Diazotrophs are overlooked contributors to carbon and nitrogen export to the deep ocean.

Diazotrophs are widespread microorganisms that alleviate nitrogen limitation in 60% of our oceans, thereby regulating marine productivity. Yet, the group-specific contribution of diazotrophs to organic matter export has not been quantified, which so far has impeded an accurate assessment of their impact on the biological carbon pump. Here, we examine the fate of five groups of globally-distributed diazotrophs by using an original combination of mesopelagic particle sampling devices across the subtropical South Pacific Ocean. We demonstrate that cyanobacterial and non-cyanobacterial diazotrophs are exported down to 1000 m depth. Surprisingly, group-specific export turnover rates point to a more efficient export of small unicellular cyanobacterial diazotrophs (UCYN) relative to the larger and filamentous Trichodesmium. Phycoerythrin-containing UCYN-B and UCYN-C-like cells were recurrently found embedded in large (>50 µm) organic aggregates or organized into clusters of tens to hundreds of cells linked by an extracellular matrix, presumably facilitating their export. Beyond the South Pacific, our data are supported by analysis of the Tara Oceans metagenomes collected in other ocean basins, extending the scope of our results globally. We show that, when diazotrophs are found in the euphotic zone, they are also systematically present in mesopelagic waters, suggesting their transport to the deep ocean. We thus conclude that diazotrophs are a significant part of the carbon sequestered in the deep ocean and, therefore, they need to be accounted in regional and global estimates of export.

Sinking Trichodesmium fixes nitrogen in the dark ocean.

The photosynthetic cyanobacterium Trichodesmium is widely distributed in the surface low latitude ocean where it contributes significantly to N2 fixation and primary productivity. Previous studies found nifH genes and intact Trichodesmium colonies in the sunlight-deprived meso- and bathypelagic layers of the ocean (200-4000 m depth). Yet, the ability of Trichodesmium to fix N2 in the dark ocean has not been explored. We performed 15N2 incubations in sediment traps at 170, 270 and 1000 m at two locations in the South Pacific. Sinking Trichodesmium colonies fixed N2 at similar rates than previously observed in the surface ocean (36-214 fmol N cell-1 d-1). This activity accounted for 40 ± 28% of the bulk N2 fixation rates measured in the traps, indicating that other diazotrophs were also active in the mesopelagic zone. Accordingly, cDNA nifH amplicon sequencing revealed that while Trichodesmium accounted for most of the expressed nifH genes in the traps, other diazotrophs such as Chlorobium and Deltaproteobacteria were also active. Laboratory experiments simulating mesopelagic conditions confirmed that increasing hydrostatic pressure and decreasing temperature reduced but did not completely inhibit N2 fixation in Trichodesmium. Finally, using a cell metabolism model we predict that Trichodesmium uses photosynthesis-derived stored carbon to sustain N2 fixation while sinking into the mesopelagic. We conclude that sinking Trichodesmium provides ammonium, dissolved organic matter and biomass to mesopelagic prokaryotes.

Anomalously high abundance of Crocosphaera in the South Pacific Gyre.

The unicellular diazotrophic cyanobacterium Crocosphaera contributes significantly to fixed nitrogen inputs in the oligotrophic ocean. In the western tropical South Pacific Ocean (WTSP), these diazotrophs abound thanks to the phosphorus-rich waters provided by the South Equatorial Current, and iron provided aeolian and subsurface volcanic activity. East of the WTSP, the South Pacific Gyre (SPG) harbors the most oligotrophic and transparent waters of the world's oceans, where only heterotrophic diazotrophs have been reported before. Here, in the SPG, we detected unexpected accumulation of Crocosphaera at 50 m with peak abundances of 5.26 × 105 nifH gene copies l-1. The abundance of Crocosphaera at 50 m was in the same order of magnitude as those detected westwards in the WTSP and represented 100% of volumetric N2 fixation rates. This accumulation at 50 m was likely due to a deeper penetration of UV light in the clear waters of the SPG being detrimental for Crocosphaera growth and N2 fixation activity. Nutrient and trace metal addition experiments did not induce any significant changes in N2 fixation or Crocosphaera abundance, indicating that this population was not limited by the resources tested and could develop in high numbers despite the oligotrophic conditions. Our findings indicate that the distribution of Crocosphaera can extend into subtropical gyres and further understanding of their controlling factors is needed.

