Krista Longnecker

Fate of Dispersants Associated with the Deepwater Horizon Oil Spill

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

Rapid microbial respiration of oil from the Deepwater Horizon spill in offshore surface waters of the Gulf of Mexico

The Deepwater Horizon oil spill was one of the largest oil spills in history, and the fate of this oil within the Gulf of Mexico ecosystem remains to be fully understood. The goal of this study—conducted in mid-June of 2010, approximately two months after the oil spill began—was to understand the key role that microbes would play in the degradation of the oil in the offshore oligotrophic surface waters near the Deepwater Horizon site. As the utilization of organic carbon by bacteria in the surface waters of the Gulf had been previously shown to be phosphorus limited, we hypothesized that bacteria would be unable to rapidly utilize the oil released from the Macondo well. Although phosphate was scarce throughout the sampling region and microbes exhibited enzymatic signs of phosphate stress within the oil slick, microbial respiration within the slick was enhanced by approximately a factor of five. An incubation experiment to determine hydrocarbon degradation rates confirmed that a large fraction of this enhanced respiration was supported by hydrocarbon degradation. Extrapolating our observations to the entire area of the slick suggests that microbes had the potential to degrade a large fraction of the oil as it arrived at the surface from the well. These observations decidedly refuted our hypothesis. However, a concomitant increase in microbial abundance or biomass was not observed in the slick, suggesting that microbial growth was nutrient limited; incubations amended with nutrients showed rapid increases in cell number and biomass, which supported this conclusion. Our study shows that the dynamic microbial community of the Gulf of Mexico supported remarkable rates of oil respiration, despite a dearth of dissolved nutrients.

Deciphering ocean carbon in a changing world

Dissolved organic matter (DOM) in the oceans is one of the largest pools of reduced carbon on Earth, comparable in size to the atmospheric CO2 reservoir. A vast number of compounds are present in DOM, and they play important roles in all major element cycles, contribute to the storage of atmospheric CO2 in the ocean, support marine ecosystems, and facilitate interactions between organisms. At the heart of the DOM cycle lie molecular-level relationships between the individual compounds in DOM and the members of the ocean microbiome that produce and consume them. In the past, these connections have eluded clear definition because of the sheer numerical complexity of both DOM molecules and microorganisms. Emerging tools in analytical chemistry, microbiology, and informatics are breaking down the barriers to a fuller appreciation of these connections. Here we highlight questions being addressed using recent methodological and technological developments in those fields and consider how these advances are transforming our understanding of some of the most important reactions of the marine carbon cycle.

Abundance and diversity of heterotrophic bacterial cells assimilating phosphate in the subtropical North Atlantic Ocean

Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of (33)P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with (33)P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using (3)H-leucine and (3)H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga-like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of (3)H-leucine or (3)H-thymidine.

Microbial Community Structure Affects Marine Dissolved Organic Matter Composition

Marine microbes are critical players in the global carbon cycle, affecting both the reduction of inorganic carbon and the remineralization of reduced organic compounds back to carbon dioxide. Members of microbial consortia all depend on marine dissolved organic matter (DOM) and in turn, affect the molecules present in this heterogeneous pool. Our understanding of DOM produced by marine microbes is biased towards single species laboratory cultures or simplified field incubations, which exclude large phototrophs and protozoan grazers. Here we explore the interdependence of DOM composition and bacterial diversity in two mixed microbial consortia from coastal seawater: a whole water community and a <1.0-μm community dominated by heterotrophic bacteria. Each consortium was incubated with isotopically-labeled glucose for 9 days. Using stable-isotope probing techniques and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry, we show that the presence of organisms larger than 1.0-μm is the dominant factor affecting bacterial diversity and low-molecular-weight (<1000 Da) DOM composition over this experiment. In the <1.0-μm community, DOM composition was dominated by compounds with lipid and peptide character at all time points, confirmed by fragmentation spectra with peptide-containing neutral losses. In contrast, DOM composition in the whole water community was nearly identical to that in the initial coastal seawater. These differences in DOM composition persisted throughout the experiment despite shifts in bacterial diversity, underscoring an unappreciated role for larger microorganisms in constraining DOM composition in the marine environment.

