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19 pages, 8 figures, 1 table.-- This is an open access article under the terms of the Creative Commons Attribution License, Seabird guano enters coastal waters providing bioavailable substrates for microbial plankton, but their role in marine ecosystem functioning remains poorly understood. Two concentrations of the water soluble fraction (WSF) of gull guano were added to different natural microbial communities collected in surface waters from the Ría de Vigo (NW Spain) in spring, summer, and winter. Samples were incubated with or without antibiotics (to block bacterial activity) to test whether gull guano stimulated phytoplankton and bacterial growth, caused changes in taxonomic composition, and altered phytoplankton–bacteria interactions. Alteromonadales, Sphingobacteriales, Verrucomicrobia and diatoms were generally stimulated by guano. Chlorophyll a (Chl a) concentration and bacterial abundance significantly increased after additions independently of the initial ambient nutrient concentrations. Our study demonstrates, for the first time, that the addition of guano altered the phytoplankton–bacteria interaction index from neutral (i.e. phytoplankton growth was not affected by bacterial activity) to positive (i.e. phytoplankton growth was stimulated by bacterial activity) in the low-nutrient environment occurring in spring. In contrast, when environmental nutrient concentrations were high, the interaction index changed from positive to neutral after guano additions, suggesting the presence of some secondary metabolite in the guano that is needed for phytoplankton growth, which would otherwise be supplied by bacteria, This research was part of the INTERES project (CTM2017-83362-R) by the Spanish Ministry of Economy and Competitiveness. Funding for open access charge: Universidade de Vigo/CISUG. Maider Justel-Díez was supported by Xunta de Galicia fellowship and Erick Delgadillo-Nuño by an FPI fellowship from the Spanish Ministry of Economy and Competitiveness

The project OLCAPP was aimed at exploring the response of a natural, eutrophic system to ocean liming by slaked lime (calcium hydroxide) dispersal in the wake of ships. The main objectives were:- to monitor and model the slaked lime dissolution kinetics and the carbonate equilibrium (alkalinity, pH spikes/alteration, Dissolved Inorganic Carbon);- to assess Dissolved Organic Matter changes in quantity and composition;- to assess possible changes in primary production, photosynthetic efficiency and phytoplankton abundance and associations;- to assess the short and medium-term response of planktonic and benthic calcareous primary producers (calcareous red algae = maerl) to alkalinization and potential precipitation of carbonate crystals induced by the treatments.In the framework of the Transnational Access provided by AQUACOSM-plus, at the ECIMAT-UVIGO facility (Vigo, Spain) we had the opportunity to test ocean liming in nine mesocosm tanks. A sediment trap and a basket of calcareous algae (maerl) were positioned at the bottom of each tank. Mearl was previously collected in the Ria de Vigo at 7m depth and prepared for the experiment. Successively, each tank was filled with natural coastal seawater (~1m3).Out of the nine mesocosms, three tanks were treated with calcium hydroxide 0.02g/L (High) and three tanks with 0.006g/L (Low) per treatment, repeated on days 1, 3 and 5 (multiple exposure). The remaining three tanks were kept as control. A record of pH, O2, salinity, temperature and PAR was performed in the mesocosms during the experiment with a ten-minutes frequency.Nutrient concentration is monitored on a long-term basis in the Ria de Vigo, and was also tested on days 1, 3 and 5 for all treatments and controls. Seawater samples were collected from the mesocosms before (pre-treatment), and after 1h, 4h, and 24h from each treatment, with a 5L Niskin bottle. Dissolved Organic Matter as Dissolved Organic Carbon (DOC) and Chromophoric Dissolved Organic Matter (CDOM) were analysed, along with the Dissolved Inorganic Carbon (DIC). Moreover, samples collected after 1h from treatments were used for assessing also the bacterial association, the size-fractionated Chl-a concentration and the plankton primary production and photosynthetic efficiency. Gross primary production, community respiration and net community production were measured by changes in oxygen concentrations after 24 h light-dark bottle incubations. Dissolved oxygen was measured by Winkler titration. A total of 165 samples were obtained from filtering 2L of mesocosm water from pre-treatments, 4h and 24h samples, for the collection of the phytoplankton community, to be analysed under optical and scanning electron microscope.Preliminary observations during the experiment and the first data on the plankton community suggest that the High treatment leads to important flocculation and sedimentation affecting both the transparency of the water and the bottom environment, with significant and stable pH increase and decrease in phytoplankton production and efficiency. The mineralogical nature of the flocculation, the response of benthic calcareous algae and phytoplankton community, in term of composition and abundance of the major components, is here discussed.

14 pages, 4 figures, 2 tables.-- This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, Prokaryotes represent a major fraction of marine biomass and play a key role in the global carbon cycle. We studied the vertical profiles (0–3500 m) of abundance, viability, and activity of prokaryotic communities along a productivity gradient in the subtropical and tropical Atlantic to assess whether there is a vertical linkage between surface productivity regimes and deep ocean prokaryotic communities. We found that latitudinal changes in the vertical patterns of cytometric variables were coupled with surface productivity: higher prokaryotic abundances and viabilities, and smaller cell sizes were observed below highly productive surface waters, an effect reaching down to the bathypelagic layer. Leucine uptake rates in deep waters showed no clear relationship with surface productivity. Changes in resource and energy allocation to growth vs. maintenance in hostile environments, cell-size-dependent metabolic requirements, and variability in leucine-to-carbon conversion may all be part of the array of factors involved in controlling the prokaryotic activity patterns that were measured. Our work adds to the recent findings that highlight the importance of vertical connectivity for prokaryotic communities in the dark ocean and unveils a remarkable impact of surface conditions in the viability of deep ocean prokaryotes. This is a key aspect when considering metabolic rates of prokaryotic communities in the bathypelagic realm, This work is a contribution to projects MAFIA (grant number CTM2012-39587-C04-01), FLUXES (grant number CTM2015-69392-C3), e-IMPACT (grant number PID2019-109084RB-C21 and -C22), and INTERES (CTM2017-83362-R) funded by the Spanish “Plan Nacional/Estatal de I+D” and cofounded with FEDER funds, and to project SUMMER (grant number AMD-817806-5) funded by the European Union's Horizon 2020 research and innovation program. Markel Gómez-Letona is supported by the Ministerio de Ciencia, Innovación y Universidades, Gobierno de España (grant number FPU17-01435) during his PhD. Marta Sebastián is supported by the Project MIAU (grant number RTI2018-101025-B-I00) and the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S)

