Mar Benavides

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.

Five decades of N2 fixation research in the North Atlantic Ocean

Dinitrogen (N2) fixation (the reduction of atmospheric N2 to ammonium by specialized prokaryotic microbes), represents an important input of fixed nitrogen and contributes significantly to primary productivity in the oceans. Marine N2 fixation was discovered in the North Atlantic Ocean (NA) in the 1960s. Ever since, the NA has been subject to numerous studies that have looked into the diversity and abundance of N2-fixing microbes (diazotrophs), the spatial and temporal variability of N2 fixation rates, and the range of physical and chemical variables that control them. The NA provides 10-25% of the globally fixed N2, ranking as the third basin with the largest N2 fixation inputs in the world’s oceans. This basin suffers a chronic depletion in phosphorus availability, more aeolian dust deposition than any other basin in the world’s oceans, and significant nutrient inputs from important rivers like the Amazon and the Congo. These characteristics make it unique in comparison with other oceanic basins. After five decades of intensive research, here we present a comprehensive review of our current understanding of diazotrophic activity in the NA from both a geochemical and biological perspective. We discuss the advantages and disadvantages of current methods, future perspectives, and questions which remain to be answered.

High‐nitrogen fixation rates in the particulate and dissolved pools in the Western Tropical Pacific (Solomon and Bismarck Seas)

Dinitrogen (N-2) fixation rates were investigated in the euphotic layer of the Bismarck and Solomon Seas using N-15(2) incubation assays taking into account both the particulate and the dissolved pools. Average depth-integrated particulate N-2 fixation rates were 203 (range 43-399) and 1396 (range 176-3132)mol N m(-2) d(-1) in the Bismarck and Solomon Seas, respectively. In both seas, N-2 fixation measured in the dissolved pool was similar to particulate N-2 fixation, highlighting the potentially substantial underestimation of N-2 fixation in oceanic budgets when only particulate N-2 fixation is considered. Among the diazotroph phylotypes targeted using quantitative polymerase chain reaction amplification of nifH genes, Trichodesmium was the most abundant. Regression analyses suggest that it accounted for the major proportion of N-2 fixation. However, unicellular cyanobacterial and non-cyanobacterial diazotrophs were also occasionally abundant. This study reports high pelagic N-2 fixation rates and confirms that the Western Tropical South Pacific is a hot spot for marine N-2 fixation.

Basin‐wide N2 fixation in the deep waters of the Mediterranean Sea

Recent findings indicate that N 2 fixation is significant in aphotic waters, presumably due to heterotrophic diazotrophs depending on organic matter for their nutrition. However, the relationship between organic matter and heterotrophic N 2 fixation remains unknown. Here we explore N 2 fixation in the deep chlorophyll maximum and underneath deep waters across the whole Mediterranean Sea and relate it to organic matter composition, characterized by optical and molecular methods. Our N 2 fixation rates were in the range of those previously reported for the euphotic zone of the Mediterranean Sea (up to 0.43 nmol N L A1 d A1) and were significantly correlated to the presence of relatively labile organic matter with fluorescence and molecular formula properties representative for peptides and unsaturated aliphatics and associated with the presence of more oxygenated ventilated water masses. Finally, and despite that the aphotic N 2 fixation contributes largely to total water column diazotrophic activity (>50%), its contribution to overall nitrogen inputs to the basin is negligible (<0.5%).

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

ABSTRACT 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. Summary: Nitrogen limits coral productivity in tropical waters; diazotrophs represent an important source of nitrogen for scleractinian corals.

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: Overlooked Key Players within the Coral Symbiosis and Tropical Reef Ecosystems?

Coral reefs are highly productive ecosystems thriving in nutrient-poor waters. Their productivity depends largely on the availability of nitrogen, the proximate limiting nutrient for primary production. In reefs, the major nitrogen pathways include regeneration, nitrification, ammonification and dinitrogen (N2) fixation. N2 fixation is performed by prokaryotes called ‘diazotrophs’ that abound in coral rubbles, sandy bottoms, microbial mats or seagrass meadows. Corals, which are the main reef builders, have developed a partnership with dinoflagellates which transform dissolved inorganic nitrogen into amino acids and protein, and with diazotrophs to gain diazotrophically-derived nitrogen (DDN). Pioneering studies found active diazotrophic cyanobacteria in the corals’ mucus and/or tissue, and later high throughput sequencing efforts have described diverse communities of non-cyanobacterial diazotrophs associated with scleractinian corals. Yet, the metabolic processes behind these associations and how they benefit corals is currently not well understood. While genomic studies describe the diversity of diazotrophs and isotopic tracer experiments quantify N2 fixation rates, combined advanced methods are needed to elucidate the mechanisms behind the transfer of DDN to the coral holobiont and whether these mechanisms change according to the identity of the diazotrophs or coral species. Here we review our current knowledge on N2 fixation in corals: the diversity and localization of diazotrophs in the coral holobiont, the environmental factors controlling N2 fixation, the fate of DDN within the coral symbiosis as well as its potential role in coral resilience. We finally summarize the unknowns: are the diversity, abundance and localization of diazotrophs within the holobiont species- and/or site-specific? Do they have an impact on DDN production? What are the metabolic mechanisms implicated? Do they change spatially, temporally or according to environmental factors? We encourage scientists to undertake research efforts to tackle these questions in order to shed light on nitrogen cycling in reef ecosystems and to understand if coral-associated N2 fixation can improve coral’s resilience in the face of climate change.

New Perspectives on Nitrogen Fixation Measurements Using 15N2 Gas

Recently, the method widely used to determine 15N2 fixation rates in marine and freshwater environments was found to underestimate rates because the dissolution of the added 15N2 gas bubble in seawater takes longer than theoretically calculated. As a solution to the potential underestimate of rate measurements, the usage of the enriched water method was proposed to provide constant 15N2 enrichment. Still, the superiority of enriched water method over the previously used bubble injection remains inconclusive. To clarify this issue, we performed laboratory based experiments and implemented the results into an error analysis of 15N2 fixation rates. Moreover, we conducted a literature search on the comparison of the two methods to calculate a mean effect size using a meta-analysis approach. Our results indicate that the error potentially introduced by an equilibrium phase of the 15N2 gas is -72% at maximum for experiments with very short incubation times of 1 hour. In contrast, the underestimation was negligible for incubations lasting 12 to 24 hours (error is -0.2%). Our meta-analysis indicates that 84 % of the measurements in the two groups will overlap and there is a 61% chance that a sample picked at random from the enriched water group will have a higher value than one picked at random from the bubble group. Overall, the underestimation of N2 fixation rates when using the bubble method relative to the enriched water method is highly dependent on incubation time and other experimental conditions and cannot be generalized.

Mixotrophic metabolism by natural communities of unicellular cyanobacteria in the western tropical South Pacific Ocean: Mixotrophy in natural marine cyanobacteria

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.

Deep Into Oceanic N2 Fixation

Deep into oceanic N2 fixation Benavides Gorostegui, Mar; Bonnet, Sophie; Berman-Frank, Ilana; Riemann, Lasse Published in: Frontiers in Marine Science DOI: 10.3389/fmars.2018.00108 Publication date: 2018 Document Version Publishers PDF, also known as Version of record Citation for published version (APA): Benavides Gorostegui, M., Bonnet, S., Berman-Frank, I., & Riemann, L. (2018). Deep into oceanic N2 fixation. Frontiers in Marine Science, 5, [108]. https://doi.org/10.3389/fmars.2018.00108