Importance of N2-Fixation on the Productivity at the North-Western Azores Current/Front System, and the Abundance of Diazotrophic Unicellular Cyanobacteria

RESEARCH ARTICLE Importance of N 2 -Fixation on the Productivity at the North-Western Azores Current/Front System, and the Abundance of Diazotrophic Unicellular Cyanobacteria Virginie Riou 1,2 *, Debany Fonseca-Batista 1,3 , Arnout Roukaerts 3 , Isabelle C. Biegala 1 , Shree Ram Prakya 2 , Clara Magalhães Loureiro 4,7 , Mariana Santos 5,6 , Angel E. MunizPiniella 3 , Mara Schmiing 2,6,7 , Marc Elskens 3 , Natacha Brion 3 , M. Ana Martins 4,7 , Frank Dehairs 3 1Aix-Marseille Université, Mediterranean Institute of Oceanography (MIO), UM 110 CNRS/INSU, IRD, 13288 Marseille, Université du Sud Toulon-Var, 83957, La Garde, France, 2IMAR—Institute of Marine Research, Centre of IMAR at the University of the Azores, Horta, Portugal, 3Analytical, Environmental and Geo-Chemistry & Earth System Sciences, Vrije Universiteit Brussel, Brussels, Belgium, 4CIBIO, Research Center in Biodiversity and Genetic Resources, InBIO Associated Laboratory, Department of Oceanography and Fisheries, Horta, Portugal, 5IPMA, I.P.—Portuguese Institute of Ocean and Atmosphere, Lisbon, Portugal, 6MARE—Marine and Environmental Sciences Centre, Lisbon, Portugal, 7DOP/UAz – Department of Oceanography and Fisheries, University of the Azores, Azores, Portugal *[email protected] Abstract To understand the impact of the northwestern Azores Current Front (NW-AzC/AzF) system on HCO 3 − -and N 2 -fixation activities and unicellular diazotrophic cyanobacteria (UCYN) distribution, we combined geochemical and biological approaches from the oligotrophic surface to upper mesopelagic waters. N 2 -fixation was observed to sustain 45–85% of the HCO 3 − -fixation in the picoplanktonic fraction performing 47% of the total C-fixation at the deep chlorophyll maximum north and south of the AzF. N 2 -fixation rates as high as 10.9 μmol N m -3 d -1 and surface nitrate δ 15 N as low as 2.7‰were found in the warm (18–24°C), most saline (36.5–37.0) and least productive waters south of the AzF, where UCYN were the least abundant. However, picoplanktonic UCYN abundances up to 55 cells mL -1 were found at 45– 200m depths in the coolest nutrient-rich waters north of the AzF. In this area, N 2 -fixation rates up to 4.5 μmol N m -3 d -1 were detected, associated with depth-integrated H 13 CO 3 − -fixation rates at least 50% higher than observed south of the AzF. The numerous eddies generated at the NW-AzC/AzF seem to enhance exchanges of plankton between water masses, as well as vertical and horizontal diapycnal diffusion of nutrients, whose increase probably enhances the growth of diazotrophs and the productivity of C-fixers. PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 1/22 OPEN ACCESS Citation: Riou V, Fonseca-Batista D, Roukaerts A, Biegala IC, Prakya SR, Magalhães Loureiro C, et al. (2016) Importance of N 2 -Fixation on the Productivity at the North-Western Azores Current/Front System, and the Abundance of Diazotrophic Unicellular Cyanobacteria. PLoS ONE 11(3): e0150827. doi:10.1371/journal.pone.0150827 Editor: Brett Neilan, University of New South Wales, AUSTRALIA Received: August 5, 2015 Accepted: February 19, 2016 Published: March 9, 2016 Copyright: © 2016 Riou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All metadata files are available from the VLIZ database (DOI: http://dx.doi. org/10.14284/40). Funding: The research leading to these results has received funding for data collection from the European Union Seventh Framework Programme [FP7/2007–2013, (http://ec.europa.eu/research/fp7/)] under grant agreement n°228344 [EUROFLEETS, (www.eurofleets.eu)]. Funding was also granted for data collection and analysis from the Portuguese Fundação para a Ciência e a Tecnologia (www.fct.pt), 1. Introduction The increase in atmospheric CO 2 concentration has stressed the need to quantify the transfer of CO 2 by the marine biological carbon pump to the deep sea, where it can be trapped for centuries [1]. Although iron (Fe) and phosphorus (P) can limit C-fixation in some regions of the ocean, nitrogen (N) is limiting or close to limiting in most of the oligotrophic oceans [2]. Once all the nitrate and nitrite have been used by phytoplankton in the euphotic zone, new primary production at the surface is only possible if N 2 -fixation occurs or if new nitrate sources appear. While upwelling provides new nitrate for phytoplankton growth, it also delivers deep ocean CO 2 , leading to less atmospheric CO 2 sequestration by the biological pump. On the other hand, if new N from N 2 -fixation is added to surface waters, net atmospheric CO 2 sequestration into export production occurs [3]. In the N depleted North Atlantic (Sub)Tropical gyre (NAST), Fe-enhanced surface N 2 -fixation, which is limited by P availability, was estimated to add the equivalent of 50–180% of the deep ocean nitrate flux into the euphotic zone [4,5]. However, the geochemical, biological, and numerical modeling estimates vary widely [6] and there is an urgent need to evaluate the impact of N 2 -fixation on sea surface productivity with more accuracy to be able to predict the future efficiency of the biological carbon pump. Hydrographic fronts are known to affect biological