Diatom-diazotroph Associations Research Articles

Marine nitrogen cycling dynamics under altering redox conditions: Insights from deposition of sapropels S1 and the ambiguous S2 in the Eastern Mediterranean Sea

The eastern Mediterranean Sea (EMS) sedimentary record is periodically interspersed with organic-rich ‘sapropel’ layers. Sapropels are characteristic of basin-wide anoxic events, triggered by precession-forced insolation maxima. Relatively subdued insolation maxima, however, are not always expressed as distinct sapropel events. The EMS sedimentary record is thus useful to investigate feedbacks between marine anoxia and the nitrogen (N) cycle and offers an analogue for modern deoxygenation and past oceanic anoxic events. To this end, we investigated a ∼68 kyr sedimentary record from the EMS containing the well-established sapropel S1 (deposited in two phases: S1a [∼10.5–8.5 ka BP] and S1b [∼7.8–6.1 ka BP]) and sediments timed to the ambiguous S2 sapropel (∼53 ka BP). We used lipid biomarkers of microorganisms to reconstruct key N-cycle components: (1) anaerobic ammonium oxidation (anammox) using ladderanes and a stereoisomer of bacteriohopanetetrol (BHT-x), (2) dinitrogen gas (N2) fixation using heterocyte glycolipids, and (3) nitrification by Thaumarchaeota using crenarchaeol. Additionally, benthic foraminifera and trace metals (U, Mo, Mn) were used to reconstruct redox conditions. During S1a, abundances of Thaumarchaeota increased, likely promoted by elevated high-nutrient freshwater discharge. At this time, a combination of phosphorus supply and intensified loss of bioavailable N via water column anammox, may have reinforced anoxia by favoring diatom-diazotroph associations. During S1b, anammox is equally intense. Yet, no positive feedback on N2-fixation is observed, likely because diazotrophs were phosphorus limited. Instead, anammox may have provided negative feedback on anoxia by quenching primary production. Ladderanes suggest additional episodes of anammox between ∼69 to 39 cal ka BP, corresponding to brief periods of water column deoxygenation. Anoxia likely occurred at the sediment–water interface in S2-timed sediments (53–51 cal ka BP). During these episodes, ladderanes co-occur with the later eluting BHT-34R stereoisomer. δ13CBHT-34R indicate an anammox source, potentially synthesized by marine sedimentary anammox bacteria. No corresponding increase in diatom-diazotroph associations is observed, likely due to the oligotrophic conditions and the limited effect of sedimentary anammox on N-availability in the euphotic zone. Our results highlight various modes of operation of the N-cycle at different degrees of deoxygenation, which depend amongst others on nutrient-availability and the niche-segregation of N-loss and N2-fixating microorganisms.

Enhancement of Summer Nitrogen Fixation by the Kuroshio Intrusion in the East China Sea and Southern Yellow Sea

AbstractThe western boundary currents are characterized by abundant diazotrophs including Trichodesmium, which may fuel N2 fixation when they intrude into marginal seas. The Kuroshio, a western boundary current in the North Pacific, flows into the East China Sea (ECS) and southern Yellow Sea (SYS), which transports abundant Trichodesmium and diatom‐diazotroph associations (DDAs). Additionally, low nitrogen:phosphorus (N:P) ratio and relatively abundant dissolved iron have been observed in the offshore ECS because of the Kuroshio intrusion as well as riverine/atmospheric inputs of P and iron. We hypothesized that the intrusion of Kuroshio greatly enhanced N2 fixation in the ECS and SYS. N2 fixation rates (NFRs) were measured using a 15N2 bubble method during summer 2013. The surface and depth‐integrated NFRs in the ECS and SYS were 1.45 nmol N L−1 d−1 and 81.7 μmol N m−2 d−1 on average, respectively, with the highest values of 13.84 nmol N L−1 d−1 and 511.8 μmol N m−2 d−1. We found that NFRs were significantly higher in the ECS oceanic (Kuroshio water) and mesohaline regions (Kuroshio‐affected water) than in the SYS and the ECS low‐salinity and coastal upwelling regions. NFR was significantly positively correlated with the densities of Trichodesmium and DDAs, salinity, and temperature but was negatively with NO3− and N:P ratio. Generalized additive models confirmed that spatial variation in NFR was overwhelmingly contributed by Trichodesmium density. These findings suggested that the Kuroshio intrusion significantly enhanced N2 fixation in the ECS through promoting growth of filamentous diazotrophs and providing appropriate nutrient environment.

