Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemical Engineering

Major Professor

George Philippidis, Ph.D.

Co-Major Professor

Ramon Gonzalez, Ph.D.

Committee Member

John Kuhn, Ph.D.

Committee Member

Aydin Sunol, Ph.D.

Committee Member

Sarina Ergas, Ph.D.

Keywords

Hydrolysate, Metabolomics, Mixotrophy, Mutagenesis, Spent Media Recycling

Abstract

The establishment of an algae industry is crucial for addressing global biofuel and bioproduct demand that meets the Sustainable Development Goals (SDG) worldwide. Photosynthetic microalgae are excellent sources for food, fiber, fuel, feed, and fertilizer, which are the 5Fs of the SDG. However, at present, algae-based materials are not cost-competitive or sufficiently sustainable. Improved productivity, lower cultivation cost, and reduced use of resources are required to transition from lab to industry, necessitating the development of superior strains that have high productivity and can tolerate environmental stress, such as temperature and salinity, so they can be cultivated at large scale outdoors using non-potable water. In this context, the present research aims at assessing a two-prong strategy for reducing the cultivation cost and enhancing the sustainability of algae cultivation: (1) using agricultural residues (sweet sorghum bagasse) as a source of renewable organic carbon and employing spent media recycling as a supplemental source of nitrogen and other nutrients; and (2) developing superior-performing algal strains that achieve high biomass and metabolite productivity, while possessing thermotolerance at high temperatures. The innovation of the study lies in the development of double-mutant algal strains using two genetic modification techniques, namely random mutagenesis and adaptive laboratory evolution, and the use of high-throughput multi-omics analysis (metabolomics and lipidomics) to elucidate the genetic modifications that occurred in the high-performing mutants. This way novel genetic targets can be identified to be used in the future to further improve strain performance. In this dissertation, four objectives were set and conducted to achieve the aforementioned goal. First, optimization of lipid productivity in Chlorella vulgaris under mixotrophic cultivation conditions using sweet sorghum bagasse hydrolysate and determination of alterations in the algal metabolism through lipidomics studies. In objective 1, C. vulgaris was successfully cultivated in SSB hydrolysate at optimized conditions achieving the highest lipid yield of 132 mg/L day, which proved that SSB hydrolysate can be used as an alternative carbon source for algae cultivation. In addition, LC-MS based lipidomics study revealed that when algal cells were cultured in SSB hydrolysate, MUFA, PUFA, and neutral lipids were enhanced, while a reduction in structural lipids was observed. In objective 2, a feasibility study was conducted to assess spent media recycling for algae cultivation in a custom-built 5-liter airlift photobioreactor and identify alterations in cell metabolism through metabolomic studies. The study demonstrated the feasibility of cultivating C. vulgaris in spent media, where cells exhibited increased carbohydrate, carotenoid, and C18:3 fatty acid content. Intracellular and extracellular metabolomics provided an insight into the algal cell and laid the groundwork for future improvements in algae tolerance to identified inhibitors. Objective 3 aimed at developing superior performing strains that can tolerate higher temperatures using UV mutagenesis and characterization of their biochemical make-up. Two UV mutants of Tetraselmis suecica were developed that outperformed the wild type and contained more lipids when grown at elevated temperatures. Objective 4 aimed at enhancing thermotolerant algal strains using EMS mutagenesis and adaptive laboratory evolution and determination of metabolic changes associated with such thermotolerance. This objective was accomplished by effectively generating high-lipid EMS mutants and adapting them to 33oC, which resulted in increased MUFA and PUFA production. Lipids produced by algae strains are viewed as promising feedstock for production of biofuels, like biodiesel and jet biofuel, and bioproducts, such as nutraceuticals, cosmetics, and pigments. Hence, this study aspires to develop algal strains tolerant to environmental stressors (sugars, recycled media, and heat) as a means of enhancing algal productivity, while improving the environmental and economic sustainability of future algal biorefineries.

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