Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We amplified both the V4 and V6 hypervariable regions of the 16S rRNA gene and clustered the sequences into operational taxonomic units (OTUs) of 97% similarity to analyze for diversity and community composition. OTU richness is much higher with the V6 tag than with the V4 tag, and subsequently OTU-level community composition is quite different between the two tags. Vertical patterns of relative diversity are broadly the same for both tags, with maximum taxonomic richness in seafloor sediment and lowest richness in subseafloor sediment at both geographic locations. Genetic dissimilarity between sample locations is also broadly the same for both tags. Community composition is very similar for both tags at the class level, but very different at the level of 97% similar OTUs. Class-level diversity and community composition of water-column samples are very similar at each water depth between the Atlantic and Pacific. However, sediment communities differ greatly from the Atlantic site to the Pacific site. Finally, for relative patterns of diversity and class-level community composition, deep sequencing and shallow sequencing provide similar results.

Microbial Selection and Survival in Subseafloor Sediment.

Many studies have examined relationships of microorganisms to geochemical zones in subseafloor sediment. However, responses to selective pressure and patterns of community succession with sediment depth have rarely been examined. Here we use 16S rDNA sequencing to examine the succession of microbial communities at sites in the Indian Ocean and the Bering Sea. The sediment ranges in depth from 0.16 to 332 m below seafloor and in age from 660 to 1,300,000 years. The majority of subseafloor taxonomic diversity is present in the shallowest depth sampled. The best predictor of sequence presence or absence in the oldest sediment is relative abundance in the near-seafloor sediment. This relationship suggests that perseverance of specific taxa into deep, old sediment is primarily controlled by the taxonomic abundance that existed when the sediment was near the seafloor. The operational taxonomic units that dominate at depth comprise a subset of the local seafloor community at each site, rather than a grown-in group of geographically widespread subseafloor specialists. At both sites, most taxa classified as abundant decrease in relative frequency with increasing sediment depth and age. Comparison of community composition to cell counts at the Bering Sea site indicates that the rise of the few dominant taxa in the deep subseafloor community does not require net replication, but might simply result from lower mortality relative to competing taxa on the long timescale of community burial.

Relationship of Bacterial Richness to Organic Degradation Rate and Sediment Age in Subseafloor Sediment.

UNLABELLED: Subseafloor sediment hosts a large, taxonomically rich, and metabolically diverse microbial ecosystem. However, the factors that control microbial diversity in subseafloor sediment have rarely been explored. Here, we show that bacterial richness varies with organic degradation rate and sediment age. At three open-ocean sites (in the Bering Sea and equatorial Pacific) and one continental margin site (Indian Ocean), richness decreases exponentially with increasing sediment depth. The rate of decrease in richness with increasing depth varies from site to site. The vertical succession of predominant terminal electron acceptors correlates with abundance-weighted community composition but does not drive the vertical decrease in richness. Vertical patterns of richness at the open-ocean sites closely match organic degradation rates; both properties are highest near the seafloor and decline together as sediment depth increases. This relationship suggests that (i) total catabolic activity and/or electron donor diversity exerts a primary influence on bacterial richness in marine sediment and (ii) many bacterial taxa that are poorly adapted for subseafloor sedimentary conditions are degraded in the geologically young sediment, where respiration rates are high. Richness consistently takes a few hundred thousand years to decline from near-seafloor values to much lower values in deep anoxic subseafloor sediment, regardless of sedimentation rate, predominant terminal electron acceptor, or oceanographic context. IMPORTANCE: Subseafloor sediment provides a wonderful opportunity to investigate the drivers of microbial diversity in communities that may have been isolated for millions of years. Our paper shows the impact of in situ conditions on bacterial community structure in subseafloor sediment. Specifically, it shows that bacterial richness in subseafloor sediment declines exponentially with sediment age, and in parallel with organic-fueled oxidation rate. This result suggests that subseafloor diversity ultimately depends on electron donor diversity and/or total community respiration. This work studied how and why biological richness changes over time in the extraordinary ecosystem of subseafloor sediment.

Bacterial diversity and community composition from seasurface to subseafloor.

We investigated compositional relationships between bacterial communities in the water column and those in deep-sea sediment at three environmentally distinct Pacific sites (two in the Equatorial Pacific and one in the North Pacific Gyre). Through pyrosequencing of the v4-v6 hypervariable regions of the 16S ribosomal RNA gene, we characterized 450,104 pyrotags representing 29,814 operational taxonomic units (OTUs, 97% similarity). Hierarchical clustering and non-metric multidimensional scaling partition the samples into four broad groups, regardless of geographic location: a photic-zone community, a subphotic community, a shallow sedimentary community and a subseafloor sedimentary community (⩾1.5 meters below seafloor). Abundance-weighted community compositions of water-column samples exhibit a similar trend with depth at all sites, with successive epipelagic, mesopelagic, bathypelagic and abyssopelagic communities. Taxonomic richness is generally highest in the water-column O2 minimum zone and lowest in the subseafloor sediment. OTUs represented by abundant tags in the subseafloor sediment are often present but represented by few tags in the water column, and represented by moderately abundant tags in the shallow sediment. In contrast, OTUs represented by abundant tags in the water are generally absent from the subseafloor sediment. These results are consistent with (i) dispersal of marine sedimentary bacteria via the ocean, and (ii) selection of the subseafloor sedimentary community from within the community present in shallow sediment.

