Biorefinery of Chlorella sp. using integrated multiphasic systems for biofuel, feed and wound healing application

Microalgae have been explored as a sustainable alternative to fuel and feed on natural resources. Microalgae possess numerous advantages over their renewable counterparts such as soybean and palm oil. It does not compete with agricultural land or freshwater for food crop production, making it a potential biofuel source. However, commercialisation of microalgae biodiesel is yet to make a presence in the billion-dollar biofuel industry due to the bottlenecks. These include rigid microalgae cell wall, low biomass concentration in the harvested culture and high downstream costs. Therefore, a fossil fuel-derived concept of refinery can be introduced to microalgae to overcome as aforementioned challenges. This project aims to focus on the algae downstream process for biorefinery applications. First, a novel biocomponent extraction method, named sugaring-out assisted liquid biphasic electric flotation (LBEF) system, for protein separation from Chlorella vulgaris was developed. High yield of proteins (69.66±0.86 %) was extracted from microalgae with a rapid and single-step process. Following this, a multiphase integrated system that focused on the extraction of two or more biomolecules in microalgae was introduced. This system focused on simultaneous component extraction rather than conventional cascade approach. The system were incorporated in two different studies. First study aimed to extract two biomolecules (protein and lipid), whereas second study focused on a concurrent three biomolecules extraction approach. The parameters of this system such as volume ratio of ammonium sulphate and t-butanol, flotation air flowrate, flotation time, ultrasound pulse settings and pH were optimised to achieve a high recovery of biomolecules. Highest yield of protein, lipids and carbohydrates were observed at 96.59±8.15 %, 61.02±0.91 % and 52.69±1.90 %, respectively. Control run without flotation technique resulted in lower yield of proteins, lipids and carbohydrates at 25.33±3.50 %, 52.96±4.59 % and 32.44±0.29 %, respectively. Whereas, control run without flotation and cell-disruption technique had lowest yield of proteins, lipids and carbohydrates at 16.73±1.26 %, 51.13±6.27 % and 26.21±0.23 %, respectively. Besides, a large-scale set-up up to 10-15 times was tested out. Recycling ability of the chemicals involved in the extraction were presented. Up to 90 % of the alcohols and salt used in the experiment were recycled. Lastly, the extracted proteins from the multiphase integrated system were purified and its application in wound healing of human keratinocyte cells was investigated. Proteins were adsorbed on a gelatine-glutaraldehyde membrane. This membrane system was used to observe the wound healing of keratinocytes. The biocompatibility, cell adhesion, proliferation and wound scratch of human keratinocyte cells were studied and presented. Overall, multiphase integrated system presented in this project serves as a successful demonstration of microalgae biorefinery concept. The improved yield of biomolecules provide potential applications of microalgae in biofuel, food and medicine field industry. Future studies should focus on analysis of life-cycle cost and optimising the operational cost required for this whole biorefinery set up. The project presented in this thesis offers a platform for future biorefinery research and possible commercial large-scale utilisation