Toward resolving disparate accounts of the extent and magnitude of nitrogen fixation in the Eastern Tropical South Pacific oxygen deficient zone

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Selden, C. R., Mulholland, M. R., Widner, B., Bernhardt, P., & Jayakumar, A. Toward resolving disparate accounts of the extent and magnitude of nitrogen fixation in the Eastern Tropical South Pacific oxygen deficient zone. Limnology and Oceanography, 66, (2021): 1950-1960, https://doi.org/10.1002/lno.11735.Examination of dinitrogen (N2) fixation in the Eastern Tropical South Pacific oxygen deficient zone has raised questions about the range of diazotrophs in the deep sea and their quantitative importance as a source of new nitrogen globally. However, technical considerations in the deployment of stable isotopes in quantifying N2 fixation rates have complicated interpretation of this research. Here, we report the findings of a comprehensive survey of N2 fixation within, above and below the Eastern Tropical South Pacific oxygen deficient zone. N2 fixation rates were measured using a robust 15N tracer method (bubble removal) that accounts for the slow dissolution of N2 gas and calculated using a conservative approach. N2 fixation was only detected in a subset of samples (8 of 125 replicated measurements) collected within suboxic waters (< 20 μmol O2 kg−1) or at the oxycline. Most of these detectable rates were measured at nearshore stations, or where surface productivity was high. These findings support the hypothesis that low oxygen/high organic carbon conditions favor non-cyanobacterial diazotrophs. Nevertheless, this study indicates that N2 fixation is neither widespread nor quantitatively important throughout this region.This work was funded by the National Science Foundation Division of Ocean Sciences (NSF-OCE) Grant OCE-1356056 to M.R.M. and A.J

Toward Resolving Disparate Accounts of the Extent and Magnitude of Nitrogen Fixation in the Eastern Tropical South Pacific Oxygen Deficient Zone

Examination of dinitrogen (N2) fixation in the Eastern Tropical South Pacific oxygen deficient zone has raised questions about the range of diazotrophs in the deep sea and their quantitative importance as a source of new nitrogen globally. However, technical considerations in the deployment of stable isotopes in quantifying N2 fixation rates have complicated interpretation of this research. Here, we report the findings of a comprehensive survey of N2 fixation within, above and below the Eastern Tropical South Pacific oxygen deficient zone. N2 fixation rates were measured using a robust 15N tracer method (bubble removal) that accounts for the slow dissolution of N2 gas and calculated using a conservative approach. N2 fixation was only detected in a subset of samples (8 of 125 replicated measurements) collected within suboxic waters (\u3c 20 μmol O2 kg−1) or at the oxycline. Most of these detectable rates were measured at nearshore stations, or where surface productivity was high. These findings support the hypothesis that low oxygen/high organic carbon conditions favor non-cyanobacterial diazotrophs. Nevertheless, this study indicates that N2 fixation is neither widespread nor quantitatively important throughout this region

Microbial niche differentiation explains nitrite oxidation in marine oxygen minimum zones

Nitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget

Diversity, Distribution, and Expression of Diazotroph nifH Genes in Oxygen-Deficient Waters of the Arabian Sea

The Arabian Sea oxygen minimum zone (OMZ), the largest suboxic region in the world\u27s oceans, is responsible for up to half of the global mesopelagic fixed nitrogen ( N ) loss from the ocean via denitrification and anammox. Dinitrogen (N2) fixation is usually attributed to cyanobacteria in the surface ocean. Model prediction and physiological inhibition of N2 fixation by oxygen, however, suggest that N2 fixation should be enhanced near the oxygen-deficient zone (ODZ) of the Arabian Sea. N2 fixation and cyanobacterial nifH genes (the gene encoding dinitrogenase reductase) have been reported in surface waters overlying the Arabian Sea ODZ. Here, water samples from depths above and within the Arabian Sea ODZ were examined to explore the distribution, diversity, and expression of nifH genes. In surface waters, nifH DNA and cDNA sequences related to Trichodesmium, a diazotroph known to occur and fix N2 in the Arabian Sea, were detected. Proteobacterial nifH phylotypes (DNA but not cDNA) were also detected in surface waters. Proteobacterial nifH DNA and cDNA sequences, as well as nifH DNA and cDNA sequences related to strictly anaerobic N -fixers, were obtained from oxygen-deficient depths. This first report of nifH gene expression in subsurface low-oxygen waters suggests that there is potential for active N2 fixation by several phylogenetically and potentially metabolically diverse microorganisms in pelagic OMZs

Dependence of nitrite oxidation on nitrite and oxygen in low-oxygen seawater

Nitrite oxidation is an essential step in transformations of fixed nitrogen. The physiology of nitrite oxidizing bacteria (NOB) implies that the rates of nitrite oxidation should be controlled by concentration of their substrate, nitrite, and the terminal electron acceptor, oxygen. The sensitivities of nitrite oxidation to oxygen and nitrite concentrations were investigated using 15N tracer incubations in the Eastern Tropical North Pacific. Nitrite stimulated nitrite oxidation under low in situ nitrite conditions, following Michaelis-Menten kinetics, indicating that nitrite was the limiting substrate. The nitrite half-saturation constant (Ks = 0.254 ± 0.161 μM) was 1–3 orders of magnitude lower than in cultivated NOB, indicating higher affinity of marine NOB for nitrite. The highest rates of nitrite oxidation were measured in the oxygen depleted zone (ODZ), and were partially inhibited by additions of oxygen. This oxygen sensitivity suggests that ODZ specialist NOB, adapted to low-oxygen conditions, are responsible for apparently anaerobic nitrite oxidation. Key Points: • Nitrite addition stimulated nitrite oxidation in both oxic and anoxic waters • Natural assemblages of marine nitrite-oxidizing bacteria have high affinity for nitrite • Addition of oxygen at μM-level inhibited nitrite oxidation in oxygen depleted water

