Bioactivity and Metabolome Mining of Deep-Sea Sediment-Derived Microorganisms Reveal New Hybrid PKS-NRPS Macrolactone from Aspergillus versicolor PS108-62.

Despite low temperatures, poor nutrient levels and high pressure, microorganisms thrive in deep-sea environments of polar regions. The adaptability to such extreme environments renders deep-sea microorganisms an encouraging source of novel, bioactive secondary metabolites. In this study, we isolated 77 microorganisms collected by a remotely operated vehicle from the seafloor in the Fram Strait, Arctic Ocean (depth of 2454 m). Thirty-two bacteria and six fungal strains that represented the phylogenetic diversity of the isolates were cultured using an One-Strain-Many-Compounds (OSMAC) approach. The crude EtOAc extracts were tested for antimicrobial and anticancer activities. While antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium was common for many isolates, only two bacteria displayed anticancer activity, and two fungi inhibited the pathogenic yeast Candida albicans. Due to bioactivity against C. albicans and rich chemical diversity based on molecular network-based untargeted metabolomics, Aspergillus versicolor PS108-62 was selected for an in-depth chemical investigation. A chemical work-up of the SPE-fractions of its dichloromethane subextract led to the isolation of a new PKS-NRPS hybrid macrolactone, versicolide A (1), a new quinazoline (-)-isoversicomide A (3), as well as three known compounds, burnettramic acid A (2), cyclopenol (4) and cyclopenin (5). Their structures were elucidated by a combination of HRMS, NMR, [α]D, FT-IR spectroscopy and computational approaches. Due to the low amounts obtained, only compounds 2 and 4 could be tested for bioactivity, with 2 inhibiting the growth of C. albicans (IC50 7.2 µg/mL). These findings highlight, on the one hand, the vast potential of the genus Aspergillus to produce novel chemistry, particularly from underexplored ecological niches such as the Arctic deep sea, and on the other, the importance of untargeted metabolomics for selection of marine extracts for downstream chemical investigations.

Dissolved Organophosphate Esters and Polybrominated Diphenyl Ethers in Remote Marine Environments: Arctic Surface Water Distributions and Net Transport through Fram Strait.

Organophosphate esters (OPEs) have been found in remote environments at unexpectedly high concentrations, but very few measurements of OPE concentrations in seawater are available, and none are available in subsurface seawater. In this study, passive polyethylene samplers (PEs) deployed on deep-water moorings in the Fram Strait and in surface waters of Canadian Arctic lakes and coastal sites were analyzed for a suite of common OPEs. Total OPEs ( ∑11OPE) at deep-water sites were dominated by chlorinated OPEs, and ranged from 6.3 to 440 pg/L. Concentrations were similar in eastern and western Fram Strait. Chlorinated OPEs were also dominant in Canadian Arctic surface waters (mean concentration ranged from < DL to 4400 pg/L), while nonhalogenated alkyl/aryl-substituted OPEs remained low (1.3-55 pg/L), possibly due to the greater long-range transport potential of chlorinated OPEs. Polybrominated diphenyl ethers (PBDEs) were found at much lower concentrations than OPEs (

Depth Profiles of Persistent Organic Pollutants in the North and Tropical Atlantic Ocean.

Little is known of the distribution of persistent organic pollutants (POPs) in the deep ocean. Polyethylene passive samplers were used to detect the vertical distribution of truly dissolved POPs at two sites in the Atlantic Ocean. Samplers were deployed at five depths covering 26-2535 m in the northern Atlantic and Tropical Atlantic, in approximately one year deployments. Samplers of different thickness were used to determine the state of equilibrium POPs reached in the passive samplers. Concentrations of POPs detected in the North Atlantic near the surface (e.g., sum of 14 polychlorinated biphenyls, PCBs: 0.84 pg L(-1)) were similar to previous measurements. At both sites, PCB concentrations showed subsurface maxima (tropical Atlantic Ocean -800 m, North Atlantic -500 m). Currents seemed more important in moving POPs to deeper water masses than the biological pump. The ratio of PCB concentrations in near surface waters (excluding PCB-28) between the two sites was inversely correlated with congeners' subcooled liquid vapor pressure, in support of the latitudinal fractionation. The results presented here implied a significant amount of HCB is stored in the Atlantic Ocean (4.8-26% of the global HCB environmental burdens), contrasting traditional beliefs that POPs do not reach the deep ocean.

