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1. Nutrient-Colimited Trichodesmium as a Nitrogen Source or Sink in a Future Ocean

   https://doi.org/10.1128/AEM.02137-17  

   Walworth, Nathan G. ; Fu, Fei-Xue ; Lee, Michael D. ; Cai, Xiaoni ; Saito, Mak A. ; Webb, Eric A. ; Hutchins, David A. ; Liu, Shuang-Jiang ( February 2018 , Applied and Environmental Microbiology)

   ABSTRACT Nitrogen-fixing (N 2 ) cyanobacteria provide bioavailable nitrogen to vast ocean regions but are in turn limited by iron (Fe) and/or phosphorus (P), which may force them to employ alternative nitrogen acquisition strategies. The adaptive responses of nitrogen fixers to global-change drivers under nutrient-limited conditions could profoundly alter the current ocean nitrogen and carbon cycles. Here, we show that the globally important N 2 fixer Trichodesmium fundamentally shifts nitrogen metabolism toward organic-nitrogen scavenging following long-term high-CO 2 adaptation under iron and/or phosphorus (co)limitation. Global shifts in transcripts and proteins under high-CO 2 /Fe-limited and/or P-limited conditions include decreases in the N 2 -fixing nitrogenase enzyme, coupled with major increases in enzymes that oxidize trimethylamine (TMA). TMA is an abundant, biogeochemically important organic nitrogen compound that supports rapid Trichodesmium growth while inhibiting N 2 fixation. In a future high-CO 2 ocean, this whole-cell energetic reallocation toward organic nitrogen scavenging and away from N 2 fixation may reduce new-nitrogen inputs by Trichodesmium while simultaneously depleting the scarce fixed-nitrogen supplies of nitrogen-limited open-ocean ecosystems. IMPORTANCE Trichodesmium is among the most biogeochemically significant microorganisms in the ocean, since it supplies up to 50% of the new nitrogen supporting open-ocean food webs. We used Trichodesmium cultures adapted to high-CO 2 conditions for 7 years, followed by additional exposure to iron and/or phosphorus (co)limitation. We show that “future ocean” conditions of high CO 2 and concurrent nutrient limitation(s) fundamentally shift nitrogen metabolism away from nitrogen fixation and instead toward upregulation of organic nitrogen-scavenging pathways. We show that the responses of Trichodesmium to projected future ocean conditions include decreases in the nitrogen-fixing nitrogenase enzymes coupled with major increases in enzymes that oxidize the abundant organic nitrogen source trimethylamine (TMA). Such a shift toward organic nitrogen uptake and away from nitrogen fixation may substantially reduce new-nitrogen inputs by Trichodesmium to the rest of the microbial community in the future high-CO 2 ocean, with potential global implications for ocean carbon and nitrogen cycling. 

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2. Defining the Sphagnum Core Microbiome across the North American Continent Reveals a Central Role for Diazotrophic Methanotrophs in the Nitrogen and Carbon Cycles of Boreal Peatland Ecosystems

   https://doi.org/10.1128/mbio.03714-21  

   Kolton, Max ; Weston, David J. ; Mayali, Xavier ; Weber, Peter K. ; McFarlane, Karis J. ; Pett-Ridge, Jennifer ; Somoza, Mark M. ; Lietard, Jory ; Glass, Jennifer B. ; Lilleskov, Erik A. ; et al ( February 2022 , mBio)

   Martiny, Jennifer B. (Ed.)

   ABSTRACT Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum -associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum -dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase ( nifH ) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales ) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere ( Methyloferula spp. of the Rhizobiales ) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands. IMPORTANCE Nitrogen availability frequently limits photosynthetic production in Sphagnum moss-dominated high-latitude peatlands, which are crucial carbon-sequestering ecosystems at risk to climate change effects. It has been previously suggested that microbial methane-fueled fixation of atmospheric nitrogen (N 2 ) may occur in these ecosystems, but this process and the organisms involved are largely uncharacterized. A combination of omics (DNA and RNA characterization) and dual-isotope incorporation approaches illuminated the functional diversity of Sphagnum -associated microbiomes and defined 12 bacterial genera in its core microbiome at the continental scale. Moreover, obligate diazotrophic methanotrophs showed high nitrogen fixation gene expression levels and incorporated a substantial amount of atmospheric nitrogen and methane-driven carbon into their biomass. Thus, these results point to a central role for members of the rare biosphere in Sphagnum microbiomes as keystone species that couple nitrogen fixation to methane oxidation in nutrient-poor peatlands. 

