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Abstract Dinitrogen (N₂) fixation supports marine life through the supply of reactive nitrogen. Recent studies suggest that particle-associated non-cyanobacterial diazotrophs (NCDs) could contribute significantly to N₂fixation contrary to the paradigm of diazotrophy as primarily driven by cyanobacterial genera. We examine the community composition of NCDs associated with suspended, slow, and fast-sinking particles in the North Pacific Subtropical Gyre. Suspended and slow-sinking particles showed a higher abundance of cyanobacterial diazotrophs than fast-sinking particles, while fast-sinking particles showed a higher diversity of NCDs includingMarinobacter,OceanobacterandPseudomonas. Using single-cell mass spectrometry we find that Gammaproteobacteria N₂fixation rates were higher on suspended and slow-sinking particles (up to 67 ± 48.54 fmol N cell⁻¹ d⁻¹), while putative NCDs’ rates were highest on fast-sinking particles (121 ± 22.02 fmol N cell⁻¹ d⁻¹). These rates are comparable to previous diazotrophic cyanobacteria observations, suggesting that particle-associated NCDs may be important contributors to pelagic N₂fixation. 

Abstract. Marine dinitrogen (N2) fixation is a globally significant biogeochemical process carried out by a specialized group of prokaryotes (diazotrophs), yet our understanding of their ecology is constantly evolving. Although marine N2 fixation is often ascribed to cyanobacterial diazotrophs, indirect evidence suggests that non-cyanobacterial diazotrophs (NCDs) might also be important. One widely used approach for understanding diazotroph diversity and biogeography is polymerase chain reaction (PCR) amplification of a portion of the nifH gene, which encodes a structural component of the N2-fixing enzyme complex, nitrogenase. An array of bioinformatic tools exists to process nifH amplicon data; however, the lack of standardized practices has hindered cross-study comparisons. This has led to a missed opportunity to more thoroughly assess diazotroph diversity and biogeography, as well as their potential contributions to the marine N cycle. To address these knowledge gaps, a bioinformatic workflow was designed that standardizes the processing of nifH amplicon datasets originating from high-throughput sequencing (HTS). Multiple datasets are efficiently and consistently processed with a specialized DADA2 pipeline to identify amplicon sequence variants (ASVs). A series of customizable post-pipeline stages then detect and discard spurious nifH sequences and annotate the subsequent quality-filtered nifH ASVs using multiple reference databases and classification approaches. This newly developed workflow was used to reprocess nearly all publicly available nifH amplicon HTS datasets from marine studies and to generate a comprehensive nifH ASV database containing 9383 ASVs aggregated from 21 studies that represent the diazotrophic populations in the global ocean. For each sample, the database includes physical and chemical metadata obtained from the Simons Collaborative Marine Atlas Project (CMAP). Here we demonstrate the utility of this database for revealing global biogeographical patterns of prominent diazotroph groups and highlight the influence of sea surface temperature. The workflow and nifH ASV database provide a robust framework for studying marine N2 fixation and diazotrophic diversity captured by nifH amplicon HTS. Future datasets that target understudied ocean regions can be added easily, and users can tune parameters and studies included for their specific focus. The workflow and database are available, respectively, on GitHub (https://github.com/jdmagasin/nifH-ASV-workflow, last access: 21 January 2025; Morando et al., 2024c) and Figshare (https://doi.org/10.6084/m9.figshare.23795943.v2; Morando et al., 2024b). 

Abstract We introduce the Global rRNA Universal Metabarcoding Plankton database (GRUMP), which consists of 1194 samples that were collected from 2003–2020 and cover extensive latitudinal and longitudinal transects, as well as depth profiles in all major ocean basins. DNA from unfractionated (>0.2 µm) seawater samples was amplified using the 515Y/926 R universal three-domain rRNA gene primers, simultaneously quantifying the relative abundance of amplicon sequencing variants (ASVs) from bacteria, archaea, eukaryotic nuclear 18S, and eukaryotic plastid 16S. Thus, the ratio between taxa in one sample is directly comparable to the ratio in any other GRUMP sample, regardless of gene copy number differences. This obviates a problem in prior global studies that used size-fractionation and different rRNA gene primers for bacteria, archaea, and eukaryotes, precluding comparisons across size fractions or domains. On average, bacteria contributed 71%, eukaryotes 19%, and archaea 8% to rRNA gene abundance, though eukaryotes contributed 32% at latitudes >40°. GRUMP is publicly available on the Simons Collaborative Marine Atlas Project (CMAP), promoting the global comparison of marine microbial dynamics. 

