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. Broader Impacts. This is an interdisciplinary and collaborative effort between US and foreign institutions, creating unique opportunities for networking and to foster international collaborations. This will also benefit the involved students (1 graduate, several undergraduates) and a postdoc. The PIs have been involved in several educational and public outreach activities over the years that have reached literally millions of individuals. Finally, the project fits with the focus of a number of multi-disciplinary and international initiatives, in which PIs are active members (e.g. the SCOR working group on Hydrothermal energy and the ocean carbon cycle; and the Deep Carbon Observatory at CIW).

Project Report

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. The group from Geophysical Laboratory, Carnegie Institution of Washington was responsible for processing the fluid and gas samples. At the site, we performed gas chromatography to determine the concentration of dissolved gases (methane, C2-C6 alkanes, H2, CO2, H2S) collected by gas-tight syringes. Samples returned to the Geophysical Lab were analyzed to determine dissolved concentrations of: dissolved inorganic carbon (DIC), organic acids and a range of major anions/cations species (e.g. SO42-, PO42-, NO3-, Cl-, Na+, K+, Ca2+, Mg2+) including trace elements and metals. Fluids were analyzed for metal and trace element concentrations at the MC-ICP-MS facility of Prof. Michael Bizimis at the University of South Carolina. In this project, we shared our samples with Dr. Bizimis and encouraged him to proceed with any further analysis that may suit his research program. We also determined the d13C composition of dissolved inorganic carbon at the stable isotope facility of the Geophysical Lab. Data have been released to the public through our Data Depository ( Furthermore, sediments and minerals collected from the submarine and subaerial vents are to be analyzed and studied as part of Joe Maloney’s M.Sc. Thesis. This is a collaborative project with Dr. Julia Nord, who is the academic advisor of Maloney at George Mason University. An important contribution to the discipline is the isolation and?characterization of a novel thermophilic Fe(III)-reducing microorganisms; that overall have been very scarcely described in microbial communities from either deep-sea or shallow-water hydrothermal vent sites. Fe(III)-reducing bacteria play a key role on the cycling of Fe between the oceanic crust and the overlying hydrosphere. This microorganism has been isolated and phylogenetically/physiologically characterized by Dr. Perez-Rodriguez (GL Postdoctoral Fellow) and Matthew Rawls (undergraduate student, George Mason University). This appears to be a thermophilic Fe(III) reducing species of Deltaproteobacteria named strain MAG-PB1. This strain was isolated from a marine sediment core located at 8 meters water depth and with a pore fluids temperature of 26oC. However, this anaerobic Fe(III) reducing bacterium attains optimum growth temperature of 65oC, which highlights the adaptation of these organisms to the strong thermal gradients of the hydrothermal fluids circulating in the shallow-water sedimentary seafloor. The organism was phylogenetically characterized revealing a ~ 97% similarity in the 16S rRNA gene to the closest relative Deferrisoma camini. D. camini is a thermophilic, anaerobic, Fe(III) reducing bacterium isolated from a deep-sea hydrothermal vent chimney at the Eastern Lau Spreading Centre, in the Pacific Ocean. Interestingly, the MAG-PB1 is directly linked to a microorganism found at significantly greater depths (2150 m) than the shallow-water marine sediments of Milos Island. However, MAG-PB1 can function as hyperthermophile (~ 70oC) and adopt autotrophic metabolism utilizing H2 as electron donor, while the D. camini is a strict heterotrophic and thermophilic Deltaproteobacteria. Research conducted involves the participation of undergraduate (Rawls), graduate (Maloney) students and postdoctoral fellows (Perez-Rodriguez). Dr. Perez-Rodriguez has been trained in aqueous geochemistry, geochemical analysis, and in sampling/processing vent fluid samples from shallow-water hydrothermal vents. Her contribution in the field was instrumental. She has also been mentoring Matthew Rawls on microbiology. Rawls worked on the project during the Fall of 2013, as part of a Biology undergrad research course at GMU, where Dr. Foustoukos was his academic advisor. Joe Maloney is involved in the mineralogical analysis of sediments and mineral deposits collected at Milos by using the USGS and GL/DTM facilities, while he has been mentored in thermodynamics and phase equilibria by Dr. Foustoukos. Finally the scientific objectives and outcome of this project have been incorporated as teaching material in the BIOL-435/CHEM-500/GEOL-500/GEO-315 Biogeochemistry class that Dr. Foustoukos delivers at the George Mason University.

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Division of Ocean Sciences (OCE)
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David L. Garrison
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Carnegie Institution of Washington
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