The production and fate of nitrogen species in deep-sea hydrothermal environments

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Applied Ocean Science & Engineering at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2019.Nitrogen (N) species in hydrothermal vent fluids serve as both a nutrient and energy source for the chemosynthetic ecosystems surrounding deep-sea vents. While numerous pathways have been identified in which N-species can be produced and consumed in the context of submarine hydrothermal vent systems, their exact nature has been largely limited to interpretation of variations in concentrations. This thesis applies stable isotope approaches to further constrain the sources and fate of N-species in deep-sea vents across a variety of geological settings. First, I discuss isotope fractionation and reaction kinetics during abiotic reduction of nitrate (NO3-) to ammonium (ΣNH4+ = NH3+NH4+) under hydrothermal conditions. Results of lab experiments conducted at high temperatures and pressures revealed a wide degree of N isotope fractionation as affected by temperature, fluid/rock ratio, and pH—all of which exert control over reaction rates. Moreover, a clear pattern in terms of reaction products can be discerned with the reaction producing ΣNH4+ only at high pH, but both ΣNH4+ and N2 at low pH. This challenges previous assumptions that O3 - is always quantitatively converted to NH4+ during submarine hydrothermal circulation. Next, I report measurements of ΣNH4+ concentrations and N isotopic composition (δ15NNH4) from vent fluid samples, together with the largest compilation to date of these measurements made from other studies of deep-sea vent systems for comparison. The importance of different processes at sediment-influenced and unsedimented systems are discussed with a focus on how they ultimately yield observed vent δ15NNH4 values. Notable findings include the role that phase separation might play under some conditions and a description of how an unsedimented site from Mid-Cayman Rise with unexpectedly high NH4+ may be uniquely influenced by N2 reduction to ΣNH4+. Lastly, I explore ΣNH4+ dynamics in the context of low-temperature vent sites at 9°50’N East Pacific Rise to investigate dynamics of microbially-mediated N transformations. Through both measurements of natural samples, as well as isotopic characterization of N species from incubation experiments and model simulations thereof, an exceptionally high variability observed in δ15NNH4 values emphasizes the complexity of these microbe-rich systems. In sum, this thesis highlights the role of microbial processes in low temperature systems, demonstrates a more mechanistic understanding of lesser-understood abiotic N reactions and improves the coverage of available data on deep-sea vent ΣNH4+ measurements.This thesis research was made possible through funding by National Science Foundation (NSF) grants OCE-1537372, OCE-1559198, OCE-1136727, OCE-1061863, OCE-0702677, and OCE-0549829, and WHOI’s Ocean Ventures Fund. Funding for Net Charoenpong was provided by the Royal Thai Government Scholarship, WHOI academic program and NSF-OCE-1537372 grant

Animal Models of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the USA and currently there are minimal therapies specific for the treatment of COPD. To advance our knowledge on COPD pathogenesis and develop new therapeutics, animal models are needed that represent key clinical and pathologic features of the human disease. The primary animal models utilized to study COPD rely on several factors associated with disease progression, i.e. genetic and epigenetic changes, environmental exposures and the microbial flora of the lungs. Here, a systematic approach was taken to summarize and evaluate the current animal models employed to study COPD pathogenesis, comorbidities and exacerbations. The strengths and limitations of these disease models are also delineated. The rodent COPD models have been extensively utilized but several studies have highlighted the potential of larger animals as an additional approach. Due to the inherent heterogeneity of COPD, the usefulness of certain animal models may be limiting but still represent helpful means to explore gene functional studies, testing new therapeutics and the exploring the significance of microbial floral changes. Therefore, interpreting the findings from animal models for the study of COPD represents a critical approach in deciding possible future human therapeutics

N-loss isotope effects in the Peru oxygen minimum zone studied using a mesoscale eddy as a natural tracer experiment

Mesoscale eddies in Oxygen Minimum Zones (OMZ's) have been identified as important fixed nitrogen (N) loss hotspots that may significantly impact both the global rate of N-loss as well as the ocean's N isotope budget. They also represent ‘natural tracer experiments’ with intensified biogeochemical signals that can be exploited to understand the large-scale processes that control N-loss and associated isotope effects (ε; the ‰ deviation from 1 in the ratio of reaction rate constants for the light versus the heavy isotopologues). We observed large ranges in the concentrations and N and O isotopic compositions of nitrate (NO3−), nitrite (NO2−) and biogenic N2 associated with an anticyclonic eddy in the Peru OMZ during two cruises in November and December 2012. In the eddy's center where NO3− was nearly exhausted, we measured the highest δ15N values for both NO3− and NO2− (up to ~70‰ and 50‰) ever reported for an OMZ. Correspondingly, N deficit and biogenic N2-N concentrations were also the highest near the eddy's center (up to ~40 µmol L−1). δ15N-N2 also varied with biogenic N2 production, following kinetic isotopic fractionation during NO2− reduction to N2 and, for the first time, provided an independent assessment of N isotope fractionation during OMZ N-loss. We found apparent variable ε for NO3− reduction (up to ~30‰ in the presence of NO2−). However, the overall ε for N-loss was calculated to be only ~13-14‰ (as compared to canonical values of ~20-30‰) assuming a closed system and only slightly higher assuming an open system (16-19‰). Our results were similar whether calculated from the disappearance of DIN (NO3− + NO2−) or from the appearance of N2 and changes in isotopic composition. Further, we calculated the separate ε for NO3− reduction to NO2− and NO2− reduction to N2 of ~16-21‰ and ~12‰, respectively, when the effect of NO2− oxidation could be removed. These results, together with the relationship between N and O of NO3− isotopes and the difference in δ15N between NO3− and NO2-, confirm a role for NO2− oxidation in increasing the apparent ε associated with NO3− reduction. The lower ε for NO3− and NO2− reduction as well as N-loss calculated in this study could help reconcile the current imbalance in the global N budget if they are representative of OMZ N-loss