Microbial activity in the marine deep biosphere: progress and prospects.

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists-all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earth's global biogeochemical cycles, and for understanding how microorganisms in these "extreme" environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) "theme team" on microbial activity (www.darkenergybiosphere.org).

Concurrent activity of anammox and denitrifying bacteria in the Black Sea.

After the discovery of ANaerobic AMMonium OXidation (anammox) in the environment, the role of heterotrophic denitrification as the main marine pathway for fixed N loss has been questioned. A 3 part, 15 month time series investigating nitrite reductase (nirS) mRNA transcripts at a single location in the Black Sea was conducted in order to better understand the activity of anammox and denitrifying bacteria. Here we show that both of these groups were active, as well as being concurrent in the lower suboxic zone over this time span. Their distributions, however, differed in that only expression of denitrification-type nirS was seen in the upper suboxic zone, where geochemistry was variable. Depth profiles covering the suboxic zone showed that the four groups of anammox-type sequences were expressed consistently in the lower suboxic zone, and were consistent with anammox 16 S rDNA gene profiles. By contrast, denitrifier-type nirS sequence groups were mixed; some groups exhibited consistent expression in the lower suboxic zone, while others appeared less consistent. Co-occurrence of both anammox and denitrifier expression was common and ongoing. Both types of transcripts were also found in samples with low concentrations of sulfide (>2 µM). Six major groups of denitrifier-type nirS transcripts were identified, and several groups of denitrifier-type nirS transcripts were closely related to sequences from the Baltic Sea. An increase in denitrifier-type nirS transcript diversity and depth range in October 2007 corresponded to a small increase in mixed layer net community productivity (NCP) as measured by O(2)/Ar gas ratios, as well as to an increase in N(2) concentrations in the suboxic zone. Taken together, the variations in expression patterns between anammox and denitrification provide one possible explanation as to how near instantaneous rate measurements, such as isotope spike experiments, may regularly detect anammox activity but underreport denitrification.

Free-living and aggregate-associated Planctomycetes in the Black Sea.

We examined the distribution of uncultured Planctomycetes phylotypes along depth profiles spanning the redox gradient of the Black Sea suboxic zone to gain insight into their respective ecological niches. Planctomycetes phylogeny correlated with depth and chemical profiles, implying similar metabolisms within phylogenetic groups. A suboxic zone sample was split into > 30 and < 30 µm fractions to examine putative aggregate-attached and free-living Planctomycetes. All identified Planctomycetes were present in the > 30 µm fraction except for members of the Scalindua genus, which were apparently free-living. Sequences from Candidatus Scalindua, known to carry out the anammox process, formed two distinct clusters with nonoverlapping depth ranges. One cluster, only 97.1% similar to the named species, was present at high nitrite/nitrate and low ammonium concentrations in the upper suboxic zone. We propose this sequence type be named 'Candidatus Scalindua richardsii'. A second cluster, containing sequences more similar to 'Candidatus Scalindua sorokinii', was present at high ammonium and low nitrite conditions in the lower suboxic zone. Sequences obtained from the sulfidic zone (1000 m depth) yielded Planctomycetes from two uncharacterized Planctomycetacia clusters and three potentially new genera as well as sequences from the uncultured OP3 phylum.

Metabolic strategies of free-living and aggregate-associated bacterial communities inferred from biologic and chemical profiles in the Black Sea suboxic zone.

The Black Sea is a permanently anoxic basin with a well-defined redox gradient. We combine environmental 16S rRNA gene data from clone libraries, terminal restriction fragment length polymorphisms, and V6 hypervariable region pyrosequences to provide the most detailed bacterial survey to date. Furthermore, this data set is informed by comprehensive geochemical data; using this combination of information, we put forward testable hypotheses regarding possible metabolisms of uncultured bacteria from the Black Sea's suboxic zone (microaerophily, nitrate reduction, manganese cycling, and oxidation of methane, ammonium, and sulfide). Dominant bacteria in the upper suboxic zone included members of the SAR11, SAR324, and Microthrix groups and in the deep suboxic zone included members of BS-GSO-2, Marine Group A, and SUP05. A particulate fraction (30 µm filter) was used to distinguish between free-living and aggregate-attached communities in the suboxic zone. The particulate fraction contained greater diversity of V6 tag sequences than the bulk water samples. Lentisphaera, Epsilonproteobacteria, WS3, Planctomycetes, and Deltaproteobacteria were enriched in the particulate fraction, whereas SAR11 relatives dominated the free-living fraction. On the basis of the bacterial assemblages and simple modeling, we find that in suboxic waters, the interior of sinking aggregates potentially support manganese reduction, sulfate reduction, and sulfur oxidation.