Community Composition of Nitrous Oxide-Related Genes in Salt Marsh Sediments Exposed to Nitrogen Enrichment

Salt marshes provide many key ecosystem services that have tremendous ecological and economic value. One critical service is the removal of fixed nitrogen from coastal waters, which limits the negative effects of eutrophication resulting from increased nutrient supply. Nutrient enrichment of salt marsh sediments results in higher rates of nitrogen cycling and, commonly, a concurrent increase in the flux of nitrous oxide, an important greenhouse gas. Little is known, however, regarding controls on the microbial communities that contribute to nitrous oxide fluxes in marsh sediments. To address this disconnect, we generated profiles of microbial communities and communities of micro-organisms containing specific nitrogen cycling genes that encode several enzymes (amoA, norB, nosZ) related to nitrous oxide flux from salt marsh sediments. We hypothesized that communities of microbes responsible for nitrogen transformations will be structured by nitrogen availability. Taxa that respond positively to high nitrogen inputs may be responsible for the elevated rates of nitrogen cycling processes measured in fertilized sediments. Our data show that, with the exception of ammonia-oxidizing archaea, the community composition of organisms involved in the production and consumption of nitrous oxide was altered under nutrient enrichment. These results suggest that previously measured rates of nitrous oxide production and consumption are likely the result of changes in community structure, not simply changes in microbial activity

Protocols for Assessing Transformation Rates of Nitrous Oxide in the Water Column

Nitrous oxide (N2O) is a potent greenhouse gas and an ozone destroying substance. Yet, clear step-by-step protocols to measure N2O transformation rates in freshwater and marine environments are still lacking, challenging inter-comparability efforts. Here we present detailed protocols currently used by leading experts in the field to measure water-column N2O production and consumption rates in both marine and other aquatic environments. We present example 15N-tracer incubation experiments in marine environments as well as templates to calculate both N2O production and consumption rates. We discuss important considerations and recommendations regarding (1) precautions to prevent oxygen (O2) contamination during low-oxygen and anoxic incubations, (2) preferred bottles and stoppers, (3) procedures for 15N-tracer addition, and (4) the choice of a fixative. We finally discuss data reporting and archiving. We expect these protocols will make 15N-labeled N2O transformation rate measurements more accessible to the wider community and facilitate future inter-comparison between different laboratories

Titanium Particles Modulate Lymphocyte and Macrophage Polarization in Peri-Implant Gingival Tissues

Titanium dental implants are one of the modalities to replace missing teeth. The release of titanium particles from the implant’s surface may modulate the immune cells, resulting in implant failure. However, little is known about the immune microenvironment that plays a role in peri-implant inflammation as a consequence of titanium particles. In this study, the peri-implant gingival tissues were collected from patients with failed implants, successful implants and no implants, and then a whole transcriptome analysis was performed. The gene set enrichment analysis confirmed that macrophage M1/M2 polarization and lymphocyte proliferation were differentially expressed between the study groups. The functional clustering and pathway analysis of the differentially expressed genes between the failed implants and successful implants versus no implants revealed that the immune response pathways were the most common in both comparisons, implying the critical role of infiltrating immune cells in the peri-implant tissues. The H&E and IHC staining confirmed the presence of titanium particles and immune cells in the tissue samples, with an increase in the infiltration of lymphocytes and macrophages in the failed implant samples. The in vitro validation showed a significant increase in the level of IL-1β, IL-8 and IL-18 expression by macrophages. Our findings showed evidence that titanium particles modulate lymphocyte and macrophage polarization in peri-implant gingival tissues, which can help in the understanding of the imbalance in osteoblast–osteoclast activity and failure of dental implant osseointegration

Nitrous oxide production by nitrification and denitrification in the Eastern Tropical South Pacific oxygen minimum zone

The Eastern Tropical South Pacific oxygen minimum zone (ETSP-OMZ) is a site of intense nitrous oxide (N2O) flux to the atmosphere. This flux results from production of N2O by nitrification and denitrification, but the contribution of the two processes is unknown. The rates of these pathways and their distributions were measured directly using 15N tracers. The highest N2O production rates occurred at the depth of peak N2O concentrations at the oxic-anoxic interface above the oxygen deficient zone (ODZ) because slightly oxygenated waters allowed (1) N2O production from both nitrification and denitrification and (2) higher nitrous oxide production yields from nitrification. Within the ODZ proper (i.e., anoxia), the only source of N2O was denitrification (i.e., nitrite and nitrate reduction), the rates of which were reflected in the abundance of nirS genes (encoding nitrite reductase). Overall, denitrification was the dominant pathway contributing the N2O production in the ETSP-OMZ