Passive Sampler Derived Profiles and Mass Flows of Perfluorinated Alkyl Substances (PFASs) across the Fram Strait in the North Atlantic.

Per- and polyfluorinated alkyl substances (PFAS) are a family of pollutants of high concern due to their ubiquity and negative human health impacts. The long-range marine transport of PFAS was observed during year-long deployments of passive tube samplers in the Fram Strait across three depth transects. Time weighted average concentrations ranged from 2.4-360 pg L-1, and 10 different PFAS were regularly observed. PFAS profiles and concentrations were generally similar to those previously characterized for polycyclic aromatic hydrocarbons (PAHs) at these sites. The detection of several anionic PFAS in "old" water demonstrated that they are not perfect water mass tracers, but are also transported to depth via settling particles. Mass flows of PFAS through the Fram Strait in and out of the Arctic Ocean were basically similar (112 ±82 Mg year-1 northward flow, 100 ±54 Mg year-1 southward flow). For FOSA, export from the Arctic Ocean via the Fram Strait exceeded import by Atlantic Water, likely due to preferential transport and deposition in the Arctic Ocean. These observations suggest PFAS in the Arctic are governed by the feedback loop previously described for PAHs in the region - with additional atmospheric transport delivering volatile PFAS to the Arctic, which then get exported further.

Sea-ice derived meltwater stratification slows the biological carbon pump: results from continuous observations.

The ocean moderates the world's climate through absorption of heat and carbon, but how much carbon the ocean will continue to absorb remains unknown. The North Atlantic Ocean west (Baffin Bay/Labrador Sea) and east (Fram Strait/Greenland Sea) of Greenland features the most intense absorption of anthropogenic carbon globally; the biological carbon pump (BCP) contributes substantially. As Arctic sea-ice melts, the BCP changes, impacting global climate and other critical ocean attributes (e.g. biodiversity). Full understanding requires year-round observations across a range of ice conditions. Here we present such observations: autonomously collected Eulerian continuous 24-month time-series in Fram Strait. We show that, compared to ice-unaffected conditions, sea-ice derived meltwater stratification slows the BCP by 4 months, a shift from an export to a retention system, with measurable impacts on benthic communities. This has implications for ecosystem dynamics in the future warmer Arctic where the seasonal ice zone is expected to expand.

Benthic Oxygen Uptake in the Arctic Ocean Margins - A Case Study at the Deep-Sea Observatory HAUSGARTEN (Fram Strait).

The past decades have seen remarkable changes in the Arctic, a hotspot for climate change. Nevertheless, impacts of such changes on the biogeochemical cycles and Arctic marine ecosystems are still largely unknown. During cruises to the deep-sea observatory HAUSGARTEN in July 2007 and 2008, we investigated the biogeochemical recycling of organic matter in Arctic margin sediments by performing shipboard measurements of oxygen profiles, bacterial activities and biogenic sediment compounds (pigment, protein, organic carbon, and phospholipid contents). Additional in situ oxygen profiles were performed at two sites. This study aims at characterizing benthic mineralization activity along local bathymetric and latitudinal transects. The spatial coverage of this study is unique since it focuses on the transition from shelf to Deep Ocean, and from close to the ice edge to more open waters. Biogeochemical recycling across the continental margin showed a classical bathymetric pattern with overall low fluxes except for the deepest station located in the Molloy Hole (5500 m), a seafloor depression acting as an organic matter depot center. A gradient in benthic mineralization rates arises along the latitudinal transect with clearly higher values at the southern stations (average diffusive oxygen uptake of 0.49 ± 0.18 mmol O2 m-2 d-1) compared to the northern sites (0.22 ± 0.09 mmol O2 m-2 d-1). The benthic mineralization activity at the HAUSGARTEN observatory thus increases southward and appears to reflect the amount of organic matter reaching the seafloor rather than its lability. Although organic matter content and potential bacterial activity clearly follow this gradient, sediment pigments and phospholipids exhibit no increase with latitude whereas satellite images of surface ocean chlorophyll a indicate local seasonal patterns of primary production. Our results suggest that predicted increases in primary production in the Arctic Ocean could induce a larger export of more refractory organic matter due to the longer production season and the extension of the ice-free zone.

