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Prokaryotic abundance and activity are commonly assessed by dividing them into two size-fractions: free-living and attached to particles. Nevertheless, organic matter, essential for the growth of heterotrophic prokaryotes, is present in the environment in a continuum of sizes, from purely dissolved to large particles. Therefore, defining the activity of the prokaryotic community would be more accurate by considering all the distinct size fractions. To achieve this, we measured prokaryotic abundance (PA), heterotrophic prokaryotic activity (as leucine incorporation) and extracellular enzyme activities at a coastal site in the NW Mediterranean Sea. We conducted measurements in both bulk seawater and size fractionated samples sequentially passing through 5 different filter types: 0.2-0.8-3-5-10 μm pore size. Our results indicate that the fraction <0.8 μm contained the highest percentage of cells (91.6 ± 1.1 %) and leucine incorporation rates (72.2 ± 3.5 %). Most of the extracellular enzyme activity appeared in the dissolved fraction (<0.2 μm; 19.8-79.4 %), yet the specific activity of the enzymes (per cell activity) was 100-1000 times higher in the particulate (>0.8 μm) than in the free-living (0.2-0.8 μm) fraction. The size fraction with highest specific activities for leucine incorporation and most of the enzyme activities (β-glucosidase, esterase, Leu-aminopeptidase and alkaline phosphatase) was the 5-10 μm fraction. In contrast, the higher specific chitobiase activity in the >10 μm fraction, suggests that the prokaryotic community colonizing large particles might be more specialized in the hydrolysis of organic matter of zooplanktonic origin than the community colonizing smaller particles., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Documenting large-scale patterns of animals in the ocean and determining the drivers of these patterns is needed for conservation efforts given the unprecedented rates of change occurring within marine ecosystems. We used existing datasets from two global expeditions, Tara Oceans and Malaspina , that circumnavigated the oceans and sampled down to 4000 m to assess metazoans from environmental DNA (eDNA) extracted from seawater. We describe patterns of taxonomic richness within metazoan phyla and orders based on metabarcoding and infer the relative abundance of phyla using metagenome datasets, and relate these data to environmental variables. Arthropods had the greatest taxonomic richness of metazoan phyla at the surface, while cnidarians had the greatest richness in pelagic zones. Half of the marine metazoan eDNA from metagenome datasets was from arthropods, followed by cnidarians and nematodes. We found that mean surface temperature and primary productivity were positively related to metazoan taxonomic richness. Our findings concur with existing knowledge that temperature and primary productivity are important drivers of taxonomic richness for specific taxa at the ocean's surface, but these correlations are less evident in the deep ocean. Massive sequencing of eDNA can improve understanding of animal distributions, particularly for the deep ocean where sampling is challenging., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

Heterotrophic Bacteria and Archaea (prokaryotes) are a major component of marine food webs and global biogeochemical cycles. Yet, there is limited understanding about how prokaryotes vary across global environmental gradients, and how their global abundance and metabolic activity (production and respiration) may be affected by climate change. Using global datasets of prokaryotic abundance, cell carbon and metabolic activity we reveal that mean prokaryotic biomass varies by just under 3-fold across the global surface ocean, while total prokaryotic metabolic activity increases by more than one order of magnitude from polar to tropical coastal and upwelling regions. Under climate change, global prokaryotic biomass in surface waters is projected to decline ~1.5% per °C of warming, while prokaryotic respiration will increase ~3.5% ( ~ 0.85 Pg C yr -1 ). The rate of prokaryotic biomass decline is one-third that of zooplankton and fish, while the rate of increase in prokaryotic respiration is double. This suggests that future, warmer oceans could be increasingly dominated by prokaryotes, diverting a growing proportion of primary production into microbial food webs and away from higher trophic levels as well as reducing the capacity of the deep ocean to sequester carbon, all else being equal., (© 2024. The Author(s).)

The ocean's mercury (Hg) content has tripled due to anthropogenic activities, and although the dark ocean (>200 m) has become an important Hg reservoir, concentrations of the toxic and bioaccumulative methylmercury (MeHg) are low and therefore very difficult to measure. As a consequence, the current understanding of the Hg cycle in the deep ocean is severely data-limited, and the factors controlling MeHg, as well as its transformation rates, remain largely unknown. By analyzing 52 globally distributed bathypelagic deep-ocean metagenomes and 26 new metatranscriptomes from the Malaspina Expedition, our study reveals the widespread distribution and expression of bacterial-coding genes merA and merB in the global bathypelagic ocean (∼4000 m depth). These genes, associated with Hg II reduction and MeHg demethylation, respectively, are particularly prevalent within the particle-attached fraction. Moreover, our results indicate that water mass age and the organic matter composition shaped the structure of the communities harboring merA and merB genes living in different particle size fractions, their abundance, and their expression levels. Members of the orders Corynebacteriales , Rhodobacterales , Alteromonadales , Oceanospirillales , Moraxellales , and Flavobacteriales were the main taxonomic players containing merA and merB genes in the deep ocean. These findings, together with our previous results of pure culture isolates of the deep bathypelagic ocean possessing the metabolic capacity to degrade MeHg, indicated that both methylmercury demethylation and Hg II reduction likely occur in the global dark ocean, the largest biome in the biosphere.