Low-Ammonium Environment Increases the Nutrient Exchange between Diatom-Diazotroph Association Cells and Facilitates Photosynthesis and N2 Fixation-a Mechanistic Modeling Analysis.

Diatom–diazotroph associations (DDAs) are one of the most important symbiotic dinitrogen (N2) fixing groups in the oligotrophic ocean. Despite their capability to fix N2, ammonium (NH4+) remains a key nitrogen (N) source for DDAs, and the effect of NH4+ on their metabolism remains elusive. Here, we developed a coarse-grained, cellular model of the DDA with NH4+ uptake and quantified how the level of extracellular NH4+ influences metabolism and nutrient exchange within the symbiosis. The model shows that, under a fixed growth rate, an increased NH4+ concentration may lower the required level of N2 fixation and photosynthesis, and decrease carbon (C) and N exchange. A low-NH4+ environment leads to more C and N in nutrient exchange and more fixed N2 to support a higher growth rate. With higher growth rates, nutrient exchange and metabolism increased. Our study shows a strong effect of NH4+ on metabolic processes within DDAs, and thus highlights the importance of in situ measurement of NH4+ concentrations.

Long-Term Eutrophication and Dynamics of Bloom-Forming Microbial Communities during Summer HAB in Large Arctic Lake

Harmful algal blooms (HABs) in arctic lakes are recent phenomena. In our study, we performed a long-term analysis (1990–2017) of the eutrophication of Lake Imandra, a large subarctic lake, and explored the biodiversity of bloom-forming microorganisms of a 2017 summer HAB. We performed a 16Sr rRNA metabarcoding study of microbial communities, analysed the associations between N, P, C, and chlorophyll concentrations in the lake water, and developed models for the prediction of HABs based on total P concentration. We have demonstrated that blooms in Lake Imandra occur outside of optimal Redfield ratios and have a nonlinear association with P concentrations. We found that recent summer HABs in a lake occur as simultaneous blooms of a diatom Aulacoseira sp. and cyanobacteria Dolichospermum sp. We have studied the temporal dynamics of microbial communities during the bloom and performed an analysis of the publicly available Dolichospermum genomes to outline potential genetic mechanisms beneath simultaneous blooming. We found genetic traits requisite for diatom-diazotroph associations, which may lay beneath the simultaneous blooming of Aulacoseira sp. and Dolichospermum sp. in Lake Imandra. Both groups of organisms have the ability to store nutrients and form a dormant stage. All of these factors will ensure the further development of the HABs in Lake Imandra and the dispersal of these bloom-forming species to neighboring lakes.

Carbon Transfer from the Host Diatom Enables Fast Growth and High Rate of N2 Fixation by Symbiotic Heterocystous Cyanobacteria.

Diatom–diazotroph associations (DDAs) are symbioses where trichome-forming cyanobacteria support the host diatom with fixed nitrogen through dinitrogen (N2) fixation. It is inferred that the growth of the trichomes is also supported by the host, but the support mechanism has not been fully quantified. Here, we develop a coarse-grained, cellular model of the symbiosis between Hemiaulus and Richelia (one of the major DDAs), which shows that carbon (C) transfer from the diatom enables a faster growth and N2 fixation rate by the trichomes. The model predicts that the rate of N2 fixation is 5.5 times that of the hypothetical case without nitrogen (N) transfer to the host diatom. The model estimates that 25% of fixed C from the host diatom is transferred to the symbiotic trichomes to support the high rate of N2 fixation. In turn, 82% of N fixed by the trichomes ends up in the host. Modeled C fixation from the vegetative cells in the trichomes supports only one-third of their total C needs. Even if we ignore the C cost for N2 fixation and for N transfer to the host, the total C cost of the trichomes is higher than the C supply by their own photosynthesis. Having more trichomes in a single host diatom decreases the demand for N2 fixation per trichome and thus decreases their cost of C. However, even with five trichomes, which is about the highest observed for Hemiaulus and Richelia symbiosis, the model still predicts a significant C transfer from the diatom host. These results help quantitatively explain the observed high rates of growth and N2 fixation in symbiotic trichomes relative to other aquatic diazotrophs.