14 pages, 3 tables, 7 figures.-- Under a Creative Commons license, In this study, the DNA metabarcoding technique was used to explore the prokaryote diversity and community structure in wastewater collected in spring and winter 2020–2021 as well as the efficiency of the treatment in a wastewater treatment plant (WWTP) in Ría de Vigo (NW Spain). The samplings included raw wastewater from the inlet stream (M1), the discharge water after the disinfection treatment (M3) and mussels used as bioindicators of possible contamination of the marine environment. Significant differences were discovered in the microbiome of each type of sample (M1, M3 and mussels), with 92 %, 45 % and 44 % of exclusive OTUs found in mussel, M3 and M1 samples respectively. Seasonal differences were also detected in wastewater samples, with which abiotic parameters (temperature, pH) could be strongly involved. Bacteria present in raw wastewater (M1) were associated with the human gut microbiome, and therefore, potential pathogens that could be circulating in the population in specific periods were detected (e.g., Arcobacter sp. and Clostridium sp.). A considerable decrease in putative pathogenic organisms from the M1 to M3 wastewater fractions and the scarce presence in mussels (, This work was supported by the Ministerio de Ciencia e Innovación [PID2021-124955OB-I00], Fondo Supera COVID of the CRUE (Spanish Universities)-Santander Foundation [project DIMCoVAR] and the EU-H2020 “CIRCLES” [818290]

Prokaryotes represent a major fraction of marine biomass and play a key role in the global carbon cycle. We studied the vertical profiles (from surface down to the bathypelagic realm) of abundance, cytometric signatures, and activity of prokaryotic communities along a productivity gradient in the subtropical and tropical Atlantic to assess whether there is a vertical linkage between surface productivity regimes and deep ocean prokaryotic communities. We found that latitudinal changes in the vertical patterns of cytometric variables were coupled with surface productivity: higher prokaryotic abundances and viabilities, and smaller cell sizes were observed below highly productive surface waters, an effect reaching down to the bathypelagic layer. On the contrary, leucine uptake rates in deep waters showed no clear relationship with surface productivity. Changes in resource and energy allocation to growth vs. maintenance in hostile environments, cell-size-dependent metabolic requirements and variability in leucine to carbon conversion may all be part of the array of factors involved in controlling prokaryotic activity patterns that were measured. Our work adds to the recent findings that highlight the importance of vertical connectivity for prokaryotic communities in the dark ocean.

We experimentally evaluated the temporal (interday and interseason) and spatial variability in microbial plankton responses to vitamin B12 and/or B1 supply (solely or in combination with inorganic nutrients) in coastal and oceanic waters of the northeast Atlantic Ocean. Phytoplankton and, to a lesser extent, prokaryotes were strongly limited by inorganic nutrients. Interday variability in microbial plankton responses to B vitamins was limited compared to interseason variability, suggesting that B-vitamin availability might be partially controlled by factors operating at seasonal scale. Chlorophyll a (Chl a) concentration and prokaryote biomass (PB) significantly increased after B-vitamin amendments in 13 % and 21 %, respectively, of the 216 cases (36 experiments × 6 treatments). Most of these positive responses were produced by treatments containing either B12 solely or B12 combined with B1 in oceanic waters, which was consistent with the significantly lower average vitamin-B12 ambient concentrations compared to that in the coastal station. Negative responses, implying a decrease in Chl a or PB, represented 21 % for phytoplankton and 26 % for prokaryotes. Growth stimulation by B1 addition was more frequent on prokaryotes than in phytoplankton, suggesting that B1 auxotrophy in the sampling area could be more widespread in prokaryotes than in phytoplankton. Negative responses to B vitamins were generalized in coastal surface waters in summer and were associated with a high contribution of Flavobacteriales to the prokaryote community. This observation suggests that the external supply of B12 and/or B1 may promote negative interactions between microbial components when B-vitamin auxotrophs are abundant. The microbial response patterns to B12 and/or B1 amendments were significantly correlated with changes in the prokaryotic community composition, highlighting the pivotal role of prokaryotes in B-vitamin cycling in marine ecosystems.

14 pages, 6 figures, 3 tables.-- Under a Creative Commons license, Organic matter is known to influence community composition and metabolism of marine prokaryotes. However, few studies have addressed this linkage in the deep ocean. We studied the relationship between fluorescent dissolved organic matter and prokaryotic community composition in meso- and bathypelagic water masses along a surface productivity gradient crossing the subtropical and tropical Atlantic Ocean. Four fluorescence components were identified, three humic-like and one protein-like. The distributions of the humic-like components were significantly explained by water mass mixing, apparent oxygen utilisation (AOU) and epipelagic productivity proxies in varying degrees, while the protein-like component was explained only by water mass mixing and epipelagic productivity. The diversity and taxonomic composition of the prokaryotic community differed between water masses: the Nitrosopumilales order dominated in water masses with high AOU and humic-like fluorescence (notably, the SubPolar Mode Water), and tended to co-occur with Marine Group II archaea, the SAR324 clade and Thiomicrospirales, while bathypelagic water masses displayed greater abundances of members of Marinimicrobia, SAR202 and SAR324. Water mass mixing regression models suggested that the distribution of some taxa (e.g., Marinimicrobia, SAR202) was dominated by mixing and selection within the water masses during ageing, while others (chiefly, Alteromonadales) were mostly influenced by local processes. Our results suggest a link between the composition of the prokaryotic community, oxygen utilisation and the signal of fluorescent dissolved organic matter, and has implications for our understanding of the processes that shape carbon cycling and prokaryotic communities in the deep ocean, This work is a contribution to projects MAFIA (CTM2012-39587-C04-01), FLUXES (CTM2015-69392-C3), e-IMPACT (PID2019-109084RB-C21), and INTERES (CTM2017-83362-R) funded by the Spanish “Plan Nacional/Estatal de I + D” and cofounded with FEDER funds, and to project SUMMER (AMD-817806-5) funded by the European Union’s Horizon 2020 research and innovation program. MGL is supported by Ministerio de Ciencia, Innovación y Universidades, Gobierno de España (FPU17-01435) during his PhD. MS is supported by the Project MIAU (RTI2018-101025-B-I00) and the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

The responses of coastal microbial communities to nutrient inputs depend on the magnitude and nature of those inputs. To understand the response of prokaryote communities to contrasting levels of nutrient inputs from different sources, three nutrient addition experiments were performed in the Ría de Vigo in spring, summer, and autumn. The experiments consisted of controlled inorganic (nitrate, ammonium and phosphate), organic (glucose and a mix of 18 amino acids), and mixed (inorganic and organic); as well as natural (atmospheric and riverine) nutrient additions to surface water samples taken from the middle of the embayment and incubated for 48h, under in situ temperature and irradiance conditions. Prokaryote community DNA was collected and analyzed using high-throughput amplicon sequencing of the 16S rRNA gene. The joint addition of inorganic and organic nutrients provoked the most pronounced changes in community composition, systematically leading to great increases in the proportion of sequence reads belonging to Vibrionales, while natural amendments only caused subtle alterations. The proportion of reads assigned to the Planktomarina genus, bacteria belonging to the order Rhodobacterales, tended to increase when natural atmospheric nutrients were added. Overall, the fraction of reads belonging to the order Flavobacteriales did not show relevant changes, although the contribution of sequences affiliated to Polaribacter and Aurantivirga genera increased after atmospheric and riverine inputs in summer, and autumn. The proportion of Vibrionales reads also increased after controlled organic amendments in autumn, and to a lesser extent after riverine additions in spring and summer. In general, our findings suggest that an increasing input of nitrogenous organic matter from allochthonous sources into this coastal environment might promote the growth of potentially pathogenic bacteria belonging to the order Vibrionales. Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Ministerio de Ciencia e Innovación | Ref. CTM2011-30155- C03-01 Xunta de Galicia | Ref. EM2013/023

