Publications

Increased accessibility of liquid chromatography mass spectrometry (LC-MS) metabolomics instrumentation and software have expanded their use in studies of dissolved organic matter (DOM) and exometabolites released by microbes. Current strategies to annotate metabolomes generally rely on matching tandem MS/MS spectra to databases of authentic standards. However, spectral matching approaches typically have low annotation rates for DOM. In this paper, we developed an alternative approach that uses accurate mass and isotopic fine structure measurements from state-of-the-art ultrahigh resolution Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS) to annotate exometabolomes obtained from lower resolution LC-MS systems. The molecular formula library approach successfully annotated 53% of exometabolome features of the marine diatom Phaeodactylum tricornutum – a nearly ten-fold increase over the 6% annotation rate achieved using a conventional MS/MS approach. Differences in the exometabolome of P. tricornutum grown under iron replete and iron limited conditions revealed 668 significant metabolites, including a suite of peptide-like molecules released by P. tricornutum in response to iron deficiency. Our findings demonstrate the utility of FT-ICR MS formula libraries for extending the accuracy and comprehensiveness of metabolome annotations.

Plant roots and associated microbes release a diverse range of functionally distinct exudates into the surrounding rhizosphere with direct impacts on soil carbon storage, nutrient availability, and contaminant dynamics. Yet mechanistic linkages between root exudation and emergent biogeochemical processes remain challenging to measure nondestructively, in real soil, over time. Here we used a novel combination of in situ microsensors with high-resolution mass spectrometry to measure, nondestructively, changing exudation and associated biogeochemical dynamics along single growing plant roots (Avena sativa). We found that metabolite and dissolved organic carbon (DOC) concentrations as well as microbial growth, redox potential (EH), and pH dynamics vary significantly among bulk soil, root tip, and more mature root zones. Surprisingly, the significant spike of rhizosphere DOC upon root tip emergence did not significantly correlate with any biogeochemical parameters. However, the presence of sugars significantly correlated with declines in EH following the arrival of the root tip, likely due to enhanced microbial oxygen demand. Similarly, the presence of organic acids significantly correlated to declines in pH upon root tip emergence. Overall, our in situ measurements highlight how different exudates released along growing roots create functionally distinct soil microenvironments that evolve over time.

Iron limitation is known to affect the phytoplankton growth in the oceanic surface waters. Less know (but also less studied!) is its role in shaping microbial production in the mesopelagic layer, also called the twilight zone (200-500m below the surface). Siderophores are bacterial metabolites that convert iron bound to proteins or water-soluble compounds into a form accessible to microorganisms. Consequently, siderophores are biomarkers for microbial iron deficiency: the less iron is available, the more efficient the uptake must be. This study established the distribution and uptake of siderophores along the GEOTRACES cruise GP15 (Pacific Meridional Transect). It revealed that concentrations are high not only in iron-limited surface waters but also in the twilight zone underlying the North and South Pacific subtropical gyres. We propose that such bacterial Fe deficiency owing to low iron availability also occurs in twilight zones of other large ocean basins, greatly expanding the region of the marine water column in which nutrients limit microbial metabolism.

Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. This work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Quinones are among the most important components in natural organic matter (NOM) for redox reactions; however, no quinones in complex environmental media have been identified. To aid the identification of quinone-containing molecules in ultracomplex environmental samples, we developed a chemical tagging method that makes use of a Michael addition reaction between quinones and thiols (–SH) in cysteine (Cys) and cysteine-contained peptides (CCP). After the tagging, candidates of quinones in representative aqueous environmental samples (water extractions of biochar) were identified through high-resolution mass spectrometry (HRMS) analysis. The MS and UV spectra analysis showed rapid reactions between Cys/CCP and model quinones with β-carbon from the same benzene ring available for Michael addition. The tagging efficiency was not influenced by other co-occurring nonquinone representative compounds, including caffeic acid, cinnamic acid, and coumaric acid. Cys and CCP were used to tag quinones in water extractions of biochars, and possible candidates of quinones (20 and 53 based on tagging with Cys and CCP, respectively) were identified based on the HRMS features for products of reactions with Cys/CCP. This study has successfully demonstrated that such a Michael addition reaction can be used to tag quinones in complex environmental media and potentially determine their identities. The method will enable an in-depth understanding of the redox chemistry of NOM and its critical chemical compositions and structures.

Understanding the chemical nature of soil organic carbon (SOC) with great potential to bind iron (Fe) minerals is critical for predicting the stability of SOC. Organic ligands of Fe are among the top candidates for SOCs able to strongly sorb on Fe minerals, but most of them are still molecularly uncharacterized. To shed insights into the chemical nature of organic ligands in soil and their fate, this study developed a protocol for identifying organic ligands using ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and metabolomic tools. The protocol was used for investigating the Fe complexes formed by model compounds of lignin-derived organic ligands, namely, caffeic acid (CA), p-coumaric acid (CMA), vanillin (VNL), and cinnamic acid (CNA). Isotopologue analysis of 54/56Fe was used to screen out the potential UHPLC-HRMS (m/z) features for complexes formed between organic ligands and Fe, with multiple features captured for CA, CMA, VNL, and CNA when 35/37Cl isotopologue analysis was used as supplementary evidence for the complexes with Cl. MS/MS spectra, fragment analysis, and structure prediction with SIRIUS were used to annotate the structures of mono/bidentate mono/biligand complexes. The analysis determined the structures of monodentate and bidentate complexes of FeLxCly (L: organic ligand, x = 1–4, y = 0–3) formed by model compounds. The protocol developed in this study can be used to identify unknown organic ligands occurring in complex environmental samples and shed light on the molecular-level processes governing the stability of the SOC.