Biomarker evidence for nitrogen-fixing cyanobacterial blooms in a brackish surface layer in the Nile River plume during sapropel deposition

Abstract Sapropels are organic-rich sediment layers deposited in the eastern Mediterranean Sea during precession minima, resulting from an increase in export productivity and/or preservation. Increased freshwater delivery from the African continent resulted in stratification, causing deepwater anoxia, while nutrient input stimulated productivity, presumably at the deep chlorophyll maximum. Previous studies have suggested that during sapropel deposition, nitrogen fixation was widespread in the highly stratified surface waters, and that cyanobacteria symbiotic with diatoms (diatom-diazotroph associations, DDAs) were responsible. Here we analyzed sapropel S5 sediments for heterocyst glycolipids (HGs) from three locations in the eastern Mediterranean. HG biomarkers can differentiate between those heterocystous cyanobacteria that are free living (found predominately in freshwater or brackish environments) and those that are from DDAs (found in marine settings). In our primary core, from a location which would have been influenced by the Nile River outflow, we detected a HG with a pentose (C5) head group specific for DDAs. However, HGs with a hexose (C6) head group, specific to free-living cyanobacteria, were present in substantially (up to 60×) higher concentration. These data suggest that at our study location, free-living cyanobacteria were the dominant diazotrophs, rather than DDAs. The C6 HGs increased substantially at the onset of sapropel S5 deposition, suggesting that substantial seasonal cyanobacterial blooms were associated with a brackish surface layer flowing from the Nile into the eastern Mediterranean. Two additional S5 sapropels were analyzed, one also from the Nile delta region and one from the region between Libya and southwestern Crete. Overall, comparison of the HG distribution in the three S5 sapropels provides evidence that all three locations were initially influenced by surface salinities that were sufficiently low to support free-living heterocystous cyanobacteria. While free-living heterocystous cyanobacteria continued to outnumber DDAs during sapropel deposition at the two Nile-influenced sites, DDAs, indicators of persistent marine salinities, were the dominant diazotrophs in the upper part of the sapropel at the more westerly site. These results indicate that N2 fixation by free-living cyanobacteria offers an important additional mechanism to stimulate productivity in regions with strong river discharge during sapropel deposition.

Seasonal modulation of mesoscale processes alters nutrient availability and plankton communities in the Red Sea

Hydrographic and atmospheric forcing set fundamental constraints on the biogeochemistry of aquatic ecosystems and manifest in the patterns of nutrient availability and recycling, species composition of communities, trophic dynamics, and ecosystem metabolism. In the Red Sea, latitudinal gradients in environmental conditions and primary production have been ascribed to fluctuations in Gulf of Aden Water inflow, upwelling/mixing, and regenerated nutrient utilization i.e. rapidly recycled nitrogen in upper layers. However, our understanding of upper layer dynamics and related changes in plankton communities, metabolism and carbon and nitrogen export is limited. We surmised that stratification and mesoscale eddies modulate the nutrient availability and taxonomic identity of plankton communities in the Red Sea. Based on remote-sensing data of sea level anomalies and high resolution in situ measurements (ScanFish) we selected stations for hydrographic CTD profiles, water sampling (nutrients, seawater oxygen stable isotopes [δ18OSW]), phytoplankton and zooplankton collections. In fall 2014, strong stratification subjected the plankton community to an overall nitrogen and phosphorus shortage. The nutrient deficiency increased numbers of heterotrophic dinoflagellates, microzooplankton, and diazotrophs (Trichodesmium, diatom-diazotroph associations [DDAs]), albeit largely decreased phytoplankton and mesozooplankton abundances. In spring 2015, mesoscale eddies increased the nutrient availability, and the thermohaline characteristics and low δ18OSW point to the interaction of eddies with Gulf of Aden Surface Water (GASW). Cyclonic eddies and, most likely, the availability of nutrients associated with the GASW, increased the abundances of autotrophs (diatoms, Prasinophytes) and supported larger numbers of zooplankton and their larvae. We demonstrate that the interplay of stratification, advection of Gulf of Aden water and mesoscale eddies are key elements to better understand changes in plankton community composition, ecosystem metabolism, and macronutrient export in the Red Sea in space and time.

