Royal Netherlands Institute for Sea Research

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This "rare biosphere" is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

Tipping points in complex systems may imply risks of unwanted collapse, but also opportunities for positive change. Our capacity to navigate such risks and opportunities can be boosted by combining emerging insights from two unconnected fields of research. One line of work is revealing fundamental architectural features that may cause ecological networks, financial markets, and other complex systems to have tipping points. Another field of research is uncovering generic empirical indicators of the proximity to such critical thresholds. Although sudden shifts in complex systems will inevitably continue to surprise us, work at the crossroads of these emerging fields offers new approaches for anticipating critical transitions.

The finding that total viral abundance is higher than total prokaryotic abundance and that a significant fraction of the prokaryotic community is infected with phages in aquatic systems has stimulated research on the ecology of prokaryotic viruses and their role in ecosystems. This review treats the ecology of prokaryotic viruses ('phages') in marine, freshwater and soil systems from a 'virus point of view'. The abundance of viruses varies strongly in different environments and is related to bacterial abundance or activity suggesting that the majority of the viruses found in the environment are typically phages. Data on phage diversity are sparse but indicate that phages are extremely diverse in natural systems. Lytic phages are predators of prokaryotes, whereas lysogenic and chronic infections represent a parasitic interaction. Some forms of lysogeny might be described best as mutualism. The little existing ecological data on phage populations indicate a large variety of environmental niches and survival strategies. The host cell is the main resource for phages and the resource quality, i.e., the metabolic state of the host cell, is a critical factor in all steps of the phage life cycle. Virus-induced mortality of prokaryotes varies strongly on a temporal and spatial scale and shows that phages can be important predators of bacterioplankton. This mortality and the release of cell lysis products into the environment can strongly influence microbial food web processes and biogeochemical cycles. Phages can also affect host diversity, e.g., by 'killing the winner' and keeping in check competitively dominant species or populations. Moreover, they mediate gene transfer between prokaryotes, but this remains largely unknown in the environment. Genomics or proteomics are providing us now with powerful tools in phage ecology, but final testing will have to be performed in the environment.

A global system of harmonized observations is needed to inform scientists and policy-makers.

The net inputs of silicic acid (dissolved silica) to the world ocean have been revised to 6.1 +/- 2.0 teramoles of silicon per year (1 teramole = 10(12) moles). The major contribution (about 80 percent) comes from rivers, whose world average silicic acid concentration is 150 micromolar. These inputs are reasonably balanced by the net ouputs of biogenic silica of 7.1 +/- 1.8 teramoles of silicon per year in modern marine sediments. The gross production of biogenic silica (the transformation of dissolved silicate to particulate skeletal material) in surface waters was estimated to be 240 +/- 40 teramoles of silicon per year, and the preservation ratio (opal accumulation in sediment/gross production in surface waters) averages 3 percent. In the world ocean the residence time of silicon, relative to total biological uptake in surface waters, is about 400 years.

Marine Crenarchaeota are the most abundant single group of prokaryotes in the ocean, but their physiology and role in marine biogeochemical cycles are unknown. Recently, a member of this clade was isolated from a sea aquarium and shown to be capable of nitrification, tentatively suggesting that Crenarchaeota may play a role in the oceanic nitrogen cycle. We enriched a crenarchaeote from North Sea water and showed that its abundance, and not that of bacteria, correlates with ammonium oxidation to nitrite. A time series study in the North Sea revealed that the abundance of the gene encoding for the archaeal ammonia monooxygenase alfa subunit (amoA) is correlated with a decline in ammonium concentrations and with the abundance of Crenarchaeota. Remarkably, the archaeal amoA abundance was 1-2 orders of magnitude higher than those of bacterial nitrifiers, which are commonly thought to mediate the oxidation of ammonium to nitrite in marine environments. Analysis of Atlantic waters of the upper 1,000 m, where most of the ammonium regeneration and oxidation takes place, showed that crenarchaeotal amoA copy numbers are also 1-3 orders of magnitude higher than those of bacterial amoA. Our data thus suggest a major role for Archaea in oceanic nitrification.