Planktonic Aggregates as Hotspots for Heterotrophic Diazotrophy: The Plot Thickens.

Biological dinitrogen (N2) fixation is performed solely by specialized bacteria and archaea termed diazotrophs, introducing new reactive nitrogen into aquatic environments. Conventionally, phototrophic cyanobacteria are considered the major diazotrophs in aquatic environments. However, accumulating evidence indicates that diverse non-cyanobacterial diazotrophs (NCDs) inhabit a wide range of aquatic ecosystems, including temperate and polar latitudes, coastal environments and the deep ocean. NCDs are thus suspected to impact global nitrogen cycling decisively, yet their ecological and quantitative importance remain unknown. Here we review recent molecular and biogeochemical evidence demonstrating that pelagic NCDs inhabit and thrive especially on aggregates in diverse aquatic ecosystems. Aggregates are characterized by reduced-oxygen microzones, high C:N ratio (above Redfield) and high availability of labile carbon as compared to the ambient water. We argue that planktonic aggregates are important loci for energetically-expensive N2 fixation by NCDs and propose a conceptual framework for aggregate-associated N2 fixation. Future studies on aggregate-associated diazotrophy, using novel methodological approaches, are encouraged to address the ecological relevance of NCDs for nitrogen cycling in aquatic environments.

Ingestion of Diazotrophs Makes Corals More Resistant to Heat Stress.

Over the past decade, coral bleaching events have continued to recur and intensify. During bleaching, corals expel millions of their symbionts, depriving the host from its main food source. One mechanism used by corals to resist bleaching consists in exploiting food sources other than autotrophy. Among the food sources available in the reefs, dinitrogen (N2)-fixing prokaryotes or planktonic diazotrophs (hereafter called 'PD') have the particularity to reduce atmospheric dinitrogen (N2) and release part of this nitrogen (diazotroph-derived nitrogen or DDN) in bioavailable form. Here, we submitted coral colonies of Stylophora pistillata, fed or not with planktonic diazotrophs, to a temperature stress of up to 31 ± 0.5 °C and measured their physiological responses (photosynthetic efficiency, symbiont density, and growth rates). Heat-unfed colonies died 8 days after the heat stress while heat-PD-fed corals remained alive after 10 days of heat stress. The supply of PD allowed corals to maintain minimal chlorophyll concentration and symbiont density, sustaining photosynthetic efficiency and stimulating coral growth of up to 48% compared to unfed ones. By providing an alternative source of bioavailable nitrogen and carbon, this specific planktonic diazotroph feeding may have a profound potential for coral bleaching recovery.

Fine-scale sampling unveils diazotroph patchiness in the South Pacific Ocean.

Diazotrophs are important contributors to nitrogen availability in the ocean. Oceanographic cruise data accumulated over the past three decades has revealed a heterogeneous distribution of diazotroph species at regional to global scales. However, dynamic fine-scale physical structures likely affect the distribution of diazotrophs at smaller spatiotemporal scales. The interaction between fine-scale ocean dynamics and diazotrophs remains poorly understood due to typically insufficient spatiotemporal sampling resolution and the lack of parallel detailed physical studies. Here we show the distribution of five groups of diazotrophs in the South Pacific at an unprecedented resolution of 7-16 km. We find a patchy distribution of diazotrophs, with each group being differentially affected by parameters describing fine-scale physical structures. The observed variability in species abundance and distribution would be masked by a coarser sampling resolution, highlighting the need to consider fine-scale physics to resolve the distribution of diazotrophs in the ocean.

Untreated sewage outfalls do not promote Trichodesmium blooms in the coasts of the Canary Islands.