The genetic diversity and evolution of diatom-diazotroph associations highlights traits favoring symbiont integration.

Diatom diazotroph associations (DDAs) are a widespread marine planktonic symbiosis between several diatom genera and di-nitrogen (N2)-fixing bacteria. Combining single cell confocal microscopy observations and molecular genetic approaches on individual field collected cells, we determined the phylogenetic diversity, distribution and evolution of the DDAs. Confocal analyses coupled with 3-D imaging re-evaluated the cellular location of DDA symbionts. DDA diversity was resolved by paired gene sequencing (18S rRNA and rbcL genes, 16S rRNA and nifH genes). A survey using the newly acquired sequences against public databases found sequences with high similarity (99–100%) to either host (18S rRNA) or symbiont (16S rRNA) in atypical regions for DDAs (high latitudes, anoxic basin and copepod gut). Concatenated phylogenies were congruent for the host and cyanobacteria sequences and implied co-evolution. Time-calibrated trees dated the appearance of N2 fixing planktonic symbiosis from 100–50Mya and were consistent with the symbiont cellular location: symbioses with internal partners are more ancient. An ancestral state reconstruction traced the evolution of traits in DDAs and highlight that the adaptive radiation to the marine environment was likely facilitated by the symbiosis. Our results present the evolutionary nature of DDAs and provide new genetic and phenotypic information for these biogeochemically relevant populations.

The Influence of Riverine Nutrients in Niche Partitioning of Phytoplankton Communities–A Contrast Between the Amazon River Plume and the Changjiang (Yangtze) River Diluted Water of the East China Sea

Riverine nutrients acting in concert with local hydrographic conditions create distinct ecological niches for phytoplankton communities across river-ocean continuums. Here we compare two of the world’s largest river-ocean systems, the Amazon River Plume (ARP) which outflows into the Western Tropical North Atlantic (WTNA) and the Changjiang Diluted Water (CDW) which empties into the East China Sea to show how distinctly different N: P ratios of their source waters, shape phytoplankton communities along the river-ocean continuum. Sampling in the relatively unpolluted surface waters of the ARP during peak river discharge revealed that phytoplankton communities along the river-ocean continuum were strongly controlled by Dissolved Inorganic Nitrogen (DIN, nitrate plus nitrite) concentrations which were low or beyond detectable, while Dissolved Inorganic Phosphorous (DIP, phosphate) was not limiting. Downstream of the plume, nutrient concentrations at the surface were far below the Redfield N:P ratio of 16 : 1, and in this severely nitrogen limited but silica replete mesohaline region, Diatom-Diazotroph Associations (DDA) such as Hemiaulus hauckii-Richelia proliferated, while offshore in nitrate poor oceanic waters, Trichodesmium spp. and Prochlorococcus thrived. In contrast, in the CDW, a system which has witnessed a substantial rise in anthropogenic nitrogen inputs from human pressures within the Changjiang River system, phytoplankton community structure was regulated primarily by DIP which was rapidly consumed in the inner shelf. Data from a series of summer-time cruises in the mid-shelf and offshore regions of the East China Sea, at peak discharge, established that when DIP limitation was ameliorated by on-shelf upwelling of DIP rich Kuroshio Intermediate Waters, diatoms and dinoflagellates dominated. Conversely, during years of heavy discharge, the westward flowing CDW plume was severely DIP limited, probably because water column stratification dampened upwelling of subsurface waters. The consequent phosphate limitation led to the proliferation of small phytoplankton such as Chlorophytes and Cyanobacteria. The absence of diazotrophs in the CDW, leads us to hypothesize that river-ocean continuums, whose source waters are heavily impacted by anthropogenic activities and with nutrient concentrations substantially in excess of Redfield ratios, may not support diatoms on account of DIP limitation nor DDAs and diazotrophs because of excess DIN.

