Development of an efficient protein recovery system using liquid biphasic flotationTools Sankaran, Revathy (2018) Development of an efficient protein recovery system using liquid biphasic flotation. PhD thesis, University of Nottingham.
AbstractExtensive development of biotechnology over the past several decades has induced a great impact in the production of biological products in various industries. To date, major challenges in the biotechnology industry includes the production process of biomolecules with high purity and low cost, whilst retaining functionality. The conventional downstream processing of valuable bioproducts which are widely employed usually involves multi-processing steps, high energy and chemical consumption and often has a large influence on the cost of the finishing product. Therefore, the demand for cost-efficient and simple downstream processes has directed towards an intensive research for exploiting novel separation tool that can achieve high level of product purity with minimum number of processing stages and greener approach. The aim of this thesis is to develop a new separation method that has minimum number of steps, environmentally friendly and with the ability to achieve maximum level of product purity. Liquid biphasic flotation system is a novel technique which incorporates the principles of aqueous two-phase systems and mass transfer mode of solvent sublation. This system has been proposed as an ideal purification technique for separation, purification and concentration of biomolecules. Liquid biphasic flotation have been utilised previously to purify several biomolecules whilst maintaining their functionality. This thesis emphasised on extending the applications, improvising and to diversify liquid biphasic flotation technique as an efficient tool for downstream processing. This thesis has four objectives, in which all the objectives highlight on the usage of liquid biphasic flotation system for maximum biomolecules extraction. The initial part of liquid biphasic flotation application study is to investigate the effect of full and continuous recycling of alcohol and salt phase components in large scale liquid biphasic flotation system for lipase extraction. In this section, main focus was to optimize operating conditions for the recycling of both phase component and to investigate the competence of recycling phase components using liquid biphasic flotation system on a large scale. The liquid biphasic flotation system investigated is composed of 1-propanol and ammonium sulphate whereby both phase components went through complete recycling process. From the results obtained, it is exhibited that by reusing the bottom phase, separation efficiency of lipase was sustained beyond 77.33 % and yield with 80%. This study showed that the recovered phase components could be recycled effectively up to four cycles and able to produce a significantly high yield of lipase. Next study was on a novel approach of liquid biphasic flotation system for lipase recovery utilizing recycling phase components comprising surfactant and sorbitol. This novel method utilized Triton X-100 and xylitol for lipase extraction from Burkholderia cepacia. The scope of this study focuses on eliminating pollution and environmentally friendly process for enzyme extraction via liquid biphasic flotation. This scope is achieved by utilising phase forming components that have recovery and recycling abilities to minimize the use of chemicals for enzyme extraction. A set of optimum conditions were identified which provides a high yield of lipase with 87.49 % and separation efficiency of 86.46%. From the recycling study, it is revealed 97.20% and 98.67% of Triton X-100 and xylitol respectively were recovered after five times of recycling and 75.87% lipase separation efficiency was obtained. Third objective is on the study of the integration process of fermentation and separation of lipase from Burkholderia cepacia using liquid biphasic flotation. Integration process exhibited high lipase separation efficiency reaching 92.29% and a yield of 95.73%. This study has proven the diversification of liquid biphasic flotation system in integration of upstream and downstream processes. Since liquid biphasic flotation system can be utilised for various type of biomolecules, the final study was done to examine the integration process of sonication and protein extraction from microalgae using sugaring-out effect. Various operating conditions were assessed for high separation efficiency and yield of protein. Maximum protein separation efficiency of 86.38% and yield with 93.33% were attained from this integration process. This study demonstrated that liquid biphasic flotation system could be integrated with ultrasound for protein separation. This thesis demonstrates the importance and diverse applications of liquid biphasic flotation for biomolecules extraction. This study has led to several novel discoveries of liquid biphasic flotation applications with economic downstream processes on an industrial scale.
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