activity and primary productivity, and therefore potentially gas exchanges at the ocean–atmosphere interface. The Azores Front (AzF) extends over the whole width of the eastern NAST basin (Fig 1). It marks the northern border of the Azores Current (AzC, 32–36°N with a main axis ~34°N, [7], Fig 1), separating cold, Eastern North Atlantic Central Waters, from the more saline, warmer 18°C Mode Water Fig 1. Map of the study area in the North Atlantic showing the main circulation patterns. The approximate locations of currents were re-drawn from [10,15]. SNAC: Southern branch of the North Atlantic Current. AzC: Azores Current. Deep MedW: Mediterranean outflow. doi:10.1371/journal.pone.0150827.g001 Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 2/22 the Research Foundation Flanders grant n °G071512N (www.fwo.be), the Research Council of Vrije Universiteit Brussel [SRP-2, (http://rd-ir.vub.ac. be/)], the Instituto do Mar [IMAR, (http://www.imar.pt/ )], the FISHBOX project [FUI 11, Institut de Recherche pour le Développement, (www.ird.fr)] and the Mediterranean Institute of Oceanography [MIO, (http://mio.pytheas.univ-amu.fr/)]. This work is also a contribution to the Labex OTMed [ANR-11-LABEX0061, (www.otmed.fr)] funded by the « Investissements d’Avenir », French Government project of the French National Research Agency [ANR, (www.agence-nationale-recherche.fr/)] through the AMidex project [ANR-11-IDEX-0001-02], funding VR during the preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. (18MW), a homogenous, well mixed water body associated with the Gulf Stream extension [8]. Previous studies showed that the North West-AzF area (NW-AzF, 30–40°W) differs markedly from the south-eastern border of the AzC-AzF system (SE-AzF, 20–25°W) regarding water mass structure, with the 18MW detected only until 30°W [9]. Although N 2 -fixation was estimated to contribute 40% of the carbon export at the SE-AzF [10], its importance in the NW-AzF area is still unknown, and direct primary production measurements are missing for this area. The passage of the AzC across the Mid-Atlantic Ridge moreover increases the formation of eddies [11,12], which are known to affect both Cand N 2 -fixations [13,14]. It is therefore crucial to assess the relative importance and spatial variability of both processes in situ at the NW-AzC/AzF system. Various methods are available to assess the amount of N 2 -fixation associated with sea surface productivity. Geochemical measurements generally reveal the weekly to seasonal integration of biological and physical processes. N 2 -fixation injects new N with low δ 15 N (-2 to 0‰, e.g. [16]) into the organic matter (OM), compared to upwelled deep ocean nitrate (δ 15 N = 5.0 ±0.5‰;[17]). Low δ 15 N signatures from particulate OM have been used to estimate the input of N 2 -fixation to microbial biomass in the NAST [18]. An increase of N 2 -fixation in the OM, followed by remineralisation and nitrification producing new nitrate, will result in a local decrease in δ 15 N NO3 . Additional information can therefore be obtained from nitrate isotopic composition, which is unaffected by the OM stoichiometry. A decrease in δ 15 N NO3 may however be masked (i) by concurrent nitrate assimilation, which leads to nitrate 15 N enrichment, and/or (ii) by mixing with abundant deep nitrate. Simultaneous analysis of the δ 18 O NO3 signature may distinguish these processes. Contrary to δ 15 N NO3 , the δ 18 O NO3 signal is not affected by the remineralised OM composition during nitrification [19], while nitrate assimilation will enrich both δ 15 N NO3 and δ 18 O NO3 signatures equally [20]. The assimilation of both deep and new nitrate will therefore result in similar δ 18 O signatures, while the difference in their δ 15 N signals will remain. Two recent studies that analysed the difference in δ 15 N NO3 and δ 18 O NO3 have revealed significant contribution of N 2 -fixation to NAST surface waters [10,21]. The geochemical indicators of N 2 -fixation (OM δ 15 N and nitrate δ 15 N/δ 18 O) can be complemented by daily-resolved activity measurements, following the incorporation of 15 N 2 -tracer in particulate OM (net N 2 -fixation). The 15 N 2 “bubble-addition method”has allowed measuring in situ N 2 -fixation rates for the last two decades [22]. Recent studies, however, showed that this method under-estimates N 2 -fixation rates by 62% to 570% in comparison to the “dissolution method”, due to slow equilibration of the 15 N 2 gas bubble with natural dissolved N 2 during incubation [23,24,25]. The magnitude of this under-estimation was observed to depend on the composition of the diazotrophic community. Buoyant Trichodesmium filamentous cyanobacteria that stay in contact with gas bubbles would thus be much less sensitive to the amount of dissolved 15 N 2 gas than filamentous Richelia cyanobacteria growing inside diatoms, diazotrophic unicellular cyanobacteria (UCYN), or γ-Proteobacteria [23,24,25]. Deciphering the composition of the diazotrophic community is also of high relevance to estimate the extent of their contribution to organic C export. While diatoms-Richelia associations are known to contribute directly to C export [26], Trichodesmium also fix CO 2 but are not known to sink beyond the euphotic zone. They would therefore rather fuel upper