Jason B. Sylvan

Microbial Ecology of the Dark Ocean above, at, and below the Seafloor

SUMMARY The majority of life on Earth—notably, microbial life—occurs in places that do not receive sunlight, with the habitats of the oceans being the largest of these reservoirs. Sunlight penetrates only a few tens to hundreds of meters into the ocean, resulting in large-scale microbial ecosystems that function in the dark. Our knowledge of microbial processes in the dark ocean—the aphotic pelagic ocean, sediments, oceanic crust, hydrothermal vents, etc.—has increased substantially in recent decades. Studies that try to decipher the activity of microorganisms in the dark ocean, where we cannot easily observe them, are yielding paradigm-shifting discoveries that are fundamentally changing our understanding of the role of the dark ocean in the global Earth system and its biogeochemical cycles. New generations of researchers and experimental tools have emerged, in the last decade in particular, owing to dedicated research programs to explore the dark ocean biosphere. This review focuses on our current understanding of microbiology in the dark ocean, outlining salient features of various habitats and discussing known and still unexplored types of microbial metabolism and their consequences in global biogeochemical cycling. We also focus on patterns of microbial diversity in the dark ocean and on processes and communities that are characteristic of the different habitats.

Life and Death of Deep-Sea Vents: Bacterial Diversity and Ecosystem Succession on Inactive Hydrothermal Sulfides

ABSTRACT Hydrothermal chimneys are a globally dispersed habitat on the seafloor associated with mid-ocean ridge (MOR) spreading centers. Active, hot, venting sulfide structures from MORs have been examined for microbial diversity and ecology since their discovery in the mid-1970s, and recent work has also begun to explore the microbiology of inactive sulfides—structures that persist for decades to millennia and form moderate to massive deposits at and below the seafloor. Here we used tag pyrosequencing of the V6 region of the 16S rRNA and full-length 16S rRNA sequencing on inactive hydrothermal sulfide chimney samples from 9°N on the East Pacific Rise to learn their bacterial composition, metabolic potential, and succession from venting to nonventing (inactive) regimes. Alpha-, beta-, delta-, and gammaproteobacteria and members of the phylum Bacteroidetes dominate all inactive sulfides. Greater than 26% of the V6 tags obtained are closely related to lineages involved in sulfur, nitrogen, iron, and methane cycling. Epsilonproteobacteria represent <4% of the V6 tags recovered from inactive sulfides and 15% of the full-length clones, despite their high abundance in active chimneys. Members of the phylum Aquificae, which are common in active vents, were absent from both the V6 tags and full-length 16S rRNA data sets. In both analyses, the proportions of alphaproteobacteria, betaproteobacteria, and members of the phylum Bacteroidetes were greater than those found on active hydrothermal sulfides. These shifts in bacterial population structure on inactive chimneys reveal ecological succession following cessation of venting and also imply a potential shift in microbial activity and metabolic guilds on hydrothermal sulfides, the dominant biome that results from seafloor venting. IMPORTANCE Hydrothermal chimneys are globally dispersed seafloor habitats associated with mid-ocean ridge spreading centers. Active, hot, venting chimneys have been examined for microbial ecology since their discovery in the late 1970s, but the microbiology of inactive chimneys, which may persist for thousands of years, has only recently been explored. We studied bacterial diversity on inactive hydrothermal sulfide chimney samples from 9°N on the East Pacific Rise to learn their bacterial community composition, potential ecological roles, and succession from active venting to inactive chimneys. Many bacteria on inactive sulfide chimneys are closely related to lineages involved in sulfur, nitrogen, iron, and methane cycling, and two common groups found on active chimneys are nearly absent from inactive vents, where they were replaced by groups likely involved in the elemental cycling mentioned above. Our findings reveal that ecological succession occurs on hydrothermal sulfides after active venting ceases and also imply a potential shift in microbial metabolic guilds. Hydrothermal chimneys are globally dispersed seafloor habitats associated with mid-ocean ridge spreading centers. Active, hot, venting chimneys have been examined for microbial ecology since their discovery in the late 1970s, but the microbiology of inactive chimneys, which may persist for thousands of years, has only recently been explored. We studied bacterial diversity on inactive hydrothermal sulfide chimney samples from 9°N on the East Pacific Rise to learn their bacterial community composition, potential ecological roles, and succession from active venting to inactive chimneys. Many bacteria on inactive sulfide chimneys are closely related to lineages involved in sulfur, nitrogen, iron, and methane cycling, and two common groups found on active chimneys are nearly absent from inactive vents, where they were replaced by groups likely involved in the elemental cycling mentioned above. Our findings reveal that ecological succession occurs on hydrothermal sulfides after active venting ceases and also imply a potential shift in microbial metabolic guilds.

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).