During the summer of 2017, recurrent extensive blooms of the diazotrophic cyanobacterium Trichodesmium invaded the beaches and coastal waters of the Canary Islands, causing great social alarm. Some local media and public sectors ascribed, without any strong scientific evidence, the origin and reactivation of these blooms to untreated sewage outfalls distributed along the coasts. In order to test whether sewage outfalls could have any influence on the metabolic activity of Trichodesmium, we performed 13C and 15N2 uptake experiments with colonies experiencing three different bloom development stages, incubated both with clear seawater and sewage water from an outfall south of Gran Canaria island. Our results showed that sewage outfalls did not promote any increase in dinitrogen (N2) fixation in Trichodesmium, supporting the hypothesis that decaying blooms were generated offshore and transported shoreward by local currents and winds, accumulating mostly leeward of the islands. The combination of unusually warm seawater temperatures, enhanced and sustained stratification of the upper water column and recurrent dust deposition events would have favored the development of the Trichodesmium blooms, which lasted for at least four months.

Dissolved organic matter stimulates N2 fixation and nifH gene expression in Trichodesmium.

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) resources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N2 fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium behaves mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes.

INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

Bleaching forces coral's heterotrophy on diazotrophs and Synechococcus.

Coral reefs are threatened by global warming, which disrupts the symbiosis between corals and their photosynthetic symbionts (Symbiodiniaceae), leading to mass coral bleaching. Planktonic diazotrophs or dinitrogen (N2)-fixing prokaryotes are abundant in coral lagoon waters and could be an alternative nutrient source for corals. Here we incubated untreated and bleached coral colonies of Stylophora pistillata with a 15N2-pre-labelled natural plankton assemblage containing diazotrophs. 15N2 assimilation rates in Symbiodiniaceae cells and tissues of bleached corals were 5- and 30-fold higher, respectively, than those measured in untreated corals, demonstrating that corals incorporate more nitrogen derived from planktonic diazotrophs under bleaching conditions. Bleached corals also preferentially fed on Synechococcus, nitrogen-rich picophytoplanktonic cells, instead of Prochlorococcus and picoeukaryotes, which have a lower cellular nitrogen content. By providing an alternative source of bioavailable nitrogen, both the incorporation of nitrogen derived from planktonic diazotrophs and the ingestion of Synechococcus may have profound consequences for coral bleaching recovery, especially for the many coral reef ecosystems characterized by high abundance and activity of planktonic diazotrophs.

Amino acids promote black carbon aggregation and microbial colonization in coastal waters off Vietnam.

The combustion of fossil fuels and biomass produces pyrogenic organic matter usually known as 'black carbon' (BC), which are transported across the atmosphere as particulate aerosol, eventually deposited on land and oceans. Soil studies have investigated the potential microbial colonization and remineralization of BC particles, but this process has been seldom studied in marine waters. BC provides a significant input of organic carbon to the oceans, yet its fate and role in biogeochemical cycling remains unknown. Here we explored the microbial colonization of BC particles in coastal seawater samples collected in Halong Bay (northern Vietnam). Using high-resolution mass spectrometry and microscopy methods, we observed an increasing colonization of BC particles by marine microbes in the presence of amino acids. Our results suggest that natural organic matter (NOM) present in seawater may promote the microbial colonization and eventual remineralization of BC particles. Future experiments should explore the potential microbial remineralization of BC particles to unveil the role of this massive source of carbon to marine ecosystems.

Mixotrophic metabolism by natural communities of unicellular cyanobacteria in the western tropical South Pacific Ocean.

Cyanobacteria are major contributors to ocean biogeochemical cycling. However, mixotrophic metabolism and the relative importance of inorganic and organic carbon assimilation within the most abundant cyanobacteria are still poorly understood. We explore the ability of Prochlorococcus and Synechococcus to assimilate organic molecules with variable C:N:P composition and its modulation by light availability and photosynthetic impairment. We used a combination of radiolabelled molecules incubations with flow cytometry cell sorting to separate picoplankton groups from the western tropical South Pacific Ocean. Prochlorococcus and Synechococcus assimilated glucose, leucine and ATP at all stations, but cell-specific assimilation rates of N and P containing molecules were significantly higher than glucose. Incubations in the dark or with an inhibitor of photosystem II resulted in reduced assimilation rates. Light-enhanced cell-specific glucose uptake was generally higher for cyanobacteria (∼50%) than for the low nucleic acid fraction of bacterioplankton (LNA, ∼35%). Our results confirm previous findings, based mainly on cultures and genomic potentials, showing that Prochlorococcus and Synechococcus have a flexible mixotrophic metabolism, but demonstrate that natural populations remain primarily photoautotrophs. Our findings indicate that mixotrophy by marine cyanobacteria is more likely to be an adaptation to low inorganic nutrient availability rather than a facultative pathway for carbon acquisition.