ocean microbial production [27] and fertilize surface waters with new N, allowing carbon export by other species. UCYN may add as much new N to the global ocean as Trichodesmium [28], which was shown to add more N to the euphotic zone than the estimated vertical flux of deep sea nitrate in the tropical North Atlantic [5]. However, UCYN-A, one of the three groups identified to date, is unable to perform C-fixation and although cultured representatives of UCYN-B and C are obligate photoautotrophs, there is no evidence for their export to depth Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 3/22 [29]. Predation, aggregation and association to calcifying or silicifying unicellular algae may, however, promote net C export and UCYN-B may excrete significant amounts of N and C to be used by surrounding larger phytoplankton (reviewed in [29]). N 2 -fixation was believed to be negligible at northern latitudes of the mid-NAST [4] until significant activity was detected at 34–42°N [30]. The subtropical mid-Atlantic north of 30°N has been much less investigated than the lower latitudes for the diversity and activity of diazotrophs [25,31,32]. Cyanobacteria-like nifH genes represented almost half of the sequences amplified from samples collected in the Azores. While filamentous cyanobacteria have not been detected north of 30°N in the mid-NAST, UCYN-A appear to dominate south of the Azores [31,33,34]. UCYN distribution is, however, barely known in the NW-AzC/AzF area. At the northern boundary of the NAST, UCYN-A abundances of up to 150 cells mL -1 have been detected using a UCYN-A-specific probe for whole-cell ribosomal RNA tyramide signal amplified-fluorescence in situ hybridization (TSA-FISH) [33,30]. This powerful technique has also been used with the Nitro821 probe targeting the three UCYN phylotypes, which allowed recognizing the worldwide importance of the picoplanktonic UCYN-A as free-living cells, or associated with inert or living particles (e.g. [33,30,35,36,37,38]). Since evidence for the presence of UCYN is growing for higher latitudes of the North Atlantic Ocean up to 42°N [33,30], it is important to assess their distribution, as well as the activity of the diazotrophic community using the “dissolution method”in that region. We focused the present study on the NW-AzF region, corresponding to the Mid-Atlantic 30–34°N latitude belt where information on Cand N 2 -fixation and UCYN distribution is limited. The upper ocean was explored down to 200m for (i) Cand N 2 -fixation activities with the “dissolution method”, and (ii) Nitro821-positive UCYN abundance by TSA-FISH. These biological data were complemented with nitrate isotopic signatures used as a geochemical tracer of N 2 -fixation, and linked to the physical-chemical conditions. This information is essential to constrain N 2 -fixation in the North Atlantic and to understand the parameters influencing UCYN density and activity in situ. 2. Materials and Methods During the DIAPICNA cruise (25 July-3 August 2011) aboard the NRP Dom Carlos I, five stations (A-E) were sampled between 31.5°N-33.0°W and 36.2°N-33.9°W, with the authorization of the portuguese Comissão Oceanografica Intersectorial–MCTES. The station positions ensured sampling of both sides of the NW-AzC-AzF system close to the Mid-Atlantic Ridge, as identified from real-time AVISO satellite altimetry-derived mean geostrophic currents (Fig 2, S1B–S1F Fig). 2.1 Water-column physical and chemical characteristics Temperature, salinity, chlorophyll (Chl) fluorescence, O 2 saturation and turbidity profiles were obtained from a SBE-9plus CTD profiler coupled with WETlabs ECO-FLrtd-deep Chl fluorometer, SBE-43-dissolved O 2 and SBE-911-turbidity sensors. Data processing and filtering were done using the Seasoft V2 software (Sea-Bird Electronics). The sensors were mounted on a SBE-32 Carousel together with 12 Niskin 2.5 L bottles used to sample seawater at 5, 25, 45, 75, 100, 125, 150, 175, 200, 250, 350 and 500m depths. For nutrient concentrations and nitrate isotopic signatures, quadruplicate 25 mL water samples were filtered onto sterile 0.45 μm porosity Acrodisc filters (Sterlitech), collected in 40 mL polypropylene vials (Nalgene) and immediately preserved at -20°C. Nitrate, ammonium and phosphate concentrations were determined back in the lab using a QuAAtro segmented flow automatic analyzing system (SEAL Analytical) with detection limits (d.l.) of 120, 120 and 140 nmol L -1 , respectively. Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 4/22 Concentrations of phosphate below the d.l. were determined on a 1 m Liquid Waveguide Capillary flow Cell (LWCC) with a QE65000 detector (Ocean Optics), down to 3 nmol L -1 concentrations (d.l.). N and O isotopic ratios of dissolved nitrate (>1μmol L -1 ) were obtained with the “denitrifier method”[39,40](seeS1 Text). 