Marine microbial communities differ genetically, metabolically, and ecologically according to their lifestyle, and they may respond differently to environmental changes. In this study, we investigated the seasonal dynamics of bacterial assemblies in the free-living (FL) and particle-associated (PA) fractions across a span of 6 years in the Blanes Bay Microbial Observatory in the Northwestern Mediterranean. Both lifestyles showed marked seasonality. The trends in alpha diversity were similar, with lower values in spring-summer than in autumn-winter. Samples from both fractions were grouped seasonally and the percentage of community variability explained by the measured environmental variables was comparable (32% in FL and 31% in PA). Canonical analyses showed that biotic interactions were determinants of bacterioplankton dynamics and that their relevance varies depending on lifestyles. Time-decay curves confirmed a high degree of predictability in both fractions. Yet, 'seasonal' Amplicon Sequence Variants (ASVs) (as defined by Lomb Scargle time series analysis) in the PA communities represented 46% of the total relative abundance while these accounted for 30% in the FL fraction. These results demonstrate that bacteria inhabiting both fractions exhibit marked seasonality, highlighting the importance of accounting for both lifestyles to fully comprehend the dynamics of marine prokaryotic communities., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Prokaryotes dominate global oceans and shape biogeochemical cycles, yet most taxa remain uncultured and uncharacterized as of today. Here we present the characterization of 26 novel marine bacterial strains from a large isolate collection obtained from Blanes Bay (NW Mediterranean) microcosm experiments made in the four seasons. Morphological, cultural, biochemical, physiological, nutritional, genomic, and phylogenomic analyses were used to characterize and phylogenetically place the novel isolates. The strains represent 23 novel bacterial species and six novel genera: three novel species pertaining to class Alphaproteobacteria (families Rhodobacteraceae and Sphingomonadaceae ), six novel species and three new genera from class Gammaproteobacteria (families Algiphilaceae , Salinispheraceae , and Alteromonadaceae ), 13 novel species and three novel genera from class Bacteroidia (family Flavobacteriaceae ), and one new species from class Rhodothermia (family Rubricoccaceae ). The bacteria described here have potentially relevant roles in the cycles of carbon (e.g., carbon fixation or energy production via proteorhodopsin), nitrogen (e.g., denitrification or use of urea), sulfur (oxidation of sulfur compounds), phosphorus (acquisition and use of different forms of phosphorus and remodeling of membrane phospholipids), and hydrogen (oxidation of hydrogen to obtain energy). We mapped the genomes of the presented strains to the Tara Oceans metagenomes to reveal that these strains were globally distributed, with those of the family Flavobacteriaceae being the most widespread and abundant, while Rhodothermia being the rarest and most localized. While molecular-only approaches are also important, our study stresses the importance of culturing as a powerful tool to further understand the functioning of marine bacterial communities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest., (Copyright © 2024 Rey-Velasco, Lucena, Belda, Gasol, Sánchez, Arahal and Pujalte.)

Marine prokaryotes play crucial roles in ocean biogeochemical cycles, being their contribution strongly influenced by their growth rates. Hence, elucidating the variability and phylogenetic imprint of marine prokaryotes' growth rates are crucial for better determining the role of individual taxa in biogeochemical cycles. Here, we estimated prokaryotic growth rates at high phylogenetic resolution in manipulation experiments using water from the northwestern Mediterranean Sea. Experiments were run in the four seasons with different treatments that reduced growth limiting factors: predators, nutrient availability, viruses, and light. Single-amplicon sequence variants (ASVs)-based growth rates were calculated from changes in estimated absolute abundances using total prokaryotic abundance and the proportion of each individual ASV. The trends obtained for growth rates in the different experiments were consistent with other estimates based on total cell-counts, catalyzed reporter deposition fluorescence in situ hybridization subcommunity cell-counts or metagenomic-operational taxonomic units (OTUs). Our calculations unveil a broad range of growth rates (0.3-10 d -1 ) with significant variability even within closely related ASVs. Likewise, the impact of growth limiting factors changed over the year for individual ASVs. High numbers of responsive ASVs were shared between winter and spring seasons, as well as throughout the year in the treatments with reduced nutrient limitation and viral pressure. The most responsive ASVs were rare in the in situ communities, comprising a large pool of taxa with the potential to rapidly respond to environmental changes. Essentially, our results highlight the lack of phylogenetic coherence in the range of growth rates observed, and differential responses to the various limiting factors, even for closely related taxa., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combined in situ sampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. The in situ decline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75-0.90 d -1 . This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d -1 . An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure., Competing Interests: The authors declare no conflict of interest.

The Ocean microbiome has a crucial role in Earth's biogeochemical cycles. During the last decade, global cruises such as Tara Oceans and the Malaspina Expedition have expanded our understanding of the diversity and genetic repertoire of marine microbes. Nevertheless, there are still knowledge gaps regarding their diversity patterns throughout depth gradients ranging from the surface to the deep ocean. Here we present a dataset of 76 microbial metagenomes (MProfile) of the picoplankton size fraction (0.2-3.0 µm) collected in 11 vertical profiles covering contrasting ocean regions sampled during the Malaspina Expedition circumnavigation (7 depths, from surface to 4,000 m deep). The MProfile dataset produced 1.66 Tbp of raw DNA sequences from which we derived: 17.4 million genes clustered at 95% sequence similarity (M-GeneDB-VP), 2,672 metagenome-assembled genomes (MAGs) of Archaea and Bacteria (Malaspina-VP-MAGs), and over 100,000 viral genomic sequences. This dataset will be a valuable resource for exploring the functional and taxonomic connectivity between the photic and bathypelagic tropical and sub-tropical ocean, while increasing our general knowledge of the Ocean microbiome., (© 2024. The Author(s).)

A persistent microbial seed bank is postulated to sustain the marine biosphere, and recent findings show that prokaryotic taxa present in the ocean's surface dominate prokaryotic communities throughout the water column. Yet, environmental conditions exert a tight control on the activity of prokaryotes, and drastic changes in these conditions are known to occur from the surface to deep waters. The simultaneous characterization of the total (DNA) and active (i.e. with potential for protein synthesis, RNA) free-living communities in 13 stations distributed across the tropical and subtropical global ocean allowed us to assess their change in structure and diversity along the water column. We observed that active communities were surprisingly more similar along the vertical gradient than total communities. Looking at the vertical connectivity of the active vs. the total communities, we found that taxa detected in the surface sometimes accounted for more than 75% of the active microbiome of bathypelagic waters (50% on average). These active taxa were generally rare in the surface, representing a small fraction of all the surface taxa. Our findings show that the drastic vertical change in environmental conditions leads to the inactivation and disappearance of a large proportion of surface taxa, but some surface-rare taxa remain active (or with potential for protein synthesis) and dominate the bathypelagic active microbiome., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The osmotrophic uptake of dissolved organic compounds in the ocean is considered to be dominated by heterotrophic prokaryotes, whereas the role of planktonic eukaryotes is still unclear. We explored the capacity of natural eukaryotic plankton communities to incorporate the synthetic amino acid L-homopropargylglycine (HPG, analogue of methionine) using biorthogonal noncanonical amino acid tagging (BONCAT), and we compared it with prokaryotic HPG use throughout a 9-day survey in the NW Mediterranean. BONCAT allows to fluorescently identify translationally active cells, but it has never been applied to natural eukaryotic communities. We found a large diversity of photosynthetic and heterotrophic eukaryotes incorporating HPG into proteins, with dinoflagellates and diatoms showing the highest percentages of BONCAT-labelled cells (49 ± 25% and 52 ± 15%, respectively). Among them, pennate diatoms exhibited higher HPG incorporation in the afternoon than in the morning, whereas small (≤5 μm) photosynthetic eukaryotes and heterotrophic nanoeukaryotes showed the opposite pattern. Centric diatoms (e.g. Chaetoceros , Thalassiosira , and Lauderia spp . ) dominated the eukaryotic HPG incorporation due to their high abundances and large sizes, accounting for up to 86% of the eukaryotic BONCAT signal and strongly correlating with bulk 3 H-leucine uptake rates. When including prokaryotes, eukaryotes were estimated to account for 19-31% of the bulk BONCAT signal. Our results evidence a large complexity in the osmotrophic uptake of HPG, which varies over time within and across eukaryotic groups and highlights the potential of BONCAT to quantify osmotrophy and protein synthesis in complex eukaryotic communities., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Microbial interactions are vital in maintaining ocean ecosystem function, yet their dynamic nature and complexity remain largely unexplored. Here, we use association networks to investigate possible ecological interactions in the marine microbiome among archaea, bacteria, and picoeukaryotes throughout different depths and geographical regions of the tropical and subtropical global ocean. Our findings reveal that potential microbial interactions change with depth and geographical scale, exhibiting highly heterogeneous distributions. A few potential interactions were global, meaning they occurred across regions at the same depth, while 11-36% were regional within specific depths. The bathypelagic zone had the lowest proportion of global associations, and regional associations increased with depth. Moreover, we observed that most surface water associations do not persist in deeper ocean layers despite microbial vertical dispersal. Our work contributes to a deeper understanding of the tropical and subtropical global ocean interactome, which is essential for addressing the challenges posed by global change., (© 2024. The Author(s).)

