30 min. The contentswere filtered with two layers of muslin cloth andthe solid residue was repeatedly washed withwater until the pH of the filtrate became neutral.The residue was dried at 40 °C for overnight andsubsequently used for enzymatic hydrolysisexperiments.Microorganisms and mediaP. stipitis NCIM3498 was procured fromNational Collection of Industrial Microorganisms(NCIM), NCL, Pune, India. It was revived andmaintained on MGYP (Malt extract 0.5%,glucose1%, Yeast extract 0.5% and Peptone0.5%, Agar 2%) slants at 30 o C. The culturecultivation conditions of P. stipitis NCIM3498were followed as described by Nigam (9).Aspergillus sp. RCAL-5 showing cellulaseactivity on 2% CMC agar (Figure 1a, 1b) wasisolated from the discarded lemon feedstock froma local vegetable market and maintained on PotatoDextrose Agar medium, subcultured at monthlyand kept at 4 o C.Fig. 1 (a). Aspergillus sp. RCAL-5, a novel isolatefrom natural resources grown on Potato Dextrose AgarmediumFig. 1(b). Culture supernatant showing Cellulaseactivity on 2%CMC agar and control without anycellulase activity.Chandra Sekhar et al
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
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