AbstractIn this study, we integrated geochemical measurements, microbial diversity surveys and physiological characterization of laboratory strains to investigate substrate-attached filamentous microbial biofilms at Tor Caldara, a shallow-water gas vent in the Tyrrhenian Sea. At this site, the venting gases are mainly composed of CO2 and H2S and the temperature at the emissions is the same as that of the surrounding water. To investigate the composition of the total and active fraction of the Tor Caldara biofilm communities, we collected established and newly formed filaments and we sequenced the 16S rRNA genes (DNA) and the 16S rRNA transcripts (cDNA). Chemoautotrophic sulfur-oxidizing members of the Gammaproteobacteria (predominantly Thiotrichales) dominate the active fraction of the established microbial filaments, while Epsilonproteobacteria (predominantly Sulfurovum spp.) are more prevalent in the young filaments. This indicates a succession of the two communities, possibly in response to age, sulfide and oxygen concentrations. Growth experiments with representative laboratory strains in sulfide gradient medium revealed that Sulfurovum riftiae (Epsilonproteobacteria) grew closer to the sulfide source than Thiomicrospirasp. (Gammaproteobacteria, Thiotrichales). Overall, our findings show that sulfur-oxidizing Epsilonproteobacteria are the dominant pioneer colonizers of the Tor Caldara biofilm communities and that Gammaproteobacteria become prevalent once the community is established. This succession pattern appears to be driven - among other factors - by the adaptation of Epsilon- and Gammaproteobacteria to different sulfide concentrations.
AbstractThe goal of this proposal is to gain insight into the metabolic and molecular adaptations of deep-sea and subseafloor bacteria exposed to crustal pressures (200-500 atm). The model organism will be an anaerobic nitrate-reducing bacterium (Nautilia strain PV-1) isolated from subseafloor fluids discharged from an active vent at the East Pacific Rise (2500 m depth). The microorganism was retrieved from a series of shipboard continuous culturing incubations conducted aboard the R/V Atlantis. We will investigate the rates of carbon fixation and anaerobic respiration of PV-1 by using our high-pressure chemostat. Further, we propose to investigate the synthrophic growth kinetics of a co-culture composed of the fermentative, hydrogen-producing piezophile, Marinitoga piezophila, and the hydrogenotrophic Nautilia strain PV-1. To our knowledge this would the first experimental study to describe the effects of pressure on chemolithoautotrophic bacteria that were sampled from deep-sea hydrothermal vents and cultured at seafloor pressure conditions prior to their isolation. We anticipate that understanding the pressure adaptations of this strain PV-1, as well as its interactions with heterotrophic bacteria with whom it shares its ecological niche, will provide a unique opportunity to define the spatial and temporal variability of the subseafloor biosphere in the Earth's oceanic crust.
AbstractNO3- reduction is a metabolism that is widespread among ε-Proteobacteria and Aquificae, two abundant classes of microorganisms found at deep-sea vents. In this study, we used Sulfurovum lithotrophicum, Caminibacter mediatlanticus and Thermovibrio ammonificans as representatives of these groups to study ecophysiological, metabolic and biogeochemical parameters associated with chemolithoautotrophic NO3- reduction under different temperature regimes. We observed that while S. lithotrophicum and C. mediatlanticus achieved higher cell densities than T. ammonificans, the overall NO3- consumption by the latter was on average ∼ 9 and ∼ 5 times faster on a per cell basis, respectively. Comparison with previously published data from other cultured vent ε-Proteobacteria and Aquificae suggests that the rate-yield trade-offs observed in our experiments are generally conserved between these two groups in line with their ecophysiologies. Kinetic isotope effects of N from NO3- reduction were 9.6 ± 2.7 ‰ for S. lithotrophicum, 6.4 ± 0.7 ‰ for C. mediatlanticus and 8.8 ± 0.6 ‰ for T. ammonificans. Our results help evaluate how metabolic partitioning between growth efficiency and reaction kinetics during chemolithoautotrophic NO3- reduction affect the concentration and isotope composition of N compounds at deep-sea hydrothermal vents.