Estuaries are among the most productive ecosystems in the world and are highly dynamic due to the interaction of freshwater and seawater, which results in strong spatial gradients in physico-chemical conditions. Bacterioplankton play a central role in these systems, driving the fluxes of carbon and energy, and being central for contaminant removal in human-impacted areas. Most studies on bacterioplankton dynamics have been carried out in temperate estuaries, and they show that salinity is a major factor driving bacterioplankton distribution. Tropical estuaries, although largely understudied, experience drastic variations in river discharge between the dry and the rainy seasons, influencing the spatial distribution of the salinity gradient and thus likely impacting bacterioplankton communities. Using Illumina sequencing of the 16S rRNA gene, here we studied bacterial communities from the Nicoya's Gulf (Costa Rica), a large tropical estuary characterized by high riverine discharge during the rainy season, to explore seasonal changes in the spatial distribution and connectivity of these communities along the Gulf. Our results show pronounced differences in bacterial diversity and community structure between seasons and zones within the estuary (the shallow upper Gulf, the middle zone and the lower zone, located in the marine end of the estuary). Bacterial communities from the different regions were more similar during the rainy season, suggesting a larger degree of microbial connectivity likely driven by the fast water circulation fueled by the riverine discharge. In the dry season, Enterobacteriales and Cyanobacteria dominated bacterial communities, whereas in the rainy season Alphaproteobacteria was the dominant group. These contrasting seasonal trends were consistent with the seasonal variations observed in bacterial assemblages during a year at a single station in the upper region of the Gulf. We conclude that the Gulf is highly dynamic in both the spatial and temporal scale and that bacterioplankton communities are strongly influenced by the riverine and tidal inputs during both seasons. This study sheds light on the sources of variability in the structure of bacterial communities in tropical estuarine systems, an understudied type of aquatic ecosystem, and sets the basis to design further comprehensive studies on their microbial diversity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Tiny ocean plankton (picoplankton) are fundamental for the functioning of the biosphere, but the ecological mechanisms shaping their biogeography were partially understood. Comprehending whether these microorganisms are structured by niche versus neutral processes is relevant in the context of global change. We investigate the ecological processes (selection, dispersal, and drift) structuring global-ocean picoplanktonic communities inhabiting the epipelagic (0 to 200 meters), mesopelagic (200 to 1000 meters), and bathypelagic (1000 to 4000 meters) zones. We found that selection decreased, while dispersal limitation increased with depth, possibly due to differences in habitat heterogeneity and dispersal barriers such as water masses and bottom topography. Picoplankton β-diversity positively correlated with environmental heterogeneity and water mass variability, but this relationship tended to be weaker for eukaryotes than for prokaryotes. Community patterns were more pronounced in the Mediterranean Sea, probably because of its cross-basin environmental heterogeneity and deep-water isolation. We conclude that different combinations of ecological mechanisms shape the biogeography of the ocean microbiome across depths.

Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes., (© 2023. The Author(s).)

Isolation of microorganisms is a useful approach to gathering knowledge about their genomic properties, physiology, and ecology, in addition to allowing the characterization of novel taxa. We performed an extensive isolation effort on samples from seawater manipulation experiments that were carried out during the four astronomical seasons in a coastal site of the northwest Mediterranean to evaluate the impact of grazing, viral mortality, resource competition reduction, and light presence/absence on bacterioplankton growth. Isolates were retrieved using two growth media, and their full 16S rRNA gene was sequenced to assess their identity and calculate their culturability across seasons and experimental conditions. A total of 1,643 isolates were obtained, mainly affiliated to the classes Gammaproteobacteria (44%), Alphaproteobacteria (26%), and Bacteroidia (17%). Isolates pertaining to class Gammaproteobacteria were the most abundant in all experiments, while Bacteroidia were preferentially enriched in the treatments with reduced grazing. Sixty-one isolates had a similarity below 97% to cultured taxa and are thus putatively novel. Comparison of isolate sequences with 16S rRNA gene amplicon sequences from the same samples showed that the percentage of reads corresponding to isolates was 21.4% within the whole data set, with dramatic increases in the summer virus-reduced (71%) and diluted (47%) treatments. In fact, we were able to isolate the top 10 abundant taxa in several experiments and from the whole data set. We also show that top-down and bottom-up controls differentially affect taxa in terms of culturability. Our results indicate that culturing marine bacteria using agar plates can be successful in certain ecological situations. IMPORTANCE Bottom-up and top-down controls greatly influence marine microbial community composition and dynamics, which in turn have effects on their culturability. We isolated a high amount of heterotrophic bacterial strains from experiments where seawater environmental conditions had been manipulated and found that decreasing grazing and viral pressure as well as rising nutrient availability are key factors increasing the success in culturing marine bacteria. Our data hint at factors influencing culturability and underpin bacterial cultures as a powerful way to discover new taxa.