Application of a rapid direct viable count method to deep-sea sediment bacteria.

For the first time, a Live/Dead (L/D) Bacterial Viability Kit (BacLight ) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250-5600 m) and sediment depth (0-5 cm). Percentage of viable (and thus potentially active) cells ranged between 20-60% within the first and 10-40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3+/-5.4 nmol cm(-3) h(-1)) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations (p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.

High biodiversity on a deep-water reef in the eastern Fram Strait.

We report on the distribution and abundance of megafauna on a deep-water rocky reef (1796-2373 m) in the Fram Strait, west of Svalbard. Biodiversity and population density are high, with a maximum average of 26.7±0.9 species m(-2) and 418.1±49.6 individuals m(-2) on the east side of the reef summit. These figures contrast with the surrounding abyssal plain fauna, with an average of only 18.1±1.4 species and 29.4±4.3 individuals m(-2) (mean ± standard error). The east side of the reef summit, where the highest richness and density of fauna are found, faces into the predominant bottom current, which likely increases in speed to the summit and serves as a source of particulate food for the numerous suspension feeders present there. We conclude that the observed faunal distribution patterns could be the result of hydrodynamic patterns and food availability above and around the reef. To our knowledge, this study is the first to describe the distribution and diversity of benthic fauna on a rocky reef in deep water.

Biogeography of Deep-sea benthic bacteria at regional scale (LTER HAUSGARTEN, Fram Strait, Arctic).

Knowledge on spatial scales of the distribution of deep-sea life is still sparse, but highly relevant to the understanding of dispersal, habitat ranges and ecological processes. We examined regional spatial distribution patterns of the benthic bacterial community and covarying environmental parameters such as water depth, biomass and energy availability at the Arctic Long-Term Ecological Research (LTER) site HAUSGARTEN (Eastern Fram Strait). Samples from 13 stations were retrieved from a bathymetric (1,284-3,535 m water depth, 54 km in length) and a latitudinal transect (∼ 2,500 m water depth; 123 km in length). 454 massively parallel tag sequencing (MPTS) and automated ribosomal intergenic spacer analysis (ARISA) were combined to describe both abundant and rare types shaping the bacterial community. This spatial sampling scheme allowed detection of up to 99% of the estimated richness on phylum and class levels. At the resolution of operational taxonomic units (97% sequence identity; OTU3%) only 36% of the Chao1 estimated richness was recovered, indicating a high diversity, mostly due to rare types (62% of all OTU3%). Accordingly, a high turnover of the bacterial community was also observed between any two sampling stations (average replacement of 79% of OTU3%), yet no direct correlation with spatial distance was observed within the region. Bacterial community composition and structure differed significantly with increasing water depth along the bathymetric transect. The relative sequence abundance of Verrucomicrobia and Planctomycetes decreased significantly with water depth, and that of Deferribacteres increased. Energy availability, estimated from phytodetrital pigment concentrations in the sediments, partly explained the variation in community structure. Overall, this study indicates a high proportion of unique bacterial types on relatively small spatial scales (tens of kilometers), and supports the sampling design of the LTER site HAUSGARTEN to study bacterial community shifts in this rapidly changing area of the world's oceans.

Small-scale heterogeneity in deep-sea nematode communities around biogenic structures.

