21Response actions to the Deepwater Horizon oil spill included the injection of ~771,000 gallons 22 (2,900,000 L) of chemical dispersant into the flow of oil near the seafloor. Prior to this incident, 23 no deepwater applications of dispersant had been conducted and thus no data exists on the 24 environmental fate of dispersants in deepwater. We used ultrahigh resolution mass 25 spectrometry and liquid chromatography with tandem mass spectrometry (LC/MS/MS) to 26 identify and quantify one key ingredient of the dispersant, the anionic surfactant DOSS (dioctyl 27 sodium sulfosuccinate), in the Gulf of Mexico deepwater during active flow and again after 28 flow had ceased. Here we show that DOSS was sequestered in deepwater hydrocarbon plumes 29 at 1000-1200m water depth and did not intermingle with surface dispersant applications. 30Further, its concentration distribution was consistent with conservative transport and dilution 31 at depth and it persisted up to 300 km from the well, 64 days after deepwater dispersant 32 applications ceased. We conclude that DOSS was selectively associated with the oil and gas 33 phases in the deepwater plume, yet underwent negligible, or slow, rates of biodegradation in 34 the affected waters. These results provide important constraints on accurate modeling of the 35 deepwater plume and critical geochemical contexts for future toxicological studies. 36 37 38

The field of metabolomics seeks to characterize the suite of small molecules that comprise the endproducts of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization-liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene-divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from < 1 pM to 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.Metabolomics is an "omics" technique that seeks to measure the small organic biomolecules produced by cells (Oliver et al. 1998;Fiehn 2002). Because these small molecules are the end-products of multiple levels of metabolic regulation, their concentrations provide a temporal snapshot of the metabolic state or phenotype of an organism. In particular, metabolites produced by nonenzymatic reactions, such as those formed by reaction with a radical oxygen species, or whose production is regulated by other small molecules, must be monitored directly because their production cannot be inferred from genomic or proteomic information. Metabolomics can be used as a diagnostic tool, identifying biomarkers of disease within the human metabolome, such as cancers (Armitage and Barbas 2014) and Crohn's Disease (Jansson et al. 2009). Metabolomics has also been applied in a wide range of organisms and environments, examining how metabolite abundances respond to environmental factors. In the oceans, marine metabolites have been a valuable source of new natural products, while other metabolomics applications are still rare but growing. For example, recent marine culture experiments have revealed metabolite production not predicted by genomic information (Baran et al. 2010;Fiore et al. 2015), metabolic shifts in response to a specific metabolite (Johnson et al. 2016), and changes in the quantity and composition of metabolite production during coculturing (Paul et al. 2012). Complementary field ...

Photoautotrophic plankton in the surface ocean release organic compounds that fuel secondary production by heterotrophic bacteria. Here we show that an abundant marine cyanobacterium, Synechococcus elongatus, contributes a variety of nitrogen-rich and sulfur-containing compounds to dissolved organic matter. A combination of targeted and untargeted metabolomics and genomic tools was used to characterize the intracellular and extracellular metabolites of S. elongatus. Aromatic compounds such as 4-hydroxybenzoic acid and phenylalanine, as well as nucleosides (e.g., thymidine, 5'-methylthioadenosine, xanthosine), the organosulfur compound 3-mercaptopropionate, and the plant auxin indole 3-acetic acid, were released by S. elongatus at multiple time points during its growth. Further, the amino acid kynurenine was found to accumulate in the media even though it was not present in the predicted metabolome of S. elongatus. This indicates that some metabolites, including those not predicted by an organism's genome, are likely excreted into the environment as waste; however, these molecules may have broader ecological relevance if they are labile to nearby microbes. The compounds described herein provide excellent targets for quantitative analysis in field settings to assess the source and lability of dissolved organic matter in situ.

Microbes, the foundation of the marine foodweb, do not function in isolation, but rather rely on molecular level interactions among species to thrive. Although certain types of interactions between autotrophic and heterotrophic microorganisms have been well documented, the role of specific organic molecules in regulating inter-species relationships and supporting growth are only beginning to be understood. Here, we examine one such interaction by characterizing the metabolic response of a heterotrophic marine bacterium, Ruegeria pomeroyi DSS-3, to growth on dimethylsulfoniopropionate (DMSP), an abundant organosulfur metabolite produced by phytoplankton. When cultivated on DMSP, R. pomeroyi synthesized a quorum-sensing molecule, N-(3-oxotetradecanoyl)-l-homoserine lactone, at significantly higher levels than during growth on propionate. Concomitant with the production of a quorum-sensing molecule, we observed differential production of intra- and extracellular metabolites including glutamine, vitamin B2 and biosynthetic intermediates of cyclic amino acids. Our metabolomics data indicate that R. pomeroyi changes regulation of its biochemical pathways in a manner that is adaptive for a cooperative lifestyle in the presence of DMSP, in anticipation of phytoplankton-derived nutrients and higher microbial density. This behavior is likely to occur on sinking marine particles, indicating that this response may impact the fate of organic matter.

