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  • GERF Bulletin of Biosciences December 2011, 2(2):29-31 Short Communication Hydrolysis of wood saw dust by combined chemical pretreatment and enzymatic methods for lignocellulosic saccharification Devendra Kumar1*, Kaushlesh K. Yadav2 and Munna Singh1 1 Department of Botany, Lucknow University, Lucknow-226007, India 2 Department of Biotechnology, Dr. Ram Manohar Lohia Avadh University, Faizabad- 224001, India AbstractWood saw dust (WSD) after lignocellulosic saccharification by different hydrolysis methods is more efficient for ethanolproduction as, its contains cellulose and hemi-cellulose at higher levels 65% (w/v) and 35% (w/v), respectively. Celluloseand hemicellulose account for about a quarter of whole biomass in all land plants. A pretreatment method using chemicalhydrolysis and enzymatic conversion from starch into fermentable sugars was investigated. The WSD was hydrolyzed at1.69 g/l, using a crude culture filtrate Aspergillus fumigatus at pH 5.0 and 30ºC in acetate buffer 50 mM, while 23.3 g/l was with 1 N sulfuric acid (H2SO4) treatment. Aonla pomace waste was used as substitute to acid because of high acidicnature. Optimum conditions for lignocellulosic saccharification is discussed in this paper.Keyword: Wood saw dust (WSD), Aspergillus fumigatus, Acid hydrolysis, Enzymatic hydrolysis. Cellulose, Hemi-cellulose.Introduction Materials and Methods High lignocellulosic agri-horticulture biomasses areemployed as alternative bio-energy (resource) to fossil Sample collectionenergy sources via lignocellulosic saccharification (Vintilaet al., 2010). Two processes used to convert cellulose and The WSD was collected in saw dust from Kakori industrialhemicellulose into biofuel (ethanol) are enzymatic and acid area. The samples were brought to room temperature washedhydrolysis (Akin-Osanaiye et al., 2005; Chandel et al., 2007; with distilled water and used in the experiment.Jurcoane et al., 2009; Karmakar et al., 2011). The mostcommonly adopted technique is acid hydrolysis (Badger et Acid -base hydrolysisal., 2002). Acidic hydrolysis is an effective method used forlignocelluloses raw material pretreatment in saccharification The WSD 25% (w/v) was hydrolyzed with 100 ml (1:2 w/which could change into ethanol. Although acids are v) of of various concentrations of H2SO4, HCl and NaOH atpowerful agents used for biomass hydrolysis, concentrated room temperature treatment for 24 hr. The hydrolysates wereacids are toxic, erosive and hazardous. Handling higher separated to obtain any suspended or unhydrolysatedconcentrations of acid requires reactors that are resistant to materials and was neutralized by 2 N NaOH and 1 N H2SO4erosion in raw material pretreatment. Diluted acid hydrolysis solution for analytical processing then autoclaved at 121­ºCespecially sulfuric acid has been successfully developed for and 15 lbs pressure for 15 min (Nat Steel Equipment Pvt. Ltd,pretreatment of cellulosic materials. India). Another method of hydrolysis is enzymatic hydrolysis. Enzymatic hydrolysisEnzymes are naturally occurring plant proteins that result in The WSD 25% (w/v) were hydrolyzed with various fungalcertain chemical reaction. However, for enzymes to work, enzymes from (10 6 spores) of Aspergillus fumigatus,they must obtain access to the molecules to be hydrolyzed Rhizopus, Trichoderma viridae and Aspergillus wenti) with(Baig et al., 2004). A combined strategy involving acid, baseand enzymatic methods in hydrolysis of saw dust is extra cellular enzymes (i.e. α-amylase, glucoamylase,investigated. cellulase and pectinase) were used in the experiment. The hydrolyate was separated by centrifugation at 12,000 rpm at room temperature.*Corresponding author: dev.biochem@gmail.comCopyright © 2011 Green Earth Research Foundation www.gerfbb.com
  • 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