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.

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.

Shallow water marine sediment bacterial community shifts along a natural CO2 gradient in the Mediterranean Sea Off vulcano, Italy

The effects of increasing atmospheric CO(2) on ocean ecosystems are a major environmental concern, as rapid shoaling of the carbonate saturation horizon is exposing vast areas of marine sediments to corrosive waters worldwide. Natural CO(2) gradients off Vulcano, Italy, have revealed profound ecosystem changes along rocky shore habitats as carbonate saturation levels decrease, but no investigations have yet been made of the sedimentary habitat. Here, we sampled the upper 2 cm of volcanic sand in three zones, ambient (median pCO(2) 419 µatm, minimum Omega (arag) 3.77), moderately CO(2)-enriched (median pCO(2) 592 µatm, minimum Omega (arag) 2.96), and highly CO(2)-enriched (median pCO(2) 1611 µatm, minimum Omega (arag) 0.35). We tested the hypothesis that increasing levels of seawater pCO(2) would cause significant shifts in sediment bacterial community composition, as shown recently in epilithic biofilms at the study site. In this study, 454 pyrosequencing of the V1 to V3 region of the 16S rRNA gene revealed a shift in community composition with increasing pCO(2). The relative abundances of most of the dominant genera were unaffected by the pCO(2) gradient, although there were significant differences for some 5 % of the genera present (viz. Georgenia, Lutibacter, Photobacterium, Acinetobacter, and Paenibacillus), and Shannon Diversity was greatest in sediments subject to long-term acidification (>100 years). Overall, this supports the view that globally increased ocean pCO(2) will be associated with changes in sediment bacterial community composition but that most of these organisms are resilient. However, further work is required to assess whether these results apply to other types of coastal sediments and whether the changes in relative abundance of bacterial taxa that we observed can significantly alter the biogeochemical functions of marine sediments.

Selected soil properties and bacterial abundance in the glacier forefields 'Black Valley Transect' and 'Glacier Transect' on Larsemann Hills, East Antarctica

Antarctic glacier forefields are extreme environments and pioneer sites for ecological succession. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats, and new terrain is becoming exposed to soil formation and microbial colonization. However, only little is known about the impact of environmental changes on microbial communities and how they develop in connection to shifting habitat characteristics. In this study, using a combination of molecular and geochemical analysis, we determine the structure and development of bacterial communities depending on soil parameters in two different glacier forefields on Larsemann Hills, East Antarctica. Our results demonstrate that deglaciation-dependent habitat formation, resulting in a gradient in soil moisture, pH and conductivity, leads to an orderly bacterial succession for some groups, for example Cyanobacteria, Bacteroidetes and Deltaproteobacteria in a transect representing 'classical' glacier forefields. A variable bacterial distribution and different composed communities were revealed according to soil heterogeneity in a slightly 'matured' glacier forefield transect, where Gemmatimonadetes, Flavobacteria, Gamma- and Deltaproteobacteria occur depending on water availability and soil depth. Actinobacteria are dominant in both sites with dominance connected to certain trace elements in the glacier forefields.

Nitrogen fixation and nitrogen gene abundance of METEOR cruises M77/3 and M77/4

Nitrogen fixation, the biological reduction of dinitrogen gas (N2) to ammonium (NH4+), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N2 fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N2 fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N2 fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N2 fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO2- and PO43- are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N2 fixation may increase in the futur