Jennifer F. Biddle

Metagenomic signatures of the Peru Margin subseafloor biosphere show a genetically distinct environment

The subseafloor marine biosphere may be one of the largest reservoirs of microbial biomass on Earth and has recently been the subject of debate in terms of the composition of its microbial inhabitants, particularly on sediments from the Peru Margin. A metagenomic analysis was made by using whole-genome amplification and pyrosequencing of sediments from Ocean Drilling Program Site 1229 on the Peru Margin to further explore the microbial diversity and overall community composition within this environment. A total of 61.9 Mb of genetic material was sequenced from sediments at horizons 1, 16, 32, and 50 m below the seafloor. These depths include sediments from both primarily sulfate-reducing methane-generating regions of the sediment column. Many genes of the annotated genes, including those encoding ribosomal proteins, corresponded to those from the Chloroflexi and Euryarchaeota. However, analysis of the 16S small-subunit ribosomal genes suggests that Crenarchaeota are the abundant microbial member. Quantitative PCR confirms that uncultivated Crenarchaeota are indeed a major microbial group in these subsurface samples. These findings show that the marine subsurface is a distinct microbial habitat and is different from environments studied by metagenomics, especially because of the predominance of uncultivated archaeal groups.

Gene expression in the deep biosphere

Scientific ocean drilling has revealed a deep biosphere of widespread microbial life in sub-seafloor sediment. Microbial metabolism in the marine subsurface probably has an important role in global biogeochemical cycles, but deep biosphere activities are not well understood. Here we describe and analyse the first sub-seafloor metatranscriptomes from anaerobic Peru Margin sediment up to 159 metres below the sea floor, represented by over 1 billion complementary DNA (cDNA) sequence reads. Anaerobic metabolism of amino acids, carbohydrates and lipids seem to be the dominant metabolic processes, and profiles of dissimilatory sulfite reductase (dsr) transcripts are consistent with pore-water sulphate concentration profiles. Moreover, transcripts involved in cell division increase as a function of microbial cell concentration, indicating that increases in sub-seafloor microbial abundance are a function of cell division across all three domains of life. These data support calculations and models of sub-seafloor microbial metabolism and represent the first holistic picture of deep biosphere activities.

Marine subsurface eukaryotes: the fungal majority

Studies on the microbial communities of deep subsurface sediments have indicated the presence of Bacteria and Archaea throughout the sediment column. Microbial eukaryotes could also be present in deep-sea subsurface sediments; either bacterivorous protists or eukaryotes capable of assimilating buried organic carbon. DNA- and RNA-based clone library analyses are used here to examine the microbial eukaryotic diversity and identify the potentially active members in deep-sea sediment cores of the Peru Margin and the Peru Trench. We compared surface communities with those much deeper in the same cores, and compared cores from different sites. Fungal sequences were most often recovered from both DNA- and RNA-based clone libraries, with variable overall abundances of different sequence types and different dominant clone types in the RNA-based and the DNA-based libraries. Surficial sediment communities were different from each other and from the deep subsurface samples. Some fungal sequences represented potentially novel organisms as well as ones with a cosmopolitan distribution in terrestrial, fresh and salt water environments. Our results indicate that fungi are the most consistently detected eukaryotes in the marine sedimentary subsurface; further, some species may be specifically adapted to the deep subsurface and may play important roles in the utilization and recycling of nutrients.

Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.

Spatial Structure and Activity of Sedimentary Microbial Communities Underlying a Beggiatoa spp. Mat in a Gulf of Mexico Hydrocarbon Seep

Background Subsurface fluids from deep-sea hydrocarbon seeps undergo methane- and sulfur-cycling microbial transformations near the sediment surface. Hydrocarbon seep habitats are naturally patchy, with a mosaic of active seep sediments and non-seep sediments. Microbial community shifts and changing activity patterns on small spatial scales from seep to non-seep sediment remain to be examined in a comprehensive habitat study. Methodology/Principal Findings We conducted a transect of biogeochemical measurements and gene expression related to methane- and sulfur-cycling at different sediment depths across a broad Beggiatoa spp. mat at Mississippi Canyon 118 (MC118) in the Gulf of Mexico. High process rates within the mat (∼400 cm and ∼10 cm from the mats edge) contrasted with sharply diminished activity at ∼50 cm outside the mat, as shown by sulfate and methane concentration profiles, radiotracer rates of sulfate reduction and methane oxidation, and stable carbon isotopes. Likewise, 16S ribosomal rRNA, dsrAB (dissimilatory sulfite reductase) and mcrA (methyl coenzyme M reductase) mRNA transcripts of sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae) and methane-cycling archaea (ANME-1 and ANME-2) were prevalent at the sediment surface under the mat and at its edge. Outside the mat at the surface, 16S rRNA sequences indicated mostly aerobes commonly found in seawater. The seep-related communities persisted at 12–20 cm depth inside and outside the mat. 16S rRNA transcripts and V6-tags reveal that bacterial and archaeal diversity underneath the mat are similar to each other, in contrast to oxic or microoxic habitats that have higher bacterial diversity. Conclusions/Significance The visual patchiness of microbial mats reflects sharp discontinuities in microbial community structure and activity over sub-meter spatial scales; these discontinuities have to be taken into account in geochemical and microbiological inventories of seep environments. In contrast, 12–20 cm deep in the sediments microbial communities performing methane-cycling and sulfate reduction persist at lower metabolic rates regardless of mat cover, and may increase activity rapidly when subsurface flow changes.