Microbial metabolism plays a primary role in shaping the marine carbon cycle through processes of carbon fixation and remineralization. Many metabolic intermediates pass through the reservoir of marine dissolved organic matter (DOM), as compounds move among microbes as part of complex ecological networks of interactions. Environmental metabolomics can be used to identify and quantify these compounds, and thus will provide insight into the chemical underpinnings of microbial networks at the foundation of global biogeochemical cycles. Here we present methods for metabolite profiling (untargeted metabolomics) and for relative quantification (targeted metabolomics) of intracellular and extracellular metabolites from marine microbes. We describe our approach to method development with regard to metabolite extraction and instrumental analysis, culminating in the methods currently in use in our laboratory.

Natural dissolved organic matter (OM) is a complex heterogeneous mixture of compounds that have defied traditional characterization using standard analytical methods. Electrospray ionization mass spectrometry, particularly ultrahigh resolution mass spectrometry, has provided a new platform for compositional assessment of this important pool of the Earth's reduced carbon. Here, we propose a framework for optimization of instrument and experiment parameters for high quality data acquisition using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). We report the impact of these parameters on reproducibility of peak detection and repeatability of peak height in replicate injections of Suwannee River fulvic acid (FA), a common terrestrial OM standard. In addition, we examine the variability in peak detection and peak height among different types of experimental replicates of dissolved OM derived from laboratory cultures of Candidatus Pelagibacter ubique, a ubiquitous marine α-proteobacterium.Kido Soule et al., Variability in FT-ICR MS spectra of DOM 3

Marine sinking particles transport carbon from the surface and bury it in deep‐sea sediments, where it can be sequestered on geologic time scales. The combination of the surface ocean food web that produces these particles and the particle‐associated microbial community that degrades them creates a complex set of variables that control organic matter cycling. We use targeted metabolomics to characterize a suite of small biomolecules, or metabolites, in sinking particles and compare their metabolite composition to that of the suspended particles in the euphotic zone from which they are likely derived. These samples were collected in the South Atlantic subtropical gyre, as well as in the equatorial Atlantic region and the Amazon River plume. The composition of targeted metabolites in the sinking particles was relatively similar throughout the transect, despite the distinct oceanic regions in which they were generated. Metabolites possibly derived from the degradation of nucleic acids and lipids, such as xanthine and glycine betaine, were an increased mole fraction of the targeted metabolites in the sinking particles relative to surface suspended particles, while algal‐derived metabolites like the osmolyte dimethylsulfoniopropionate were a smaller fraction of the observed metabolites on the sinking particles. These compositional changes are shaped both by the removal of metabolites associated with detritus delivered from the surface ocean and by production of metabolites by the sinking particle‐associated microbial communities. Furthermore, they provide a basis for examining the types and quantities of metabolites that may be delivered to the deep sea by sinking particles.

13Phytoplankton are significant producers of dissolved organic matter (DOM) in marine 14 ecosystems but the identity and dynamics of this DOM remain poorly constrained. Knowledge 15 on the identity and dynamics of DOM are crucial for understanding the molecular-level reactions 16 at the base of the global carbon cycle. Here we apply emerging analytical and computational 17 tools from metabolomics to investigate the composition of DOM produced by the centric diatom 18 Thalassiosira pseudonana. We assessed both intracellular metabolites within T. pseudonana (the 19 endo-metabolome) and extracellular metabolites released by T. pseudonana (the exo-20 metabolome). The intracellular metabolites had a more variable composition than the 21 extracellular metabolites. We putatively identified novel compounds not previously associated 22 with T. pseudonana as well as compounds that have previously been identified within T. 23 pseudonana's metabolic capacity (e.g. dimethylsulfoniopropionate and degradation products of 24 chitin). The resulting information will provide the basis for future experiments to assess the 25 impact of T. pseudonana on the composition of dissolved organic matter in marine 26 environments. 27 3