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Oxygen minimum zones (OMZs) are expanding due to increased sea surface temperatures, subsequent increased oxygen demand through respiration, reduced oxygen solubility, and thermal stratification driven in part by anthropogenic climate change. Devil's Hole, Bermuda is a model ecosystem to study OMZ microbial biogeochemistry because the formation and subsequent overturn of the suboxic zone occur annually. During thermally driven stratification, suboxic conditions develop, with organic matter and nutrients accumulating at depth. In this study, the bioavailability of the accumulated dissolved organic carbon (DOC) and the microbial community response to reoxygenation of suboxic waters was assessed using a simulated overturn experiment. The surface inoculated prokaryotic community responded to the deep (formerly suboxic) 0.2 µm filtrate with cell densities increasing 2.5-fold over 6 days while removing 5 µmol L-1 of DOC. After 12 days, the surface community began to shift, and DOC quality became less diagenetically altered along with an increase in SAR202, a Chloroflexi that can degrade recalcitrant dissolved organic matter (DOM). Labile DOC production after 12 days coincided with an increase of Nitrosopumilales, a chemoautotrophic ammonia oxidizing archaea (AOA) that converts ammonia to nitrite based on the ammonia monooxygenase (amoA) gene copy number and nutrient data. In comparison, the inoculation of the deep anaerobic prokaryotic community into surface 0.2 µm filtrate demonstrated a die-off of 25.5% of the initial inoculum community followed by a 1.5-fold increase in cell densities over 6 days. Within 2 days, the prokaryotic community shifted from a Chlorobiales dominated assemblage to a surface-like heterotrophic community devoid of Chlorobiales. The DOM quality changed to less diagenetically altered material and coincided with an increase in the ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) gene number followed by an influx of labile DOM. Upon reoxygenation, the deep DOM that accumulated under suboxic conditions is bioavailable to surface prokaryotes that utilize the accumulated DOC initially before switching to a community that can both produce labile DOM via chemoautotrophy and degrade the more recalcitrant DOM. [ABSTRACT FROM AUTHOR]

Sulfur (S) is a major heteroatom in organic matter. This project evaluated spatial variability in the concentration and molecular‐level composition of organic sulfur along gradients of depth and latitude. We measured the concentration of total organic sulfur (TOS) directly from whole seawater. Our data reveal high variability in organic sulfur, relative to established variability in total organic carbon or nitrogen. The deep ocean contained significant amounts of organic sulfur, and the concentration of TOS in North Atlantic Deep Water (NADW) decreased with increasing age while total organic carbon remained stable. Analysis of dissolved organic matter extracts by ultrahigh resolution mass spectrometry revealed that 6% of elemental formulas contained sulfur. The sulfur‐containing compounds were structurally diverse, and showed higher numbers of sulfur‐containing elemental formulas as NADW moved southward. These measurements of organic sulfur in seawater provide the foundation needed to define the factors controlling organic sulfur in the global ocean. [ABSTRACT FROM AUTHOR]

Untargeted metabolomics datasets provide ample opportunity for discovery of novel metabolites. The major challenge is focusing data analysis on a short list of metabolites. Here, we apply a combination of computational tools that serve to reduce complex mass spectrometry data in order allow us to focus on new environmentally-relevant metabolites. In the first portion of the project, we explored mass spectrometry data from intracellular metabolites extracted from a model marine diatom, Thalassiosira pseudonana . The fragmentation data from these samples were analyzed using molecular networking, an on-line tool that clusters metabolites based on shared structural similarities. The features within each metabolite cluster were then putatively annotated using MetFrag, an in silico fragmentation tool. Using this combination of computational tools, we observed multiple lyso-sulfolipids, organic compounds not previously known to exist within cultured marine diatoms. In the second stage of the project, we searched our environmental data for these lyso-sulfolipids. The lyso-sulfolipid with a C14:0 fatty acid was found in dissolved and particulate samples from the western Atlantic Ocean, and a culture of cyanobacteria grown in our laboratory. Thus, the putative lyso-sulfolipids are present in both laboratory experiments and environmental samples. This project highlights the value of combining computational tools to detect and putatively identify organic compounds not previously recognized as important within T. pseudonana or the marine environment. Future applications of these tools to emerging metabolomics data will further open the black box of natural organic matter, identifying molecules that can be used to understand and monitor the global carbon cycle. [ABSTRACT FROM AUTHOR]

