(Table S2) Relative abundance of archaeal 16S rRNA genes in sediment cores

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells (Parkes et al., 1994, doi:10.1038/371410a0; D'Hondt et al., 2004, doi:10.1126/science.1101155). Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (Parkes et al., 1994, doi:10.1038/371410a0) and 303 Pg (Whitman et al., 1998) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem (Schippers et al., doi:10.1038/nature03302; Inagaki et al., doi:10.1073/pnas.0511033103 ; Mauclaire et al., doi:10.1111/j.1472-4677.2004.00035.x; Biddle et al., doi:10.1073/pnas.0600035103). Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells (Biddle et al., doi:10.1073/pnas.0600035103; Sturt et al., 2004, doi:10.1002/rcm.1378), are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

Table 2: Bacterial diversity in Ny-Ålesund, Spitzbergen

We describe the antibiotic resistance profiling of bacterial isolates collected from Ny-Alesund, Arctic, as part of the Indian Arctic Summer Expedition 2009. It was interesting to note that the bacterial isolates collected from the Arctic showed multidrug resistance. 32% of the isolates were found to be multi- drug resistant with several combinations of antibiotics. The 16S rRNA sequencing results shows a diverse group of bacteria belonging to Phyla Proteobacteria, Actinobacteria and Bacteriodetes and their relatedness was studied by phylogenetic analysis. While analysing the plasmid profiling, the most resistant two strains of Pseudomonas migulae showed multiple plasmids of varying sizes ~5.2-5.3 kb and ~9.5 kb. The extent and frequency of multidrug resistance in the polar bacteria deserves close monitoring and efforts to understand the various molecular mechanisms of drug resistance and control the spread of antibiotic resistance in polar environment is called for.

Molecular fingerprinting and amplicon sequencing of the bacterial biofilm on settlement tiles deployed along natural pH gradients in Papua New Guinea

Bacterial biofilms provide cues for the settlement of marine invertebrates such as coral larvae, and are therefore important for the resilience and recovery of coral reefs. This study aimed to better understand how ocean acidification may affect the community composition and diversity of bacterial biofilms on surfaces under naturally reduced pH conditions. Settlement tiles were deployed at coral reefs in Papua New Guinea along pH gradients created by two CO2 seeps, and upper and lower tiles surfaces were sampled 5 and 13 months after deployment. Automated Ribosomal Intergenic Spacer Analysis were used to characterize more than 200 separate bacterial communities, complemented by amplicon sequencing of the bacterial 16S rRNA gene of 16 samples. The bacterial biofilm consisted predominantly of Alpha-, Gamma- and Deltaproteobacteria, as well as Cyanobacteria, Flavobacteriia and Cytophaga, whereas putative settlement-inducing taxa only accounted for a small fraction of the community. Bacterial biofilm composition was heterogeneous with approximately 25% shared operational taxonomic units between samples. Among the observed environmental parameters, pH only had a weak effect on community composition (R² ~ 1%) and did not affect community richness and evenness. In contrast, there were strong differences between upper and lower surfaces (contrasting in light exposure and grazing intensity). There also appeared to be a strong interaction between bacterial biofilm composition and the macroscopic components of the tile community. Our results suggest that on mature settlement surfaces in situ, pH does not have a strong impact on the composition of bacterial biofilms. Other abiotic and biotic factors such as light exposure and interactions with other organisms may be more important in shaping bacterial biofilms than changes in seawater pH.

Microbiota and Microsatellite genotypes of Pacific oysters stemming from three oyster bed in the Northern Wadden Sea

Background: Studies of oyster microbiomes have revealed that a limited number of microbes, including pathogens, can dominate microbial communities in host tissues such as gills and gut. Much of the bacterial diversity however remains underexplored and unexplained, although environmental conditions and host genetics have been implicated. We used 454 next generation 16S rRNA amplicon sequencing of individually tagged PCR reactions to explore the diversity of bacterial communities in gill tissue of the invasive Pacific oyster Crassostrea gigas stemming from genetically differentiated beds under ambient outdoor conditions and after a multifaceted disturbance treatment imposing stress on the host. Results: While the gill associated microbial communities in oysters were dominated by few abundant taxa (i.e. Sphingomonas, Mycoplasma) the distribution of rare bacterial groups correlated to relatedness between the hosts under ambient conditions. Exposing the host to disturbance broke apart this relationship by removing rare phylotypes thereby reducing overall microbial diversity. Shifts in the microbiome composition in response to stress did not result in a net increase in genera known to contain potentially pathogenic strains. Conclusion: The decrease in microbial diversity and the disassociation between population genetic structure of the hosts and their associated microbiome suggest that disturbance (i.e. stress) may play a significant role for the assembly of the natural microbiome. Such community shifts may in turn also feed back on the course of disease and the occurrence of mass mortality events in oyster populations.