New Insight into Microbial Iron Oxidation as Revealed by the Proteomic Profile of an Obligate Iron-Oxidizing Chemolithoautotroph

ABSTRACT Microaerophilic, neutrophilic, iron-oxidizing bacteria (FeOB) grow via the oxidation of reduced Fe(II) at or near neutral pH, in the presence of oxygen, making them relevant in numerous environments with elevated Fe(II) concentrations. However, the biochemical mechanisms for Fe(II) oxidation by these neutrophilic FeOB are unknown, and genetic markers for this process are unavailable. In the ocean, microaerophilic microorganisms in the genus Mariprofundus of the class Zetaproteobacteria are the only organisms known to chemolithoautotrophically oxidize Fe and concurrently biomineralize it in the form of twisted stalks of iron oxyhydroxides. The aim of this study was to identify highly expressed proteins associated with the electron transport chain of microaerophilic, neutrophilic FeOB. To this end, Mariprofundus ferrooxydans PV-1 was cultivated, and its proteins were extracted, assayed for redox activity, and analyzed via liquid chromatography-tandem mass spectrometry for identification of peptides. The results indicate that a cytochrome c 4, cbb 3-type cytochrome oxidase subunits, and an outer membrane cytochrome c were among the most highly expressed proteins and suggest an involvement in the process of aerobic, neutrophilic bacterial Fe oxidation. Proteins associated with alternative complex III, phosphate transport, carbon fixation, and biofilm formation were abundant, consistent with the lifestyle of Mariprofundus.

Low temperature geomicrobiology follows host rock composition along a geochemical gradient in lau basin.

The East Lau Spreading Center (ELSC) and Valu Fa Ridge (VFR) comprise a ridge segment in the southwest Pacific Ocean where rapid transitions in the underlying mantle chemistry manifest themselves as gradients in seafloor rock geochemistry. We studied the geology and microbial diversity of three silicate rock samples and three inactive sulfide chimney samples collected, from north to south, at the vent fields Kilo Moana, ABE, Tui Malila, and Mariner. This is the first study of microbial populations on basaltic andesite, which was sampled at Mariner vent field. Silicate rock geochemistry exhibits clear latitudinal trends that are mirrored by changes in bacterial community composition. α-proteobacteria, ε-proteobacteria, and Bacteroidetes are most common on a silicate collected from Kilo Moana and their proportions decrease linearly on silicates collected further south. Conversely, a silicate from Mariner vent field hosts high proportions of a unique lineage of Chloroflexi unrelated (<90% sequence similarity) to previously recovered environmental clones or isolates, which decrease at ABE and are absent at Kilo Moana. The exteriors of inactive sulfide structures are dominated by lineages of sulfur oxidizing α-proteobacteria, γ-proteobacteria, and ε-proteobacteria, while the interior of one chimney is dominated by putative sulfur-reducing δ-proteobacteria. A comparison of bacterial communities on inactive sulfides from this and previous studies reveals the presence of a clade of uncultured Bacteroidetes exclusive to sulfidic environments, and a high degree of heterogeneity in bacterial community composition from one sulfide structure to another. In light of the heterogeneous nature of bacterial communities observed here and in previous studies of both active and inactive hydrothermal sulfide structures, the presence of numerous niches may be detected on these structures in the future by finer scale sampling and analysis.

Spatially resolved sampling reveals dynamic microbial communities in rising hydrothermal plumes across a back-arc basin

Within hydrothermal plumes, chemosynthetic processes and microbe–mineral interactions drive primary productivity in deep-ocean food webs and may influence transport of elements such as iron. However, the source of microorganisms in plumes and the factors governing how these communities assemble are poorly understood, in part due to lack of data from early stages of plume formation. In this study, we examined microbial community composition of rising hydrothermal plumes from five vent fields along the Eastern Lau Spreading Center. Seafloor and plume microbial communities were significantly dissimilar and shared few phylotypes. Plume communities were highly similar to each other with significant differences in community membership only between Kilo Moana and Mariner, two vents that are separated by extremes in depth, latitude and geochemistry. Systematic sampling of waters surrounding the vents revealed that species richness and phylogenetic diversity was typically highest near the vent orifice, implying mixing of microbial communities from the surrounding habitats. Above-plume background communities were primarily dominated by SAR11, SAR324 and MG-I Archaea, while SUP05, Sulfurovum, Sulfurimonas, SAR324 and Alteromonas were abundant in plume and near-bottom background communities. These results show that the ubiquitous water-column microorganisms populate plume communities, and that the composition of background seawater exerts primary influence on plume community composition, with secondary influence from geochemical and/or physical properties of vents. Many of these pervasive deep-ocean organisms are capable of lithotrophy, suggesting that they are poised to use inorganic electron donors encountered in hydrothermal plumes.