Traditional culture techniques usually retrieve a small fraction of the marine microbial diversity, which mainly belong to the so-called rare biosphere. However, this paradigm has not been fully tested at a broad scale, especially in the deep ocean. Here, we examined the fraction of heterotrophic bacterial communities in photic and deep ocean layers that could be recovered by culture-dependent techniques at a large scale. We compared 16S rRNA gene sequences from a collection of 2003 cultured heterotrophic marine bacteria with global 16S rRNA metabarcoding datasets (16S TAGs) covering surface, mesopelagic and bathypelagic ocean samples that included 16 of the 23 samples used for isolation. These global datasets represent 60 322 unique 16S amplicon sequence variants (ASVs). Our results reveal a significantly higher proportion of isolates identical to ASVs in deeper ocean layers reaching up to 28% of the 16S TAGs of the bathypelagic microbial communities, which included the isolation of 3 of the top 10 most abundant 16S ASVs in the global bathypelagic ocean, related to the genera Sulfitobacter, Halomonas and Erythrobacter. These isolates contributed differently to the prokaryotic communities across different plankton size fractions, recruiting between 38% in the free-living fraction (0.2-0.8 µm) and up to 45% in the largest particles (20-200 µm) in the bathypelagic ocean. Our findings support the hypothesis that sinking particles in the bathypelagic act as resource-rich habitats, suitable for the growth of heterotrophic bacteria with a copiotroph lifestyle that can be cultured, and that these cultivable bacteria can also thrive as free-living bacteria., (© 2023. ISME Publications B.V.)

Microbes drive the biogeochemical cycles of marine ecosystems through their vast metabolic diversity. While we have a fairly good understanding of the spatial distribution of these metabolic processes in various ecosystems, less is known about their seasonal dynamics. We investigated the annual patterns of 21 biogeochemical relevant functions in an oligotrophic coastal ocean site by analysing the presence of key genes, analysing high-rank gene taxonomy and the dynamics of nucleotide variants. Most genes presented seasonality: photoheterotrophic processes were enriched during spring, phosphorous-related genes were dominant during summer, coinciding with potential phosphate limitation, and assimilatory nitrate reductases appeared mostly during summer and autumn, correlating negatively with nitrate availability. Additionally, we identified the main taxa driving each function at each season and described the role of underrecognized taxa such as Litoricolaceae in carbon fixation (rbcL), urea degradation (ureC), and CO oxidation (coxL). Finally, the seasonality of single variants of some families presented a decoupling between the taxonomic abundance patterns and the functional gene patterns, implying functional specialization of the different genera. Our study unveils the seasonality of key biogeochemical functions and the main taxonomic groups that harbour these relevant functions in a coastal ocean ecosystem., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Background: Viruses play important roles in the ocean's biogeochemical cycles. Yet, deep ocean viruses are one of the most under-explored fractions of the global biosphere. Little is known about the environmental factors that control the composition and functioning of their communities or how they interact with their free-living or particle-attached microbial hosts., Results: We analysed 58 viral communities associated with size-fractionated free-living (0.2-0.8 μm) and particle-attached (0.8-20 μm) cellular metagenomes from bathypelagic (2150-4018 m deep) microbiomes obtained during the Malaspina expedition. These metagenomes yielded 6631 viral sequences, 91% of which were novel, and 67 represented high-quality genomes. Taxonomic classification assigned 53% of the viral sequences to families of tailed viruses from the order Caudovirales. Computational host prediction associated 886 viral sequences to dominant members of the deep ocean microbiome, such as Alphaproteobacteria (284), Gammaproteobacteria (241), SAR324 (23), Marinisomatota (39), and Chloroflexota (61). Free-living and particle-attached viral communities had markedly distinct taxonomic composition, host prevalence, and auxiliary metabolic gene content, which led to the discovery of novel viral-encoded metabolic genes involved in the folate and nucleotide metabolisms. Water mass age emerged as an important factor driving viral community composition. We postulated this was due to changes in quality and concentration of dissolved organic matter acting on the host communities, leading to an increase of viral auxiliary metabolic genes associated with energy metabolism among older water masses., Conclusions: These results shed light on the mechanisms by which environmental gradients of deep ocean ecosystems structure the composition and functioning of free-living and particle-attached viral communities. Video Abstract., (© 2023. The Author(s).)

Background: Microbial interactions are fundamental for Earth's ecosystem functioning and biogeochemical cycling. Nevertheless, they are challenging to identify and remain barely known. Omics-based censuses are helpful in predicting microbial interactions through the statistical inference of single (static) association networks. Yet, microbial interactions are dynamic and we have limited knowledge of how they change over time. Here, we investigate the dynamics of microbial associations in a 10-year marine time series in the Mediterranean Sea using an approach inferring a time-resolved (temporal) network from a single static network., Results: A single static network including microbial eukaryotes and bacteria was built using metabarcoding data derived from 120 monthly samples. For the decade, we aimed to identify persistent, seasonal, and temporary microbial associations by determining a temporal network that captures the interactome of each individual sample. We found that the temporal network appears to follow an annual cycle, collapsing, and reassembling when transiting between colder and warmer waters. We observed higher association repeatability in colder than in warmer months. Only 16 associations could be validated using observations reported in literature, underlining our knowledge gap in marine microbial ecological interactions., Conclusions: Our results indicate that marine microbial associations follow recurrent temporal dynamics in temperate zones, which need to be accounted for to better understand the functioning of the ocean microbiome. The constructed marine temporal network may serve as a resource for testing season-specific microbial interaction hypotheses. The applied approach can be transferred to microbiome studies in other ecosystems. Video Abstract., (© 2023. The Author(s).)

Strong purifying selection is considered a major evolutionary force behind small microbial genomes in the resource-poor photic ocean. However, very little is currently known about how the size of prokaryotic genomes evolves in the global ocean and whether patterns reflect shifts in resource availability in the epipelagic and relatively stable deep-sea environmental conditions. Using 364 marine microbial metagenomes, we investigate how the average genome size of uncultured planktonic prokaryotes varies across the tropical and polar oceans to the hadal realm. We find that genome size is highest in the perennially cold polar ocean, reflecting elongation of coding genes and gene dosage effects due to duplications in the interior ocean microbiome. Moreover, the rate of change in genome size due to temperature is 16-fold higher than with depth up to 200 m. Our results demonstrate how environmental factors can influence marine microbial genome size selection and ecological strategies of the microbiome., (© 2023. The Author(s).)