AbstractWe report measurements of resolved 12CH2D2 and 13CH3D at natural abundances in a variety of methane gases produced naturally and in the laboratory. The ability to resolve 12CH2D2 from 13CH3D provides unprecedented insights into the origin and evolution of CH4. The results identify conditions under which either isotopic bond order disequilibrium or equilibrium are expected. Where equilibrium obtains, concordant Δ12CH2D2 and Δ13CH3D temperatures can be used reliably for thermometry. We find that concordant temperatures do not always match previous hypotheses based on indirect estimates of temperature of formation nor temperatures derived from CH4/H2 D/H exchange, underscoring the importance of reliable thermometry based on the CH4 molecules themselves. Where Δ12CH2D2 and Δ13CH3D values are inconsistent with thermodynamic equilibrium, temperatures of formation derived from these species are spurious. In such situations, while formation temperatures are unavailable, disequilibrium isotopologue ratios nonetheless provide novel information about the formation mechanism of the gas and the presence or absence of multiple sources or sinks. In particular, disequilibrium isotopologue ratios may provide the means for differentiating between methane produced by abiotic synthesis vs. biological processes. Deficits in 12CH2D2 compared with equilibrium values in CH4 gas made by surface-catalyzed abiotic reactions are so large as to point towards a quantum tunneling origin. Tunneling also accounts for the more moderate depletions in 13CH3D that accompany the low 12CH2D2 abundances produced by abiotic reactions. The tunneling signature may prove to be an important tracer of abiotic methane formation, especially where it is preserved by dissolution of gas in cool hydrothermal systems (e.g., Mars). Isotopologue signatures of abiotic methane production can be erased by infiltration of microbial communities, and Δ12CH2D2 values are a key tracer of microbial recycling.
|Project Title||Autotrophic carbon fixation at a shallow-water hydrothermal system: Constraining microbial activity, isotopic and geochemical regimes|
|Acronym||Hydrothermal Autotrophic Carbon Fixation|
|Created||January 8, 2014|
|Modified||June 3, 2015|
In this project we studied the shallow-water hydrothermal vent sites at Milos Island (Greece) to better understand the extent of autotrophic carbon fixation and its chemical and isotopic signature along environmental (redox/thermal) gradients. This was a 12-day long expedition (May 18 to 30, 2012) to sample vent fluids, gases and retrieve sediment cores at Paleochori Bay by using SCUBA diving at 8-10 m depth. In addition to the submarine vent sites, two subaerial locations of venting were identified at 36o 40' 28"N - 24o 31' 14" E and 36o 40' 25" N - 24o 30' 44" E. Both the subaerial and submarine sites are located on the same fracture zone that likely controls the hydrothermal circulation of evolved meteoritic water and seawater within the magmatic zone of Milos Island. To this end, the geochemistry of the fluids and gases emitted from subaerial sites provide important information towards identifying the linkage between the subaerial and submarine magmatic activity and provide insights on the metabolic functions (e.g. H2 oxidation, Fe(III) reduction, C and S cycling) of the subsurface microbial community.
Currently, there is only limited information on the identity and activity of the microorganisms carrying out CO2-fixation in situ, despite the fact that these organisms form the basis of their respective ecosystems. Representatives that are able to grow autotrophically are known to exist in almost all major groups of prokaryotes, and these organisms play essential roles in ecosystems by providing a continuous supply of organic carbon for heterotrophs. Microorganisms present in extreme environments utilize CO2- fixation pathways other than the Calvin-Benson-Bassham (CBB) cycle. At present, five alternative autotrophic CO2 fixation pathways are known. Different carbon fixation pathways result in distinct isotopic signatures of the produced biomass due to the isotopic discrimination between light (12C) and heavy (13C) carbon by the carboxylating enzymes. Thus, inferences about the carbon fixation pathway predominantly utilized by the microbial community can also be made based on the stable carbon isotopic composition of the organic matter, in extant systems as well as in the geological record. However, at present little is known about the systematics and extents of fractionation during carbon fixation by prokaryotic organisms, and to our knowledge no studies exist that have systematically studied the relationship between the operation of different carbon fixation pathways and how this is reflected in the stable carbon isotopic composition in a natural system. This is a 2-year interdisciplinary, international research program that employs a powerful combination of cutting-edge research tools aiming to improve our understanding of autotrophic carbon fixation and its chemical and isotopic signature along environmental gradients in a natural hydrothermal system. The following hypotheses are addressed:
1. The diversity of microorganisms present along a thermal and redox gradient, and rates of CO2 fixation, will reflect adaptation to in situ temperatures and geochemical conditions