Periodic and coordinated gene expression between a diazotroph and its diatom host.

In the surface ocean, light fuels photosynthetic carbon fixation of phytoplankton, playing a critical role in ecosystem processes including carbon export to the deep sea. In oligotrophic oceans, diatom-diazotroph associations (DDAs) play a keystone role in ecosystem function because diazotrophs can provide otherwise scarce biologically available nitrogen to the diatom host, fueling growth and subsequent carbon sequestration. Despite their importance, relatively little is known about the nature of these associations in situ. Here we used metatranscriptomic sequencing of surface samples from the North Pacific Subtropical Gyre (NPSG) to reconstruct patterns of gene expression for the diazotrophic symbiont Richelia and we examined how these patterns were integrated with those of the diatom host over day-night transitions. Richelia exhibited significant diel signals for genes related to photosynthesis, N2 fixation, and resource acquisition, among other processes. N2 fixation genes were significantly co-expressed with host nitrogen uptake and metabolism, as well as potential genes involved in carbon transport, which may underpin the exchange of nitrogen and carbon within this association. Patterns of expression suggested cell division was integrated between the host and symbiont across the diel cycle. Collectively these data suggest that symbiont-host physiological ecology is strongly interconnected in the NPSG.

Programmed cell death in diazotrophs and the fate of organic matter in the western tropical South Pacific Ocean during the OUTPACE cruise

Abstract. The fate of diazotroph (N2 fixers) derived carbon (C) and nitrogen (N) and their contribution to vertical export of C and N in the western tropical South Pacific Ocean was studied during OUTPACE (Oligotrophy to UlTra-oligotrophy PACific Experiment). Our specific objective during OUTPACE was to determine whether autocatalytic programmed cell death (PCD), occurring in some diazotrophs, is an important mechanism affecting diazotroph mortality and a factor regulating the vertical flux of organic matter and, thus, the fate of the blooms. We sampled at three long duration (LD) stations of 5 days each (LDA, LDB and LDC) where drifting sediment traps were deployed at 150, 325 and 500 m depth. LDA and LDB were characterized by high chlorophyll a (Chl a) concentrations (0.2–0.6 µg L−1) and dominated by dense biomass of the filamentous cyanobacterium Trichodesmium as well as UCYN-B and diatom–diazotroph associations (Rhizosolenia with Richelia-detected by microscopy and het-1 nifH copies). Station LDC was located at an ultra-oligotrophic area of the South Pacific gyre with extremely low Chl a concentration (∼ 0.02 µg L−1) with limited biomass of diazotrophs predominantly the unicellular UCYN-B. Our measurements of biomass from LDA and LDB yielded high activities of caspase-like and metacaspase proteases that are indicative of PCD in Trichodesmium and other phytoplankton. Metacaspase activity, reported here for the first time from oceanic populations, was highest at the surface of both LDA and LDB, where we also obtained high concentrations of transparent exopolymeric particles (TEP). TEP were negatively correlated with dissolved inorganic phosphorus and positively coupled to both the dissolved and particulate organic carbon pools. Our results reflect the increase in TEP production under nutrient stress and its role as a source of sticky carbon facilitating aggregation and rapid vertical sinking. Evidence for bloom decline was observed at both LDA and LDB. However, the physiological status and rates of decline of the blooms differed between the stations, influencing the amount of accumulated diazotrophic organic matter and mass flux observed in the traps during our experimental time frame. At LDA sediment traps contained the greatest export of particulate matter and significant numbers of both intact and decaying Trichodesmium, UCYN-B and het-1 compared to LDB where the bloom decline began only 2 days prior to leaving the station and to LDC where no evidence for bloom or bloom decline was seen. Substantiating previous findings from laboratory cultures linking PCD to carbon export in Trichodesmium, our results from OUTPACE indicate that nutrient limitation may induce PCD in high biomass blooms such as displayed by Trichodesmium or diatom–diazotroph associations. Furthermore, PCD combined with high TEP production will tend to facilitate cellular aggregation and bloom termination and will expedite vertical flux to depth.