Coastal upwelling zones are hotspots of oceanic productivity, driven by phytoplankton photosynthesis. Bacteria, in turn, grow on and are the principal remineralizers of dissolved organic matter (DOM) produced in aquatic ecosystems. However, the molecular processes that key bacterial taxa employ to regulate the turnover of phytoplankton-derived DOM are not well understood. We therefore carried out comparative time-series metatranscriptome analyses of bacterioplankton in the Northwest Iberian upwelling system, using parallel sampling of seawater and mesocosms with in situ-like conditions. The mesocosm experiment uncovered a taxon-specific progression of transcriptional responses from bloom development (characterized by a diverse set of taxa in the orders Cellvibrionales, Rhodobacterales, and Pelagibacterales), over early decay (mainly taxa in the Alteromonadales and Flavobacteriales), to senescence phases (Flavobacteriales and Saprospirales taxa). Pronounced order-specific differences in the transcription of glycoside hydrolases, peptidases, and transporters were found, supporting that functional resource partitioning is dynamically structured by temporal changes in available DOM. In addition, comparative analysis of mesocosm and field samples revealed a high degree of metabolic plasticity in the degradation and uptake of carbohydrates and nitrogen-rich compounds, suggesting these gene systems critically contribute to modulating the stoichiometry of the labile DOM pool. Our findings suggest that cascades of transcriptional responses in gene systems for the utilization of organic matter and nutrients largely shape the fate of organic matter on the time scales typical of upwelling-driven phytoplankton blooms.

25 pages, 1 table, 5 figures, Cobalamin (B12) is an essential cofactor that is exclusively synthesized by some prokaryotes while many prokaryotes and eukaryotes require an external supply of B12. The spatial and temporal availability of B12 is poorly understood in marine ecosystems. Field measurements of B12 along with a large set of ancillary biotic and abiotic factors were obtained during three oceanographic cruises in the NW Iberian Peninsula, covering different spatial and temporal scales. B12 concentrations were remarkably low (, The scintillation counter was supported by the 2006‐06 ICTS project of the Ministry of Science and Innovation at the Spanish Institute of Oceanography. This research was supported by the Spanish Ministry of Economy and Competitiveness through the ENVISION (CTM2014‐59031‐P) and INTERES (CTM2017‐83362‐R) projects. Further support was provided by the Swedish Research Council VR. Vanessa Joglar was supported by an FPI fellowship from the Spanish Ministry of Economy and Competitiveness

11 pages, 5 figures, 2 tables, supplementary data https://doi.org/10.1016/j.ecss.2019.106565, Organic nutrient loading in coastal waters has considerably increased due to anthropogenic activities over the last decades. In particular, the increase in the proportion of organic vs. inorganic nitrogen may have an impact on microbial plankton communities, particularly within the small size classes, where mixotrophy is likely to be widespread. Here we explore the change in taxonomic composition of small eukaryotes (, This research was supported by the Xunta de Galicia through DIMENSION project (grant EM2013/023) and by Spanish Ministry of Economy, Industry and Competitivity (MINECO) trough ENVISION project (grant CTM2014-59031-P) in collaboration with the “Instituto Español de Oceanografía” (IEO) RADIALES project. Esther Barber-Lluch was funded by an F.P.I. MINECO fellowship. [...] R. Logares was supported by a Ramón y Cajal fellowship (RYC-2013-12554, MINECO, Spain), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

10 pages, 7 figures, 2 tables, The chemical composition of the seawater soluble fraction (WSF) of yellow-legged gulls and harbour seal faeces and their impact on microbial plankton communities from an eutrophic coastal area have been tested. After characterisation of the C:N:P stoichiometry, trace metals content and organic molecular composition of the faeces, significant differences between species have been observed in all parameters. Seagull faeces present about three times larger N content than seal faeces and are also richer in trace elements except for Cu and Zn. Organic nitrogen in seagull faeces is dominated by uric acid, while the proteins are the main N source in seal faeces. It is remarkable that seagull faeces are five times more soluble in seawater than seal faeces and present a much higher N content (48.0 versus 3.5 mg N in the WSF per gram of dry faeces), >85% of which as dissolved organic nitrogen, with C:N molar ratios of 2.4 and 13 for seagull and seal faeces, respectively. Based on this contrasting N content, large differences were expected in their impact on microbial populations. To test this hypothesis, a 3-day microcosm incubation experiment was performed, in which coastal seawater was amended with realistic concentrations of the WSF of seagull or seal faeces. A significant and similar increase in bacterial biomass occurred in response to both treatments. In the case of phytoplankton, the impact of the treatment with seagull faeces was significantly larger that the effect of the treatment with seal faeces. Our data suggest that the distinct competitive abilities of phytoplankton and bacteria largely influence the potential impact of distinct animal faeces on primary productivity in coastal ecosystems. Impacts on the microbial plankton communities do not affect only this trophic level, but the whole trophic chain, contributing to nutrient recycling in coastal areas where large populations of these species are settled, This research was partially supported by the Spanish Ministry of Economy and Competitiveness through the project INTERES (CTM2017-83362-R)

The capability of different bacterial populations to degrade abundant polymers, such as algal-derived polysaccharides, or to utilize preferentially polymers over monomers, remains largely unknown. In this study, microautoradiography was combined with fluorescence in situ hybridization (MAR-FISH) to evaluate the ability of Bacteroidetes, SAR11, Roseobacter spp., Gammaproteobacteria and SAR86 cells to use bicarbonate, leucine and starch under natural light conditions at two locations in shelf surface waters off NW Spain. The percentage of cells incorporating bicarbonate was relatively high (mean 32% ± 4%) and was positively correlated with the intensity of solar radiation. The proportion of cells using starch (mean 56% ± 4%) or leucine (mean 47% ± 4%) was significantly higher than that using bicarbonate. On average, SAR11, Roseobacter spp. and Gammaproteobacteria showed a similarly high percentage of cells using leucine (47%-65% of hybridized cells) than using starch (51%-64% of hybridized cells), while Bacteroidetes and SAR86 cells preferentially used starch (53% of hybridized cells) over leucine (34%-40% of hybridized cells). We suggest that the great percentage of bacteria using starch is related to a high ambient availability of polymers associated to algal cell lysis, which, in turn, weakens the short-term coupling between phytoplankton release and bacterial production.