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3. Quantification of biological nitrogen fixation by Mo-independent complementary nitrogenases in environmental samples with low nitrogen fixation activity

   https://doi.org/10.1038/s41598-022-24860-9  

   Haynes, Shannon J. ; Darnajoux, Romain ; Han, Eunah ; Oleynik, Sergey ; Zimble, Ezra ; Zhang, Xinning ( December 2022 , Scientific Reports)

   Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (¹³C/¹²C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. Here we describe a high sensitivity method to measure ethylene δ¹³C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoformAzotobacter vinelandiimutants and environmental sample assays rely on a common acetylene source for ethylene production. Application of the Low BNF activity ISARA (LISARA) method to low nitrogen-fixing activity soils, leaf litter, decayed wood, cryptogams, and termites indicates complementary BNF in most sample types, calling for additional studies of isoform-specific BNF.

    

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4. Lagrangian Studies of Net Community Production: The Effect of Diel and Multiday Nonsteady State Factors and Vertical Fluxes on O 2 /Ar in a Dynamic Upwelling Region

   https://doi.org/10.1029/2019JG005569  

   Wang, Seaver ; Kranz, Sven A. ; Kelly, Thomas B. ; Song, Hajoon ; Stukel, Michael R. ; Cassar, Nicolas ( June 2020 , Journal of Geophysical Research: Biogeosciences)

   The ratio of dissolved oxygen to argon in seawater is frequently employed to estimate rates of net community production (NCP) in the oceanic mixed layer. The in situ O₂/Ar‐based method accounts for many physical factors that influence oxygen concentrations, permitting isolation of the biological oxygen signal produced by the balance of photosynthesis and respiration. However, this technique traditionally relies upon several assumptions when calculating the mixed‐layer O₂/Ar budget, most notably the absence of vertical fluxes of O₂/Ar and the principle that the air‐sea gas exchange of biological oxygen closely approximates net productivity rates. Employing a Lagrangian study design and leveraging data outputs from a regional physical oceanographic model, we conducted in situ measurements of O₂/Ar in the California Current Ecosystem in spring 2016 and summer 2017 to evaluate these assumptions within a “worst‐case” field environment. Quantifying vertical fluxes, incorporating nonsteady state changes in O₂/Ar, and comparing NCP estimates evaluated over several day versus longer timescales, we find differences in NCP metrics calculated over different time intervals to be considerable, also observing significant potential effects from vertical fluxes, particularly advection. Additionally, we observe strong diel variability in O₂/Ar and NCP rates at multiple stations. Our results reemphasize the importance of accounting for vertical fluxes when interpreting O₂/Ar‐derived NCP data and the potentially large effect of nonsteady state conditions on NCP evaluated over shorter timescales. In addition, diel cycles in surface O₂/Ar can also bias interpretation of NCP data based on local productivity and the time of day when measurements were made.

    

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5. Nitrogen fixation associated with epiphytes on the seagrass Zostera marina in a temperate lagoon with moderate to high nitrogen loads

   https://doi.org/10.1007/s10533-023-01083-2  

   Marino, Roxanne ; Hayn, Melanie ; Howarth, Robert W. ; Giblin, Anne E. ; McGlathery, Karen J. ; Berg, Peter ( October 2023 , Biogeochemistry)

   As part of a long-term study on the effects of nitrogen (N) loading in a shallow temperate lagoon, we measured rates of N₂fixation associated with seagrass (Zostera marina) epiphytes during the summer from 2005 to 2019, at two sites along a gradient from where high N groundwater enters the system (denoted SH) to a more well-flushed outer harbor (OH). The data presented here are the first such long-term N₂fixation estimates for any seagrass system and one of the very few reported for the phyllosphere in a temperate system. Mean daily N₂fixation was estimated from light and dark measurements using the acetylene reduction assay intercalibrated using both incorporation of¹⁵N₂into biomass and a novel application of the N₂:Ar method. Surprisingly, despite a large inorganic N input from a N-contaminated groundwater plume, epiphytic N₂fixation rates were moderately to very high for a seagrass system (OH site 14-year mean of 0.94 mmol N m⁻² d⁻¹), with the highest rates (2.6 mmol N m⁻² d⁻¹) measured at the more N-loaded eutrophic site (SH) where dissolved inorganic N was higher and soluble reactive phosphorus was lower than in the better-flushed OH. Over 95% of the total N₂fixation measured was in the light, suggesting the importance of cyanobacteria in the epiphyte assemblages. We observed large inter-annual variation both within and across the two study sites (range from 0.1 to 2.6 mmol N fixed m⁻²d⁻¹), which we suggest is in part related to climatic variation. We estimate that input from phyllosphere N₂fixation over the study period contributes on average an additional 20% to the total daily N load per area within the seagrass meadow.

    

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