C<sub>5</sub> glycolipids of heterocystous cyanobacteria track symbiont abundance in the diatom <i>Hemiaulus hauckii</i> across the tropical North Atlantic

Abstract. Diatom–diazotroph associations (DDAs) include marine heterocystous cyanobacteria found as exosymbionts and endosymbionts in multiple diatom species. Heterocysts are the site of N2 fixation and have thickened cell walls containing unique heterocyst glycolipids which maintain a low oxygen environment within the heterocyst. The endosymbiotic cyanobacterium Richelia intracellularis found in species of the diatom genus Hemiaulus and Rhizosolenia makes heterocyst glycolipids (HGs) which are composed of C30 and C32 diols and triols with pentose (C5) moieties that are distinct from limnetic cyanobacterial HGs with predominantly hexose (C6) moieties. Here we applied a method for analysis of intact polar lipids to the study of HGs in suspended particulate matter (SPM) and surface sediment from across the tropical North Atlantic. The study focused on the Amazon plume region, where DDAs are documented to form extensive surface blooms, in order to examine the utility of C5 HGs as markers for DDAs as well as their transportation to underlying sediments. C30 and C32 triols with C5 pentose moieties were detected in both marine SPM and surface sediments. We found a significant correlation between the water column concentration of these long-chain C5 HGs and DDA symbiont counts. In particular, the concentrations of both the C5 HGs (1-(O-ribose)-3,27,29-triacontanetriol (C5 HG30 triol) and 1-(O-ribose)-3,29,31-dotriacontanetriol (C5 HG32 triol)) in SPM exhibited a significant correlation with the number of Hemiaulus hauckii symbionts. This result strengthens the idea that long-chain C5 HGs can be applied as biomarkers for marine endosymbiotic heterocystous cyanobacteria. The presence of the same C5 HGs in surface sediment provides evidence that they are effectively transported to the sediment and hence have potential as biomarkers for studies of the contribution of DDAs to the paleo-marine N cycle.

Seasonal resource conditions favor a summertime increase in North Pacific diatom\u2013diazotroph associations

In the North Pacific Subtropical Gyre (NPSG), an annual pulse of sinking organic carbon is observed at 4000 m between July and August, driven by large diatoms found in association with nitrogen fixing, heterocystous, cyanobacteria: Diatom–Diazotroph Associations (DDAs). Here we ask what drives the bloom of DDAs and present a simplified trait-based model of subtropical phototroph populations driven by observed, monthly averaged, environmental characteristics. The ratio of resource supply rates favors nitrogen fixation year round. The relative fitness of DDA traits is most competitive in early summer when the mixed layer is shallow, solar irradiance is high, and phosphorus and iron are relatively abundant. Later in the season, as light intensity drops and phosphorus is depleted, the traits of small unicellular diazotrophs become more competitive. The competitive transition happens in August, at the time when the DDA export event occurs. This seasonal dynamic is maintained when embedded in a more complex, global-scale, ecological model, and provides predictions for the extent of the North Pacific DDA bloom. The model provides a parsimonious and testable hypothesis for the stimulation of DDA blooms.

A Short Comparison of Two Marine Planktonic Diazotrophic Symbioses Highlights an Un-quantified Disparity

Some N2-fixing cyanobacteria form symbiosis with diverse protists. In the plankton two groups of diazotrophic symbioses are described: (1) a collective group of diatoms which associate with heterocystous cyanobacteria (Diatom Diazotroph Associations, DDA), and (2) the microalgal prymnesiophyte Braarudosphaera bigelowii and its relatives which associate with the unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (hereafter as UCYN-A). Both symbiotic systems co-occur, and in both partnerships the symbionts function as a nitrogen (N) source. In this perspective, we provide a brief comparison between the DDAs and the prymnesiophyte- UCYN-A symbioses highlighting similarities and differences in both systems, and present a bias in the attention and current methodology that has led to an under-detection and under-estimation of the DDAs.

