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. SCOR working group on Hydrothermal energy and the ocean carbon cycle;and Deep Carbon Observatory at CIW).
Currently, there is only limited information on the identity and activity of the microorganisms carrying out CO2-fixation in the natural environment, despite the fact that these organisms form the basis of their respective ecosystems. To this end, we carried out an interdisciplinary, international research program aiming to improve our understanding of autotrophic carbon fixation and its chemical and isotopic signature along environmental gradients in a natural hydrothermal system. To perform these studies, we chose a shallow-water vent system off Paleochori Bay (Milos Island, Greece) as a natural laboratory. In May 2012, we conducted a 12-day long expedition to the shallow water vent sites in Paleochori Bay to sample vent fluids and sediments using SCUBA diving. The scientific party included the three PIs (Foustoukos, Sievert, Vetriani), international collaborators (Le Bris [UPMC, Banyuls-sur-Mer, France], Bühring [Bremen University, Germany]) and members of the respective labs. This expedition was highly successful and resulted in the collection of sediment, fluid and volatile samples from different areas of venting (subaerial and submarine). In addition, we successfully deployed in situ injection core units that are used to identify the microorganisms actively performing autotrophic carbon fixation. The spatial and temporal geochemical dynamics of a shallow-water vent site in Paleochori Bay has been described (Yücel et al., 2013, Chemical Geology 356:11-20). This includes a 6-day long, high-resolution time series, which is the first of its kind for such an environment. This data set provides the framework for assessing the microbial communities inhabiting the sediments along the studied environmental gradient. To this end, we have extracted nucleic acids from the various frozen samples and performed a survey based on the gene coding for the 16S ribosomal RNA specific for Bacteria and Archaea to assess the general diversity. These data are being analyzed in the context of the geochemcial parameters to obtain further insights into the controls of the distribution and abundance of specific microbial groups along the gradient. Already, we can see that both temperature and distance from the center of the vent exert a strong influence. Clear trends are apparent, with Epsilonproteobacteria being the dominant groups in the more strongly hydrothermally influenced sediments and Gammaproteobacteria being more dominant in the periphery. These two groups are known to utilize different pathways for autotrophic carbon fixation, i.e., the reductive TCA cycle vs. the Calvin cycle, which result in distinct stable carbon isotopic composition of the produced biomass. To test the hypothesis that this would be reflected in the stable carbon isotopic composition of the organic carbon along the transect, we analyzed the concentration and the stable carbon isotopic composition of the total organic carbon (TOC) in the sediments. Surprisingly and in contrast to the microbial community analyses, no obvious trend emerged from these data, with TOC being consistently low and the isotopic composition staying constant around -18 permille, in line with the use of the rTCA cycle for autotrophic carbon fixation. Analyzing the stable carbon isotopic composition of specific lipid biomarkers (in collaboration with Dr. Bühring) as well as mRNA analyses of indicative functional genes (in collaboration with Co-PI Vetriani) will provide further insights to explain this pattern. The data generated in this study will allow us to constrain the relationship between autotrophic carbon fixation and the resulting isotopic signatures of biomass and specific biomarker molecules, such as lipids, in a natural system, which does not only have implications for assessing the importance of carbon fixation in extant ecosystems, but will also provide a tool to improve the interpretation of isotopic values in the geological record. This project provided opportunities for research, teaching and mentoring in science and engineering areas for an undergraduate student, research associate, and postdoc. Sievert incorporated aspects of the research into a course entitled 'Marine Microbiology and Biogeochemistry' that he taught in the WHOI/MIT Joint Graduate Program in Oceanography/Applied Ocean Science & Engineering.