2. Microorganisms utilizing the CBB cycle for autotrophic CO2-fixation will represent a smaller percentage of the chemolithoautotrophic community at higher temperatures, where microorganisms utilizing alternative CO2-fixation pathways dominate
3. Isotopic values of biomass and specific biomarker molecules will vary along a thermal and redox gradient from zones characterized by a higher hydrothermal fluid flux and thus higher temperatures to the surrounding, cooler areas, corresponding to the physiology of the microorganisms utilizing different pathways for carbon fixation
The PIs will use a multidisciplinary approach to delineate the relative contribution of the different carbon fixation pathways along an environmental gradient by combining metagenomic analyses coupled with: 1) an assessment of the frequency and the expression of specific key genes involved in carbon fixation, and 2) with the measurement of carbon fixation rates. These data will be integrated with the determination of stable C isotopic composition of biomass, DIC, and specific hydrocarbons/lipids. Due to its easy accessibility, well-established environmental gradients, and extensive background information, the shallow-water vents off Milos (Greece) will be used as a natural laboratory to perform these studies.
Intellectual Merit. The data generated in this study will allow constraints on the relationship between autotrophic carbon fixation and the resulting isotopic signatures of biomass and specific biomarker molecules (e.g. CH4, C2+ alkanes, lipids) in a natural system. This has implications for assessing the importance of carbon fixation in extant ecosystems, and it will also provide a tool to improve the interpretation of isotopic values in the geological record.
|Costantino Vetriani||Rutgers University||Co-Principal Investigator|
|Stefan M. Sievert||Woods Hole Oceanographic Institution (WHOI)||Co-Principal Investigator|
|Dionysis I. Foustoukos||Carnegie Institution for Science (CIS)||Co-Principal Investigator|
|Project Title||An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents|
|Acronym||Microbial Communities at Deep-Sea Vents|
|Created||June 11, 2012|
|Modified||June 11, 2012|
Deep-sea hydrothermal vents, first discovered in 1977, are poster child ecosystems where microbial chemosynthesis rather than photosynthesis is the primary source of organic carbon. Significant gaps remain in our understanding of the underlying microbiology and biogeochemistry of these fascinating ecosystems. Missing are the identification of specific microorganisms mediating critical reactions in various geothermal systems, metabolic pathways used by the microbes, rates of the catalyzed reactions, amounts of organic carbon being produced, and the larger role of these ecosystems in global biogeochemical cycles. To fill these gaps, the investigators will conduct a 3-year interdisciplinary, international hypothesis-driven research program to understand microbial processes and their quantitative importance at deep-sea vents. Specifically, the investigators will address the following objectives: 1. Determine key relationships between the taxonomic, genetic and functional diversity, as well as the mechanisms of energy and carbon transfer, in deep-sea hydrothermal vent microbial communities. 2. Identify the predominant metabolic pathways and thus the main energy sources driving chemoautotrophic production in high and low temperature diffuse flow vents. 3. Determine energy conservation efficiency and rates of aerobic and anaerobic chemosynthetic primary productivity in high and low temperature diffuse flow vents. 4. Determine gene expression patterns in diffuse-flow vent microbial communities during attachment to substrates and the development of biofilms.
Integration: To address these objectives and to characterize the complexity of microbially-catalyzed processes at deep-sea vents at a qualitatively new level, we will pursue an integrated approach that couples an assessment of taxonomic diversity using cultivation-dependent and -independent approaches with methodologies that address genetic diversity, including a) metagenomics (genetic potential and diversity of community), b) single cell genomics (genetic potential and diversity of uncultured single cells), c) meta-transcriptomics and -proteomics (identification and function of active community members, realized potential of the community). To assess function and response to the environment, these approaches will be combined with 1) measurement of in situ rates of chemoautotrophic production, 2) geochemical characterization of microbial habitats, and 3) shipboard incubations under simulated in situ conditions (hypothesis testing under controlled physicochemical conditions). Network approaches and mathematical simulation will be used to reconstruct the metabolic network of the natural communities. A 3-day long project meeting towards the end of the second year will take place in Woods Hole. This Data Integration and Synthesis meeting will allow for progress reports and presentations from each PI, postdoc, and/or student, with the aim of synthesizing data generated to facilitate the preparation of manuscripts.