Metabolomics is the study of small molecules, or ‘metabolites’, that are the end products of biological processes. While -omics technologies such as genomics, transcriptomics, and proteomics measure the metabolic potential of organisms, metabolomics provides detailed information on the organic compounds produced during metabolism and found within cells and in the environment. Improvements in analytical techniques have expanded our understanding of metabolomics and developments in computational tools have made metabolomics data accessible to a broad segment of the scientific community. Yet, metabolomics methods have only been applied to a limited number of projects in the marine environment. Here, we review analysis techniques for mass spectrometry data and summarize the current state of metabolomics databases. We then describe a boutique database developed in our laboratory for efficient data analysis and selection of mass spectral targets for metabolite identification. The code to implement the database is freely available on GitHub ( https://github.com/joefutrelle/domdb ). Data organization and analysis are critical, but often under-appreciated, components of metabolomics research. Future advances in environmental metabolomics will take advantage of continued development of new tools that facilitate analysis of large metabolomics datasets. [ABSTRACT FROM AUTHOR]

Abstract: Groundwater constitutes a globally important source of freshwater for drinking water and other agricultural and industrial purposes, and is a prominent source of freshwater flowing into the coastal ocean. Therefore, understanding the chemical components of groundwater is relevant to both coastal and inland communities. We used electrospray ionization coupled with Fourier-transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to examine dissolved organic compounds in groundwater prior to and after passage through a sediment-filled column containing microorganisms. The data revealed that an unexpectedly high proportion of organic compounds contained nitrogen and sulfur, possibly due to transport of surface waters from septic systems and rain events. We matched 292 chemical features, based on measured mass:charge (m/z) values, to compounds stored in the Kyoto Encyclopedia of Genes and Genomes (KEGG). A subset of these compounds (88) had only one structural isomer in KEGG, thus supporting tentative identification. Most identified elemental formulas were linked with metabolic pathways that produce polyketides or with secondary metabolites produced by plants. The presence of polyketides in groundwater is notable because of their anti-bacterial and anti-cancer properties. However, their relative abundance must be quantified with appropriate analyses to assess any implications for public health. [Copyright &y& Elsevier]

Abstract: 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 in ultrahigh resolution mode, provides 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. [Copyright &y& Elsevier]

Abstract: With the advent of ultrahigh resolution mass spectrometry (MS), recent studies have begun to resolve molecular relationships between terrestrial and aquatic dissolved organic matter (DOM) in rivers, estuaries, mangrove swamps and their receiving oceans and lakes. Here, we extend ultrahigh resolution MS techniques to Lake Superior, the largest freshwater lake in the world by area. Solid-phase extracted samples from the western arm of the lake and its watershed, including swamp, creek, river, lake–river confluence and offshore lake sites were compared using electrospray ionization (ESI), Fourier transform ion cyclotron resonance MS (FT-ICR-MS). Results were analyzed using cluster analysis and van Krevelen diagrams. Chemical similarity appears related to hydrological proximity, terrestrial impact and flow conditions. For example, higher and lower flow samples from the same stream differ from one another. Toivola Swamp, Lake Superior and the south shore river have diverse arrays of unique molecular formulae relative to the north shore river and stream sampled in the data set. The lake’s unique elemental formulae, relative to its watershed samples, are primarily in the lignin-like and reduced hydrocarbon regions of van Krevelen diagrams. The ESI-amenable lake DOM also has a higher proportion of formulae containing nitrogen or sulfur relative to the other samples. The degree of overlap among formulae within the data set is consistent with previous ESI FT-ICR-MS characterization of terrestrial, estuarine and marine OM. There appears to be a conserved portion of formulae across natural OM samples, perhaps because these compounds are intrinsically refractory or because they are commonly generated as products of natural reworking processes. [Copyright &y& Elsevier]

Ultrahigh resolution mass spectrometry, such as Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), can resolve thousands of molecular ions in complex organic matrices. A Compound Identification Algorithm (CIA) was previously developed for automated elemental formula assignment for natural organic matter (NOM). In this work, we describe software Formularity with a user-friendly interface for CIA function and newly developed search function Isotopic Pattern Algorithm (IPA). While CIA assigns elemental formulas for compounds containing C, H, O, N, S, and P, IPA is capable of assigning formulas for compounds containing other elements. We used halogenated organic compounds (HOC), a chemical class that is ubiquitous in nature as well as anthropogenic systems, as an example to demonstrate the capability of Formularity with IPA. A HOC standard mix was used to evaluate the identification confidence of IPA. Tap water and HOC spike in Suwannee River NOM were used to assess HOC identification in complex environmental samples. Strategies for reconciliation of CIA and IPA assignments were discussed. Software and sample databases with documentation are freely available. [ABSTRACT FROM AUTHOR]