LC-MS- based metabolomics analysis to determine the effect that varying growth conditions have on microalgae
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Oleaginous, green microalgae are an advantageous source of triacylglycerols (TAGs) for biodiesel production along with other value-added products such as phytosterols, vitamins, and terpenoids. When microalgae are grown under nutrient stressed conditions the TAGs and lipids in general accumulate. Most of the previous analysis on microalgae have focused on how the nitrate concentration affects the production of TAGs and other lipids. These analyses are static, in that they typically focus on the effect of one nutrient and at one time point. These previous analyses are also limited since they analyze lipids as families without providing species-specific information. Also, most of the previous lipidomic analysis of microalgae have focused on model organisms. Our aim was to develop a method to determine the effect of varying nutrient concentrations and growth periods on the hydrophobic metabolites produced by an oleaginous, non-model, green microalgae species, Ettlia oleoabundans. This method was twofold and included sample preparation and LC-MS-based metabolomics analysis. Initially, the growth conditions of interest were determined based on the growth curve and concentrations of nitrate and phosphate remaining in the media at each day. These conditions chosen were nitrogen deplete, nitrogen replete, phosphorous deplete and phosphorous replete at days 2, 4, 7, and 10 which represented early and late exponential and stationary phase, respectively. The method for the microalgae metabolite extraction and sample preparation was then developed and tested based on the extraction efficiency of surrogates and biomass-based normalization. Then a previously optimized LC-MS-based analysis method was tested for a few representative endogenous plant lipids to determine its efficiency in detection and separation. In the next part of the dissertation, this experimental design and methods were used to determine the effect of varying nutrient concentration and growth phases on the hydrophobic metabolites and lipids produced by the microalgae. From this analysis it was determined that TAGs accumulated when the nitrate and phosphate were completely internalized to be used as energy sources during the stationary phase of growth. It was also shown that phosphatidylglycerols and galactolipids, involved in chloroplast and thylakoid membrane structure, were depleted under the nutrient deplete conditions. This trend was in correlation with the depletion of chlorophylls grown under nitrogen deplete conditions. These correlations seem to link the effect of the depletion of membrane structural lipids on the photosynthetic efficiency. Lastly, another group of galactolipids, sulfoquinovosyldiacylglycerols, were shown to be depleted in the late stages of growth. It is proposed that their break down facilitates the use of sulfur to form other important biochemicals such as proteins for cell survival. In the last part of the dissertation, a method was developed for the sample preparation and detection of phytosterols which were below the detection limit in the previous analyses. Solubility-based and solid phase extraction-based separations along with chemical derivatizations were tested. The best method for the preparation and improved signal to noise of phytosterols was the dimethylglycine-based derivatization. In the future this optimized method will be used to determine the effects that the varied growth conditions have on the production of phytosterols in microalgae. Overall this dissertation provides a method for the preparation and analysis of hydrophobic metabolites, lipids and phytosterols in microalgae by LC-MS, along with the analysis of the changes in these metabolites in the microalgae resulting from the varying growth conditions. In the future these metabolomics results will be correlated with the regulations at the transcriptional level to obtain a comprehensive understanding of the biochemical processes in microalgae cells.