Geochemistry of pockmark sediments from the inner Oslofjord

Pockmarks are geological features that are found on the bottom of lakes and oceans all over the globe. Some are active, seeping oil or methane, while others are inactive. Active pockmarks are well studied since they harbor specialized microbial communities that proliferate on the seeping compounds. Such communities are not found in inactive pockmarks. Interestingly, inactive pockmarks are known to have different macrofaunal communities compared to the surrounding sediments. It is undetermined what the microbial composition of inactive pockmarks is and if it shows a similar pattern as the macrofauna. The Norwegian Oslo Fjord contains many inactive pockmarks and they are well suited to study the influence of these geological features on the microbial community in the sediment. Here we present a detailed analysis of the microbial communities found in three inactive pockmarks and two control samples at two core depth intervals. The communities were analyzed using high-throughput amplicon sequencing of the 16S rRNA V3 region. Microbial communities of surface pockmark sediments were indistinguishable from communities found in the surrounding seabed. In contrast, pockmark communities at 40 cm sediment depth had a significantly different community structure from normal sediments at the same depth. Statistical analysis of chemical variables indicated significant differences in the concentrations of total carbon and non-particulate organic carbon between 40 cm pockmark and reference sample sediments. We discuss these results in comparison with the taxonomic classification of the OTUs identified in our samples. Our results indicate that microbial surface sediment communities are affect by the water column, while the 40 cm communities are affect by local conditions within the sediment.

Metadata und statistic analysis of archael and bacterial sequences originating from sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

DNA extraction was carried out as described on the MICROBIS project pages (http://icomm.mbl.edu/microbis ) using a commercially available extraction kit. We amplified the hypervariable regions V4-V6 of archaeal and bacterial 16S rRNA genes using PCR and several sets of forward and reverse primers (http://vamps.mbl.edu/resources/primers.php). Massively parallel tag sequencing of the PCR products was carried out on a 454 Life Sciences GS FLX sequencer at Marine Biological Laboratory, Woods Hole, MA, following the same experimental conditions for all samples. Sequence reads were submitted to a rigorous quality control procedure based on mothur v30 (doi:10.1128/AEM.01541-09) including denoising of the flow grams using an algorithm based on PyroNoise (doi:10.1038/nmeth.1361), removal of PCR errors and a chimera check using uchime (doi:10.1093/bioinformatics/btr381). The reads were taxonomically assigned according to the SILVA taxonomy (SSURef v119, 07-2014; doi:10.1093/nar/gks1219) implemented in mothur and clustered at 98% ribosomal RNA gene V4-V6 sequence identity. V4-V6 amplicon sequence abundance tables were standardized to account for unequal sampling effort using 1000 (Archaea) and 2300 (Bacteria) randomly chosen sequences without replacement using mothur and then used to calculate inverse Simpson diversity indices and Chao1 richness (doi:10.2307/4615964). Bray-Curtis dissimilarities (doi:10.2307/1942268) between all samples were calculated and used for 2-dimensional non metric multidimensional scaling (NMDS) ordinations with 20 random starts (doi:10.1007/BF02289694). Stress values below 0.2 indicated that the multidimensional dataset was well represented by the 2D ordination. NMDS ordinations were compared and tested using Procrustes correlation analysis (doi:10.1007/BF02291478). All analyses were carried out with the R statistical environment and the packages vegan (available at: http://cran.r-project.org/package=vegan), labdsv (available at: http://cran.r-project.org/package=labdsv), as well as with custom R scripts. Operational taxonomic units at 98% sequence identity (OTU0.03) that occurred only once in the whole dataset were termed absolute single sequence OTUs (SSOabs; doi:10.1038/ismej.2011.132). OTU0.03 sequences that occurred only once in at least one sample, but may occur more often in other samples were termed relative single sequence OTUs (SSOrel). SSOrel are particularly interesting for community ecology, since they comprise rare organisms that might become abundant when conditions change.16S rRNA amplicons and metagenomic reads have been stored in the sequence read archive under SRA project accession number SRP042162.

