RESUMEN
In this research, optimization of the production medium to enhance tannase production by Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor was studied. Amount of substrate Triphala, moisture content, aeration rate, and fermentation period was chosen for optimization study. During one variable at a time optimization, the highest tannase activity of 0.226 U/gds was shown with Triphala as a substrate at the fermentation period of 32 h. Furthermore, the optimum conditions predicted by response surface methodology (RSM) and genetic algorithm (GA) were found to be 11.532 g of substrate Triphala, 47.071% of the moisture content, and 1.188 L/min of an aeration rate with uppermost tannase activity of 0.262 U/gds. In addition, the single hidden layer feedforward neural network (SLFNN) and the radial basis function neural network (RBFNN) of an artificial neural network (ANN) were adopted to compare the prediction performances of the RSM and GA. It revealed that the ANN models (SLFNN, R 2 = 0.9930; and RBFNN, R 2 = 0.9949) were better predictors than the RSM (R 2 = 0.9864). Finally, the validation experiment exhibited 0.265 U/gds of tannase activity at the optimized conditions, which is an 11-fold increase compared to unoptimized media in shake flask.
RESUMEN
Production of tannase was performed in packed bed reactor filled with an inert support polyurethane foam (PUF) using Bacillus gottheilii M2S2. The influence of process parameters such as fermentation time (24-72 h), tannic acid concentration (0.5-2.5% w/v), inoculum size (7-12% v/v), and aeration rate (0-0.2 L/min) on tannase production with PUF were analyzed using one variable at a time (OVAT) approach. The outcome of OVAT was optimized by central composite design. Based on the statistical investigation, the proposed mathematical model recommends 1% (w/v) of tannic acid, 10% (v/v) of inoculum size and 0.13 L/min of aeration rate for maximum production (76.57 ± 1.25 U/L). The crude enzyme was purified using ammonium sulfate salt precipitation method followed by dialysis. The biochemical properties of partially purified tannase were analyzed and found the optimum pH (4.0), temperature (40 °C) for activity, and Km (1.077 mM) and Vmax (1.11 µM/min) with methyl gallate as a substrate. Based on the SDS-PAGE analysis, tannase exhibited two bands with molecular weights of 57.5 and 42.3 kDa. Briefly, the partially purified tannase showed 4.2 fold increase (63 ± 1.60 U/L) in comparison to the submerged fermentation and the production of tannase was validated by using NMR spectrometer.
Asunto(s)
Bacillus/metabolismo , Proteínas Bacterianas/biosíntesis , Hidrolasas de Éster Carboxílico/biosíntesis , Técnicas de Cultivo de Célula/métodos , Fermentación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Taninos/química , TemperaturaRESUMEN
Production of tannase from B. gottheilii M2S2 was studied under solid-state fermentation with an optimized medium consisting of polyurethane foam matrix of dimension 40 × 40 × 5 mm, impregnated with a liquid medium comprising (w/v): 4% tannic acid; 2% NH4NO3; 0.1% KH2PO4; 0.2% MgSO4; 0.1% NaCl and 0.05% CaCl2·2H2O in distilled water, having a pH of 4.7. Maximum tannase production of 56.87 U/L was obtained after 32 h of fermentation at 32 °C in static condition. This study deals with the evaluation of unstructured kinetic models to understand the behavior of biomass, tannase production and tannic acid degradation, with the fermentation time. The growth rate of B. gottheilii M2S2 was 0.0703 h-1 at 32 h of fermentation. Product (Yx/s) and biomass yield (Yp/s) coefficients were estimated as 1.77 U/g of tannic acid and 0.276 g of biomass/g of tannic acid. All the kinetic constants µ, α, ß, m and n were evaluated using MATLAB 2015Rb program. The experimental and model-generated data showed a good correlation, which indicated that these models will describe tannase production and fermentation process.