(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.

Seagrass biofilm communities at a naturally CO2-rich vent at Papua New Guinea

Seagrass meadows are a crucial component of tropical marine reef ecosystems. The seagrass plants are colonized by a multitude of epiphytic organisms that contribute to determining the ecological role of seagrasses. To better understand how environmental changes like ocean acidification might affect epiphytic assemblages, the microbial community composition of the epiphytic biofilm of Enhalus acroides was investigated at a natural CO2 vent in Papua New Guinea using molecular fingerprinting and next generation sequencing of 16S and 18S rRNA genes. Both bacterial and eukaryotic epiphytes formed distinct communities at the CO2-impacted site compared to the control site. This site-related CO2 effect was also visible in the succession pattern of microbial epiphytes. We further found an increased abundance of bacterial types associated with coral diseases at the CO2-impacted site (Fusobacteria, Thalassomonas) whereas eukaryotes such as certain crustose coralline algae commonly related to healthy reefs were less diverse. These trends in the epiphytic community of E. acroides suggest a potential role of seagrasses as vectors of coral pathogens and may support previous predictions of a decrease in reef health and prevalence of diseases under future ocean acidification scenarios.

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.

Ladderane lipid analysis and pore water and sediment chemistry of sediment cores from the continental shelf and slope off northwest Africa

The presence and abundance of anaerobic ammonium-oxidizing (anammox) bacteria was investigated in continental shelf and slope sediments (300-3000 m water depth) off northwest Africa in a combined approach applying quantitative polymerase chain reaction (q-PCR) analysis of anammox-specific 16S rRNA genes and anammox-specific ladderane biomarker lipids. We used the presence of an intact ladderane monoether lipid with a phosphocholine (PC) headgroup as a direct indicator for living anammox bacteria and compared it with the abundance of ladderane core lipids derived from both living and dead bacterial biomass. All investigated sediments contained ladderane lipids, both intact and core lipids, in agreement with the presence of anammoxspecific 16S rRNA gene copies, indicating that anammox occurs at all sites. Concentrations of ladderane core lipids in core top sediments varied between 0.3 and 97 ng g**-1 sediment, with the highest concentrations detected at the sites located on the shelf at shallower water depths between 300 and 500 m. In contrast, the C20 [3]-ladderane monoether-PC lipid was most abundant in a core top sediment from 1500 m water depth. Both anammox-specific 16S rRNA gene copy numbers and the concentration of the C20 [3]-ladderane monoether-PC lipid increased downcore in sediments located at greater water depths, showing highest concentrations of 1.2 x 10**8 copies g**-1 sediment and 30 pg g**-1 sediment, respectively, at the deepest station of 3000 m water depth. This suggests that the relative abundance of anammox bacteria is higher in sediments at intermediate to deep water depths where carbon mineralization rates are lower but where anammox is probably more important than denitrification.

Seawater carbonate chemistry, nitrogen concentration and macro community analyse, 2011

Rising anthropogenic CO2 emissions acidify the oceans, and cause changes to seawater carbon chemistry. Bacterial biofilm communities reflect environmental disturbances and may rapidly respond to ocean acidification. This study investigates community composition and activity responses to experimental ocean acidification in biofilms from the Australian Great Barrier Reef. Natural biofilms grown on glass slides were exposed for 11 d to four controlled pCO2 concentrations representing the following scenarios: A) pre-industrial (~300 ppm), B) present-day (~400 ppm), C) mid century (~560 ppm) and D) late century (~1140 ppm). Terminal restriction fragment length polymorphism and clone library analyses of 16S rRNA genes revealed CO2-correlated bacterial community shifts between treatments A, B and D. Observed bacterial community shifts were driven by decreases in the relative abundance of Alphaproteobacteria and increases of Flavobacteriales (Bacteroidetes) at increased CO2 concentrations, indicating pH sensitivity of specific bacterial groups. Elevated pCO2 (C + D) shifted biofilm algal communities and significantly increased C and N contents, yet O2 fluxes, measured using in light and dark incubations, remained unchanged. Our findings suggest that bacterial biofilm communities rapidly adapt and reorganize in response to high pCO2 to maintain activity such as oxygen production.

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.