Antarctic polynyas are highly productive open water areas surrounded by ice where extensive phytoplankton blooms occur, but little is known about how these surface blooms influence carbon fluxes and prokaryotic communities from deeper waters. By sequencing the 16S rRNA gene, we explored the vertical connectivity of the prokaryotic assemblages associated with particles of three different sizes in two polynyas with different surface productivity, and we linked it to the magnitude of the particle export fluxes measured using thorium-234 ( 234 Th) as particle tracer. Between the sunlit and the mesopelagic layers (700 m depth), we observed compositional changes in the prokaryotic communities associated with the three size-fractions, which were mostly dominated by Flavobacteriia , Alphaproteobacteria , and Gammaproteobacteria . Interestingly, the vertical differences between bacterial communities attached to the largest particles decreased with increasing 234 Th export fluxes, indicating a more intense downward transport of surface prokaryotes in the most productive polynya. This was accompanied by a higher proportion of surface prokaryotic taxa detected in deep particle-attached microbial communities in the station with the highest 234 Th export flux. Our results support recent studies evidencing links between surface productivity and deep prokaryotic communities and provide the first evidence of sinking particles acting as vectors of microbial diversity to depth in Antarctic polynyas, highlighting the direct influence of particle export in shaping the prokaryotic communities of mesopelagic waters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Puigcorbé, Ruiz-González, Masqué and Gasol.)

On a standard oceanographic cruise, flow cytometry data are usually collected sparsely through a bottle-based sampling and with stations separated by kilometers leading to a fragmented view of the ecosystem; to improve the resolution of the datasets produced by this technique here it is proposed the application of an automatic method of sampling and staining. The system used consists of a flow-cytometer (Accuri-C6) connected to an automated continuous sampler (OC-300) that collects samples of marine surface waters every 15 min. We tested this system for five days during a brief Mediterranean cruise with the aim of estimating the abundance, relative size and phenotypic diversity of prokaryotes. Seawater was taken by a faucet linked to an inlet pump ( ca. 5 m depth). Once the sample was taken, the Oncyt-300 stained it and sent it to the flow cytometer. A total of 366 samples were collected, effectively achieving a fine-grained scale view of microbial community composition both through space and time. A significative positive relationship was found comparing data obtained with the automatic method and 10 samples collected from the faucet but processed with the standard protocol. Abundance values retrieved varied from 3.56·10 5 cell mL -1 in the coastal area till 6.87 10 5 cell mL -1 in open waters, exceptional values were reached in the harbor area where abundances peaked to 1.28 10 6 cell mL -1 . The measured features (abundance and size) were associated with metadata (temperature, salinity, conductivity) also taken in continuous, of which conductivity was the one that better explained the variability of abundance. A full 24 h measurement cycle was performed resulting in slightly higher median bacterial abundances values during daylight hours compared to night. Alpha diversity, calculated using computational cytometry techniques, showed a higher value in the coastal area above 41° of latitude and had a strong inverse relationship with both salinity and conductivity. This is the first time to our knowledge that the OC-300 is directly applied to the marine environment during an oceanographic cruise; due to its high-resolution, this set-up shows great potential both to cover large sampling areas, and to monitor day-night cycles in situ ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pernice and Gasol.)

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities., (© 2022 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinhassi, Farnelid, García, Teira, Galand, Obernosterer, Quince, Vila-Costa, Gasol, Lundin, Andersson, Labrenz and Riemann.)

Subterranean estuaries are biogeochemically active coastal sites resulting from the underground mixing of fresh aquifer groundwater and seawater. In these systems, microbial activity can largely transform the chemical elements that may reach the sea through submarine groundwater discharge (SGD), but little is known about the microorganisms thriving in these land-sea transition zones. We present the first spatially-resolved characterization of the bacterial assemblages along a coastal aquifer in the NW Mediterranean, considering the entire subsurface salinity gradient. Combining bulk heterotrophic activity measurements, flow cytometry, microscopy and 16S rRNA gene sequencing we find large variations in prokaryotic abundances, cell size, activity and diversity at both the horizontal and vertical scales that reflect the pronounced physicochemical gradients. The parts of the transect most influenced by freshwater were characterized by smaller cells and lower prokaryotic abundances and heterotrophic production, but some activity hotspots were found at deep low-oxygen saline groundwater sites enriched in nitrite and ammonium. Diverse, heterogeneous and highly endemic communities dominated by Proteobacteria, Patescibacteria, Desulfobacterota and Bacteroidota were observed throughout the aquifer, pointing to clearly differentiated prokaryotic niches across these transition zones and little microbial connectivity between groundwater and Mediterranean seawater habitats. Finally, experimental manipulations unveiled large increases in community heterotrophic activity driven by fast growth of some rare and site-specific groundwater Proteobacteria. Our results indicate that prokaryotic communities within subterranean estuaries are highly heterogeneous in terms of biomass, activity and diversity, suggesting that their role in transforming nutrients will also vary spatially within these terrestrial-marine transition zones., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups 1 , this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds 2,3 . However, studying this diversity to identify genomic pathways for the synthesis of such compounds 4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments., (© 2022. The Author(s).)

Background: Ocean microbes constitute ~ 70% of the marine biomass, are responsible for ~ 50% of the Earth's primary production and are crucial for global biogeochemical cycles. Marine microbiotas include core taxa that are usually key for ecosystem function. Despite their importance, core marine microbes are relatively unknown, which reflects the lack of consensus on how to identify them. So far, most core microbiotas have been defined based on species occurrence and abundance. Yet, species interactions are also important to identify core microbes, as communities include interacting species. Here, we investigate interconnected bacteria and small protists of the core pelagic microbiota populating a long-term marine-coastal observatory in the Mediterranean Sea over a decade., Results: Core microbes were defined as those present in > 30% of the monthly samples over 10 years, with the strongest associations. The core microbiota included 259 Operational Taxonomic Units (OTUs) including 182 bacteria, 77 protists, and 1411 strong and mostly positive (~ 95%) associations. Core bacteria tended to be associated with other bacteria, while core protists tended to be associated with bacteria. The richness and abundance of core OTUs varied annually, decreasing in stratified warmers waters and increasing in colder mixed waters. Most core OTUs had a preference for one season, mostly winter, which featured subnetworks with the highest connectivity. Groups of highly associated taxa tended to include protists and bacteria with predominance in the same season, particularly winter. A group of 13 highly-connected hub-OTUs, with potentially important ecological roles dominated in winter and spring. Similarly, 18 connector OTUs with a low degree but high centrality were mostly associated with summer or autumn and may represent transitions between seasonal communities., Conclusions: We found a relatively small and dynamic interconnected core microbiota in a model temperate marine-coastal site, with potential interactions being more deterministic in winter than in other seasons. These core microbes would be essential for the functioning of this ecosystem over the year. Other non-core taxa may also carry out important functions but would be redundant and non-essential. Our work contributes to the understanding of the dynamics and potential interactions of core microbes possibly sustaining ocean ecosystem function., (© 2022. The Author(s).)

