Abstract:
overall objective of this research is to develop a simple, effective and superior protocol to immobilize lipase on woollen cloth, and then combine this immobilized lipase with the spinning disc reactor (SDR) concept to develop a novel type of rotating reactor system: the spinning cloth disc reactor (SCDR). An improved, simple, effective and superior protocol has been developed to immobilize lipase on woollen cloth using polyethyleneimine (PEI) with glutaraldehyde (GA) cross-linking. The success of immobilization was confirmed by FTIR and confocal laser scanning microscope (CLSM), the latter proving that enzyme is well distributed across the wool fibre surfaces throughout the cloth. The optimal protocol (GA at 0.5 % and pH 6, lipase solution pH 6) gave an enzyme loading of 46.8 mg per gram dry cloth with expressed activity of 178.3 U. Zeta potential measurements showed that PEI significantly enhanced the positive charge on woollen cloth and shifted the isoelectric point to approximately 7. Therefore at a lipase solution pH of around 6, the wool-PEI and lipase are oppositely charged, leading to a maximal adsorption of lipase to the wool surface. The performance of the immobilized lipase on woollen cloth was investigated to determine its suitability for industrial application. The immobilized lipase had good reusability maintaining 81.3% of its original activity after 10 runs. The immobilized lipase also displayed good storage stability maintaining 75.8% of the initial activity after storage of 40 weeks at 4 °C. The optimal pH for immobilized lipase in tributyrin hydrolysis was 7, slightly lower than that of the free lipase (pH 8). The optimal temperature for both free and immobilized lipase was 45 °C. The thermal stability of lipase was significantly improved after immobilization. Kinetic studies were conducted by both Lineweaver-Burk plot and nonlinear regression, and the obtained Km was very similar. Km of lipase increased by around 3 times (e.g. from 1.49 mM to 4.60 mM derived from nonlinear regression) after immobilization. The thermal deactivation rate of immobilized lipase followed the Arrhenius law with the thermal deactivation energy of 198.9 kJ mol−1. The immobilized lipase can be applied in biodiesel production, and under optimal conditions (8:1 molar ratio of ethanol to oil, and no water), biodiesel conversion of 76.6% was achieved after 24 h. The immobilized lipase on woollen cloth was successfully combined with the SDR concept to form a novel rotating reactor system: SCDR. The SCDR was characterized using tributyrin emulsion hydrolysis as a model reaction. Both the conversion and reaction rate were improved in the SCDR compared to that in a conventional BSTR under comparable conditions, indicating the reaction intensification has occurred. Conversion increased by approximately 7% on average as the flow rate increased from 2 to 5 mL s-1, and the highest conversion (72.1%) occurred at 400 rpm. The loss of enzyme from the spinning woollen support increased with an increase in surface shear, and this phenomenon was more evident after the surface shear reached around 9,500 s-1 which is hence considered to be the ‘critical shear’ below which this SCDR should be operated. The immobilized lipase showed excellent stability to repeat reactions in the SCDR: 80% of the original activity was retained after 15 consecutive runs. The SCDR was also successfully applied for the hydrolysis of different vegetable oils at reaction rates higher than other reactors in the literature. The flow regimes in the SCDR were characterized by means of residence time distribution (RTD) and visual study tracking dye staining. RTD analysis showed that the flow pattern on the spinning disc with/without cloth became close to plug flow with the increase of spinning speed and flow rate. With the cloth, the number of tanks-in-series (N) was half of that without cloth, indicating that the flow is better mixed, in contrast to the typical plug flow found for conventional SDR. Two flow regimes were observed from the visual dye study: radial finger-like flow and concentric flow. At low spinning speed and high flow rate, the flow was in the form of a few random and uneven radial streams. At higher spinning speed and lower flow rate, the unevenly radial flow was replaced by an even centric flow. Using tributyrin hydrolysis as a model reaction, a SCDR mathematical model based on perfectly mixed model and Ping Pong Bi Bi kinetics was developed to simulate the conversion in SCDR with spinning speed and flow rate, and the model fitted well with the experimental data. The effect of the number of cloth layers in the SCDR on reaction conversion and rate was investigated using tributyrin emulsion hydrolysis as a model reaction. Within the conversion limit, the effect of multi-cloth was quite efficient: both conversion and reaction rate increased significantly with the increase of cloth number. The mean residence time increased with the increase in the number of cloths, due to flow existing inside the volume of multiple cloths. The number of tanks-in-series (N) showed a slight decrease with the increase in the number of cloths, indicating that more cloths increased the well-mixed behaviour of SCDR. Visual study showed that the dye can penetrate through the three layers of multi-cloth at both low (100 rpm) and high (400 rpm) spinning speeds. Generally, the dye did not spread synchronously on all the three layers: very fast on the first layer and slowly on the third layer.