We evaluated the temporal (inter-day and inter-season) and spatial variability in microbial plankton responses to vitamins B12 and B1 supply in coastal and oceanic waters. Inter-day variability in microbial plankton responses to B-vitamins was not of great concern, suggesting that B-vitamins availability was controlled by factors operating at larger temporal scales, such as those driving microbial community seasonal succession. Most positive responses were produced by treatments containing either B12 alone or B12 combined with B1 in oceanic waters, which was consistent with the significantly lower average vitamin B12 ambient concentrations compared to that in the coastal station. Growth stimulation by B1 addition was more frequent on bacteria, which is coherent with their widespread dependence on exogenous sources for this growth factor. Negative responses to B-vitamins were generalized in coastal waters in summer, and were associated to a high contribution of Flavobacteriales to the prokaryote community. This observation suggests that the external supply of B12 and/or B1 may promote negative interactions between microbial components when B-vitamins auxotrophs are abundant. The microbial response patterns to B12 and/or B1 amendments were significantly correlated with changes in the prokaryotic community composition, highlighting the pivotal role of prokaryotes in B-vitamins cycling in marine ecosystems.

Vitamin B12 (cobalamin), a large and complex organometallic molecule composed by a corrin ring with a central cobalt atom, is an essential coenzyme for nearly half of phytoplankton species but its production in marine ecosystems is restricted to prokaryotic organisms. Vitamin B 12 auxotrophy is widespread in the marine microbial community not being linked to any species lineage. Knowledge about the role of this organic compound on the control of the structure, composition and growth of natural marine microbial assemblages is scarce, but recent studies carried out in shelf waters off NW Iberian Peninsula have demonstrated that growth of phytoplankton and heterotrophic bacteria can be affected by the availability of this vitamin. Liquid Chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been set up as an analytical tool for the first direct determination of vitamin B 12 present in these coastal waters enabling the separation of two different B 12 analogs: hydroxocobalamin (HyB12) and cyanocobalamin (CyB12). In this study, samples were collected from shelf (coastal station) and shelf-break waters of the NW Iberian Peninsula in February, April and August 2016 in order to encompass both the spatial and temporal variability of vitamin B 12 concentrations in seawater. Concentrations of dissolved vitamin B 12 varied from 0.15 pM, at the shelf-break station, to 2.7 pM at the coastal station, HyB12 being the most abundant congener. Overall, the low concentrations of dissolved vitamin B 12 and the significant and positive relationship between depth-weighted water column averages of Chl-a and vitamin B 12 (r 2 = 0 . 80 , p>0.001), suggest that this micronutrient might play a significant role in phytoplankton biomass production during the spring productive period of this coastal ecosystem. The temporal cycle elucidated from this data described an exhaustion of vitamin B 12 winter inventory by direct consumption during the phytoplankton productive period and a stock recovery in August.

Even though compelling evidences indicate that marine microbes show biogeographic patterns, very little is known on the mechanisms driving those patterns in aquatic ecosystems. Here we examined bacterial community structure in epipelagic waters of a highly hydrodynamic area of the Southern Ocean to gain insight into the role of biogeochemical factors and water mass mixing, as a proxy for dispersal, on microbial biogeography. We targeted four water masses that converge and mix around the South Shetland Islands (northern tip of the Antarctic Peninsula). Bacterioplankton communities were water-mass specific and best explained by dispersal rather than by biogeochemical factors, which we attribute to the relatively reduced environmental gradients found in these cold and nutrient rich waters. Our results support the notion that currents and water mixing may have a considerable effect in connecting and transforming different water bodies, and consequently, in shaping communities of microorganisms. Considering the multidimensional and dynamic nature of our oceans, we recommend the analysis of water mass mixing as a more suitable approach to investigate the role of dispersal on microbial plankton biogeography rather than geographical distance. This article is protected by copyright. All rights reserved.

Microbial plankton communities do not always respond to inorganic nutrient additions in coastal waters off NW Spain. However, enhanced growth of phytoplankton and heterotrophic bacteria has been ob ...

Teira, Eva ... et. al.-- 9 pages, 6 figures, 2 tables, The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean, This study was supported by the by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) and project DIFUNCAR (CTM2008-03790/MAR)

Ecophysiology of Nitrogen‐Metabolising Microbes.-- 15 pages, 7 figures, 1 table, supporting information https://doi.org/10.1111/1462-2920.14581, The impact of grazing, resource competition and light on prokaryotic growth and taxonomic composition in subtropical and tropical surface waters were studied through 10 microcosm experiments conducted between 30°N and 30°S in the Atlantic, Pacific and Indian oceans. Under natural sunlight conditions, significant changes in taxonomic composition were only observed after the reduction of grazing by sample filtration in combination with a decrease in resource competition by sample dilution. Sunlight exposure significantly reduced prokaryote growth (11 ± 6%) and community richness (14 ± 4%) compared to continuous darkness but did not significantly change community composition. The largest growth inhibition after sunlight exposure occurred at locations showing deep mixed layers. The reduction of grazing had an expected and significant positive effect on growth, but caused a significant decrease in community richness (16 ± 6%), suggesting that the coexistence of many different OTUs is partly promoted by the presence of predators. Dilution of the grazer‐free prokaryotic community significantly enhanced growth at the level of community, but consistently and sharply reduced the abundance of Prochlorococcus and SAR11 populations. The decline of these oligotrophic bacterial taxa following an increase in resource availability is consistent with their high specialization for exploiting the limited resources available in the oligotrophic warm ocean, This study was supported by the by the Spanish Ministerio de Ciencia e Innovación through project Consolider‐Ingenio Malaspina 2010 (CSD2008‐00077). Sample sequencing was funded by grant M'OMICS‐MALAN (CTM2011‐15461‐E PI) of the Spanish Ministerio de Economía y Competitividad