Using network analysis to discern compositional patterns in ultrahigh resolution mass spectrometry data of dissolved organic matter

RATIONALE Marine dissolved organic matter (DOM) has long been recognized as a large and dynamic component of the global carbon cycle. Yet, DOM is chemically varied and complex and these attributes present challenges to researchers interested in addressing questions about the role of DOM in global biogeochemical cycles. METHODS Organic matter extracts from seawater were analyzed by direct infusion with electrospray ionization into a Fourier transform ion cyclotron resonance mass spectrometer. Network analysis was used to quantify the number of chemical transformations between mass-to-charge values in each sample. The network of chemical transformations was calculated using the MetaNetter plug-in within Cytoscape. The chemical transformations serve as markers for the shared structural characteristics of compounds within complex DOM. RESULTS Network analysis revealed that transformations involving selected sulfur-containing moieties and isomers of amino acids were more prevalent in the deep sea than in the surface ocean. Common chemical transformations were not significantly different between the deep sea and surface ocean. Network analysis complements existing computational tools used to analyze ultrahigh-resolution mass spectrometry data. CONCLUSIONS This combination of ultrahigh-resolution mass spectrometry with novel computational tools has identified new potential building blocks of organic compounds in the deep sea, including the unexpected importance of dissolved organic sulfur components. The method described here can be readily applied by researchers to analyze heterogeneous and complex DOM. Copyright

Effect of carbon addition and predation on acetate-assimilating bacterial cells in groundwater

Groundwater microbial community dynamics are poorly understood due to the challenges associated with accessing subsurface environments. In particular, microbial interactions and their impact on the subsurface carbon cycle remain unclear. In the present project, stable isotope probing with uniformly labeled [(13)C]-acetate was used to identify metabolically active and inactive bacterial populations based on their ability to assimilate acetate and/or its metabolites. Furthermore, we assessed whether substrate availability (bottom-up control) or grazing mortality (top-down control) played a greater role in shaping bacterial community composition by separately manipulating the organic carbon supply and the protozoan grazer population. A community fingerprinting technique, terminal restriction fragment length polymorphism, revealed that the bacterial community was not affected by changes in acetate availability but was significantly altered by the removal of protozoan grazers. In silico identification of terminal restriction fragments and 16S rRNA gene sequences from clone libraries revealed a bacterial community dominated by Proteobacteria, Firmicutes, and Bacteroidetes. Elucidation of the factors that structure the bacterial community will improve our understanding of the bacterial role in the carbon cycle of this important subterranean environment.

Using Stable Isotope Probing to Characterize Differences Between Free-Living and Sediment-Associated Microorganisms in the Subsurface

Aquifers are subterranean reservoirs of freshwater with heterotrophic bacterial communities attached to the sediments and free-living in the groundwater. In the present study, mesocosms were used to assess factors controlling the diversity and activity of the subsurface bacterial community. The assimilation of 13C, derived from 13C-acetate, was monitored to determine whether the sediment-associated and free-living bacterial community would respond similarly to the presence of protozoan grazers. We observed a dynamic response in the sediment-associated bacterial community and none in the free-living community. The disparity in these observations highlights the importance of the sediment-associated bacterial community in the subsurface carbon cycle.

Formularity: Software for Automated Formula Assignment of Natural and Other Organic Matter from Ultrahigh-Resolution Mass Spectra

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.

Targeted metabolomics reveals proline as a major osmolyte in the chemolithoautotroph Sulfurimonas denitrificans

Chemoautotrophic bacteria belonging to the genus Sulfurimonas in the class Campylobacteria are widespread in many marine environments characterized by redox interfaces, yet little is known about their physiological adaptations to different environmental conditions. Here, we used liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) in a targeted metabolomics approach to study the adaptations of Sulfurimonas denitrificans to varying salt concentrations that are found in its natural habitat of tidal mudflats. Proline was identified as one of the most abundant internal metabolites and its concentration showed a strong positive correlation with ionic strength, suggesting that it acts as an important osmolyte in S. denitrificans. 2,3‐dihydroxypropane‐1‐sulfonate was also positively correlated with ionic strength, indicating it might play a previously unrecognized role in osmoregulation. Furthermore, the detection of metabolites from the reductive tricarboxylic acid cycle at high internal concentrations reinforces the importance of this pathway for carbon fixation in Campylobacteria and as a hub for biosynthesis. As the first report of metabolomic data for an campylobacterial chemolithoautotroph, this study provides data that will be useful to understand the adaptations of Campylobacteria to their natural habitat at redox interfaces.