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GERF Bulletin of Biosciences 2011, 2(2):29-31 30Reducing sugar quantification by DNS method Table 2: Effect of wood saw dust (WSD) fungal enzyme One gm of 3, 5 Dinitro salicylic acid (DNS) was mixed and chemical treatment for saccharification for bio-energywith 20 ml of 2 N NaOH. Thirty gm of sodium potassium productiontartrate was added and volume was made up to 100 ml. Enzymatic and Autoclaved Un-AutoclavedSubstrate (0.4 ml) was taken in a fresh tube and 0.1 ml of gm % (w/v) gm% (w/v) Chemical treatmentenzyme was added into it, then 1 ml of 3, 5 DNS was mixed in Control 0.60±0.05 0.07±0.03the solution and kept in boiling water bath for 10 min. The Aspergillus fumigatus 1.69±0.01 1.32±0.04samples were with drawn and cooled under running tap water. NaOH (1N) 0.57±0.02 0.072±0.02Ten ml of distilled water was added and reading was taken at H2SO4(1N) 5.52±0.05 1.1±0.02546 nm (Jurcoane et al., 2009).The amount of reducing sugar HCl(1N) 4.67±0.08 0.86±0.01was determination as per method described by Sadasivamand Manickam (1996). Values are presented as mean + standard deviation (n=3)Results and Discussion 2 .5 0 1 4 .0 0 Enzyme hydrolysis from several fungal strains was tested. Hydrolysis of Enzyme (mg/ml) 2 .2 01 2 .0 0 Hydrolysis with acid (mg/ml) 2 .0 0 1 1 .0 0It was found that the values of the reducing sugars obtained 1 0 .0 0 1 .7 2from the WSD are shown in Table 1. T. viride produced 1 .5 0 8 .0 0 7 .0 0enzymes showed lowest value (0.022±0.002 g/l) for 6 .0 0 5 .0 9hydrolysis as well as a saccharification and maximum 1 .0 0 4 .0 7 4 .0 0saccharification was observed (0.119±0.136 g/l) with A. wentii 0 .7 2 2 .0 0generated microbial enzyme. 0 .5 0 1 .3 0 0 .2 5 0 .4 1 0 .02 0 .0 0 0 .1 3 Treatment with 1 N H2SO4 after A. fumigatus extracellular 0 .0 0 0 .03 0 .0 5 -2 .0 0 0 1 3 5 7 17 21enzymatic hydrolysis showed higher value (0.99±0.001g/l). Tim e Inter va l (hour)It increases 24% more than enzymatic saccharification. Most En zym e Su lfu ric a cidlignocellulosic wastes, due to the presence of cellulosecrystallinity, the chemical attack on the cellulose is retarded Fig1: Effect of enzyme and sulfuric acid on hydrolysis of(Mosier et al., 2002). Therefore, chemical pretreatment was wood saw at dust different time interval.necessary to increase the susceptibility of lignocellulose forhydrolysis reaction. Chemical treatment may accelerate the significant effect for saccharification in horticulture waste.rate of reaction and the extent of cellulose hydrolysis Earlier (Nzelibe et al., 2007) also reported that sulfuric acid(Najafpour et al., 2007). hydrolysis was better than alkaline hydrolysis. Perhaps WSD waste might have high cellulose and hemicellulose contentsTable1: Effect of wood saw dust (WSD) fungal enzyme and low lignin content. Enzyme is placed beneath the networkand chemical treatment for saccharification. of lignin and hemicellulose components. Pretreatment or hydrolysis with sulphuric acid might have removed and Hydrolysis of wood saw dust Sugar gm% hydrolysed hemicellulose to their monomeric constituent waste (WSD) and lignin hemicellulose cellulose interactions partially Aspergillus fumigatus 0.024±0.001 disrupted. Compared to acid hydrolysis 11.0±0.75 g/l was Rhizopus 0.026±0.005 found better than enzyme hydrolysis (2.20±0.08 g/l) in Fig.1. Trichoderma viride 0.022±0.002 This showed acid hydrolysis significantly (P<0.01) enhanced Aspergillus wenti 0.119±0.136 saccharification of saw dust waste. Increasing their Aspergillus fumigatus+ HCl (1N) 0.990±0.001 concentration (1, 3 and 5 N) sulfuric acid lowered hydrolysis Rhizopus+ HCl (1N) 0.893±0.001 (7.7±0.1 g/l) at unautoclaved condition but maximum Trichoderma viride+ HCl (1N) 0.025±0.002 hydrolysis was found same concentration (1 N sulfuric acid) Aspergillus wenti+ HCl (1N) 0.029±0.003 at autoclaved condition (23.4375±0.2 g/l) and 5 N sulfuricValues are presented as mean + standard deviation (n=3) acid does not shows any significant result for hydrolysisBy comparison of enzyme and chemical hydrolysis, it was compared to low acid concentration (1N and 3 N). As clearlyfound that autoclaved enzyme treatment followed by stated by the numbers, the sugar concentration wassulphuric acid hydrolysis resulted in maximum saccharifica- increased with an increase in the acid concentration thattion (5.52±0.05 g/l) in Table 2. It was approximate increase of was applicable to the acid, catalyzed the hydrolysis process.5% than unautoclaved but sodium hydroxide showed no The catalyst activity was proportional to H+ concentration. www.gerfbb.com
31 GERF Bulletin of Biosciences 2011, 2(2):29-31The more hydrogen ions formed in the solution, the more hydrolysis of pretreated palm oil lignocellulosicrapid the hydrolysis process occurred (Mosier et al., 2002). wastes. IJE Transactions. 20(2): 147-156.Aonla pomace was used as strong hydrolyser because it 9. Nzelibe HC and Okafoagu CU (2007). Optimizationwas acidic in pH (>2) which help saccharification of wood. of ethanol production from Garcinia kola (bitterThe WSD hydrolyzed with extracellular enzyme, dilute kola) pulp agrowaste. Afr. J. Biotechnol. 6(17):sulfuric acid (1 N) and aonla pomace waste as hydrolyser 2033-2037.produced sugars, 3.28, 23.11 and 2.61 g/l, respectively. It’s 10. Sadasivam S and Manickam A (1996). Biochemicalshowed 11.29% hydrolysis compared to dilute sulfuric acid. Methods, New Age. International Publishers (P) Ltd., New Delhi, India.Conclusion 11. Vintila T, Dragomirescu M, Croitoriu V, Vintila C, Barbu H and Sand C (2010 ). Saccharification of This study revealed that WSD was hydrolyzed at 1.69 g/ lignocellulose using different cellulases. Romanianl, using a A fumigatus extracted crude culture filtrate at pH Biotechnol. Lett. 15(4): 5498-5504.5.0, 30 ºC in acetate buffer 50 mM, while when using 1 Nsulfuric acid at a temperature of 121ºC for 20 min, was 23.3 g/l but in 5 N there was no significant effect. This study alsosuggested that aonla pomace waste could be used ashydrolyser.Reference 1. Akin-Osanaiye BC, Nzelibe HC and Agbaji AS (2005). Production of ethanol from Carica papaya (pawpaw) agro waste: effect of saccharification and different treatments on ethanol yield. Afr. J. Biotechnol. 4(7): 657-659. 2. Badger PC (2002). Ethanol from cellulose: A general review. In: Trends in new crops and new uses (Eds. Janick J. and Whipkey A.). ASHS Press, Alexandria., pp. 17-21. 3. Baig MMV, Baig MLB, Baig MIA and Yasmeen M (2004). Saccharification of banana agro-waste by cellulolytic enzymes. Afr. J. Biotechnol. 3(9): 447- 450. 4. Chandel AK, Chan ES, Rudravaram R, Narasu, Rao LV and Ravindra P (2007). Economics and environmental impact of bioethanol production technologies: An appraisal. Biotechnol. Mol. Bio. Rev. 2(1): 14-32. 5. Jurcoane S, Radoi-Matei F, Toma R, Stelian P, Vintiloiu A and Diguta C (2009). Hydrolysis of agricultural biomass by combined pretreatment and enzymatic methods in order to produce biofuels (ethanol, biogas). Zootehnie si Biotehnol. 42(1): 58-63. 6. Karmakar M and Ray RR (2011). Saccharification of agro wastes by the endoglucanase of Rhizopus oryzae. Ann. Bio. Rech. 2(1): 20-208. 7. Mosier NS, Ladisch CM and Ladich MR (2002). Characterization of acid catalytic domains for cellulosehydrolysis and glucose degradation. Biotechnol. Bioeng. 79(6): 610-618. 8. Najafpour G, Ideris A, and Salmanpour S (2007). Acid www.gerfbb.com