The aerobic anoxygenic phototrophic (AAP) bacteria are common in most marine environments but their global diversity and biogeography remain poorly characterized. Here, we analyzed AAP communities across 113 globally-distributed surface ocean stations sampled during the Malaspina Expedition in the tropical and subtropical ocean. By means of amplicon sequencing of the pufM gene, a genetic marker for this functional group, we show that AAP communities along the surface ocean were mainly composed of members of the Halieaceae (Gammaproteobacteria), which were adapted to a large range of environmental conditions, and of different clades of the Alphaproteobacteria, which seemed to dominate under particular circumstances, such as in the oligotrophic gyres. AAP taxa were spatially structured within each of the studied oceans, with communities from adjacent stations sharing more taxonomic similarities. AAP communities were composed of a large pool of rare members and several habitat specialists. When compared to the surface ocean prokaryotic and picoeukaryotic communities, it appears that AAP communities display an idiosyncratic global biogeographical pattern, dominated by selection processes and less influenced by dispersal limitation. Our study contributes to the understanding of how AAP communities are distributed in the horizontal dimension and the mechanisms underlying their distribution across the global surface ocean., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

COVID-19 has led to global population lockdowns that have had indirect effects on terrestrial and marine fauna, yet little is known on their effects on marine planktonic communities. We analysed the effect of the spring 2020 lockdown in a marine coastal area in Blanes Bay, NW Mediterranean. We compared a set of 23 oceanographic, microbial and biogeochemical variables sampled right after the strict lockdown in Spain, with data from the previous 15 years after correcting for long-term trends. Our analysis shows a series of changes in the microbial communities which may have been induced by the combination of the decreased nitrogen atmospheric load, the lower wastewater flux and the reduced fishing activity in the area, among other factors. In particular, we detected a slight decrease beyond the long-term trend in chlorophyll a, in the abundance of several microbial groups (phototrophic nanoflagellates and total prokaryotes) and in prokaryotic activity (heterotrophic prokaryotic production and β-glucosidase activity) which, as a whole, resulted in a moderate increase of oligotrophy in Blanes Bay after the lockdown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacteria display dynamic abundance fluctuations over time in marine environments, where they play key biogeochemical roles. Here, we characterized the seasonal dynamics of marine bacteria in a coastal oligotrophic time series station, tested how similar the temporal niche of closely related taxa is, and what are the environmental parameters modulating their seasonal abundance patterns. We further explored how conserved the niche is at higher taxonomic levels. The community presented recurrent patterns of seasonality for 297 out of 6825 amplicon sequence variants (ASVs), which constituted almost half of the total relative abundance (47%). For certain genera, niche similarity decreased as nucleotide divergence in the 16S rRNA gene increased, a pattern compatible with the selection of similar taxa through environmental filtering. Additionally, we observed evidence of seasonal differentiation within various genera as seen by the distinct seasonal patterns of closely related taxa. At broader taxonomic levels, coherent seasonal trends did not exist at the class level, while the order and family ranks depended on the patterns that existed at the genus level. This study identifies the coexistence of closely related taxa for some bacterial groups and seasonal differentiation for others in a coastal marine environment subjected to a strong seasonality., (© 2021. The Author(s).)

The deep sea, the largest ocean's compartment, drives planetary-scale biogeochemical cycling. Yet, the functional exploration of its microbial communities lags far behind other environments. Here we analyze 58 metagenomes from tropical and subtropical deep oceans to generate the Malaspina Gene Database. Free-living or particle-attached lifestyles drive functional differences in bathypelagic prokaryotic communities, regardless of their biogeography. Ammonia and CO oxidation pathways are enriched in the free-living microbial communities and dissimilatory nitrate reduction to ammonium and H 2 oxidation pathways in the particle-attached, while the Calvin Benson-Bassham cycle is the most prevalent inorganic carbon fixation pathway in both size fractions. Reconstruction of the Malaspina Deep Metagenome-Assembled Genomes reveals unique non-cyanobacterial diazotrophic bacteria and chemolithoautotrophic prokaryotes. The widespread potential to grow both autotrophically and heterotrophically suggests that mixotrophy is an ecologically relevant trait in the deep ocean. These results expand our understanding of the functional microbial structure and metabolic capabilities of the largest Earth aquatic ecosystem.

Anthropogenic carbon emissions are causing changes in seawater carbonate chemistry including a decline in the pH of the oceans. While its aftermath for calcifying microbes has been widely studied, the effect of ocean acidification (OA) on marine viruses and their microbial hosts is controversial, and even more in combination with another anthropogenic stressor, i.e., human-induced nutrient loads. In this study, two mesocosm acidification experiments with Mediterranean waters from different seasons revealed distinct effects of OA on viruses and viral-mediated prokaryotic mortality depending on the trophic state and the successional stage of the plankton community. In the winter bloom situation, low fluorescence viruses, the most abundant virus-like particle (VLP) subpopulation comprising mostly bacteriophages, were negatively affected by lowered pH with nutrient addition, while the bacterial host abundance was stimulated. High fluorescence viruses, containing cyanophages, were stimulated by OA regardless of the nutrient conditions, while cyanobacteria of the genus Synechococcus were negatively affected by OA. Moreover, the abundance of very high fluorescence viruses infecting small haptophytes tended to be lower under acidification while their putative hosts' abundance was enhanced, suggesting a direct and negative effect of OA on viral-host interactions. In the oligotrophic summer situation, we found a stimulating effect of OA on total viral abundance and the viral populations, suggesting a cascading effect of the elevated p CO 2 stimulating autotrophic and heterotrophic production. In winter, viral lysis accounted for 30 ± 16% of the loss of bacterial standing stock per day (VMM BSS ) under increased p CO 2 compared to 53 ± 35% in the control treatments, without effects of nutrient additions while in summer, OA had no significant effects on VMM BSS (35 ± 20% and 38 ± 5% per day in the OA and control treatments, respectively). We found that phage production and resulting organic carbon release rates significantly reduced under OA in the nutrient replete winter situation, but it was also observed that high nutrient loads lowered the negative effect of OA on viral lysis, suggesting an antagonistic interplay between these two major global ocean stressors in the Anthropocene. In summer, however, viral-mediated carbon release rates were lower and not affected by lowered pH. Eutrophication consistently stimulated viral production regardless of the season or initial conditions. Given the relevant role of viruses for marine carbon cycling and the biological carbon pump, these two anthropogenic stressors may modulate carbon fluxes through their effect on viruses at the base of the pelagic food web in a future global change scenario., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Malits, Boras, Balagué, Calvo, Gasol, Marrasé, Pelejero, Pinhassi, Sala and Vaqué.)