10 pages, 6 figures, 5 tables, Over the past decades, as a consequence of human activity, there was an increase in nutrient inputs to the ocean and they are expected to enhance even more in the future. Coastal areas, accounting for a significant proportion of marine primary productivity, are the most vulnerable zones to anthropogenic impacts. The response of phytoplankton communities to an increase in organic and inorganic nutrients levels from natural allochthonous sources was assessed in microcosm experiments conducted in a coastal system affected by intermittent upwelling events (Ría de Vigo, NW Iberia). Three nutrient addition experiments were performed in spring, summer and autumn, when surface water was supplemented with 5 and 10% of atmospheric and riverine matter. Pico-, nano- and microphytoplankton abundances, chlorophyll a concentration (Chl a) and primary production rates (PP) were measured and compared with those in the control seawater sample (without additions) after 48 h of incubation. Simultaneous experiments with controlled additions of inorganic and organic nutrients were also performed in order to describe the limiting nutrient for phytoplankton growth at each experiment. The composition of the matter inputs and the structure of the phytoplankton communities determined the type of response observed. Phytoplankton responses varied among seasons, being positively correlated with dissolved inorganic nitrogen (DIN) concentrations. As expected, the phytoplankton responses to external nutrient inputs were stronger under low nutrient levels (summer) than when phytoplankton was already growing in nutrient replete conditions (spring). Null and negative responses to the natural inputs were observed in autumn, which suggests that the oceanic phytoplankton advected to this coastal system during downwelling events could be occasionally inhibited by these nutrient inputs. In a future global change scenario, characterized by enhanced nutrient inputs from riverine and atmospheric origin, the response of phytoplankton communities will strongly depend on the concentration and chemical composition of these inputs and on the structure of phytoplankton communities able to respond to them., This work was funded by the Spanish Ministry of Science and Innovation under the project REIMAGE (CTM2011-30155-C03-03) and the Spanish Ministry of Economy and Competitivity under the project iSmall (CTM2014-56119-R). I.G.T was funded by a Post-doctoral grant by the Portuguese Foundation for Science and Technology (SFRH/BPD/108485/2015) co-financed by national and European funds (POCH programme) and M.N.-C. was supported by the CSIC Program "Junta para la Ampliación de Estudios" co-financed by the ESF (reference JAE DOC 040) and the project FERMIO (MINECO, CTM2014-57334-JIN)

19 pages, 7 figures, 1 table, Small eukaryotes (0.2–20 μm cell‐size) represent a significant fraction of the microbial plankton community in shelf waters of NW‐Spain. The community composition of small eukaryotes living at the surface and at the base of the photic zone was analysed by means of 18S rDNA high‐throughput sequencing on a circa‐monthly basis over a 23 months period. Ostreococcus was the most abundant taxon in surface waters, showing marked peaks in read abundance in spring and late summer, while Syndiniales dominated at the base of the photic zone. A well‐defined seasonal pattern of community composition, linked to the succession of the dominant taxa, was found in surface waters. Seasonality was less apparent at the base of the euphotic zone. Temporal changes in abiotic factors significantly correlated with changes in community composition in surface (r = 0.71) and at the base of the photic zone (r = 0.38). Changes in community composition significantly correlated with changes in community function‐related variables (including biomass, primary production and respiration) only in surface water (r = 0.36). Co‐occurrence network analyses revealed 45 significant interspecies associations among the 50 most abundant taxa with highly connected OTUs belonging to cryptophyceans. The network topology, with small‐world characteristics, suggests a stabilizing role of biotic interactions to environmental disturbance, This research was supported by the Xunta the Galicia through DIMENSION project (grant EM2013/023) and by Spanish Ministry of Economy, Industry and Competitivity (MINECO) trough ENVISION project (grant CTM2014‐59031‐P) in collaboration with the ‘Instituto Español de Oceanografía’ (IEO) RADIALES project. Esther Barber‐Lluch was funded by an F.P.I. MINECO fellowship

Lara, Elena ... et al.-- 12 pages, 4 figures, 3 tables, supplementary material http://advances.sciencemag.org/content/suppl/2017/09/01/3.9.e1602565.DC1, Viruses are a key component of marine ecosystems, but the assessment of their global role in regulating microbial communities and the flux of carbon is precluded by a paucity of data, particularly in the deep ocean. We assessed patterns in viral abundance and production and the role of viral lysis as a driver of prokaryote mortality, from surface to bathypelagic layers, across the tropical and subtropical oceans. Viral abundance showed significant differences between oceans in the epipelagic and mesopelagic, but not in the bathypelagic, and decreased with depth, with an average power-law scaling exponent of −1.03 km−1 from an average of 7.76 × 106 viruses ml−1 in the epipelagic to 0.62 × 106 viruses ml−1 in the bathypelagic layer with an average integrated (0 to 4000 m) viral stock of about 0.004 to 0.044 g C m−2, half of which is found below 775 m. Lysogenic viral production was higher than lytic viral production in surface waters, whereas the opposite was found in the bathypelagic, where prokaryotic mortality due to viruses was estimated to be 60 times higher than grazing. Free viruses had turnover times of 0.1 days in the bathypelagic, revealing that viruses in the bathypelagic are highly dynamic. On the basis of the rates of lysed prokaryotic cells, we estimated that viruses release 145 Gt C year−1 in the global tropical and subtropical oceans. The active viral processes reported here demonstrate the importance of viruses in the production of dissolved organic carbon in the dark ocean, a major pathway in carbon cycling, This work was supported by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) and funding received through grant FCS/1/2449-01-01 of the Office of Sponsored Research of the King Abdullah University of Science and Technology., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Analysis of seasonal patterns of marine bacterial community structure along horizontal and vertical spatial scales can help to predict long-term responses to climate change. Several recent studies have shown predictable seasonal reoccurrence of bacterial assemblages. However, only a few have assessed temporal variability over both horizontal and vertical spatial scales. Here, we simultaneously studied the bacterial community structure at two different locations and depths in shelf waters of a coastal upwelling system during an annual cycle. The most noticeable biogeographic patterns observed were seasonality, horizontal homogeneity, and spatial synchrony in bacterial diversity and community structure related with regional upwelling–downwelling dynamics. Water column mixing eventually disrupted bacterial community structure vertical heterogeneity. Our results are consistent with previous temporal studies of marine bacterioplankton in other temperate regions and also suggest a marked influence of regional factors on the bacterial communities inhabiting this coastal upwelling system. Bacterial-mediated carbon fluxes in this productive region appear to be mainly controlled by community structure dynamics in surface waters, and local environmental factors at the base of the euphotic zone.

Even though compelling evidences indicate that marine microbes show biogeographic patterns, very little is known on the mechanisms driving those patterns in aquatic ecosystems. In the present study, bacterial community structure was examined in epipelagic waters of a highly hydrodynamic area of the Southern Ocean to gain insight into the role that biogeochemical factors and water mass mixing (a proxy of dispersal) have on microbial biogeography. Four water masses that converge and mix around the South Shetland Islands (northern tip of the Antarctic Peninsula) were investigated. Bacterioplankton communities were water-mass specific, and were best explained by dispersal rather than by biogeochemical factors, which is attributed to the relatively reduced environmental gradients found in these cold and nutrient rich waters. These results support the notion that currents and water mixing may have a considerable effect in connecting and transforming different water bodies, and consequently, in shaping communities of microorganisms. Considering the multidimensional and dynamic nature of the ocean, analysis of water mass mixing is a more suitable approach to investigate the role of dispersal on the biogeography of planktonic microorganisms rather than geographical distance.