Hydrogen-isotopic abundances of lipid biomarkers are emerging as important proxies in the study of ancient environments and ecosystems. A decade ago, pioneering studies made use of new analytical methods and demonstrated that the hydrogen-isotopic composition of individual lipids from aquatic and terrestrial organisms can be related to the composition of their growth (i.e., environmental) water. Subsequently, compound-specific deuterium/hydrogen (D/H) ratios of sedimentary biomarkers have been increasingly used as paleohydrological proxies over a range of geological timescales. Isotopic fractionation observed between hydrogen in environmental water and hydrogen in lipids, however, is sensitive to biochemical, physiological, and environmental influences on the composition of hydrogen available for biosynthesis in cells. Here we review the factors and processes that are known to influence the hydrogen-isotopic compositions of lipids—especially n-alkanes—from photosynthesizing organisms, and we provide a framework for interpreting their D/H ratios from ancient sediments and identify future research opportunities.

Comparison of eight iron experiments shows that maximum Chl a , the maximum DIC removal, and the overall DIC/Fe efficiency all scale inversely with depth of the wind mixed layer (WML) defining the light environment. Moreover, lateral patch dilution, sea surface irradiance, temperature, and grazing play additional roles. The Southern Ocean experiments were most influenced by very deep WMLs. In contrast, light conditions were most favorable during SEEDS and SERIES as well as during IronEx‐2. The two extreme experiments, EisenEx and SEEDS, can be linked via EisenEx bottle incubations with shallower simulated WML depth. Large diatoms always benefit the most from Fe addition, where a remarkably small group of thriving diatom species is dominated by universal response of Pseudo ‐ nitzschia spp. Significant response of these moderate (10–30 μm), medium (30–60 μm), and large (>60 μm) diatoms is consistent with growth physiology determined for single species in natural seawater. The minimum level of “dissolved” Fe (filtrate < 0.2 μm) maintained during an experiment determines the dominant diatom size class. However, this is further complicated by continuous transfer of original truly dissolved reduced Fe(II) into the colloidal pool, which may constitute some 75% of the “dissolved” pool. Depth integration of carbon inventory changes partly compensates the adverse effects of a deep WML due to its greater integration depths, decreasing the differences in responses between the eight experiments. About half of depth‐integrated overall primary productivity is reflected in a decrease of DIC. The overall C/Fe efficiency of DIC uptake is DIC/Fe ∼ 5600 for all eight experiments. The increase of particulate organic carbon is about a quarter of the primary production, suggesting food web losses for the other three quarters. Replenishment of DIC by air/sea exchange tends to be a minor few percent of primary CO 2 fixation but will continue well after observations have stopped. Export of carbon into deeper waters is difficult to assess and is until now firmly proven and quite modest in only two experiments.

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

Denitrifying and anammox bacteria are involved in the nitrogen cycling in marine sediments but the environmental factors that regulate the relative importance of these processes are not well constrained. Here, we evaluated the abundance, diversity and potential activity of denitrifying, anammox, and sulfide-dependent denitrifying bacteria in the sediments of the seasonally hypoxic saline Lake Grevelingen, known to harbor an active microbial community involved in sulfur oxidation pathways. Depth distributions of 16S rRNA gene, nirS gene of denitrifying and anammox bacteria, aprA gene of sulfur-oxidizing and sulfate-reducing bacteria, and ladderane lipids of anammox bacteria were studied in sediments impacted by seasonally hypoxic bottom waters. Samples were collected down to 5 cm depth (1 cm resolution) at three different locations before (March) and during summer hypoxia (August). The abundance of denitrifying bacteria did not vary despite of differences in oxygen and sulfide availability in the sediments, whereas anammox bacteria were more abundant in the summer hypoxia but in those sediments with lower sulfide concentrations. The potential activity of denitrifying and anammox bacteria as well as of sulfur-oxidizing, including sulfide-dependent denitrifiers and sulfate-reducing bacteria, was potentially inhibited by the competition for nitrate and nitrite with cable and/or Beggiatoa-like bacteria in March and by the accumulation of sulfide in the summer hypoxia. The simultaneous presence and activity of organoheterotrophic denitrifying bacteria, sulfide-dependent denitrifiers and anammox bacteria suggests a tight network of bacteria coupling carbon-, nitrogen- and sulfur cycling in Lake Grevelingen sediments.