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ13C signatures of CH4, dissolved inorganic carbon and porewater concentration profiles of CH4 and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.

Global dispersion and local diversification of the methane seep microbiome

Significance Methane seeps are natural gas leaks at the seafloor that emit methane to the hydrosphere. The emission rates are controlled by methane-oxidizing microorganisms, which shape the ecosystem by supplying energy sources to other microorganisms and animal symbioses. We provide evidence that methane seeps are island-like habitats, harboring distinct microbial communities that share few organisms with other seafloor ecosystems. The seep communities comprise bacteria and archaea that occur worldwide but are locally selected by the environment. These microorganisms show high relative sequence abundances, suggesting high population densities and global relevance for the control of methane emission from the seafloor. At individual seeps, the cosmopolitan microorganisms are associated with a substantial diversity of rare relatives, turning seeps into hotspots of microbial biodiversity. Methane seeps are widespread seafloor ecosystems shaped by the emission of gas from seabed reservoirs. The microorganisms inhabiting methane seeps transform the chemical energy in methane to products that sustain rich benthic communities around the gas leaks. Despite the biogeochemical relevance of microbial methane removal at seeps, the global diversity and dispersion of seep microbiota remain unknown. Here we determined the microbial diversity and community structure of 23 globally distributed methane seeps and compared these to the microbial communities of 54 other seafloor ecosystems, including sulfate–methane transition zones, hydrothermal vents, coastal sediments, and deep-sea surface and subsurface sediments. We found that methane seep communities show moderate levels of microbial richness compared with other seafloor ecosystems and harbor distinct bacterial and archaeal taxa with cosmopolitan distribution and key biogeochemical functions. The high relative sequence abundance of ANME (anaerobic methanotrophic archaea), as well as aerobic Methylococcales, sulfate-reducing Desulfobacterales, and sulfide-oxidizing Thiotrichales, matches the most favorable microbial metabolisms at methane seeps in terms of substrate supply and distinguishes the seep microbiome from other seafloor microbiomes. The key functional taxa varied in relative sequence abundance between different seeps due to the environmental factors, sediment depth and seafloor temperature. The degree of endemism of the methane seep microbiome suggests a high local diversification in these heterogeneous but long-lived ecosystems. Our results indicate that the seep microbiome is structured according to metacommunity processes and that few cosmopolitan microbial taxa mediate the bulk of methane oxidation, with global relevance to methane emission in the ocean.

Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed “Mat Mound”) were characterized by porewater geochemistry of methane, C2–C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.

Metagenomics of the subsurface Brazos-Trinity Basin (IODP site 1320): Comparison with other sediment and pyrosequenced metagenomes

The Brazos-Trinity Basin on the slope of the Gulf of Mexico passive margin was drilled during Integrated Ocean Drilling Progam Expedition 308. The buried anaerobic sediments of this basin are largely organic-poor and have few microbial inhabitants compared with the organic-rich sediments with high cell counts from the Peru Margin that were drilled during Ocean Drilling Program Leg 201. Nucleic acids were extracted from Brazos-Trinity Basin sediments and were subjected to whole-genome amplification and pyrosequencing. A comparison of the Brazos-Trinity Basin metagenome, consisting of 105 Mbp, and the existing Peru Margin metagenome revealed trends linking gene content, phylogenetic content, geological location and geochemical regime. The major microbial groups (Proteobacteria, Firmicutes, Euryarchaeota and Chloroflexi) occur consistently throughout all samples, yet their shifting abundances allow for discrimination between samples. The cluster of orthologous groups category abundances for some classes of genes are correlated with geochemical factors, such as the level of ammonia. Here we describe the sediment metagenome from the oligotrophic Brazos-Trinity Basin (Site 1320) and show similarities and differences with the dataset from the Pacific Peru Margin (Site 1229) and other pyrosequenced datasets. The microbial community found at Integrated Ocean Drilling Program Site 1320 likely represents the subsurface microbial inhabitants of turbiditic slopes that lack substantial upwelling.

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 Earths 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).