Dinitrogen (N₂) fixation is carried out by specialized microbes, called diazotrophs, and is a major source of nitrogen supporting primary production in oligotrophic oceans. One of the best-characterized diazotroph habitats is the North Pacific Subtropical Gyre (NPSG), where warm, chronically N-limited surface waters promote year-round N₂fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N₂fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (TrichodesmiumandRichelia), unicellular free-livingCrocosphaera, and the UCYN-A/haptophyte symbiosis, based on using microscopy and quantitative PCR (qPCR). However, the diazotroph community in this ecosystem is diverse and includes non-cyanobacterial diazotrophs (NCDs). We investigated the diversity, depth distributions, and seasonality of diazotroph communities at Stn. ALOHA using high throughput sequencing (HTS) ofnifHgene fragments from samples collected throughout the euphotic zone (0-175 m) at near-monthly intervals from June 2013 to July 2016. The UCYN-A symbioses andTrichodesmiumsp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a newCrocosphaera-like species, and several NCDs affiliated with γ- and δ-proteobacteria. Notably, some of the NCDs appear to be stable components of the community at Stn. ALOHA, having also been reported in prior studies. Depth and temporal patterns in microdiversity within two major diazotroph groups (Trichodesmiumand UCYN-A) suggested that sub-populations are adapted to time- and depth-dependent environmental variation. A network analysis of the upper euphotic (0-75 m) HTS data identified two modules that reflect a diazotroph community structure with seasonal turnover between UCYN-A/Gamma A, andTrichodesmium/Crocosphaera. It also reveals the seasonality of several important cyanobacteria and NCDs about which little is known, including a putative δ-proteobacterial phylotype originally discovered at Stn. ALOHA. Collectively, these results underscore the importance of couplingnifHgene HTS with other molecular techniques to obtain a comprehensive view of diazotroph community composition in the marine environment and reveal several understudied diazotroph groups that may contribute to N₂fixation in the NPSG. 

Abstract Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research. 

Abstract Biological nitrogen fixation is a major important source of nitrogen for low-nutrient surface oceanic waters. Nitrogen-fixing (diazotrophic) cyanobacteria are believed to be the primary contributors to this process, but the contribution of non-cyanobacterial diazotrophic organisms in oxygenated surface water, while hypothesized to be important, has yet to be demonstrated. In this study, we used simultaneous¹⁵N-dinitrogen and¹³C-bicarbonate incubations combined with nanoscale secondary ion mass spectrometry analysis to screen tens of thousands of mostly particle-associated, cell-like regions of interest collected from the North Pacific Subtropical Gyre. These dual isotope incubations allow us to distinguish between non-cyanobacterial and cyanobacterial nitrogen-fixing microorganisms and to measure putative cell-specific nitrogen fixation rates. With this approach, we detect nitrogen fixation by putative non-cyanobacterial diazotrophs in the oxygenated surface ocean, which are associated with organic-rich particles (<210 µm size fraction) at two out of seven locations sampled. When present, up to 4.1% of the analyzed particles contain at least one active putative non-cyanobacterial diazotroph. The putative non-cyanobacterial diazotroph nitrogen fixation rates (0.76 ± 1.60 fmol N cell⁻¹d⁻¹) suggest that these organisms are capable of fixing dinitrogen in oxygenated surface water, at least when attached to particles, and may contribute to oceanic nitrogen fixation. 

Abstract Persistent nitrogen depletion in sunlit open ocean waters provides a favorable ecological niche for nitrogen-fixing (diazotrophic) cyanobacteria, some of which associate symbiotically with eukaryotic algae. All known marine examples of these symbioses have involved either centric diatom or haptophyte hosts. We report here the discovery and characterization of two distinct marine pennate diatom-diazotroph symbioses, which until now had only been observed in freshwater environments. Rhopalodiaceae diatomsEpithemia pelagicasp. nov. andEpithemia catenatasp. nov. were isolated repeatedly from the subtropical North Pacific Ocean, and analysis of sequence libraries reveals a global distribution. These symbioses likely escaped attention because the endosymbionts lack fluorescent photopigments, havenifHgene sequences similar to those of free-living unicellular cyanobacteria, and are lost in nitrogen-replete medium. Marine Rhopalodiaceae-diazotroph symbioses are a previously overlooked but widespread source of bioavailable nitrogen in marine habitats and provide new, easily cultured model organisms for the study of organelle evolution. 

Abstract The availability of fixed nitrogen (N) is an important factor limiting biological productivity in the oceans. In coastal waters, high dissolved inorganic N concentrations were historically thought to inhibit dinitrogen (N2) fixation, however, recent N2 fixation measurements and the presence of the N2-fixing UCYN-A/haptophyte symbiosis in nearshore waters challenge this paradigm. We characterized the contribution of UCYN-A symbioses to nearshore N2 fixation in the Southern California Current System (SCCS) by measuring bulk community and single-cell N2 fixation rates, as well as diazotroph community composition and abundance. UCYN-A1 and UCYN-A2 symbioses dominated diazotroph communities throughout the region during upwelling and oceanic seasons. Bulk N2 fixation was detected in most surface samples, with rates up to 23.0 ± 3.8 nmol N l−1 d−1, and was often detected at the deep chlorophyll maximum in the presence of nitrate (>1 µM). UCYN-A2 symbiosis N2 fixation rates were higher (151.1 ± 112.7 fmol N cell−1 d−1) than the UCYN-A1 symbiosis (6.6 ± 8.8 fmol N cell−1 d−1). N2 fixation by the UCYN-A1 symbiosis accounted for a majority of the measured bulk rates at two offshore stations, while the UCYN-A2 symbiosis was an important contributor in three nearshore stations. This report of active UCYN-A symbioses and broad mesoscale distribution patterns establishes UCYN-A symbioses as the dominant diazotrophs in the SCCS, where heterocyst-forming and unicellular cyanobacteria are less prevalent, and provides evidence that the two dominant UCYN-A sublineages are separate ecotypes.