Intellectual Merit. Combining the community expression profile with diversity and metagenomic analyses as well as process and habitat characterization will be unique to hydrothermal vent microbiology. The approach will provide new insights into the functioning of deep-sea vent microbial communities and the constraints regulating the interactions between the microbes and their abiotic and biotic environment, ultimately enabling us to put these systems into a quantitative framework and thus a larger global context.
Broader Impacts. This is an interdisciplinary and collaborative effort between 4 US and 4 foreign institutions, creating unique opportunities for networking and fostering international collaborations. This will also benefit the involved students (2 graduate, several undergraduate) and 2 postdoctoral associates. This project will directly contribute to many educational and public outreach activities of the involved PIs, including the WHOI Dive & Discover program; single cell genomics workshops and Cafe Scientifique (Bigelow); REU (WHOI, Bigelow, CIW); COSEE and RIOS (Rutgers), and others. The proposed research fits with the focus of a number of multidisciplinary and international initiatives, in which PIs are active members (SCOR working group on Hydrothermal energy and the ocean carbon cycle, http://www.scorint. org/Working_Groups/wg135.htm; Deep Carbon Observatory at CIW, https://dco.gl.ciw.edu/; Global Biogeochemical Flux (GBF) component of the Ocean Observatories Initiative (OOI), http://www.whoi.edu/GBF-OOI/page.do?pid=41475)
|Stefan M. Sievert||Woods Hole Oceanographic Institution (WHOI)||Lead Principal Investigator|
|Costantino Vetriani||Rutgers University||Principal Investigator|
|Dionysis I. Foustoukos||Carnegie Institution for Science (CIS)||Principal Investigator|
|Ramunas Stepanauskas||Bigelow Laboratory for Ocean Sciences||Principal Investigator|
|Craig Taylor||Woods Hole Oceanographic Institution (WHOI)||Co-Principal Investigator|
|Jeffrey S. Seewald||Woods Hole Oceanographic Institution (WHOI)||Co-Principal Investigator|
|Nadine Le Bris||Laboratoire d'Écogéochimie des Environnements Benthiques (LECOB)||International Collaborator|
|Niculina Musat||Max Planck Institute for Marine Microbiology (MPI)||International Collaborator|
|Thomas Schweder||University of Greifswald||International Collaborator|
|Fengping Wang||Shanghai Jiao Tong University (SJTU)||International Collaborator|
A novel thermophilic, anaerobic, mixotrophic bacterium, designated strain MAG-PB1T, was isolated from a shallow-water hydrothermal vent system in Palaeochori Bay off the coast of the island of Milos, Greece. The cells were Gram-negative, rugose, short rods, approximately 1.0 μm long and 0.5 μm wide. Strain MAG-PB1T grew at 30–70 °C (optimum 60 °C), 0–50 g NaCl l− 1 (optimum 15–20 g l− 1) and pH 5.5–8.0 (optimum pH 6.0). Generation time under optimal conditions was 2.5 h. Optimal growth occurred under chemolithoautotrophic conditions with H2 as the energy source and CO2 as the carbon source. Fe(III), Mn(IV), arsenate and selenate were used as electron acceptors. Peptone, tryptone, Casamino acids, sucrose, yeast extract, d-fructose, α-d-glucose and ( − )-d-arabinose also served as electron donors. No growth occurred in the presence of lactate or formate. The G+C content of the genomic DNA was 66.7 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that this organism is closely related to Deferrisoma camini, the first species of a recently described genus in the Deltaproteobacteria. Based on the 16S rRNA gene phylogenetic analysis and on physiological, biochemical and structural characteristics, the strain was found to represent a novel species, for which the name Deferrisoma palaeochoriense sp. nov. is proposed. The type strain is MAG-PB1T ( = JCM 30394T = DSM 29363T).