The NDR/LATS Family Kinase Cbk1 Directly Controls Transcriptional Asymmetry

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase

Evidence for microbial mediation of subseafloor nitrogen redox processes at Loihi Seamount, Hawaii

© The Author(s), 2016. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 198 (2017): 131-150, doi:10.1016/j.gca.2016.10.029.The role of nitrogen cycling in submarine hydrothermal systems is far less studied than that of other biologically reactive elements such as sulfur and iron. In order to address this knowledge gap, we investigated nitrogen redox processes at Loihi Seamount, Hawaii, using a combination of biogeochemical and isotopic measurements, bioenergetic calculations and analysis of the prokaryotic community composition in venting fluids sampled during four cruises in 2006, 2008, 2009 and 2013. Concentrations of NH4+ were positively correlated to dissolved Si and negatively correlated to NO3-+NO2-, while NO2- was not correlated to NO3-+NO2-, dissolved Si or NH4+. This is indicative of hydrothermal input of NH4+ and biological mediation influencing NO2- concentrations. The stable isotope ratios of NO3- (d15N and d18O) was elevated with respect to background seawater, with d18O values exhibiting larger changes than corresponding d15N values, reflecting the occurrence of both production and reduction of NO3- by an active microbial community. d15N-NH4+ values ranged from 0‰ to +16.7‰, suggesting fractionation during consumption and potentially N-fixation as well. Bioenergetic calculations reveal that several catabolic strategies involving the reduction of NO3- and NO2- coupled to sulfide and iron oxidation could provide energy to microbes in Loihi fluids, while 16S rRNA gene sequencing of Archaea and Bacteria in the fluids reveals groups known to participate in denitrification and N-fixation. Taken together, our data support the hypothesis that microbes are mediating N-based redox processes in venting hydrothermal fluids at Loihi Seamount.This work was supported by the NSF Microbial Observatories program (MCB 0653265), the Gordon and Betty Moore Foundation (GBMF1609), NSF-OCE 0648287, the Center for Dark Energy Biosphere Investigations (C-DEBI) and the NASA Astrobiology Institute — Life Underground (NAI-LU). Sequence data was generated as part of the Alfred P. Sloan Foundation's ICoMM field project and the W. M. Keck Foundation

N2 fixation in eddies of the eastern tropical South Pacific Ocean

Mesoscale eddies play a major role in controlling ocean biogeochemistry. By impacting nutrient availability and water column ventilation, they are of critical importance for oceanic primary production. In the eastern tropical South Pacific Ocean off Peru, where a large and persistent oxygen deficient zone is present, mesoscale processes have been reported to occur frequently. However, investigations on their biological activity are mostly based on model simulations, and direct measurements of carbon and dinitrogen (N2) fixation are scarce. We examined an open ocean cyclonic eddy and two anticyclonic mode water eddies: a coastal one and an open ocean one in the waters off Peru along a section at 16° S in austral summer 2012. Molecular data and bioassay incubations point towards a difference between the active diazotrophic communities present in the cyclonic eddy and the anticyclonic mode water eddies. In the cyclonic eddy, highest rates of N2 fixation were measured in surface waters but no N2 fixation signal was detected at intermediate water depths. In contrast, both anticyclonic mode water eddies showed pronounced maxima in N2 fixation below the euphotic zone as evidenced by rate measurements and geochemical data. N2 fixation and carbon (C) fixation were higher in the young coastal mode water eddy compared to the older offshore mode water eddy. A co-occurrence between N2 fixation and biogenic N2, an indicator for N loss, indicated a link between N loss and N2 fixation in the mode water eddies, which was not observed for the cyclonic eddy. The comparison of two consecutive surveys of the coastal mode water eddy in November and December 2012 revealed also a reduction of N2 and C fixation at intermediate depths along with a reduction in chlorophyll by half, mirroring an aging effect in this eddy. Our data indicate an important role for anticyclonic mode water eddies in stimulating N2 fixation and thus supplying N offshore