The main objectives of the KM3NeT Collaboration are (i) the discovery and subsequent observation of high-energy neutrino sources in the Universe and (ii) the determination of the mass hierarchy of neutrinos. These objectives are strongly motivated by two recent important discoveries, namely: (1) the highenergy astrophysical neutrino signal reported by IceCube and (2) the sizable contribution of electron neutrinos to the third neutrino mass eigenstate as reported by Daya Bay, Reno and others. To meet these objectives, the KM3NeT Collaboration plans to build a new Research Infrastructure consisting of a network of deep-sea neutrino telescopes in the Mediterranean Sea. A phased and distributed implementation is pursued which maximises the access to regional funds, the availability of human resources and the synergistic opportunities for the Earth and sea sciences community. Three suitable deep-sea sites are selected, namely off-shore Toulon (France), Capo Passero (Sicily, Italy) and Pylos (Peloponnese, Greece). The infrastructure will consist of three so-called building blocks. A building block comprises 115 strings, each string comprises 18 optical modules and each optical module comprises 31 photo-multiplier tubes. Each building block thus constitutes a threedimensional array of photo sensors that can be used to detect the Cherenkov light produced by relativistic particles emerging from neutrino interactions. Two building blocks will be sparsely configured to fully explore the IceCube signal with similar instrumented volume, different methodology, improved resolution and complementary field of view, including the galactic plane. One building block will be densely configured to precisely measure atmospheric neutrino oscillations.

Ever since Darwin's early descriptions of coral reefs, scientists have debated how one of the world's most productive and diverse ecosystems can thrive in the marine equivalent of a desert. It is an enigma how the flux of dissolved organic matter (DOM), the largest resource produced on reefs, is transferred to higher trophic levels. Here we show that sponges make DOM available to fauna by rapidly expelling filter cells as detritus that is subsequently consumed by reef fauna. This "sponge loop" was confirmed in aquarium and in situ food web experiments, using (13)C- and (15)N-enriched DOM. The DOM-sponge-fauna pathway explains why biological hot spots such as coral reefs persist in oligotrophic seas--the reef's paradox--and has implications for reef ecosystem functioning and conservation strategies.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

