Microalgae Cultivation on Wastewater and Genetic Modifications to Increase Triacylglycerol Production

Abstract

Microalgae are photosynthetic microorganisms that can grow in a variety of environments. They have become a popular feedstock for the production of biofuels, high-value chemicals, and nutraceuticals. One of the main products obtained from microalgae is biodiesel: a mix of fatty acid methyl esters (FAMEs) obtained from the transesterification of triacylglycerol (TAG). However, microalgal biodiesel still cannot compete economically with petroleoum diesel. Therefore, the objective of this thesis was to explore alternatives to make microalgal cultivation for biodiesel more efficient. Microalgal treatment of municipal wastewater has been discussed as a strategy for the simultaneous removal of excess nutrients and the generation of microalgal biomass. This thesis evaluated the performance of Scenedesmus sp. and Chlorella sorokiniana in the removal of nutrients from non-sterile, highly concentrated synthetic-wastewater. These microalgae removed up to 60% NH4+ and 44% PO43- in a semi-continuous cultivation mode without negatively affecting effluent quality, demonstrating that microalgal growth can be coupled with wastewater treatment. Microalgal research is growing, and efforts to enhance TAG production are the focus of many studies. Reliable genetic modification protocols for many species, such as Chlorella vulgaris, are still not available. Therefore, work with the model Chlamydomonas reinhardtii is a practical alternative for proof-of-concept studies. The work in this thesis generated five Chlorella vulgaris starch mutants through UV-mutagenesis: four low-starch producing and one starch-overproducer. Additionally, four Chlamydomonas reinhardtii transgenic lines expressing Chlorella vulgaris genes involved in glycerolipid metabolism were developed. Mutant st27, with lower starch production than wildtype, displayed up to 3.8-fold increase in TAG, illustrating the importance of the organic carbon source in the medium to provide enough carbon precursors for TAG biosynthesis. Similarly, the transgenic lines increased lipid content more than 100-fold, suggesting that increasing glycerol-3-phosphate availability is crucial to generate oleaginous feedstock. This thesis illustrates the possibility of generating microalgal strains rich in both starch and TAG, which would be a promising feedstock for microalgal biorefineries. Additionally, two new gene candidates to improve TAG yields in microalgal strains are suggested, expanding on the knowledge available for metabolic engineering of microalgal crops for biodiesel production.