Different factors affect the way dissolved organic matter (DOM) is processed in the ocean water column, including environmental conditions and the functional capabilities of the communities. Recent studies have shown that bathypelagic prokaryotes are metabolically flexible, but whether this versatility translates into a higher ability to process DOM has been barely explored. Here we performed a multifactorial transplant experiment to compare the growth, activity and changes in DOM quality in surface and bathypelagic waters inoculated with either surface or bathypelagic prokaryotic communities. The effect of nutrient additions to surface waters was also explored. Despite no differences in the cell abundance of surface and deep ocean prokaryotes were observed in any of the treatments, in surface waters with nutrients the heterotrophic production of surface prokaryotes rapidly decreased. Conversely, bathypelagic communities displayed a sustained production throughout the experiment. Incubations with surface prokaryotes always led to a significant accumulation of recalcitrant compounds, which did not occur with bathypelagic prokaryotes, suggesting they have a higher ability to process DOM. These contrasting abilities could be explained by the recruitment of a comparatively larger number of opportunistic taxa within the bathypelagic assemblages, which likely resulted in a broader community capability of substrate utilization., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Estimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict.

High-throughput sequencing of microbial assemblages has been proposed as an alternative methodology to the traditional ones used in marine monitoring and environmental assessment. Here, we evaluated pico- and nanoplankton diversity as ecological indicators in NW Mediterranean coastal waters by comparing their diversity in samples subjected to varying degrees of continental pressures. Using metabarcoding of the 16S and 18S rRNA genes, we explored whether alphadiversity indices, abundance of Operational Taxonomic Units and taxonomic groups (and their ratios) provide information on the ecological quality of coastal waters. Our results revealed that only eukaryotic diversity metrics and a limited number of prokaryotic and eukaryotic taxa displayed potential in assessing continental influences in our surveyed area, resulting thus in a restrained potential of microbial plankton diversity as an ecological indicator. Therefore, incorporating microbial plankton diversity in environmental assessment could not always result in a significant improvement of current marine monitoring strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Massive metagenomic sequencing combined with gene prediction methods were previously used to compile the gene catalogue of the ocean and host-associated microbes. Global expeditions conducted over the past 15 years have sampled the ocean to build a catalogue of genes from pelagic microbes. Here we undertook a large sequencing effort of a perturbed Red Sea plankton community to uncover that the rate of gene discovery increases continuously with sequencing effort, with no indication that the retrieved 2.83 million non-redundant (complete) genes predicted from the experiment represented a nearly complete inventory of the genes present in the sampled community (i.e., no evidence of saturation). The underlying reason is the Pareto-like distribution of the abundance of genes in the plankton community, resulting in a very long tail of millions of genes present at remarkably low abundances, which can only be retrieved through massive sequencing. Microbial metagenomic projects retrieve a variable number of unique genes per Tera base-pair (Tbp), with a median value of 14.7 million unique genes per Tbp sequenced across projects. The increase in the rate of gene discovery in microbial metagenomes with sequencing effort implies that there is ample room for new gene discovery in further ocean and holobiont sequencing studies., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Seasonal dynamics of ocean prokaryotic communities in the free-living fraction have been widely described, but less is known about the seasonality of prokaryotes inhabiting marine particles. We describe the seasonality of bacterial communities in the particulate matter continuum by sampling monthly over two years in a temperate oligotrophic coastal ecosystem and using a serial filtration (including six size-fractions spanning from 0.2 to 200 μm). We observed that bacterial communities in the particulate matter continuum had annual changes following harmonic seasonal oscillations, where alpha, beta, and gamma diversity increased during the warm period and decreased during the cold period. Communities in each size-fraction changed gradually over time, being the communities in larger size-fractions the ones with stronger annual changes. Annual community changes were driven mainly by day length and sea surface temperature, and each size-fraction was additionally affected by other variables (e.g., smaller size-fractions by dissolved PO 4 and larger size-fractions by turbidity). While some taxonomic groups mantained their preference for a given size fraction during most of the year, others varied their distribution into different size fractions over time, as e.g., SAR11, which increased its presence in particles during the cold period. Our results indicate that the size-fractionation scheme provides novel seasonal patterns that are not possible to unveil by analyzing only free-living bacteria, and that help to better understand the temporal dynamics of prokaryotes., (Copyright © 2020 Mestre, Höfer, Sala and Gasol.)

Microbes associated with sinking marine particles play key roles in carbon sequestration in the ocean. The sampling of particle-attached microorganisms is often done with sediment traps or by filtration of water collected with oceanographic bottles, both involving a certain time lapse between collection and processing of samples that may result in changes in particle-attached microbial communities. Conversely, in situ water filtration through submersible pumps allows a faster storage of sampled particles, but it has rarely been used to study the associated microbial communities and has never been compared to other particle-sampling methods in terms of the recovery of particle microbial diversity. Here we compared the prokaryotic communities attached to small (1-53 μm) and large (>53 μm) particles collected from the mesopelagic zone (100-300 m) of two Antarctic polynyas using in situ pumps (ISP) and oceanographic bottles (BTL). Each sampling method retrieved largely different particle-attached communities, suggesting that they capture different kinds of particles. These device-driven differences were greater for large particles than for small particles. Overall, the ISP recovered 1.5- to 3-fold more particle-attached bacterial taxa than the BTL, and different taxonomic groups were preferentially recovered by each method. In particular, typical particle-attached groups such as Planctomycetes and Deltaproteobacteria recovered with ISP were nearly absent from BTL samples. Our results suggest that the method used to sample marine particles has a strong influence in our view of their associated microbial communities., (Copyright © 2020 Puigcorbé, Ruiz-González, Masqué and Gasol.)