Original research paper, The impact of solar radiation on dissolved organic matter (DOM) derived from 3 different sources (seawater, eelgrass leaves and river water) and the effect on the bacterial carbon cycling and diversity were investigated. Seawater with DOM from the sources was first either kept in the dark or exposed to sunlight (4 days), after which a bacterial inoculum was added and incubated for 4 additional days. Sunlight exposure reduced the coloured DOM and carbon signals, which was followed by a production of inorganic nutrients. Bacterial carbon cycling was higher in the dark compared with the light treatment in seawater and river samples, while higher levels were found in the sunlight-exposed eelgrass experiment. Sunlight pre-exposure stimulated the bacterial growth efficiency in the seawater experiments, while no impact was found in the other experiments. We suggest that these responses are connected to differences in substrate composition and the production of free radicals. The bacterial community that developed in the dark and sunlight pre-treated samples differed in the seawater and river experiments. Our findings suggest that impact of sunlight exposure on the bacterial carbon transfer and diversity depends on the DOM source and on the sunlight-induced production of inorganic nutrients., EUROMARINE, CSIC-ESF, 3,408

11 páginas, 5 tablas, 5 figuras, The processes mediated by microbial planktonic communities occur along the entire water column, yet the microbial activity and composition have been studied mainly in surface waters. This research examined the vertical variation in bacterial abundance, activity and community composition and structure from surface down to 5000 m depth following a longitudinal transect off the Galician coast (NW Iberian margin, from 43°N, 9°W to 43°N, 15°W). Community activity and composition changed with depth. The leucine incorporation rates decreased from the euphotic layer to the bathypelagic waters by three orders of magnitude, whereas prokaryotic abundance decreased only by one order of magnitude. The relative abundance of SAR11 and Alteromonas, determined by catalyzed reported deposition fluorescence in situ hybridization (CARD-FISH), decreased with depth. Meanwhile, the contribution of SAR 202 and SAR324 was significantly higher in the deeper layers (i.e. NEADW, North East Atlantic Deep Water and LDW, Lower Deep Water) than in the euphotic zone. Bacterial community structure, assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA), was depth-specific. A distance based linear model (DistLM) revealed that the variability found in bacterial community structure was mainly explained by temperature nitrate, phosphate, dissolved organic matter (DOM) fluorescence, prokaryotic abundance, leucine incorporation and to a lesser extent salinity, oxygen, CDOM absorbance and dissolved organic carbon concentration. Our results displayed a bacterial community structure shaped not only by depth-related physicochemical features but also by DOM quality, indicating that different prokaryotic taxa have the potential to metabolize particular DOM sources, Funding for the sampling and analysis was supported by the Local Research Program of Xunta de Galicia grant “Biodiversidade Funcional do Microplancton nas Profundidades Mariñas de Galicia” (BIO-PROF, Ref. 10MMA604024PR, 2010–2012) and “Relación Entre la Diversidad Funcional del Bacterioplancton y la Circulación de Carbono en el Océano Costero en el Contexto del Cambio Global” (DIFUNCAR, Ref. CTM2008–03790). V. D-A. was funded by a Campus do Mar Master Scholarship. M. N.-C. was funded by the project FERMIO (MINECO, CTM2014-57334-JIN). Hernando-Morales V. was supported by the MICINN program “Formación de Personal Investigador” (FPI), Ref. grant BES-2009–028186

12 páginas, 5 figuras, 2 tablas, Anthropogenic pressures are changing the magnitude and nature of matter inputs into the ocean. The Ría de Vigo (NW Spain) is a highly productive and dynamic coastal system that is likely affected by such alterations. Previous nutrient-addition microcosm experiments conducted during contrasting hydrographic conditions suggested that heterotrophic bacteria are limited by organic carbon (C) and occasionally co-limited by inorganic nutrients in this coastal area. In order to assess short-term responses in biomass, production, and respiration of heterotrophic bacteria from the Ría de Vigo to increasing amounts of natural inputs of matter, we conducted 6 microcosm experiments, wherein surface seawater collected in spring, summer, and autumn was mixed with increasing amounts of dissolved natural matter concentrates from riverine and atmospheric origin. Simultaneous experiments with controlled inorganic and/or organic additions indicated that bacteria were co-limited by inorganic nutrients and C in spring and summer and primarily limited by C in autumn. Production responded more than biomass to increasing inputs of matter, whereas respiration did not change. The bacterial production response to increasing dissolved organic C load associated with riverine and atmospheric inputs was strongly related to the relative phosphorus (P) content of the dissolved matter concentrates. Our data suggest that bacterial production might decrease with the increase of P-deficient allochthonous matter inputs, which would have important biogeochemical consequences for C cycling in coastal areas, This research was supported by the Spanish Ministry of Science and Innovation through project REIMAGE (grants CTM2011-30155-C03-01 and CTM2011-30155-C03-02) and by the Xunta the Galicia trough project DIMENSION (grant EM2013/023). E. B-L was funded by a F.P.I. MICINN fellowship. M.N.-C. was funded by the CSIC Program "Junta para la Ampliación de Estudios" co-financed by the ESF. S.M.G. was funded by a Marie Curie fellowship

Phytoplankton and bacterioplankton dynamics were studied around South Shetland Islands (Antarctica) with special emphasis on the Drake Passage region, during austral summer, in order to expand our knowledge on the coupling between the autotrophic and heterotrophic microbial plankton compartments in polar ecosystems. In addition, we directly estimated bacterial growth efficiency in the Drake Passage with the aim of better constraining total bacterial carbon utilization in this important polar ecosystem. Integrated chlorophyll-a concentration (21–86 mg m � 2 ), primary production rates (0.7– 19.3 mg C m � 3 d � 1 ) and mean water-column photochemical efficiency (0.24–0.60) were significantly correlated with Si n tracer (r 2 ¼ 0.55, 0.46 and 0.64, respectively), which indirectly points to iron as the

Adequate bacterial respiration (BR) estimations are necessary to understand the flow of carbon through marine plankton food webs. A considerably higher bacterial contribution to total microbial plankton community respiration (CR) has been observed in oligotrophic systems compared to more productive systems. Classical BR estimation procedures, typically oxygen consumption mea- surements, comprise pre-incubation filtration to separate bacteria from the rest of the plankton community and long incubation times (24 h). The resulting disruption of the community linkages over long time periods might affect BR measure- ments, especially in oligotrophic systems charac- terized by tightly coupled microbial food webs. In this study, BR data were compiled from 2 con - trasting environments: the highly productive Ria de Vigo (NW Spain) and the North Atlantic oligotrophic gyre. Standard procedures or in vivo electron transport system (ETS) activity (non pre- filtered and short-time incubation) procedures were used to obtain a total of 209 BR estimations. Bacterial contribution to plankton CR was signifi- cantly higher in oligotrophic than in highly pro- ductive conditions (155% and 42%, respectively) when using standard procedures, while similar values were observed (31% and 30%) with in vivo ETS procedures. The relation between plankton CR and BR along the studied productivity gradient suggests that bacterial contribution to total CR varies less than previously assumed with an aver- age value approximating 30% through different trophic situations.

