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Multifaceted analysis of algal biomolecules: from intra-organism imaging to separations of extracellular polymers
Lesco, Kaitlin Cherry
Lesco, Kaitlin Cherry
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2024
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2026-04-09
Abstract
The success of algal biotechnology hinges on the ability to efficiently cultivate algae at scale and subsequently convert the biomass into high-value products. Despite the vast potential of microalgae in applications ranging from biofuels to pharmaceuticals, significant challenges in growth, harvesting, and product conversion persist, limiting the widespread adoption of algal biotechnology. A promising avenue for addressing these challenges lies in enhancing our understanding of the culture biology, ecology, and the macromolecular composition of algae. To achieve this, the application of analytical chemistry is vital.
A critical issue in analytical practices when trying to characterize the compounds inherent to microalgae is the challenge of performing analyses in native environments, which often leads to gaps in our knowledge regarding the structures, functions, and in situ interactions of biomolecules. Specifically, ion diversity, and high ionic strengths which make up the native environment of algal microorganisms’ complicate analyses. Therefore, adopting analytical methods capable of preserving the native structures of algal macromolecules is essential for supporting the growth of the algal biotechnology sector, facilitating more efficient product development, scale-up, and application implementation.
This thesis presents a series of studies that develop and optimize analytical methodologies designed to investigate the molecular properties of microalgae and their secreted extracellular polymeric substances (EPS) while preserving their native structures and conformations. This approach facilitates the analysis of the biochemistry in a manner that closely resembles in vivo conditions.
A novel approach to lipidomic analysis of Arthrospira platensis is explored through the application of ultra-high-resolution imaging mass spectrometry. In Chapter 3, the optimization of matrix composition and application facilitated untargeted in situ lipidomics and enabled the spatial resolution of the lipidome. This spatial mapping of lipids enhances the understanding of their distribution and localization within the organism, providing insights into cellular functions and metabolic pathways.
Challenges in microalgae characterization extend beyond the microorganisms to their secreted polymers. Chapters 4 and 5 address analytical challenges in EPS characterization through the use of asymmetrical flow field-flow fractionation (AF4) coupled with multiple online detectors. An analytical pipeline consisting of AF4 separation with online multi-angle light scattering detection (MALS), followed by offline high-performance anion exchange chromatography (HPAEC) and liquid chromatography-mass spectrometry (LC-MS), was established to investigate the molecular weight distributions and aggregation behavior of these complex hydrocolloid polymers. Analysis of EPS from Chlorella vulgaris revealed different polymers with distinct primary structures and molecular weight distributions. This project further investigates polymer aggregation behavior in varying ionic environments, observing that elution patterns are influenced by ionic strength and electrolyte chaotropicity/kosmotropicity. These findings highlight how EPS physical properties change under ideal separation conditions compared to those that more closely resemble native high-ionic-strength environments, indicating larger molecular weights and a greater propensity for aggregation at native ionic strengths.
The final chapter expands upon the analytical methodology developed in Chapter 4 to analyze the structural properties of EPS. The physical and structural properties of EPS secreted by Picochlorum celeri were investigated as a function of harvest time and nutrient conditions using AF4-MALS. This work reveals for the first time the distribution of conformations that comprise EPS, demonstrating how molecular weight, size, and shape are subject to change when the polymer is produced under different physiological conditions. The data trends not only support that EPS is sensitive to environmental stimuli but also suggest that EPS is deliberately produced to serve biological functions rather than merely acting as a carbon sink when algal growth is stunted by environmental stress.
The overarching goal of this thesis is to develop analytical tools that preserve the native structures and conformations of microalgae and their macromolecules, enabling analyses that closely mimic in vivo conditions. By addressing characterization challenges and laying the groundwork for analyses compatible with the marine environment, this research advances foundational knowledge of the structure and molecular properties of microalgae and their secreted polymers.
 
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