Background: Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. We have created a marine culture collection of heterotrophic bacteria (MARINHET) using a standard marine medium comprising a total of 1561 bacterial strains, and covering a variety of oceanographic regions from different seasons and years, from 2009 to 2015. Specifically, our marine collection contains isolates from both photic (817) and aphotic layers (744), including the mesopelagic (362) and the bathypelagic (382), from the North Western Mediterranean Sea, the North and South Atlantic Ocean, the Indian, the Pacific, and the Arctic Oceans. We described the taxonomy, the phylogenetic diversity and the biogeography of a fraction of the marine culturable microorganisms to enhance our knowledge about which heterotrophic marine isolates are recurrently retrieved across oceans and along different depths., Results: The partial sequencing of the 16S rRNA gene of all isolates revealed that they mainly affiliate with the classes Alphaproteobacteria (35.9%), Gammaproteobacteria (38.6%), and phylum Bacteroidetes (16.5%). In addition, Alteromonas and Erythrobacter genera were found the most common heterotrophic bacteria in the ocean growing in solid agar medium. When comparing all photic, mesopelagic, and bathypelagic isolates sequences retrieved from different stations, 37% of them were 100% identical. This percentage increased up to 59% when mesopelagic and bathypelagic strains were grouped as the aphotic dataset and compared to the photic dataset of isolates, indicating the ubiquity of some bacterial isolates along different ocean depths. Finally, we isolated three strains that represent a new species, and the genome comparison and phenotypic characterization of two of these strains (ISS653 and ISS1889) concluded that they belong to a new species within the genus Mesonia., Conclusions: Overall, this study highlights the relevance of culture-dependent studies, with focus on marine isolated bacteria from different oceanographic regions and depths, to provide a more comprehensive view of the culturable marine bacteria as part of the total marine microbial diversity.

Prokaryotes play a fundamental role in decomposing organic matter in the ocean, but little is known about how microbial metabolic capabilities vary at the global ocean scale and what are the drivers causing this variation. We aimed at obtaining the first global exploration of the functional capabilities of prokaryotes in the ocean, with emphasis on the under-sampled meso- and bathypelagic layers. We explored the potential utilization of 95 carbon sources with Biolog GN2 plates ® in 441 prokaryotic communities sampled from surface to bathypelagic waters (down to 4,000 m) at 111 stations distributed across the tropical and subtropical Atlantic, Indian, and Pacific oceans. The resulting metabolic profiles were compared with biological and physico-chemical properties such as fluorescent dissolved organic matter (DOM) or temperature. The relative use of the individual substrates was remarkably consistent across oceanic regions and layers, and only the Equatorial Pacific Ocean showed a different metabolic structure. When grouping substrates by categories, we observed some vertical variations, such as an increased relative utilization of polymers in bathypelagic layers or a higher relative use of P-compounds or amino acids in the surface ocean. The increased relative use of polymers with depth, together with the increases in humic DOM, suggest that deep ocean communities have the capability to process complex DOM. Overall, the main identified driver of the metabolic structure of ocean prokaryotic communities was temperature. Our results represent the first global depiction of the potential use of a variety of carbon sources by prokaryotic communities across the tropical and the subtropical ocean and show that acetic acid clearly emerges as one of the most widely potentially used carbon sources in the ocean., (Copyright © 2020 Sala, Ruiz-González, Borrull, Azúa, Baña, Ayo, Álvarez-Salgado, Gasol and Duarte.)

Deep ocean microbial communities rely on the organic carbon produced in the sunlit ocean, yet it remains unknown whether surface processes determine the assembly and function of bathypelagic prokaryotes to a larger extent than deep-sea physicochemical conditions. Here, we explored whether variations in surface phytoplankton assemblages across Atlantic, Pacific and Indian ocean stations can explain structural changes in bathypelagic (ca. 4,000 m) free-living and particle-attached prokaryotic communities (characterized through 16S rRNA gene sequencing), as well as changes in prokaryotic activity and dissolved organic matter (DOM) quality. We show that the spatial structuring of prokaryotic communities in the bathypelagic strongly followed variations in the abundances of surface dinoflagellates and ciliates, as well as gradients in surface primary productivity, but were less influenced by bathypelagic physicochemical conditions. Amino acid-like DOM components in the bathypelagic reflected variations of those components in surface waters, and seemed to control bathypelagic prokaryotic activity. The imprint of surface conditions was more evident in bathypelagic than in shallower mesopelagic (200-1,000 m) communities, suggesting a direct connectivity through fast-sinking particles that escape mesopelagic transformations. Finally, we identified a pool of endemic deep-sea prokaryotic taxa (including potentially chemoautotrophic groups) that appear less connected to surface processes than those bathypelagic taxa with a widespread vertical distribution. Our results suggest that surface planktonic communities shape the spatial structure of the bathypelagic microbiome to a larger extent than the local physicochemical environment, likely through determining the nature of the sinking particles and the associated prokaryotes reaching bathypelagic waters., (© 2020 John Wiley & Sons Ltd.)

Background: The ocean microbiota modulates global biogeochemical cycles and changes in its configuration may have large-scale consequences. Yet, the underlying ecological mechanisms structuring it are unclear. Here, we investigate how fundamental ecological mechanisms (selection, dispersal and ecological drift) shape the smallest members of the tropical and subtropical surface-ocean microbiota: prokaryotes and minute eukaryotes (picoeukaryotes). Furthermore, we investigate the agents exerting abiotic selection on this assemblage as well as the spatial patterns emerging from the action of ecological mechanisms. To explore this, we analysed the composition of surface-ocean prokaryotic and picoeukaryotic communities using DNA-sequence data (16S- and 18S-rRNA genes) collected during the circumglobal expeditions Malaspina-2010 and TARA-Oceans., Results: We found that the two main components of the tropical and subtropical surface-ocean microbiota, prokaryotes and picoeukaryotes, appear to be structured by different ecological mechanisms. Picoeukaryotic communities were predominantly structured by dispersal-limitation, while prokaryotic counterparts appeared to be shaped by the combined action of dispersal-limitation, selection and drift. Temperature-driven selection appeared as a major factor, out of a few selected factors, influencing species co-occurrence networks in prokaryotes but not in picoeukaryotes, indicating that association patterns may contribute to understand ocean microbiota structure and response to selection. Other measured abiotic variables seemed to have limited selective effects on community structure in the tropical and subtropical ocean. Picoeukaryotes displayed a higher spatial differentiation between communities and a higher distance decay when compared to prokaryotes, consistent with a scenario of higher dispersal limitation in the former after considering environmental heterogeneity. Lastly, random dynamics or drift seemed to have a more important role in structuring prokaryotic communities than picoeukaryotic counterparts., Conclusions: The differential action of ecological mechanisms seems to cause contrasting biogeography, in the tropical and subtropical ocean, among the smallest surface plankton, prokaryotes and picoeukaryotes. This suggests that the idiosyncrasy of the main constituents of the ocean microbiota should be considered in order to understand its current and future configuration, which is especially relevant in a context of global change, where the reaction of surface ocean plankton to temperature increase is still unclear. Video Abstract.