Molecular-level characterization of microbial interactions with labile dissolved organic matter

Kathryn H. Halloran, Ph.D., 2024
Dr. Elizabeth Kujawinski, Advisor

Submitted to the Department of Earth, Atmospheric, and Planetary Sciences on May 30th 2024, in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy in Chemical Oceanography.

Marine microbes produce and consume labile dissolved organic matter (DOM), generating a carbon flux with significant implications for global carbon cycling and microbial ecosystems. Better understanding this flux requires direct and compound-specific characterization of metabolites, the small organic biomolecules that make up labile DOM. However, these direct measurements are challenging due to low metabolite concentrations, high ambient salt concentrations, and the complexity of labile DOM. More complete characterization of dissolved metabolites is therefore a standing challenge in the field. This in turn leaves many open questions with respect to the specificity of microbe-DOM interactions and the biotic and abiotic drivers of those interactions. This thesis addresses those challenges and questions. In Chapter 2, I explore the compound-specific uptake of metabolites by the copiotrophic gamma-proteobacterium Alteromonas macleodii, with a focus on metabolites derived from the cyanobacteria Prochlorococcus. I find that Alteromonas responds specifically to 3-methyl-2-oxobutanoic acid, a valine intermediate, but not to the other cognate branched chain amino acid intermediate. This substrate selectivity is likely driven by transporter specificity. The distinct fate of these structurally similar molecules emphasizes the importance of compound-specific characterization of labile DOM. To expand our ability to make these compound-specific measurements, in Chapter 3 I develop a method for derivatizing carboxylate-, carbonyl-, and phosphate-containing molecules via aniline derivatization, solid phase extraction, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is able to quantify 51 different metabolites dissolved in seawater, with pM to nM detection limits. I verify the utility of this method by applying aniline derivatization to phytoplankton culture filtrates and field samples. Additionally, I show that the measurements obtained by aniline derivatization are in good agreement with measurements yielded by other methods. Finally, in Chapter 4 I apply aniline derivatization to filtrate from cultures of Prochlorococcus grown under diel light conditions and sampled every two hours. I find that Prochlorococcus not only release metabolites into solution, but also takes those metabolites up again, with diel rhythmicity. Together, this thesis offers new insights into the specificity and subtleties of microbe-DOM interactions, and expands our ability to quantify the metabolites that make up labile DOM.