Marine dissolved organic matter (DOM) is a complex mixture of molecules with varying persistence within the ocean. This study investigated the relationship between DOM composition and its degradation rate in the North Atlantic Gyre. Employing advanced mass spectrometry techniques (LC-Orbitrap with CoreMS data processing), we identified distinct DOM fractions based on their depth distribution and molecular characteristics. Results indicate that more labile DOM, enriched in aliphatic, hydrophobic compounds, is concentrated near the surface, while refractory DOM with distinct chemical properties is distributed uniformly throughout the water column. These findings suggest that processes affecting the removal of hydrophobic compounds, such as aggregation and particle sorption, influence the overall persistence of marine DOM.

This review article summarizes recent advancements in our understanding of trace element speciation in the ocean learned through the GEOTRACES program, an international research program focused on understanding the biogeochemical cycles of trace elements and isotopes in the ocean. Organic ligands are crucial in controlling trace metal speciation and bioavailability, particularly for iron. By studying ligand distribution, sources, and sinks, GEOTRACES has contributed to understanding metal cycling. Recent analytical advancements have provided molecular insights into metal speciation and bioavailability, highlighting knowledge gaps in how these factors relate to marine productivity and the global carbon cycle.

The marine cyanobacterium Trichodesmium benefits from airborne dust as a nutrient source but also faces potential risks, including buoyancy loss and exposure to toxic metals. This study investigated the effects of desert dust on Trichodesmium, finding that high dust loads above a threshold resulted in sinking colonies that would no longer be able to stay within the sunlit surface ocean. While dust-borne metals can be toxic, the lethal dose was significantly higher than observed environmental concentrations. Although the potential for particle removal as a detoxification mechanism was explored, evidence did not support this hypothesis. Overall, the study suggests that the nutritional benefits of dust acquisition outweigh the associated risks for Trichodesmium, highlighting the importance of dust in marine productivity.

The speciation of most biologically active trace metals in seawater is dominated by complexation by organic ligands. This review traces the history of work in this area, from the early observations that showed surprisingly poor recoveries using metal preconcentration protocols to the present day, where advances in mass spectroscopy and stable isotope geochemistry are providing new insights into the structure, origin, fate, and biogeochemical impact of organic ligands. 

Subtle variations in stable isotope ratios at natural abundance are challenging to measure but can yield critical insights into biological, physical, and geochemical processes. Well-established methods, particularly multicollector, gas-source, or plasma isotope ratio mass spectrometry, are the gold standard for stable isotope measurement, but inherent limitations in these approaches make them ill-suited to determining site-specific and multiply substituted isotopic abundances. Here, we report the first use of Fourier transform ion cyclotron resonance mass spectrometry for the accurate and precise determination of δ13C and δ15N in caffeine isotopologues. We further report the ability to make these measurements with online liquid chromatography, enabling applications of this approach to complex mixtures.

The photosynthetic and diazotrophic cyanobacterium Trichodesmium is a critical marine organism responsible for up to half of ocean nitrogen fixation. Trichodesmium form colonies that harbor a distinct microbiome that expand Trichodesmium’s functional potential and is predicted to influence the cycling of carbon, nitrogen, phosphorus, and iron (C, N, P, and Fe). To link the bacteria associated with Trichodesmium to key functional traits and elucidate how community structure can influence nutrient cycling, we characterized Red Sea Trichodesmium colonies using metagenomics and metaproteomics. The analysis supports Trichodesmium as an active hotspot for C, N, P, Fe, and vitamin exchange. In turn, Trichodesmium may rely on associated bacteria to meet its high Fe demand as several can synthesize photolabile siderophores which can enhance the bioavailability of particulate Fe to the entire consortium. Trichodesmium’s reliance on its microbiome and the observed redundancy of key functional traits likely underpins the dominance and resileincy of Trichodesmium.

Accurately quantifying metal-organic species by LC-ICPMS has remained an analytical challenge due to drifting sensitivities during chromatographic separations. This paper developed a new 'gold standard' methods for separating and quantifying metal species from environmental samples that yields near-quantitative recoveries for a wide range of transition metals. 

Marine dissolved organic carbon and nitrogen (DOC and DON) are major global carbon and nutrient reservoirs, and their characterization relies on extraction methods for preconcentration and salt removal. Existing methods optimize for capturing and describing DOC. This study reports an optimized solid phase extraction strategy to recover marine DON for subsequent molecular characterization.  The approach provides a methodological basis for understanding how DON composition varies across the ocean and determine the processes that govern the supply and removal of this critical organic nutrient. 

Current understanding of dissolved iron (Fe) speciation in the ocean is largely based on liquid chromatography mass spectrometry methods that characterize ligands at a molecular level, but the kinetic and thermodynamic metal-binding properties of these metal species has remained difficult to determine.  This paper describes a method for determining Fe–ligand dissociation rate constants (kd) of suites of naturally occurring ligands in seawater by monitoring the exchange of ligand-bound 56Fe with 57Fe using liquid chromatography–inductively coupled mass spectrometry. These measurements provide critical information needed to develop metal speciation models that can deconvolve the molecular complexity found in the environment.

Phytoplankton serve as the foundation of marine ecosystems. A curious aspect of  the most abundant phytoplantkon, prochlorococcus, is their very small genomes and significant diversity across environmental gradients, reflecting genome streamlining that optimizes cellular fitness.  To interpret the structuring role of variations in genetic potential, as well as metabolic and physiological acclimation, we developed a mechanistic constraint-based modeling framework that incorporates the full suite of genes, proteins, metabolic reactions, pigments, and biochemical compositions of 69 sequenced isolates spanning the Prochlorococcus pangenome. Predicted growth rates covaried with observed ecotype abundances, affirming their significance as a measure of fitness. Our study demonstrates the potential to interpret global-scale ecosystem organization in terms of cellular-scale metabolic processes.