Employing 13C tracers to elucidate bacterial and microalgal metabolism in dynamic systems
Date
2019
Authors
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Publisher
University of Delaware
Abstract
Climate change is one of the most threatening problems facing humanity. Instead of burning fossil fuels and increasing the atmospheric carbon dioxide (CO2) concentration, an alternative is to recycle CO2 by fixing it as cellular biomass and converting it to biofuels. One promising biofuel production strategy is to use Chlorella vulgaris, a photosynthetic microalga, to capture CO2 from large point-source emitters and produce biodiesel from its lipids or use its carbohydrates to feed heterotrophic fermentation processes. However, due to inefficiencies in carbon metabolism, critical technical bottlenecks remain to economic production of microalgal biomass. Therefore, the first aim of this dissertation is to use 13C tracers to obtain a quantitative understanding of Chlorella vulgaris’s metabolism in multiple trophic conditions while being subjected to cycling substrate availability and nitrogen depletion stress. ☐ In addition to fuels, many chemicals are made from petroleum so there is a need to develop renewable chemical production systems. Escherichia coli has been a biotechnological workhorse since the development of recombinant DNA technology. Many strains have been engineered to produce useful metabolites and it is common that they require complex media to achieve industrially relevant rates and titers. To support further metabolic engineering of these strains, it would be advantageous to have a quantitative understanding of the flow of carbon through their metabolic pathways. However, the existing methodologies to quantify in vivo metabolic fluxes using 13C metabolic flux analysis necessitate growth in minimal media. Therefore, the second aim of this work is to develop a 13C metabolic flux analysis methodology for elucidating fluxes of cells grown in complex media. ☐ This dissertation begins with the development of a method to measure the carbohydrate composition and stable-isotope labeling in biomass using GC/MS. The method consists of two-stage hydrochloric acid hydrolysis, followed by chemical derivatization of the released monomer sugars, and then quantification by GC/MS. ☐ Next, the metabolism of C. vulgaris is characterized in auto-, hetero-, and mixotrophic conditions. Light cycling and nitrogen depletion are major determinants of biomass composition. In the dark, the autotrophic cells cannot fix CO2, so they must consume their starch reserves for maintenance energy. The cells change starch production/consumption mode within one hour of light condition change. Growing C. vulgaris cells (nitrogen replete phase) are determined to mostly be composed of protein. Upon nitrogen depletion, however, the cells change their composition and the dominant macromolecules become carbohydrates and fatty acids. ☐ Polysaccharide secretion is a major source of starch turnover in C. vulgaris. Culturing heterotrophic microbes (isolated from soil) with C. vulgaris leads to increased biomass production. Understanding the interaction between autotrophs and heterotrophs is important because large-scale microalgal production facilities will likely be open ponds where contaminating organisms could easily enter the culture from the air. ☐ Towards the second aim, tracer experiments with [U-13C]glucose and yeast extract are used to determine the contributions of each substrate to biomass formation. The results suggest that the proteinogenic amino acids glycine, alanine, aspartate/ asparagine, and tyrosine are made from glucose even if these amino acids are present in the medium. A novel methodology to perform 13C-metabolic flux analysis of E. coli in the presence of glucose and yeast extract is finally presented and used to analyze wild-type E. coli.