2.2 Particle isotopic composition and Cand N 2 - fixation activities Seawater samples were taken from each station at the surface (11–16m), above the Deep Chlorophyll Maximum (above DCM; 45–48m), at the DCM (86–112m) and in the upper mesopelagic zone (200–217m). A subsample of 4.5 L was immediately filtered for natural particulate organic carbon and nitrogen concentrations (POC/PN) and isotopic compositions (δ 13 C POC / δ 15 N PN ). Rates of Cand N 2 -fixation were measured with the dissolved NaH 13 CO 3 and 15 N 2 tracer method, as detailed in the supplementary material (S1 Text). A recent study showed that some batches of commercial 15 N 2 gas could contain 15 N-labeled contaminants such as nitrate, nitrite and ammonium which could have greatly biased past estimates [41]. The influence of potential contaminants associated with the dissolution of the same 15 N 2 gas reference as used in the present study (Eurisotop 15 N 2 98+ atom%), was tested in low nutrient waters amended with IAEA nitrate and ammonium reference compounds. No significant difference was detected in the δ 15 N signatures of the latter compounds, indicating that potential contamination of the gas used for the present study was negligible (Fonseca-Batista et al., unpublished data). Incubations were performed in duplicates in 4.5 L Nalgene polycarbonate bottles. These were filled to the very rim with the sample after enrichment with a NaH 13 CO 3 (Eurisotop 99 atom%) spiking solution and 285 mL of degassed (by vacuum pumping under magnetic stirring) 0.2 μm-filtered low nutrient seawater (Osil) containing dissolved 15 N 2 . Both tracers (Eurisotop 99 and +98 atom%, respectively) were added to reach theoretical final enrichments of 10 13 C atom% and 5 15 N atom%. The incubations were performed for 24 h in on-deck incubators flushed with flowing surface seawater at around 24°C, and wrapped in blue filters (Rosco), selected to simulate 0% (surface), 70% (subsurface), 97% (DCM) and 99.9% (200–217m depths) daylight attenuation, according to Piazena et al.[42]. At the end of the incubation period, aliquots were withdrawn from the samples under helium pressure to measure the Fig 2. Sea Surface Height and geostrophic currents during the DIAPICNA cruise. Station locations (open circles) overlaid on AVISO altimetry-derived geostrophic currents centered between 26/07/2011 and 01/08/2011, with color scale indicating the Sea Surface Height (SSH) in m. doi:10.1371/journal.pone.0150827.g002 Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 5/22 Dissolved Inorganic Carbon (DIC) 13 C atom% and dissolved N 215 N atom%. The aliquots were transferred through the PTFE septa, to 12 mL exetainers (Labco) poisoned with HgCl 2 , and measured on a Flash EA 1112 Elemental Analyzer coupled to a DELTA V Isotope Ratio Mass Spectrometer via a Conflo III interface (EA-IRMS, Thermo Instruments) equipped with a custom made manual gas injection port (see S1 Text). Aliquots of the incubations were also filtered on 0.45 μm Acrodiscs and kept at -20°C for nutrient analysis. Natural and enriched particles were subsequently size-fractionated by serial filtration onto 25 mm diameter membranes of 3.0 μm and 0.3 μm porosities, made of silver (Sterlitech) and pre-combusted glass fiber (GF75, Advantec MFS Inc.), respectively. They were treated and analyzed for POC/PN and δ 13 C POC and δ 15 N PN using the EA-IRMS, as detailed in the S1 Text. 2.3 Cand N 2 - fixation rates and error calculations Cand N 2 -uptakes (Uptake C and N in nmol L -1 ) were calculated as: Uptake X ¼ðfinalAparticle t¼0AparticleÞConcentration finalAsubtrate t¼0Aparticle ð1Þ where final A particle is the 13 Cor 15 N atom% measured in the particles after incubation, t=0 A particle is the natural 13 Cor 15 N atom% measured in particles without incubation, Concentration is the POC or PN content after incubation (μmol L –1 )and final A substrate is the DIC 13 C atom% or dissolved N 215 N atom% after incubation. The assumption was made that the 24 h H 13 CO 3and 15 N 2 -fixation activities did not significantly affect A DIC and A N2 . Uptake rates (μmol X m -3 d -1 ) were obtained by dividing the uptakes with the incubation duration. Data correction and selection procedure is detailed in the S1 Text. All C-uptake rates were above background, while 70 of the 94 15 N 2 uptake values were at or below background and were reported as d.l. (below detection). 2.4 Unicellular cyanobacterial diazotroph (UCYN) cellular abundance Samples from the surface, above DCM, DCM and upper mesopelagic casts were size-fractionated on 47 mm diameter polycarbonate membranes (PCTE, Sterlitech) of different porosities connected in series. Filters of 0.2, 3 and 10 μm porosity were used to collect the cells present in 250 mL, 2 L and 4.5 L of the sample, respectively. Cell fixation, preservation and TSA-FISH were done according to [35] (detailed in S1 Text), hybridising16S rRNAs with the horseradish peroxidase (HRP) labeled Nitro821 probe (5’-CAAGCCACACCTAGTTTC-3’, ThermoFisherScientific GmbH) specific for UCYN [43]. The hybridized cells were stained with fluorescein-tyramide using the TSA system (TSA kit, PerkinElmer), followed by DNA counter-staining of all the cells using DAPI (4’,6’DiAmidino-2-PhenylIndole, Sigma-Aldrich). Nitro821 positive cells were counted with a 40X objective (NA 0.75N Plan Fluor WD 0.72mm, Nikon) on an epifluorescence ECLIPSE 50i microscope (Nikon) using a Halogen lamp (H65761, Orbitec) and dichroïc filters for DAPI (Excitation 365±10 nm, Emission 400 nm) and Fluorescein IsoThioCyanate (Ex. 480 ±40 nm, Em. 510 nm long pass). UCYN cells detected in the <3μmand>3μm size fractions were grouped into three categories: (1) free picoplanktonic cells (<3μm), (2) picoplanktonic cells attached to particles or larger algae, and (3) nano-planktonic cells (3–10 μm). The entire surface of each 1/16 th filter portion was counted following [37], after we validated that it was representative for the whole sample. Validation was done by counting triplicate portions of the same sample, resulting in relatively low standard deviations (e.g. 24.45 ± 1.98 cell mL -1 in the <3μm size fraction, see also [37]). Relatively high triplicate variability was observed for cell counts below 0.2 cell mL -1 (0.12 ± 0.09 cell mL -1 for the <3μm size fraction; 0.09 ± 0.07 cell mL -1 for the >10 μm size fraction). Filters of all porosities (0.2, 3 and 10 μm) were counted, and the results for the 3 and 10 μm porosity filters were pooled to compare with the >3μm fraction 15 N 2 uptake rates. Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 6/22 2.5 Statistical analysis and data availability For each depth strata, pairplots were done for the response variables (POC, PN, Cand N 2 -fixation, Pico-UCYN abundance) and selected environmental variables (% surface photosynthetically active radiation-PAR, potential temperature, salinity, % O 2 saturation, nitrate, phosphate, ammonium and silicate concentrations). The plots were done with the software R (version 3.1.3, [44]). In addition, the Spearman rank correlation coefficient was calculated for the same variables using a Bonferroni correction to account for multiple comparisons. The metadata acquired during DIAPICNA are publically available from the Marine Data Archive at the Belgian VLIZ institute under the following DOI: http://dx.doi.org/10.14284/40 3. Results 3.1 Water-column physical properties across the NW Azores Current The AzF was located between stations C and D, as derived from the 16°C isotherm at 200m depth [11](Fig 3A). Below 80m depth, the AzF separated warmer and more saline waters to the south, from cooler and fresher waters to the north. Isotherms and PAR (S2 Fig) reached deeper inside the AzC (stations B-C). Anticyclonic features drove downwelling at stations B and C, but also at station E (Fig 2;S1C, S1D and S1F Fig). On the contrary stations A south of the AzC and D north of the AzF, did not seem to be associated to any particular hydrological feature and may be considered as “reference”stations for the physical-chemical conditions met south and north of the NW-AzC, respectively. Surface waters from 0–300m consisted of North Atlantic Central Water (NACW). South of the AzF (stations A-C), the surface T-S diagram patterns were typical of the 18°C Mode Water of subtropical origin (18MW, σ ϴ = 26.5, Fig 3B). These waters were consistently more saline by 0.38–0.61 units than the northern NACW surface waters (stations D-E, S3A Fig). The T-S relationship at station D (0–100m) was characteristic of the 15°C Mode Water of subpolar origin (15MW, σ ϴ = 26.9). Surface waters at station E were more saline (0–200m) and more than 1°C warmer down to 10m depth (25.2°C) than at all other stations (23.5–24.2°C). The euphotic zone, with its lower boundary at 1% of surface PAR, extended to 85–103m at station A, 106– 112m at stations B and C, and 76–98m at stations D and E. The upper mixed layer reached down to 25–40m at station A, and shoaled towards the north (20–25m at stations B-C, and 5–13m at stations D-E). The seasonal thermocline also shoaled from 60m in the south to 30m northward. In situ Chl fluorescence profiles indicated the presence of DCM at 87–109m south of the AzF, and at 65–85m in stations D and E (S3B Fig). These DCM were associated with O 2 concentrations close to saturation (S3C Fig). Below 300m, two main layers were identified at all stations: (i) the main thermocline layer of the NACW (26.8<σ ϴ <27.2, 300–600m) and (ii) intermediate levels (27.2<σ ϴ <27.9) of the SubArctic Intermediate Water (500–800m, SAIW), Mediterranean Water (MedW) and Labrador Sea Water. The T-S diagram from station E appeared to be more influenced by MedW at about 800–1000m (as in station D, Fig 3B), although there is no conclusive evidence that the nearby anticyclonic eddy was in fact a MedW eddy (no O 2 minimum layer expected from MedW). In addition, due to the persistence of this eddy in the region, it is expected that waters from station E have received some influence from waters further south (AzC and 18MW, S1A Fig). 3.2 Nutrient concentrations The top of the nutricline corresponded to the depth of the DCM (Fig 4A,S3B and S3D Fig). Above it, low phosphate (<30 nmol L -1 ) and nitrate (<d.l. = 120 nmol L -1 ) concentrations Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 7/22 down to 75m (except at station D) indicated that the area was oligotrophic. Values measured 125–200m deep, respectively south (stations A-C) and north of the front (stations D-E), were 1.5–4.3 and 3.1–6.5 μmol L -1 for nitrate (except for station B at 125m: <d.l.) and 0.02–0.24 and 0.17–0.28 μmol L -1 for phosphate. The nutricline deepened at station B (close to 32°N), with phosphate levels below the d.l. down to 200m depth, associated with lower nitrate concentrations. In contrast, the uplift of isotherms and isohalines at station D indicated the presence of deeper waters, richer in nutrients north of the AzF (Fig 3A,S3A Fig). Overall ammonium concentrations were below 0.27 μmol L -1 , showing no real trend with depth (S3E Fig). Higher concentrations of up to 0.49 μmol L -1 Fig 3. Physical parameters monitored along the DIAPICNA cruise track. (A) Temperature (°C) longitudinal cross-section, with the position of the Azores Front deduced from the 16°C isotherm at 200m depth (dotted lines); (B) TS diagrams for stations A-E, with overlaid σ θ density isolines. NACW: North Atlantic Central Water; 15MW: 15°C Mode Water; 18MW: 18°C Mode Water; SAIW: SubArctic Intermediate Water; MedW: Mediterranean Water; LSW: Labrador Sea Water. The black box indicates possible increased influence of Mediterranean Waters at 800–1000m in stations D and E. doi:10.1371/journal.pone.0150827.g003 Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 8/22 were detected at station C (above and below the DCM), station D (75–125m) and station E (125–175m). 