Defence chemistry modulation by light and temperature (results in 4 excel files, zipped)

The goals of this study were (1) to investigate whether Fucus vesiculosus regulates the production of its antifouling defence chemicals against microfoulers in response to light limitation and temperature shifts and (2) to investigate if different surface concentrations of defence compounds shape epibacterial communities. F. vesiculosus was incubated in indoor mesocosms at five different temperature conditions (5 to 25°C) and in outdoor mesocosms under six differently reduced sunlight conditions (0 to 100%), respectively. Algal surface concentrations of previously identified antifouling compounds - dimethylsulphopropionate (DMSP), fucoxanthin and proline - were determined and the bacterial community composition was characterized by in-depth sequencing of the 16S-rRNA gene. Altogether, the effect of different treatment levels upon defence compound concentrations was limited. Under all conditions DMSP alone appeared to be sufficiently concentrated to warrant for at least a partial inhibitory action against epibiotic bacteria of F. vesiculosus. In contrast, proline and fucoxanthin rarely reached the necessary concentration ranges for self-contained inhibition. Nonetheless, in both experiments along with the direct influence of temperature and light, all three compounds apparently affected (and thereby shaped) the overall bacterial community composition associated with F. vesiculosus since tendencies for insensitivity towards all three compounds were observed among bacterial taxa that typically dominate those communities. Given that the concentrations of at least one of the compounds (in most cases DMSP) were always high enough to inhibit bacterial settlement, we conclude that the capacity of F. vesiculosus for such defence will hardly be compromised by shading or warming to temperatures up to 25°C.

Environmental measurements from the Black Sea in Odessa region, Ukraine, July 2014

The Black Sea is the unique ecosystem with lots of geological, ecological and biological features. For full understanding of these systems it is very important to investigate and indentify the microbial communities, including how the environment shapes its genome. Despite the data obtained by different investigations about the certain groups of microorganisms, isolated as pure cultures on nutritive mediums the total microbial metagenome hasn't been analysed. During July 2014 the 9 sites along the coast in Odessa region were selected for sampling of surface marine water, isolation of total DNA and further sequence 16S rRNA analysis. The water sampling and filtration were accompanied by measurement of metadata for evaluation of how the environment influences the present microbial biodiversity.

Soil characteristics and abundance of archaea in samples from the Ross Sea Region, Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (>99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.

Spatial and seasonal distribution of Thaumarchaeota in the water column and sediment of the southern North Sea

We have examined the spatial and seasonal distribution of Thaumarchaeota in the water column and sediment of the southern North Sea using the specific intact polar lipid (IPL) hexose, phosphohexose (HPH) crenarchaeol, as well as thaumarchaeotal 16S rRNA gene abundances and expression. In the water column, a higher abundance of Thaumarchaeota was observed in the winter season than in the summer, which is in agreement with previous studies, but this was not the case in the sediment where Thaumarchaeota were most abundant in spring and summer. This observation corresponds well with the idea that ammonia availability is a key factor in thaumarchaeotal niche determination. In the surface waters of the southern North Sea, we observed a spatial variability in HPH crenarchaeol, thaumarchaeotal 16S rRNA gene abundance and transcriptional activity that corresponded well with the different water masses present. In bottom waters, a clear differentiation based on water masses was not observed; instead, we suggest that observed differences in thaumarchaeotal abundance with depth may be related to resuspension from the sediment. This could be due to suspension of benthic Thaumarchaeota to the water column or due to delivery of e.g. resuspended sediment or ammonium to the water column, which could be utilized by pelagic Thaumarchaeota. This study has shown that the seasonality of Thaumarchaeota in water and sediment is different and highlights the importance of water masses, currents and sedimentary processes in determining the spatial abundance of Thaumarchaeota in the southern North Sea.