Mesozooplankton Graze on Cyanobacteria in the Amazon River Plume and Western Tropical North Atlantic.

Diazotrophic cyanobacteria, those capable of fixing di-nitrogen (N2), are considered one of the major sources of new nitrogen (N) in the oligotrophic tropical ocean, but direct incorporation of diazotrophic N into food webs has not been fully examined. In the Amazon River-influenced western tropical North Atlantic (WTNA), diatom diazotroph associations (DDAs) and the filamentous colonial diazotrophs Trichodesmium have seasonally high abundances. We sampled epipelagic mesozooplankton in the Amazon River plume and WTNA in May–June 2010 to investigate direct grazing by mesozooplankton on two DDA populations: Richelia associated with Rhizosolenia diatoms (het-1) and Hemiaulus diatoms (het-2), and on Trichodesmium using highly specific qPCR assays targeting nitrogenase genes (nifH). Both DDAs and Trichodesmium occurred in zooplankton gut contents, with higher detection of het-2 predominantly in calanoid copepods (2.33–16.76 nifH copies organism-1). Abundance of Trichodesmium was low (2.21–4.03 nifH copies organism-1), but they were consistently detected at high salinity stations (>35) in calanoid copepods. This suggests direct grazing on DDAs, Trichodesmium filaments and colonies, or consumption as part of sinking aggregates, is common. In parallel with the qPCR approach, a next generation sequencing analysis of 16S rRNA genes identified that cyanobacterial assemblage associated with zooplankton guts was dominated by the non-diazotrophic unicellular phylotypes Synechococcus (56%) and Prochlorococcus (26%). However, in two separate calanoid copepod samples, two unicellular diazotrophs Candidatus Atelocyanobacterium thalassa (UCYN-A) and Crocosphaera watsonii (UCYN-B) were present, respectively, as a small component of cyanobacterial assemblages (<2%). This study represents the first evidence of consumption of DDAs, Trichodesmium, and unicellular cyanobacteria by calanoid copepods in an area of the WTNA known for high carbon export. These diazotroph populations are quantitatively important in the global N budget, widespread and hence, the next step is to accurately quantify grazing. Nonetheless, these results highlight a direct pathway of diazotrophic N into the food web and have important implications for biogeochemical cycles, particularly oligotrophic regions where N2 fixation is the main source of new nitrogen.

Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA).

Diatom diazotroph associations (DDAs) are important components in the world’s oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont’s nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (>106 cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30–35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (>0.1 μmol L-1) and Si (>1.0 μmol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 × 105 nifH copies L-1) than het-2 and limited to regions of oceanic SSS (>36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (>103 nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8–34.3), immeasurable DIN, moderate DIP (0.1–0.60 μmol L-1) and higher Si (4.19–22.1 μmol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations.

Amazon River influence on nitrogen fixation and export production in the western tropical North Atlantic

AbstractAs part of the ANACONDAS program, we investigated the role of the Amazon plume in stimulating offshore nitrogen fixation and export production during the river's high‐discharge period (May–June 2010). Using the shipboard underway system, we performed high‐resolution sampling of over 450,000 km2 of surface waters, characterizing the distribution of nutrients, phytoplankton, particulate organic matter (POM), and stable carbon and nitrogen isotopes of POM in the offshore plume. We found distinct regional variations in diazotroph communities, with the Diatom‐Diazotroph Associations (DDA) Hemiaulus hauckii – Richelia intracellularis dominating the low N : P mesohaline waters to the northwest of the plume axis and Trichodesmium spp. primarily occupying oceanic waters to the east. Nutrient availability broadly shaped diazotroph distributions along the salinity gradient, but habitat longevity may also play a role in the finer‐scale distributions of communities, particularly of DDAs. H. hauckii and Trichodesmium spp. affected the nitrogen and carbon budgets in fundamentally different ways within the plume‐influenced regions, with H. hauckii making much greater contributions to the particulate nitrogen pool and to CO2 drawdown than Trichodesmium spp., leading to much higher export fluxes. Our findings provide an important constraint on the role of the Amazon plume in creating distinct niches and roles for diazotrophs in the nutrient and carbon budgets of the western tropical North Atlantic.

