Although they are the base of the food-web in deep sea hydrothermal systems, the growth efficiencies and other essential metabolic characteristics of microorganisms that live off chemical energy from geothermal/mineralogical sources in hydrothermal vent areas in the deep ocean are poorly known. This research uses a novel, flow through, laboratory reactor apparatus to run first-of-a-kind experiments looking at microbial growth rates and metabolic efficiencies at the temperatures, pressures (100 to 250 bars), and chemical conditions found in hydrothermal systems on the deep seafloor. Experiments will target the metabolic processes of two common deep sea vent microorganisms by measuring, in the reactor solutions, nitrogen-bearing respiratory products of nitrate-reducing and hydrogen-oxidizing microbes. Research goals are to determine the role of redox gradients on nitrate reduction and ammonification in hydrothermal systems, use isotopes to determine metabolic rates using kinetic isotope effects, and determine the growth efficiency of mesophilic and thermophilic anaerobic microbes at a range of H2/CO2 molar ratios. Broader impacts of the work include cross-training of a graduate student in two disciplines and laboratories and inclusion of a student from an under-represented minority in research.
In the great depths of the Earth's oceans, forces capable of moving continents sculpt the seafloor and form volcanoes transferring materials and energy to the ocean. It is under these extreme conditions that, by a synergy of chemistry and biology, life thrives in spite of the darkness and sparse nutrients. At pressures and temperatures resembling these environments, we investigated the cycle of nitrogen and carbon through the metabolism of nitrate-reducing anaerobic chemolithoautotrophic bacteria, and we constrained the fate and decomposition of biomolecules. Novel experimental techniques have been developed to grow microbes at pressures similar to those at 4 miles deep in the ocean. In detail, we used Caminibacter mediatlanticus and Thermovibrio ammonificans as model organisms to constrain physiological parameters associated with dissimilatory nitrate reduction to ammonium (DNRA) in deep-sea vent Epsilonproteobacteria and Aquificaceae. We accessed the 15N/14N isotopic signatures of DNRA during growth of nitrate reducers at atmospheric and seafloor pressures. DNRA kinetic constants and cell specific nitrate reduction rates (csNRR) obtained from our data showed that within similar time frames T. ammonificans used 2.5 to 3 times as much nitrate than C. mediatlanticus and it did so ~3 times faster. However, the increased consumption of nitrate in T. ammonificans did not translate into higher growth yield. This is suggestive of either differential efficiencies in energy generating pathways or differential production of organic matter (cell biomass versus extracellular organic material). Nitrogen isotope fractionation for nitrate was similar for both organisms, with discrimination factors of -5 to -6‰ for C. mediatlanticus and -7 to -8‰ for T. ammonificans. Similar experiments performed under high hydrostatic pressure conditions (50 and 200 bar) showed that changes in pressure greatly affected both growth rates and DNRA kinetic rate constants in both microorganisms; however, δ15N discrimination factors for nitrate were not affected. Our high pressure culture experiments place more realistic constraints on the growth efficiency and metabolic rates of anaerobic nitrate reducing chemolithoautotrophs than those estimated from serum vial-1 atm experiments. We also focused on understanding the fate of biomolecules under geothermal conditions. The stability of amino acids at elevated temperatures and pressures is of great interest for the cycling of C and N within the crust and the overlying oceanic water column. We conducted a series of hydrothermal experiments to investigate the effect of temperature, pH and redox on the kinetics of glutamic acid decomposition at 100-250 °C and pressures of ~ 150 bar. Overall, the experimental conditions of T, P, pH or H2 are similar to those in some natural ultramafic-hosted hydrothermal systems such as the Lost City hydrothermal vents at 30° N on the Mid-Atlantic Ridge. In these experiments, we followed the reaction pathway of glutamic acid decomposition, and documented the metastable equilibria conditions (reducing, alkaline) that enhance the stability of this molecule at elevated temperatures. In short, results support the important role of H2 on reducing the extent of glutamic acid decomposition. This enhanced stability of amino acids under H2-enriched conditions is expected to play an important role on supporting the microbial ecosystems at deep-sea hydrothermal environments through heterotrophic metabolism. This award supported the development of procedures and techniques to culture microorganisms under high hydrostatic pressures by utilizing batch and continuous culture approaches. For example, the CIW high-pressure continuous culture system allows for experimentation of microbial processes associated with a broad array of unexplored environmental regimes. The continuous culture bioreactor is capable of withstanding temperatures ranging from 25 to 120 °C and pressures up to 69 MPa. Our experimental setup has been fully integrated with isobaric gas tight samplers that collect and transfer natural microbial communities from deep-sea vents under high pressure conditions. This technological advancement, which allows the onsite incubation of microorganism without depressurization or under simulated in-situ physical (temperature, pressure) and chemical conditions, will help determine the relevance of experimental pressure-based physiological responses to their in-situ growth in nature. This project involved the participation of two undergraduate students from George Mason University, a graduate student from Johns Hopkins University and a postdoctoral fellow. Opportunities for training and professional development were given to women and minority students/researchers. We provided tours of our hydrothermal laboratory to visitors, school teachers and undergraduate students from the George Mason University. This project was highlighted in the 2nd USA Science and Engineering Festival in Washington (DC), and in 'Chemical & Engineering News' with a series of on-site interviews and lab demonstrations. We anticipate that the experimental protocols developed for high-pressure microbial culturing will have practical implications for: i) the synthesis of microbial products in industrial biotechnology, ii) improving biofuel/ bioremediation procedures and iii) contributing to high-pressure food processing research.
