Subseafloor sedimentary environments harbor a remarkable number of microorganisms that constitute anaerobic and aerobic microbial ecosystems beneath the ocean margins and open-ocean gyres, ...respectively. Microbial biomass and diversity richness generally decrease with increasing sediment depth and burial time. However, there has been a long-standing debate over the contribution and distribution of Archaea in the subseafloor sedimentary biosphere. Here we show the global quantification of archaeal and bacterial 16S rRNA genes in 221 sediment core samples obtained from diverse oceanographic settings through scientific ocean drilling using microfluidic digital PCR. We estimated that archaeal cells constitute 37.3% of the total microbial cells (40.0% and 12.8% in the ocean margin and open-ocean sites, respectively), corresponding to 1.1 × 10
cells on Earth. In addition, the relative abundance of archaeal 16S rRNA genes generally decreased with the depth of water in the overlying sedimentary habitat, suggesting that Archaea may be more sensitive to nutrient quality and quantity supplied from the overlying ocean.
Abstract
Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these ...communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.
Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ...ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ~47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species–area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 10³ to 6.10 × 10⁵ and 3.28 × 10⁴ to 2.46 × 10⁶ amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth’s global biosphere.
Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of ...target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL−1 humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL−1. The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×103copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×102copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.
Next-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular ...sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5' end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103 to 5.0 × 104 copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.
Summary
Development of an improved technique for separating microbial cells from marine sediments and standardization of a high‐throughput and discriminative cell enumeration method were conducted. ...We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104–108 cells cm−3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high‐throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single‐cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.
Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells. Extrapolation of direct counts of stained microbial cells to the total volume of habitable ...marine subsurface sediments suggests that between 56 Pg (ref. 1) and 303 Pg (ref. 3) 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. 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, 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.
The analysis of eukaryotic environmental DNA (eDNA) in sediment has the potential for understanding past ecosystems, even for taxa lacking skeletons or preserved only as a part of necromass. Despite ...the paleoenvironmental and ecological importance of eukaryotic eDNA in marine sediment, the duration of remaining eDNA and the species of eDNA present in marine sediment has not been well investigated. Here, we analyzed eDNA extracted from 299 sediment samples down to 678 m below the seafloor at 40 geologically distinct sites. The results showed that eukaryotic eDNA was amplified from more than 80% of the sediments with a depositional age of less than 100,000 years. The eDNA was well conserved in anoxic sediments than in oxic sediments, with PCR success rates of 48% and 18%, respectively. The eukaryotic communities include non-benthic organisms such as marine plankton, including diatoms, dinoflagellates, and coccolithophores. A freshwater diatom genus
Aulacoseira
was detected in the Baltic Sea sediments from the last glacial lacustrine environment. These results provide new insights into the global-scale distribution of the past eukaryotic eDNA preserved in marine sediment.
The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 ...expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of 13C- or 15N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.
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