Phytoplankton community structure in the VAHINE mesocosm experiment

Abstract. The VAHINE mesocosm experiment was designed to trigger a diazotroph bloom and to follow the subsequent transfer of diazotroph-derived nitrogen (DDN) in the rest of the food web. Three mesocosms (50 m3) located inside the Nouméa lagoon (New Caledonia, southwestern Pacific) were enriched with dissolved inorganic phosphorus (DIP) in order to promote N2 fixation in these low-nutrient, low-chlorophyll (LNLC) waters. Initially, the diazotrophic community was dominated by diatom diazotroph associations (DDAs), mainly by Rhizosolenia/Richelia intracellularis, and by Trichodesmium, which fueled enough DDN to sustain the growth of other diverse diatom species and Synechococcus populations that were well adapted to limiting DIP levels. After DIP fertilization (1 µM) on day 4, an initial lag time of 10 days was necessary for the mesocosm ecosystems to start building up biomass. However, changes in community structure were already observed during this first period, with a significant drop of both Synechococcus and diatom populations, while Prochlorococcus benefited from DIP addition. At the end of this first period, corresponding to when most added DIP was consumed, the diazotroph community changed drastically and became dominated by Cyanothece-like (UCYN-C) populations, which were accompanied by a monospecific bloom of the diatom Cylindrotheca closterium. During the second period, biomass increased sharply together with primary production and N2-fixation fluxes near tripled. Diatom populations, as well as Synechococcus and nanophytoeukaryotes, showed a re-increase towards the end of the experiment, showing efficient transfer of DDN to non-diazotrophic phytoplankton.

Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

Biogeochemical and biological impacts of diazotroph blooms in a low-nutrient, low-chlorophyll ecosystem: synthesis from the VAHINE mesocosm experiment (New Caledonia)

Abstract. In marine ecosystems, biological N2 fixation provides the predominant external source of nitrogen (N; 140 ± 50 Tg N yr−1), contributing more than atmospheric and riverine inputs to the N supply. Yet the fate and magnitude of the newly fixed N, or diazotroph-derived N (hereafter named DDN) in marine ecosystems is poorly understood. Moreover, whether the DDN is preferentially and directly exported out of the photic zone, recycled by the microbial loop and/or transferred into larger organisms remains unclear. These questions were investigated in the framework of the VAHINE (VAriability of vertical and tropHIc transfer of diazotroph derived N in the south wEst Pacific) project. Triplicate large volume ( ∼ 50 m3) mesocosms were deployed in the tropical south-west Pacific coastal ocean (New Caledonia). The mesocosms were intentionally fertilized with ∼ 0.8 µM dissolved inorganic phosphorus (DIP) at the start of the experiment to stimulate diazotrophy. A total of 47 stocks, fluxes, enzymatic activities and diversity parameters were measured daily inside and outside the mesocosms by the 40 scientists involved in the project. The experiment lasted for 23 days and was characterized by two distinct and successive diazotroph blooms: a dominance of diatom-diazotroph associations (DDAs) during the first half of the experiment (days 2–14) followed by a bloom of unicellular cyanobacterial lineage C (UCYN-C during the second half of the experiment (days 15–23). These conditions provided a unique opportunity to compare the DDN transfer and export efficiency associated with different diazotrophs. Here we summarize the major experimental and modelling results obtained during the project and described in the VAHINE special issue, in particular those regarding the evolution of the main standing stocks, fluxes and biological characteristics over the 23-day experiment, the contribution of N2 fixation to export fluxes, the DDN released to dissolved pool and its transfer to the planktonic food web (bacteria, phytoplankton, zooplankton). We then apply our Eco3M modelling platform to further infer the fate of DDN in the ecosystem and the role of N2 fixation on productivity, food web structure and carbon export. Recommendations for future work are finally provided in the conclusion section.