Chasing after Non-cyanobacterial Nitrogen Fixation in Marine Pelagic Environments.

Traditionally, cyanobacterial activity in oceanic photic layers was considered responsible for the marine pelagic dinitrogen (N2) fixation. Other potentially N2-fixing bacteria and archaea have also been detected in the pelagic water column, however, the activity and importance of these non-cyanobacterial diazotrophs (NCDs) remain poorly constrained. In this perspective we summarize the N2 fixation rates from recently published studies on photic and aphotic layers that have been attributed to NCD activity via parallel molecular measurements, and discuss the status, challenges, and data gaps in estimating non-cyanobacterial N2 fixation NCNF in the ocean. Rates attributed to NCNF have generally been near the detection limit thus far (<1 nmol N L-1 d-1). Yet, if considering the large volume of the dark ocean, even low rates of NCNF could make a significant contribution to the new nitrogen input to the ocean. The synthesis here shows that nifH transcription data for NCDs have been reported in only a few studies where N2 fixation rates were detected in the absence of diazotrophic cyanobacteria. In addition, high apparent diversity and regional variability in the NCDs complicate investigations of these communities. Future studies should focus on further investigating impacts of environmental drivers including oxygen, dissolved organic matter, and dissolved inorganic nitrogen on NCNF. Describing the ecology of NCDs and accurately measuring NCNF rates, are critical for a future evaluation of the contribution of NCNF to the marine nitrogen budget.

Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific.

The globally distributed diazotroph Trichodesmium contributes importantly to nitrogen inputs in the oligotrophic oceans. Sites of dissolved organic matter (DOM) accumulation could promote the mixotrophic nutrition of Trichodesmium when inorganic nutrients are scarce. Nano-scale secondary ion mass spectrometry (nanoSIMS) analyses of individual trichomes sampled in the South Pacific Ocean, showed significant 13C-enrichments after incubation with either 13C-labeled carbohydrates or amino acids. These results suggest that DOM could be directly taken up by Trichodesmium or primarily consumed by heterotrophic epibiont bacteria that ultimately transfer reduced DOM compounds to their host trichomes. Although the addition of carbohydrates or amino acids did not significantly affect bulk N2 fixation rates, N2 fixation was enhanced by amino acids in individual colonies of Trichodesmium. We discuss the ecological advantages of DOM use by Trichodesmium as an alternative to autotrophic nutrition in oligotrophic open ocean waters.

Diazotrophs: a non-negligible source of nitrogen for the tropical coral Stylophora pistillata.

Corals are mixotrophs: they are able to fix inorganic carbon through the activity of their symbiotic dinoflagellates and to gain nitrogen from predation on plankton and uptake of dissolved organic and inorganic nutrients. They also live in close association with diverse diazotrophic communities, inhabiting their skeleton, tissue and mucus layer, which are able to fix dinitrogen (N2). The quantity of fixed N2 transferred to the corals and its distribution within coral compartments as well as the quantity of nitrogen assimilated through the ingestion of planktonic diazotrophs are still unknown. Here, we quantified nitrogen assimilation via (i) N2 fixation by symbiont diazotrophs, (ii) ingestion of cultured unicellular diazotrophs and (iii) ingestion of natural planktonic diazotrophs. We estimate that the ingestion of diazotrophs provides 0.76±0.15 µg N cm(-2) h(-1), suggesting that diazotrophs represent a non-negligible source of nitrogen for scleractinian corals.

Mesopelagic N2 Fixation Related to Organic Matter Composition in the Solomon and Bismarck Seas (Southwest Pacific).

Dinitrogen (N2) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N2 fixation rates (up to ~1 nmol N L-1 d-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N2 fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N2 fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 103 nifH gene copies L-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.