Experimental results related to the effects of ocean acidification on planktonic marine microbes are still rather inconsistent and occasionally contradictory. Moreover, laboratory or field experiments that address the effects of changes in CO2 concentrations on heterotrophic microbes are very scarce, despite the major role of these organisms in the marine carbon cycle. We tested the direct effect of an elevated CO2 concentration (1000 ppmv) on the biomass and meta- bolic rates (leucine incorporation, CO2 fixation and respiration) of 2 isolates belonging to 2 rele- vant marine bacterial families, Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217). Our results demonstrate that, contrary to some expectations, high pCO2 did not nega- tively affect bacterial growth but increased growth efficiency in the case of MED217. The elevated partial pressure of CO2 (pCO2) caused, in both cases, higher rates of CO2 fixation in the dissolved fraction and, in the case of MED217, lower respiration rates. Both responses would tend to increase the pH of seawater acting as a negative feedback between elevated atmospheric CO2 concentrations and ocean acidification.

17 páginas, 7 figuras, 2 tablas, The short-termeffects of inorganic N and P (nitrate, ammonium, phosphate) and organic C and N (glucose, amino acids) input, added separately as well as jointly, on microplankton community structure and metabolism were studied in 6 microcosm experiments conducted from February to July 2008 in a eutrophic coastal embayment in NW Spain under contrasting hydrographic conditions. The responses of planktonic communities were highly variable. Strong positive effects of combined inorganic and organic nutrient enrichments for phytoplankton and heterotrophic bacteria were found. In most experiments enhanced phytoplankton biomass and productivity (primary production increased up to 5.5-fold) was measured after mixed (inorganic plus organic) additions but phytoplankton responded only in one of the experiments after inorganic additions. Heterotrophic bacteria responded faster than phytoplankton to the additions but were never affected by inorganic nutrient additions, suggesting a C limitation of bacterial activity in this coastal environment. Heterotrophic bacteria biomass, productivity, and respiration responded to mixed additions (containing C, N, and P) in all the experiments (bacterial production increased from 14- to 34-fold) whereas its response to organic (containing C and N) additions greatly differed among experiments (bacterial production increased from 0.6- to 21-fold), which could be partially related to P availability. A general increase in bacterial growth efficiency occurred only after mixed additions (up to 2-fold). The tendency towards heterotrophy of the microbial plankton community was weaker after the mixed (Production/Respiration (P/R) ratio changes from 0.34- to 1.6-fold) than after the organic additions (P/R changes from 0.16- to 0.8-fold). Our results suggest that phytoplankton requirememnts for organic nutrients for growth and P deficiency of bacterial populations intermittently occur in this coastal area., This research was supported by the Xunta de Galicia through projects AddEx (grant PGIDIT06PXIB312222PR) and IMAN (grant 07MMA002402PR). S.M.-G. was funded by an FPU MEC fellowship. E.T. was funded by a Juan de la Cierva and a Ramón y Cajal-MEC contract. C.L. was funded by a fellowship from the Early Stage Training site ECOSUMMER (MEST-CT-2004-0205019) and the Carlsberg Foundation.

Bacterioplankton are responsible for a large fraction of respiration and dissolved organic matter (DOM) remineralisation in the ocean; therefore, changes in their taxonomic composition due to changes in nutrient inputs may have important biogeochemical implications. The impact of nutrient loading will depend on the type of input and the initial environmental conditions. We conducted a set of 5 microcosm experiments along a broad latitudinal transect in the Atlantic Ocean (26°N to 29°S) in order to assess the effects of inorganic (nitrate, ammonium, phosphate, silica) and organic (glucose, amino acids) inputs, added separately as well as jointly, on the bacterioplankton community composition at the level of major groups. We followed changes in the relative abundance of important bacterial phylogenetic (Roseobacter spp., SAR11, Gammaproteobacteria, Bacteroidetes) and cytometric (low, high and very high nucleic acid content) groups. We observed that distinct groups responded differently to nutrient additions: SAR11 and Bacteroidetes responded negatively to organic and mixed additions, while Roseobacter spp. and Gammaproteobacteria responded positively. Only the group Roseobacter spp. responded positively to inorganic additions. The abundance of very high nucleic acid content bacteria was low in the control and the inorganic treatment; however, they reached very high numbers in the organic and mixed treatments. There was a clear increasing north to south gradient in the magnitude of response of Gammaproteobacteria to organic inputs (containing carbon and nitrogen), likely related to an increasing north to south gradient in phosphate concentration. The magnitude of response of Gammaproteobacteria explained 89% of the observed variability in the magnitude of bacterial production response, which suggests a strong link between bacterial community composition and carbon cycling in the oligotrophic ocean., Sí

Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2-para (iodo-phenyl)-3(nitrophenyl)-5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2-6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

The input of allochthonous matter of continental origin to coastal zones globally may cause changes in the activity (function) as well as the taxonomic composition (structure) of the micro- bial plankton community. The goal of the present study was the simultaneous analysis of microbial plankton community structure (size-fractionated phytoplankton biomass, bacterial community com- position) and function (particulate and dissolved primary production, bacterial production, microbial plankton community respiration) in the northwest Iberian coastal transition zone during a dry (Feb- ruary 2005) and a rainy (October 2005) period. An influence of freshwater input was observed in October, even at an offshore site, but not in February. We found an autotrophic community domi- nated by picophytoplankton during both sampling periods. In contrast, the bacterial groups Beta- and Gammaproteobacteria were significantly more abundant during the high precipitation period. Pri- mary production rates were low and similar during both sampling periods; however, bacterial pro- duction was 8-fold and community respiration was 3-fold higher in October than in February. Conse- quently, the microbial community metabolism was net autotrophic in February and net heterotrophic in October. The high precipitation and the significant presence of bacteria belonging to the Betapro- teobacteria, typical for freshwater systems, in October compared to February, strongly suggest an influence of material of continental origin on microbial metabolism in this coastal transition zone.

Aims: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water Methods and Results: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338 ⁄ EUB338-II ⁄ EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77–96%) bacteria and 11% (range 3–22%) identified as Archaea. Conclusions: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. Significance and Impact of the Study: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.