Abstract. Accurate assessment of anthropogenic carbon dioxide (CO2) emissions and their redistribution among the atmosphere, ocean, and terrestrial biosphere is important to better understand the global carbon cycle, support the development of climate policies, and project future climate change. Here we describe data sets and a methodology to quantify all major components of the global carbon budget, including their uncertainties, based on the combination of a range of data, algorithms, statistics, and model estimates and their interpretation by a broad scientific community. We discuss changes compared to previous estimates as well as consistency within and among components, alongside methodology and data limitations. CO2 emissions from fossil fuels and industry (EFF) are based on energy statistics and cement production data, while emissions from land-use change (ELUC), mainly deforestation, are based on combined evidence from land-cover-change data, fire activity associated with deforestation, and models. The global atmospheric CO2 concentration is measured directly and its rate of growth (GATM) is computed from the annual changes in concentration. The mean ocean CO2 sink (SOCEAN) is based on observations from the 1990s, while the annual anomalies and trends are estimated with ocean models. The variability in SOCEAN is evaluated with data products based on surveys of ocean CO2 measurements. The global residual terrestrial CO2 sink (SLAND) is estimated by the difference of the other terms of the global carbon budget and compared to results of independent dynamic global vegetation models forced by observed climate, CO2, and land-cover change (some including nitrogen–carbon interactions). We compare the mean land and ocean fluxes and their variability to estimates from three atmospheric inverse methods for three broad latitude bands. All uncertainties are reported as ±1σ, reflecting the current capacity to characterise the annual estimates of each component of the global carbon budget. For the last decade available (2005–2014), EFF was 9.0 ± 0.5 GtC yr−1, ELUC was 0.9 ± 0.5 GtC yr−1, GATM was 4.4 ± 0.1 GtC yr−1, SOCEAN was 2.6 ± 0.5 GtC yr−1, and SLAND was 3.0 ± 0.8 GtC yr−1. For the year 2014 alone, EFF grew to 9.8 ± 0.5 GtC yr−1, 0.6 % above 2013, continuing the growth trend in these emissions, albeit at a slower rate compared to the average growth of 2.2 % yr−1 that took place during 2005–2014. Also, for 2014, ELUC was 1.1 ± 0.5 GtC yr−1, GATM was 3.9 ± 0.2 GtC yr−1, SOCEAN was 2.9 ± 0.5 GtC yr−1, and SLAND was 4.1 ± 0.9 GtC yr−1. GATM was lower in 2014 compared to the past decade (2005–2014), reflecting a larger SLAND for that year. The global atmospheric CO2 concentration reached 397.15 ± 0.10 ppm averaged over 2014. For 2015, preliminary data indicate that the growth in EFF will be near or slightly below zero, with a projection of −0.6 [range of −1.6 to +0.5] %, based on national emissions projections for China and the USA, and projections of gross domestic product corrected for recent changes in the carbon intensity of the global economy for the rest of the world. From this projection of EFF and assumed constant ELUC for 2015, cumulative emissions of CO2 will reach about 555 ± 55 GtC (2035 ± 205 GtCO2) for 1870–2015, about 75 % from EFF and 25 % from ELUC. This living data update documents changes in the methods and data sets used in this new carbon budget compared with previous publications of this data set (Le Quéré et al., 2015, 2014, 2013). All observations presented here can be downloaded from the Carbon Dioxide Information Analysis Center (doi:10.3334/CDIAC/GCP_2015).

Abstract. The Surface Ocean CO2 Atlas (SOCAT) is a synthesis of quality-controlled fCO2 (fugacity of carbon dioxide) values for the global surface oceans and coastal seas with regular updates. Version 3 of SOCAT has 14.7 million fCO2 values from 3646 data sets covering the years 1957 to 2014. This latest version has an additional 4.6 million fCO2 values relative to version 2 and extends the record from 2011 to 2014. Version 3 also significantly increases the data availability for 2005 to 2013. SOCAT has an average of approximately 1.2 million surface water fCO2 values per year for the years 2006 to 2012. Quality and documentation of the data has improved. A new feature is the data set quality control (QC) flag of E for data from alternative sensors and platforms. The accuracy of surface water fCO2 has been defined for all data set QC flags. Automated range checking has been carried out for all data sets during their upload into SOCAT. The upgrade of the interactive Data Set Viewer (previously known as the Cruise Data Viewer) allows better interrogation of the SOCAT data collection and rapid creation of high-quality figures for scientific presentations. Automated data upload has been launched for version 4 and will enable more frequent SOCAT releases in the future. High-profile scientific applications of SOCAT include quantification of the ocean sink for atmospheric carbon dioxide and its long-term variation, detection of ocean acidification, as well as evaluation of coupled-climate and ocean-only biogeochemical models. Users of SOCAT data products are urged to acknowledge the contribution of data providers, as stated in the SOCAT Fair Data Use Statement. This ESSD (Earth System Science Data) "living data" publication documents the methods and data sets used for the assembly of this new version of the SOCAT data collection and compares these with those used for earlier versions of the data collection (Pfeil et al., 2013; Sabine et al., 2013; Bakker et al., 2014). Individual data set files, included in the synthesis product, can be downloaded here: doi:10.1594/PANGAEA.849770. The gridded products are available here: doi:10.3334/CDIAC/OTG.SOCAT_V3_GRID.