Prospects for the Study of Evolution in the Deep Biosphere

Since the days of Darwin, scientists have used the framework of the theory of evolution to explore the interconnectedness of life on Earth and adaptation of organisms to the ever-changing environment. The advent of molecular biology has advanced and accelerated the study of evolution by allowing direct examination of the genetic material that ultimately determines the phenotypes upon which selection acts. The study of evolution has been furthered through examination of microbial evolution, with large population numbers, short generation times, and easily extractable DNA. Such work has spawned the study of microbial biogeography, with the realization that concepts developed in population genetics may be applicable to microbial genomes (Martiny et al., 2006; Manhes and Velicer, 2011). Microbial biogeography and adaptation has been examined in many different environments. Here we argue that the deep biosphere is a unique environment for the study of evolution and list specific factors that can be considered and where the studies may be performed. This publication is the result of the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) theme team on Evolution (www.darkenergybiosphere.org).

Bacillus rigiliprofundi sp. nov., an endospore-forming, Mn-oxidizing, moderately halophilic bacterium isolated from deep subseafloor basaltic crust

A facultatively anaerobic bacterium, designated strain 1MBB1T, was isolated from basaltic breccia collected from 341 m below the seafloor by seafloor drilling of Rigil Guyot during Integrated Ocean Drilling Program Expedition 330. The cells were straight rods, 0.5 μm wide and 1-3 μm long, that occurred singly and in chains. Strain 1MBB1T stained Gram-positive. Catalase and oxidase were produced. The isolate grew optimally at 30 °C and pH 7.5, and could grow with up to 12 % (w/v) NaCl. The DNA G+C content was 40.5 mol%. The major cellular fatty acids were C16:1ω11c (26.5 %), anteiso-C15:0 (19.5 %), C16:0 (18.7 %) and iso-C15:0 (10.4 %), and the cell-wall diamino acid was meso-diaminopimelic acid. Endospores of strain 1MBB1T oxidized Mn(II) to Mn(IV), and siderophore production by vegetative cells was positive. Phylogenetic analysis of the 16S rRNA gene indicated that strain 1MBB1T was a member of the family Bacillaceae, with Bacillus foraminis CV53T and Bacillus novalis LMG 21837T being the closest phylogenetic neighbours (96.5 and 96.2 % similarity, respectively). This is the first novel species described from deep subseafloor basaltic crust. On the basis of our polyphasic analysis, we conclude that strain 1MBB1T represents a novel species of the genus Bacillus, for which we propose the name Bacillus rigiliprofundi sp. nov. The type strain is 1MBB1T ( = NCMA B78T = LMG 28275T).

Carbon fixation by basalt-hosted microbial communities.

Oceanic crust is a massive potential habitat for microbial life on Earth, yet our understanding of this ecosystem is limited due to difficulty in access. In particular, measurements of rates of microbial activity are sparse. We used stable carbon isotope incubations of crustal samples, coupled with functional gene analyses, to examine the potential for carbon fixation on oceanic crust. Both seafloor-exposed and subseafloor basalts were recovered from different mid-ocean ridge and hot spot environments (i.e., the Juan de Fuca Ridge, the Mid-Atlantic Ridge, and the Loihi Seamount) and incubated with 13C-labeled bicarbonate. Seafloor-exposed basalts revealed incorporation of 13C-label into organic matter over time, though the degree of incorporation was heterogeneous. The incorporation of 13C into biomass was inconclusive in subseafloor basalts. Translating these measurements into potential rates of carbon fixation indicated that 0.1–10 nmol C g-1rock d-1 could be fixed by seafloor-exposed rocks. When scaled to the global production of oceanic crust, this suggests carbon fixation rates of 109–1012 g C year-1, which matches earlier predictions based on thermodynamic calculations. Functional gene analyses indicate that the Calvin cycle is likely the dominant biochemical mechanism for carbon fixation in basalt-hosted biofilms, although the reductive acetyl-CoA pathway and reverse TCA cycle likely play some role in net carbon fixation. These results provide empirical evidence for autotrophy in oceanic crust, suggesting that basalt-hosted autotrophy could be a significant contributor of organic matter in this remote and vast environment.

Extracellular enzyme activity and microbial diversity measured on seafloor exposed basalts from Loihi seamount indicate the importance of basalts to global biogeochemical cycling.

ABSTRACT Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (V max) of LAP ranged from 0.47 to 0.90 nmol (g rock)−1 h−1; the V max for AP was 28 to 60 nmol (g rock)−1 h−1. The Km of LAP ranged from 26 to 33 μM, while the Km for AP was 2 to 7 μM. Bacterial communities on Loihi basalts were comprised primarily of Alpha-, Delta-, andGammaproteobacteria, Bacteroidetes, and Planctomycetes. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders Xanthomonadales, Flavobacteriales, and Planctomycetales. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes.