Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean.
The isotopic composition of lipid biomarkers and biomass in sedimentary environments are widely used to infer microbial metabolisms and constrain carbon cycling processes. It has been observed that metabolic energy availability in the form of H2 impacts the stable carbon isotopes of CH4 during hydrogenotrophic methanogenesis, but it is unknown whether this relationship extends to lipids and amino acids. Since lipids and amino acids are long-lived, they can be used to reconstruct past conditions over geologic timescales. Therefore, the controls on their isotopic signatures are important to constrain to better interpret past environments.
In this study, the isotopic distributions of carbon metabolized and reduced by the hyperthermophile Methanocaldococcus jannaschii were quantified following growth at 82 °C in a chemostat with high (80–83 µM) and low (15–27 µM) H2 concentrations. As has been shown previously, the stable carbon isotope fractionation factors for CH4 were >15‰ larger in low H2 experiments than in high H2 experiments. Lipid biomarkers and amino acids were similarly impacted with approximately 10‰ larger fractionation factors under low H2 conditions. The increase in fractionation factors can be related to the lower availability of thermodynamic energy, suggesting that even larger fractionation factors would be observed in methanogens living close to their threshold energy needs, as they do in most environments. The resulting isotopic signatures of long-lived lipid biomarkers synthesized by hydrogenotrophic methanogens may become as ‘superlight’ as those synthesized by archaea carrying out the anaerobic oxidation of methane. These results help to describe the underlying mechanisms that determine the isotopic composition of long-lived biomarkers and provide constraints for interpreting these signatures in the environment.
The size and biogeochemical impact of the subseafloor biosphere in oceanic crust remain largely unknown due to sampling limitations. We used reactive transport modeling to estimate the size of the subseafloor methanogen population, volume of crust occupied, fluid residence time, and nature of the subsurface mixing zone for two low-temperature hydrothermal vents at Axial Seamount. Monod CH4 production kinetics based on chemostat H2 availability and batch-culture Arrhenius growth kinetics for the hyperthermophile Methanocaldococcus jannaschii and thermophile Methanothermococcus thermolithotrophicus were used to develop and parameterize a reactive transport model, which was constrained by field measurements of H2, CH4, and metagenome methanogen concentration estimates in 20–40 °C hydrothermal fluids. Model results showed that hyperthermophilic methanogens dominate in systems where a narrow flow path geometry is maintained, while thermophilic methanogens dominate in systems where the flow geometry expands. At Axial Seamount, the residence time of fluid below the surface was 29–33 h. Only 1011 methanogenic cells occupying 1.8–18 m3 of ocean crust per m2 of vent seafloor area were needed to produce the observed CH4 anomalies. We show that variations in local geology at diffuse vents can create fluid flow paths that are stable over space and time, harboring persistent and distinct microbial communities.
Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80-83 μM) and low (15-27 μM) aqueous H2 concentrations and in co-culture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in co-culture. However, cell yield (cells produced per mole of CH4 produced) increased six-fold when M. jannaschii was grown in co-culture relative to monoculture. Relative to high H2concentrations, isotopic fractionation of CO2 to CH4 (ϵCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in co-culture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in co-culture growth. In co-culture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats.
| Project Title | Modeling how virus-microbe interactions influence carbon flow at a deep-sea volcano |
|---|---|
| Acronym | Subseafloor Life |
| URL | http://www.bco-dmo.org/project/711112 |
| Created | August 1, 2017 |
| Modified | August 3, 2017 |
Abstract:
In order to break open the black box of deep-sea hydrothermal vent microbiology and take our understanding of subseafloor microbial processes and the carbon cycle to a new level, we propose to investigate autotrophy in the rocky subseafloor using molecular biological, cultivation, and geochemical techniques at Axial Seamount, an active submarine volcano that will soon be part of a seafloor cabled observatory. In line with the Moore Foundation’s MMI objectives, this project will address the functional roles of various autotrophic subseafloor microbial community members across temperature and metabolism classifications; their relationships with each other, with viruses, and with other sources of syntrophic metabolic energy; and their collective impact on carbon biogeochemistry as dictated by environmental gradients in temperature and geochemistry. Of particular importance is the inclusion of seafloor experimentation at Axial, which will not only yield data on microbial activity and carbon transformations in situ, but will also set the stage for integration of a microbial instrument at the Axial cabled observatory. Our comprehensive suite of land-based, shipboard, and in situ analyses will yield cross-disciplinary advances in our understanding of the microbial ecology and geochemistry of carbon cycling in the subseafloor biosphere at mid-ocean ridges.
| Name | Affiliation | Role |
|---|---|---|
| Julie A. Huber | Woods Hole Oceanographic Institution (WHOI) | Lead Principle Investigator |
| David A. Butterfield | National Oceanic and Atmospheric Administration (NOAA-PMEL) | Co-Principal Investigator |
| James F. Holden | University of Massachusetts Dartmouth (UMass Dartmouth) | Co-Principal Investigator |
| Jullie Zeigler Allen | J. Craig Venter Institute (JCVI) | Co-Principal Investigator |
| Joseph J. Vallino | Marine Biological Laboratory (MBL) | Co-Principal Investigator |
Articles in this special issue of Oceanography represent a compendium of research that spans the disciplinary and thematic breadth of the National Science Foundation’s Ridge 2000 Program, as well as its geographic focal points. The mid-ocean ridge (MOR) crest is where much of Earth’s volcanism is focused and where most submarine volcanic activity occurs. If we could look down from space at our planet with the ocean drained, the MOR’s topography and shape, along with its intervening fracture zones, would resemble the seams on a baseball, with the ocean basins dominating our planetary panorama. The volcanic seafloor is hidden beneath the green-blue waters of the world’s ocean, yet therein lie fundamental clues to how our planet works and has evolved over billions of years, something that was not clearly understood 65 years ago—witness the following quote from H.H. Hess (1962) in his essay on “geopoetry” and commentary on J.H.F. Umbgrove’s (1947) comprehensive summary of Earth and ocean history:
The birth of the oceans is a matter of conjecture, the subsequent history is obscure, and the present structure is just beginning to be understood. Fascinating speculation on these subjects has been plentiful, but not much of it predating the last decade [the 1950s] holds water.