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Approximately 5.25 trillion plastic pieces are floating at the sea surface. The impact of plastic pollution on the lowest trophic levels of the food web, however, remains unknown. Here we show that plastics release dissolved organic carbon (DOC) into the ambient seawater stimulating the activity of heterotrophic microbes. Our estimates indicate that globally up to 23,600 metric tons of DOC are leaching from marine plastics annually. About 60% of it is available to microbial utilization in less than 5 days. If exposed to solar radiation, however, this DOC becomes less labile. Thus, plastic pollution of marine surface waters likely alters the composition and activity of the base of the marine food webs. It is predicted that plastic waste entering the ocean will increase by a factor of ten within the next decade, resulting in an increase in plastic-derived DOC that might have unaccounted consequences for marine microbes and for the ocean system.

Abstract. Version 2 of the Global Ocean Data Analysis Project (GLODAPv2) data product is composed of data from 724 scientific cruises covering the global ocean. It includes data assembled during the previous efforts GLODAPv1.1 (Global Ocean Data Analysis Project version 1.1) in 2004, CARINA (CARbon IN the Atlantic) in 2009/2010, and PACIFICA (PACIFic ocean Interior CArbon) in 2013, as well as data from an additional 168 cruises. Data for 12 core variables (salinity, oxygen, nitrate, silicate, phosphate, dissolved inorganic carbon, total alkalinity, pH, CFC-11, CFC-12, CFC-113, and CCl4) have been subjected to extensive quality control, including systematic evaluation of bias. The data are available in two formats: (i) as submitted but updated to WOCE exchange format and (ii) as a merged and internally consistent data product. In the latter, adjustments have been applied to remove significant biases, respecting occurrences of any known or likely time trends or variations. Adjustments applied by previous efforts were re-evaluated. Hence, GLODAPv2 is not a simple merging of previous products with some new data added but a unique, internally consistent data product. This compiled and adjusted data product is believed to be consistent to better than 0.005 in salinity, 1 % in oxygen, 2 % in nitrate, 2 % in silicate, 2 % in phosphate, 4 µmol kg−1 in dissolved inorganic carbon, 6 µmol kg−1 in total alkalinity, 0.005 in pH, and 5 % for the halogenated transient tracers.The original data and their documentation and doi codes are available at the Carbon Dioxide Information Analysis Center (http://cdiac.ornl.gov/oceans/GLODAPv2/). This site also provides access to the calibrated data product, which is provided as a single global file or four regional ones – the Arctic, Atlantic, Indian, and Pacific oceans – under the doi:10.3334/CDIAC/OTG.NDP093_GLODAPv2. The product files also include significant ancillary and approximated data. These were obtained by interpolation of, or calculation from, measured data. This paper documents the GLODAPv2 methods and products and includes a broad overview of the secondary quality control results. The magnitude of and reasoning behind each adjustment is available on a per-cruise and per-variable basis in the online Adjustment Table.

Anthropogenic litter is present in all marine habitats, from beaches to the most remote points in the oceans. On the seafloor, marine litter, particularly plastic, can accumulate in high densities with deleterious consequences for its inhabitants. Yet, because of the high cost involved with sampling the seafloor, no large-scale assessment of distribution patterns was available to date. Here, we present data on litter distribution and density collected during 588 video and trawl surveys across 32 sites in European waters. We found litter to be present in the deepest areas and at locations as remote from land as the Charlie-Gibbs Fracture Zone across the Mid-Atlantic Ridge. The highest litter density occurs in submarine canyons, whilst the lowest density can be found on continental shelves and on ocean ridges. Plastic was the most prevalent litter item found on the seafloor. Litter from fishing activities (derelict fishing lines and nets) was particularly common on seamounts, banks, mounds and ocean ridges. Our results highlight the extent of the problem and the need for action to prevent increasing accumulation of litter in marine environments.

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