Production of thermostable pullulanase from Bacillus Flavothermus KWF-1 in fed-batch culture

Abstract

Optimization of pullulanase production by Bacillus flavothermus KWF-1 in fed batch cultures was carried out using 2 L bioreactor with working volume of 1.5 L. Fermentation was initiated with batch culture at 50'C and agitation speed of 200 rpm using PYE medium consisted of 2.0% (w/v) sago statch, 1.75% (w/v) peptone, 0.5 (w/v) yeast extract, 0.1% (w/v) KH2PO4 and 0.02% (w/v) MgSO4.7H2O. The initial pH of hte medium was adjusted to 7.5 using 0.1M of NaOH. During batch culture fermentation the highest pullulanase activity (0.080. U/ml) was detected at early stationary phase (t=76h) with specific activity of 0.0213 U/mg. Fed batch culture was initiated after 96 hour when starch was completely depleted (S=0). Optimization of pullulanase production in fed batch culture was performed based on feeding mode, carbon concentration and nitrogen source. Exponential feeding mode with flow rate of 0.01 L/h-1 and sago starch at concentration of 2% (w/v) resulted in highest pullulanase activity with 0.171 U/ml and specific activity of 0.066 U/mg which was respectively 2.1 and 3.1 fold higher than in batch culture. Screening of suitable single nitrogen source (organic and inorganic) for enhancement of pullulanase production shown addition of 0.5% (w/v) yeast extract as single nitrogen source gave highest pullulanase activity of 0.133 U/ml which was 1.7 fold higher than in batch culture as compared to other organic and inorganic nitrogen sources. Feeding medium supplemented with 0.5% (w/v) of (NH4)2SO4 enhanced pullulanase specific activity by 3.2 fold as compared to batch culture. The optimization of carbon an nitrogen concentration using sago starch and (NH4)2SO4 enhanced pullulanase specific activity by 3.2 fold as compared to batch culture. The optimization of carbon and nitrogen concentration using sago starch and (NH4)2AO4 was carried out using Response Surface Methodology (RSM). The optimum conditions obtained were 2.01% (w/v) of sago starch and 0.41 (w/v) of (NH4)2SO4. The optimized medium improved pullulanase activity up to 68.8% (0.13557 U/ml) as compared to batch culture.