Niche differentiation among mat-forming, sulfide-oxidizing bacteria in chemosynthetic ecosystems of the Nile Deep Sea Fan (Eastern Mediterranean Sea)

Sulfidic muds of cold seeps on the Nile Deep Sea Fan are populated by different types of mat-forming sulfide-oxidizing bacteria. The predominant sulfide oxidizers of three different mats were identified by microscopic and phylogenetic analyses as (i) Arcobacter species producing cotton-ball-like sulfur precipitates, (ii) large filamentous sulfur bacteria including Beggiatoa species, or (iii) single, spherical cells resembling Thiomargarita species. High resolution in situ microprofiles revealed different geochemical settings selecting for different mat types. Arcobacter mats occurred where oxygen and sulfide overlapped at the bottom water interface. Filamentous sulfide oxidizers were associated with non-overlapping, steep gradients of oxygen and sulfide. A dense population of Thiomargarita was favored by temporarily changing supplies of oxygen and sulfide. These results indicate that the decisive factors in selecting for different mat-forming bacteria within one deep-sea province are spatial or temporal variations in energy supply. Furthermore, the occurrence of Arcobacter spp.-related 16S rRNA genes in the sediments below all three types of mats, as well as on top of brine lakes of the Nile Deep Sea Fan, indicates that this group of sulfide oxidizers can switch between different life modes depending on the geobiochemical habitat setting.

Molecular composition in surface and subsurface sediments of the Helgoland mud area, North Sea

The role of microorganisms in the cycling of sedimentary organic carbon is a crucial one. To better understand relationships between molecular composition of a potentially bioavailable fraction of organic matter and microbial populations, bacterial and archaeal communities were characterized using pyrosequencing-based 16S rRNA gene analysis in surface (top 30 cm) and subsurface/deeper sediments (30-530 cm) of the Helgoland mud area, North Sea. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) was used to characterize a potentially bioavailable organic matter fraction (hot-water extractable organic matter, WE-OM). Algal polymer-associated microbial populations such as members of the Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia were dominant in surface sediments while members of the Chloroflexi (Dehalococcoidales and candidate order GIF9) and Miscellaneous Crenarchaeota Groups (MCG), both of which are linked to degradation of more recalcitrant, aromatic compounds and detrital proteins, were dominant in subsurface sediments. Microbial populations dominant in subsurface sediments (Chloroflexi, members of MCG, and Thermoplasmata) showed strong correlations to total organic carbon (TOC) content. Changes of WE-OM with sediment depth reveal molecular transformations from oxygen-rich [high oxygen to carbon (O/C), low hydrogen to carbon (H/C) ratios] aromatic compounds and highly unsaturated compounds toward compounds with lower O/C and higher H/C ratios. The observed molecular changes were most pronounced in organic compounds containing only CHO atoms. Our data thus, highlights classes of sedimentary organic compounds that may serve as microbial energy sources in methanic marine subsurface environments.

Environmental characteristics and gas composition of Lost Hammer, Canadian Arctic

We report the first microbiological characterization of a terrestrial methane seep in a cryo-environment in the form of an Arctic hypersaline (~24% salinity), subzero (-5 C), perennial spring, arising through thick permafrost in an area with an average annual air temperature of -15 C. Bacterial and archaeal 16S rRNA gene clone libraries indicated a relatively low diversity of phylotypes within the spring sediment (Shannon index values of 1.65 and 1.39, respectively). Bacterial phylotypes were related to microorganisms such as Loktanella, Gillisia, Halomonas and Marinobacter spp. previously recovered from cold, saline habitats. A proportion of the bacterial phylotypes were cultured, including Marinobacter and Halomonas, with all isolates capable of growth at the in situ temperature (-5 C). Archaeal phylotypes were related to signatures from hypersaline deep-sea methane-seep sediments and were dominated by the anaerobic methane group 1a (ANME-1a) clade of anaerobic methane oxidizing archaea. CARD-FISH analyses indicated that cells within the spring sediment consisted of ~84.0% bacterial and 3.8% archaeal cells with ANME-1 cells accounting for most of the archaeal cells. The major gas discharging from the spring was methane (~50%) with the low CH4/C2 + ratio and hydrogen and carbon isotope signatures consistent with a thermogenic origin of the methane. Overall, this hypersaline, subzero environment supports a viable microbial community capable of activity at in situ temperature and where methane may behave as an energy and carbon source for sustaining anaerobic oxidation of methane-based microbial metabolism. This site also provides a model of how a methane seep can form in a cryo-environment as well as a mechanism for the hypothesized Martian methane plumes.