3.3 Particulate Nitrogen (PN) and nitrate natural isotopic compositions In the euphotic zone (from the surface to the DCM), average natural δ 15 N PN signatures of particles within the <3μm and >3μm size classes were 1.9±1.6‰and 2.1±1.2‰, respectively (weighted means, Fig 4C). In <3μm particles, they varied between -0.3 and +3.1‰in stations A, B and E, were around +2.5‰in station C (with an unrealistic high value of +11.0‰at 47m, omitted from the weighted means calculations, and probably due to the presence of a large aggregate or copepod on the filter) and varied from +0.7 to +5.4‰in station D. Particles >3μm had δ 15 N PN signatures ranging from +0.2 to +4.3‰in all stations except station D, where they were rather constant at +2.2 to +3.2‰. In the upper mesopelagic (200–217m), suspended particles were relatively enriched in 15 N (+4.3 to +14.0‰), except in particles >3μmat stations A, B and D (-0.3 to +2.8‰,S1 Table). In the top 500m of the water column, nitrate δ 15 N signature was slightly depleted in 15 N south of the AzF, in comparison to waters north of the front (Figs 4B and 5A). At 500m, δ 15 N NO3 signatures were close to 4.0‰in all stations and decreased by around 1.0‰from 500m to 200m depth (δ 15 N NO3 = 2.9–3.6‰). The only exception was in station D, where δ 15 N NO3 decreased by <0.5‰towards 200m (δ 15 N NO3 = 3.6–4.0‰) and increased to 5.6– 6.4‰in the DCM. At these shallower depths, values were generally lower south (2.7–3.3‰) than north of the AzF (3.4–4.0‰below the DCM at station E), although larger fluctuations were observed among stations. In contrast, values of δ 18 O NO3 displayed a similar trend south and north of the AzF (Fig 5B). They ranged from 2.0–3.9‰between 500m and 200m, increased to 3.4–5.1‰above 200m (except at station E DCM: 2.8‰), and even reached 6.5‰ at station D (75m). The δ 18 O NO3 was strongly negatively correlated with nitrate concentrations over the 75–1900m depth range at station D only (δ 18 O NO3 = 6.4 e -0.1[NO3-] , r² = 0.82). At Fig 4. Geochemical tracers of N 2 -fixation along the DIAPICNA cruise transect. Longitudinal cross-sections of (A) nitrate concentration, (B) δ 15 N NO3 signal, (C) PN isotopic signature and (D) Δ(15,18)—see eq (2). The position of the Azores Front is indicated by a dashed line, and station i.d. is given on top. doi:10.1371/journal.pone.0150827.g004 Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 9/22 their abundance and activity (nutrients, trace metals, O 2 , temperature, [52]). In the present study, the variations in the abundance of UCYN in the smaller size fraction (<3μm) at the surface (11–16m) were linked to salinity variations, with higher UCYN concentrations in the saltier waters south of the AzF, where the lowest surface C-fixation and the only surface N 2 - fixation rates of the transect were detected. This could be an indication that UCYN may have performed a significant part of the N 2 -fixation observed in surface waters south of the AzF. The highest UCYN counts were however found 45–200m deep, north of the AzF, in 14.5– 20.5°C, oxygenated and nitrate-enriched waters (1–6μM) with phosphate concentrations of 8–207 nM. Increased UCYN abundance in nutrient-enriched waters has already been observed in earlier studies (e.g. [60,61]). Amendments of 1 μM nitrate alone, or in combination with 200 nM phosphate have also been found to induce UCYN-A nifH transcript increases in tropical Atlantic waters [62]. Our results contradict previous conclusions for the North Atlantic regarding the restriction of UCYN to warm waters (>18°C) with sub-micromolar nitrate concentrations [30,31]. However, they are supported by the presence of active UCYN in 14.5–19.0°C Pacific open ocean and North Atlantic shelf waters [61,63], and even in 2.5°C cold waters in the area between the North and Baltic Seas [64]. In the DCM, the picoplanktonic fraction was responsible for half of the total POC production along the transect, except in the least oligrotrophic station D (30%). N 2 -fixation rates sustained on average 45–64% of this production south of the AzF and up to 85% at the northernmost station (using the average 6.2 C/N ratio measured in the picoplanktonic fraction at the DCM along the transect). Since picoplanktonic POC and PN concentrations were directly linked to C-fixation in this size fraction and strongly correlated with total in situ Chl fluorescence, we argue that the latter is mainly an indicator of deep picoplanktonic productivity maxima in that area. Chl fluorescence increased with decreasing salinity, which was significantly correlated with lower temperatures that were associated with higher phosphate concentrations. Picoplankton productivity may have therefore been bound to phosphate availability in the DCM, which may also have limited N 2 -fixation and UCYN growth. In the upper mesopelagic zone, no N 2 -fixation was measured north of the AzF. However, south of the AzF in the larger size fraction, Cand N 2 -fixation might have presented a co-variation, with no relationship to the environmental variables. This might indicate that N 2 -fixation provided new N in association with dark CO 2 -fixation in sinking particles. Future investigations in this area should therefore focus on understanding temporal patterns of N 2 -fixation and obtaining more accurate activity measurements at in situ temperature (and hydrostatic pressure). 4.5 Impact of the hydrology at the NW-AzC/AzF system The high shear experienced at the edge of eddies most probably enhances horizontal diapycnal exchange [65], although the mechanisms are still poorly understood. This might increase nutrient supply for new production, particularly in oligotrophic regions where the vertical nitrate flux effectively constrains C-uptake [66]. At the time of sampling, the AzC at 33°W consisted of a large anticyclonic feature sampled at stations B and C, with lowest nutrient concentrations, productivity, biomass, Chl fluorescence and turbidity. An intrusion of northern waters richer in phosphate through the AzF (also observed by Macedo et al.[8]) might have induced the slightly increased UCYN counts and N 2 -fixation activity observed in the >3μm size fraction at station C. The highest UCYN abundances, however, were measured at 45–200m in station E and at 200m in station D. Horizontal diapycnal exchange of subsurface waters from the periphery of the anticyclonic eddy sampled in station E might explain the presence of abundant UCYN at 200m in station D. In addition, surface salinity at station E was between the salinities detected at station D and south of the AzF. This might indicate that waters at station E had been Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 16 / 22 transported from the south and mixed with northern waters (cross-frontal exchange seen in S1A Fig). The transport of Trichodesmium filaments from southern waters could explain their presence in northern, colder waters (DCM station E), where they have been rarely reported [31,67]. Enhanced N 2 -fixation rates and UCYN abundances in deep samples at station E may have resulted from the influence of the anticyclonic eddy driving downwelling in its center [14] resulting in slightly higher water temperatures (~1°C) at station E in comparison to station D. Atmospheric P and Fe deposition can also drive increased C-, N 2 -fixation and UCYN abundance, as observed in the tropical North Atlantic (e.g. [62]). It is likely that any influence of a dust deposition event on N 2 -fixation would have been detected in surface waters at both stations D and E, but this was not the case. Moreover, station E was located on the Mid-Atlantic Ridge, on top of a 2300m deep Fe-rich hydrothermal vent field [68]. This Fe source is remote from the surface where N 2 -fixation was detected, but stabilized Fe can be transported over long distances and brought up to shallower water masses [69]. Although we have too little evidence to relate increased N 2 -fixation to a potential influence of hydrothermal Fe, these processes merit further attention in future studies. Conclusion In the present study, marked differences in summer Cand N 2 -fixations were observed across the NW-AzC/AzF system close to the Mid-Atlantic Ridge, presenting contrasted physicalchemical conditions. These first direct measurements of H 13 CO 3 − -fixation in the area confirmed previous estimates, with productivity north of the AzF being twice that observed to the South, and nutrient limitation in the euphotic zone over the whole area. Geochemical measurements revealed the importance of N 2 -fixation in the area, reflected by low particle and nitrate δ 15 N signatures observed down to 200 m depth, resulting from N 2 incorporation into the particles, followed by remineralisation-nitrification in subsurface waters. South of the front (as well as in an anticyclonic eddy north of the AzF), picoplankton in the DCM performed half of the C-fixation, which was mostly supported by N 2 -fixation. Higher pico-UCYN abundances were however detected in the DCM only in the northern station, where abundances increased down to 200m depth in cool and nutrient-replete waters. At all other stations, the high <3μmN 2 -fixation activity in the DCM was detected in the presence of low UCYN abundance. Other types of prokaryotes might thus contribute to the picoplanktonic diazotrophic activity in the area. Further research is needed to identify the actors of this important activity that sustains C-fixation at the NW-AzC/AzF system. In upper mesopelagic waters (200–217m depth), Cand N 2 -fixation might have been linked in the >3μm particles south of the AzF, which could suggest a coupling between N 2 -fixation and dark CO 2 -fixation in sinking particles. This aspect of the dark end of the biological carbon pump should be examined in more detail in future studies. The intense hydrological dynamics related to the NW-AzC/AzF system appear to influence the biogeochemical processes in the area. The intrusion of southern waters, past the AzF into the nutrient-rich northern waters, associated with eddy-driven downwelling, for instance, corresponded to large increases in UCYN abundances and N 2 -fixation activity in the <3μm size fraction on the northern side of the front. North-south water mass exchanges as well as diapycnal transfers therefore probably influence the distribution and activities of plankton species in the NW-AzC/AzF area. Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 17 / 22 Supporting Information S1 Fig. Real-time AVISO satellite altimetry derived mean geostrophic currents and Sea Surface Height. Day by day sea level anomalies before (A) and at the time of each DIAPICNA station sampling (B, C, D, E, F). Weekly-integrated sea level anomalies during the August 2011 DIAPICNA (G) and September 2006 MSM03/01 VISION cruises (H) indicate that the hydrological setting was similar during both cruises and that the southernmost stations sampled during the VISION cruise were located north of the AzF (see manuscript discussion 4.3). Station locations are marked with dots or stars and the approximate position of the Azores CurrentFront system is indicated as a black line. (PDF) S2 Fig. Depth profiles (m) of physical-chemical parameters. Stations A (day and night), B, C, D and E in situ fluorescence (FlECO-AFL), density (Sigma-θ), O 2 (Oxsol ML/L), salinity, temperature and photo-active radiation (PAR, purple curves) profiles. (PDF) S3 Fig. Physical-Chemical properties of the water column over the DIAPICNA transect (Yaxis: depth in m). Longitudinal cross-sections of A) Salinity, B) in situ chlorophyll fluorescence (mg m -3 ), C) O 2 % saturation and concentrations of D) phosphate in nmol L -1 , and E) ammonium in μmol L -1 . The dotted line indicates the position of the AzF. (PDF) S4 Fig. Spearman correlation coefficient matrixes of the waters’physico-chemical and biogeochemical properties. Samples collected (A) at the surface (n = 18), (B) above the DCM (n = 18), (C) in the DCM (n = 18) and (D) in the upper mesopelagic (n = 18). The upper right panels show the pairwise scatterplots. A smoothing curve (LOESS) with a span of 0.66 was added for visual interpretation. The lower left panels show the correlation coefficient (Spearman rank), including significant p-values. Histograms of the variables are included in the diagonal. Significant correlations at p<0.001, p<0.01 and p<0.05 are indicated with , and , and highlighted in red, orange and yellow, respectively. The numbers at the top, bottom and sides of the multipanel figure are the units of the respective variable. (PDF) S1 Table. Particulate Nitrogen enrichment for <3μm and >3μm particles. Particles collected during the day in the euphotic zone. Corrected (i.e. given the value of natural SD/2) if <Depth 3xSD. Flagged in grey if <0.0908 (highest error from 15 N 2 replicates). Flagged in black if N 2 fixation <propagated error E. (PDF) S2 Table. Particulate organic carbon enrichment for <3μm and >3μm particles. Particles collected during the day in the euphotic zone. Evalues represent calibration uncertainties and propagated errors at each step of the calculation. (PDF) S3 Table. Particulate organic carbon and nitrogen enrichments for the night cast at station 1. Corrected (i.e. given the value of natural SD/2) if <Depth 3xSD. Flagged in grey if <0.0908 (highest error from 15 N 2 replicates). Flagged in black if N 2 fixation <propagated error E. (PDF) S4 Table. Correlations between the physical-chemical and biogeochemical variables. Spearman correlations’significance at p<0.001, p<0.01 and p<0.05 are shown with , and , respectively. O 2 : oxygen saturation %; Temp: potential temperature; Sal: salinity; PAR: % Cand N 2 -Fixation around the North-Western Azores Current PLOS ONE | DOI:10.1371/journal.pone.0150827 March 9, 2016 18 / 22 surface photoactive radiation; Chl: in situ Chlorophyll fluorescence. POM, POC, PN: Particulate Organic Matter, Carbon, Nitrogen concentrations; C-Fix, N 2 -Fix: Cand N 2 -fixation and Pico-UCYN abundance are examined for the small (<3μm, s) and larger (>3μm, L) size fractions. (PDF) S5 Table. Comparison of N 2 -fixation volumetric rates measured in the Subtropical MidAtlantic between 2006 and 2011. (PDF) S1 Text. Supporting material and methods, results and references. (PDF) Acknowledgments We thank H. Lopes, S. Gomes, A. Medeiros, L. Rymenans and M. Korntheuer for the experimental help, as well as L. Pinheiro, R.S. Santos, E. Isidro, P. Bonin and R. Sempéré for their organizational support, and H. Diogo, I. Martins and C. Tamburini for useful advices. We would also like to thank the anonymous reviewers for their comments and helpful suggestions for improving our manuscript. This work benefited from the technical facilities of the MIO microscopy platform for oceanography. We are grateful to Cpt. Moreira Pinto, Lt. Cardoso Jeronimo, Cpl. Arrojado Oliveira, Lt A.V. Alves and the crew of “NRP Dom Carlos I”for their skilfull assistance during work at sea. Author Contributions Conceived and designed the experiments: VR ICB FD. Performed the experiments: VR DFB AR SRP CML M. 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