The unexpected high species richness of deep-sea sediments gives rise to the questions, which processes produce and maintain diversity in the deep sea, and at what spatial scales do these processes operate? The idea of a small-scale habitat structure at the deep-sea floor provides the background for this study. At small scales biogenic structures create a heterogeneous environment that influences the structure of the surrounding communities and the dynamics of the meiobenthic populations. As an example for biogenic structures, small deep-sea sponges (Tentorium semisuberites Schmidt 1870) and their sedimentary environment were investigated for small-scale distribution patterns of benthic deep-sea nematodes. Sampling was carried out with the remotely operated vehicle Victor 6000 at the Arctic deep-sea observatory HAUSGARTEN. In order to investigate nematode community patterns sediment cores around three small sponges and corresponding control cores were analysed. A total of approx. 5800 nematodes were identified. The comparison of the nematode communities from sponge and control samples indicated an influence of the biogenic structure "sponge" on diversity patterns and habitat heterogeneity. The increased number of nematode species and functional groups found in the sediments around the sponges suggest that on a small scale the sponge acts as a gradient and creates a more divers habitat structure. The nematode community from the sponge sediments shows a greater taxonomic variance and species richness together with lower relative abundances of the species compared to those from control sediments. Obviously, the more homogeneous habitat conditions of the control sediments offer less micro-habitats than the sediments around the sponges. This seems to reduce the number of functional groups and species coexisting in the control sediments.

Deep-sea nematodes actively colonise sediments, irrespective of the presence of a pulse of organic matter: results from an in-situ experiment.

A colonisation experiment was performed in situ at 2500 m water depth at the Arctic deep-sea long-term observatory HAUSGARTEN to determine the response of deep-sea nematodes to disturbed, newly available patches, enriched with organic matter. Cylindrical tubes,laterally covered with a 500 µm mesh, were filled with azoic deep-sea sediment and (13)C-labelled food sources (diatoms and bacteria). After 10 days of incubation the tubes were analysed for nematode response in terms of colonisation and uptake. Nematodes actively colonised the tubes, however with densities that only accounted for a maximum of 2.13% (51 ind.10 cm(-2)) of the ambient nematode assemblages. Densities did not differ according to the presence or absence of organic matter, nor according to the type of organic matter added. The fact that the organic matter did not function as an attractant to nematodes was confirmed by the absence of notable (13)C assimilation by the colonising nematodes. Overall, colonisation appears to be a process that yields reproducible abundance and diversity patterns, with certain taxa showing more efficiency. Together with the high variability between the colonising nematode assemblages, this lends experimental support to the existence of a spatio-temporal mosaic that emerges from highly localised, partially stochastic community dynamics.

Global patterns and predictions of seafloor biomass using random forests.

A comprehensive seafloor biomass and abundance database has been constructed from 24 oceanographic institutions worldwide within the Census of Marine Life (CoML) field projects. The machine-learning algorithm, Random Forests, was employed to model and predict seafloor standing stocks from surface primary production, water-column integrated and export particulate organic matter (POM), seafloor relief, and bottom water properties. The predictive models explain 63% to 88% of stock variance among the major size groups. Individual and composite maps of predicted global seafloor biomass and abundance are generated for bacteria, meiofauna, macrofauna, and megafauna (invertebrates and fishes). Patterns of benthic standing stocks were positive functions of surface primary production and delivery of the particulate organic carbon (POC) flux to the seafloor. At a regional scale, the census maps illustrate that integrated biomass is highest at the poles, on continental margins associated with coastal upwelling and with broad zones associated with equatorial divergence. Lowest values are consistently encountered on the central abyssal plains of major ocean basins The shift of biomass dominance groups with depth is shown to be affected by the decrease in average body size rather than abundance, presumably due to decrease in quantity and quality of food supply. This biomass census and associated maps are vital components of mechanistic deep-sea food web models and global carbon cycling, and as such provide fundamental information that can be incorporated into evidence-based management.