Optimization of sulphuric acid pre-treatment of Acacia saw dust through box-bhenken design for cellulase production by B. Subtilis

Aasiya Anjum, Muhammad Irfan, Fouzia Tabbsum, Hafiz Abdullah Shakir, Javed Iqbal Qazi


Background: Cellulases are enzymes which are capable of degrading lignocellulosic biomass. The current study is centred on optimization of dilute sulphuric acid pre-treatment of Acacia saw dust for maximizing cellulase production (CMCase and FPase). Hydrolysis or saccharification of lignocellulosic biomass is brought about by cellulases and the sugar thus released can be used for further bioethanol production.

Methods: Box- Bhenken design (BBD) was employed for optimization of pre-treatment conditions for Acacia saw dust. Three variables i.e. sulphuric acid concentration (0.6%, 0.8% and 1.0% v/v), substrate concentration (5%,10% and15%)  and reaction time (4h,6h and 8h) was optimized. The pre-treated saw dust was used in the study as a substrate for producing cellulase enzyme through submerged fermentation by Bacillus subtilis (K-18).

Results: An optimum conditions i.e. (0.8% H2SO4 conc., 15% substrate conc. and 4h of reaction time) yielded highest filter paper activity (1.3617 IU/ml/min) and CMCase activity (0.7783 IU/ml/min). The suggested model was significant as revealed by F-value, coefficient of determination (R2) andP-value.

Conclusion: Results concluded that pre-treated substrate (Acacia sawdust) significantly increased cellulase production as compared to untreated substrate that could be utilized for further biofuel production.

Full Text:



Yaliwal VS, Adaganti SY, Banapurmath MR, Tewari PG. Renewable and sustainable fuel production from woody biomass. Indian Journal of Chemical Technology, (2015); 22: 61-66.

Kuhad RC, Gupta R, Singh A. Microbial cellulases and their industrial applications. Enzyme Research, (2011); doi:10.4061/2011/280696.

Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. International Journal of Molecular Sciences, (2008); 9: 1621–1651.

Zhang PH, Himmel ME, Mielenz JR. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, (2006); 24: 452 – 481.

Kirk OT, Borchert V, Fuglsang CC. Industrial enzyme applications. Current Opinion in Biotechnology, (2002); 13: 345–51.

Maki M, Leung KT, Qin W. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. International Journal of Biological Sciences, (2009); 5:500-516.

Sadhu S, Maiti TK. Cellulase production by bacteria: a review. British Microbiology Research Journal, (2013); 3: 235–258.

Tarek AAM, Nagwa AT. Optimization of cellulase and glucosidase induction by sugarbeet pathogen Sclerotium rolfsii. African Journal of Biotechnology, (2007); 6(8): 1048-1054.

Li W, Du W, Liu DH. Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology. Journal of Molecular Catalaysis B Enzymatic, (2007); 45:12–127.

Arooj A, Irfan M, Tabsum F, Shakir HA, Qazi JI. Effect of dilute sulphuric acid pretreatment on cellulase production by Bacillus subtilis K-18 through response surface methodology. Proceedings of the Pakistan Academy of Science B; Life and environmental Science, (2017); 54(1): 11-20.

Acharya PB, Acharya DK, Modi HA. Optimization for cellulase production by Aspergillus nigerusing saw dust as substrate. African Journal of Biotechnology, (2008); 7(22): 4147-4152.

Ojumu TV, Solomon BO, Betiku E, Layokun SK, Amigun B. Cellulase production by Aspergillus flavus Linn Isolate NSPR 101 fermented in sawdust, bagasse and corncob. African Journal of biotechnology, (2003); 2(6):150-152.

Padilha QM, Carvalho LCT, Dias PVS, Grisi CSL, Honorato da Silva FL, Santos SFM, Araujo DAM. Production and characterization of thermophilic carboxymethyl cellulase synthesized byBacillus sp. growing in sugarcane bagasse in submerged fermentation. Brazilian Journal of Chemical Engineering, (2015); 32(1).

Patagundi BI, Shivasharan CT, Kaliwal BB. Isolation and characterization of cellulase producing bacteria from Soil. International Journal of Current Microbiology and Applied Sciences, (2014); 3 (5): 59-69.

Gilna VV, Khalil KM. Cellulase enzyme activity of Aspergillus fumigatus from mangrove soil on lignocellulosic substrate. Recent Research in Science and Technology, (2011); 3(1): 132-134.

Iqbal S, Irfan M, Tabassum F, Shakir HA, Qazi JI. Application of Box-Behnken design for optimization of different pre-treatments conditions for cellulase production. Journal of Northeast Agricultural University, (2017); 24(3): 51-59

Arshad F, Irfan M, Shakir HA, Tabbsum F, Qazi JI. Optimization of dilute sulphuric acid pre-treatments of peanut shells through Box- Bhenken design for cellulase production by Bacillus subtilis K-18. Punjab University Journal of Zoology, (2017); 32 (1): 81-90.

Irfan M. Mushtaq Q, Tabassum F, Shakir HA, Qazi JI Carboxymethyl Cellulase production optimization from newly isolated thermophilic Bacillus subtilis K-18 for saccharification using response surface methodology. AMB Express, (2017); 7:29.

Khalid S, Irfan M, Shakir HA, Qazi JI. Endoglucanase producing potential of Bacillus species isolated from the gut of Labeo rohita. Journal of Marine Science & Technology, (2017); 25(5): 581-587.


  • There are currently no refbacks.