Pernice, Massimo ... et. al.-- 11 pages, 7 figures, 3 tables, The dark ocean is one of the largest biomes on Earth, with critical roles in organic matter remineralization and global carbon sequestration. Despite its recognized importance, little is known about some key microbial players, such as the community of heterotrophic protists (HP), which are likely the main consumers of prokaryotic biomass. To investigate this microbial component at a global scale, we determined their abundance and biomass in deepwater column samples from the Malaspina 2010 circumnavigation using a combination of epifluorescence microscopy and flow cytometry. HP were ubiquitously found at all depths investigated down to 4000 m. HP abundances decreased with depth, from an average of 72±19 cells ml-1 in mesopelagic waters down to 11±1 cells ml-1 in bathypelagic waters, whereas their total biomass decreased from 280±46 to 50±14 pg C ml-1. The parameters that better explained the variance of HP abundance were depth and prokaryote abundance, and to lesser extent oxygen concentration. The generally good correlation with prokaryotic abundance suggested active grazing of HP on prokaryotes. On a finer scale, the prokaryote:HP abundance ratio varied at a regional scale, and sites with the highest ratios exhibited a larger contribution of fungi molecular signal. Our study is a step forward towards determining the relationship between HP and their environment, unveiling their importance as players in the dark ocean's microbial food web. © 2015 International Society for Microbial Ecology All rights reserved, This study was supported by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) (to CMD) and FLAME (CGL2010-16304) (to RM)

17 páginas, 8 figuras, 2 tablas, We simultaneously studied microbial-mediated carbon fluxes at 2 contrasting sites within the coastal upwelling system off Galicia, near A Coruña and Vigo, Spain, over an annual cycle in order to compare the fraction of primary production released as dissolved organic carbon (DOC) and the degree of coupling between bacteria and phytoplankton. A significant fraction of primary production was released as DOC at both sites, averaging ~30%. DOC release accounted for, on average, 30% of the total bacterial carbon demand, which is indicative of a moderate trophic dependence of bacteria on phytoplankton in these coastal ecosystems. Nevertheless, differences in hydrographic conditions associated with stronger upwelling pulses off Vigo, and deeper upper mixed layers during the downwelling period off A Coruña, led to significant differences in phytoplankton dynamics and the subsequent direct coupling with heterotrophic bacteria. Strong direct coupling between phytoplankton extracellular release and bacterial production (BP) was found off Vigo, which could be related to the quality of the DOC produced by actively growing phytoplankton. By contrast, DOC release and BP rates were decoupled off A Coruña, likely due to unaccounted DOC associated with indirect trophic processes or to the low availability of freshly produced exudates associated with overflow or photoinhibition mechanisms, This research was supported in part by the MEC project DIFUNCAR (CTM2008-03790/MAR) and by IEO project RADIALES. S.M-G. was funded by a FPU– MEC fellowship. V.H-M. was funded by a FPI– MEC fellowship. E.T. was funded by a Ramón y Cajal-MEC con- tract

11 páginas, 3 tablas, 5 figuras, The impact of rain water collected at marine, urban and rural sites on coastal phytoplankton biomass, primary production and community composition as well as the effect on microbial plankton metabolism was studied in 3 microcosm experiments conducted under contrasting spring, autumn and winter conditions. The measured responses were highly variable. Rainwater additions increased chlorophyll a (Chl a) concentration (5–68% difference between rainwater treatments relative to the control) in all experiments and reduced or stimulated primary production (PP) depending on the treatment and the experiment (from −10 to +169% relative to the control). Autotrophic stimulation was highest in spring, probably related to the low initial natural nutrient concentrations. Under winter nutrient replete conditions, rainwater inputs changed the phytoplankton community although this change did not promote increases in primary production. Enhancement of net autotrophy (increase of net oxygen production up to 227%) after rainwater inputs were only found during the period of low nutrient availability. Inputs of dissolved organic nitrogen (DON) explained a large fraction of the variability in the response of PP, Chl a, community respiration (CR) and net community production (NCP). Our results suggest that differences in the initial environmental conditions (i.e. nutrient availability), rainwater composition and the ability of the present autotrophic communities to utilize the new nutrients result in substantial changes in the microbial responses and associated biologically-mediated carbon fluxes. As atmospheric nutrient inputs into coastal oceans are increasing rapidly, our results help to understand the effects of different inputs on the metabolism of distinct microbial communities, This research was supported by the Galician Government (Xunta de Galicia) through the grants 07MMA002402PR (IMAN) and PGIDIT06PXIB312222PR (AddEx). S.M-G. and E.E.G-M. were funded by F.P.U. fellowships and E.T. by a Ramón y Cajal contract of the Spanish Ministry of Science and Innovation

We determined the distribution and activity of the major prokaryotic groups (Bacteria, Cren-, and Euryarchaeota) inhabiting the deep water masses of the North Atlantic by following the path of the North Atlantic Deep Water (NADW) from its formation in the Greenland-Iceland-Norwegian (GIN) Sea along its two major branches covering approximately the first 50 yr of the NADW in the oceanic conveyor belt system. The relative abundance of Eury- and Crenarchaeota, assessed by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), was significantly higher in the western branch (17% and 24% of 49,69-diamidino-2- phenylindole (DAPI)-stained cells, respectively) than in the eastern (9% and 17%, respectively) branch of the NADW. In contrast, the relative abundance of Bacteria (30% of DAPI-stained cells) did not differ between the western and the eastern basin. Prokaryotic production and turnover rates, however, were higher in the western than the eastern basin. Generally, the contribution of Euryarchaeota to total picoplankton was correlated positively with oxygen concentrations ( p < 0.001) and negatively with salinity ( p < 0.001) and temperature ( p < 0.001). The contribution of Crenarchaeota to total picoplankton correlated positively with oxygen ( p < 0.05) and negatively with salinity ( p < 0.001). There was a positive correlation between the crenarchaeotal contribution to picoplankton and nitrite concentration ( p < 0.001), especially in the oxygen minimum layer, suggesting their potential involvement in the marine nitrogen cycle as nitrifiers. The observed variability in archaeal abundance in relation to bulk prokaryotic activity supports the emerging notion that Archaea are a highly dynamic and metabolically active component of the deep ocean prokaryotic community.

We determined the contribution of the three major prokaryotic groups (Bacteria, Crenarchaeota, and Euryarchaeota) on the uptake of D- and L-aspartic acid (Asp) in the major water masses of the North Atlantic (from 100to 4,000-m depth) with the use of microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH). The percentage of prokaryotic cells that assimilated D- and L-Asp ranged from ,5% to 25%. In the meso- and bathypelagic waters of the North Atlantic, Archaea are more abundant (42% 6 2% of 49,69-diamino-2-phenylindole [DAPI]‐stained cells) than Bacteria (30% 6 1% of DAPI-stained cells), and more archaeal than bacterial cells are actively incorporating D-Asp (62% 6 2% vs. 38% 6 2% of total D-Asp active cells). In contrast, Bacteria and Archaea almost equally contribute to L-Asp use in the deep waters of the North Atlantic (47% 6 2% vs. 53% 6 2% of total L-Asp active cells). The increase in the D-Asp : L-Asp uptake ratio in the prokaryotic community with depth appears to be driven by the efficient uptake of D-Asp by, especially, the Crenarchaeota in the deep waters. Because Archaea, and particularly Crenarchaeota, commonly dominate the prokaryotic communities in the ocean’s interior, we suggest that they represent a previously unrecognized sink of D‐amino acids in the deep ocean.