Microbial Growth of a Denitrifying Bacteria at In-situ Hydrothermal Conditions: Implications for Biomineralization at Mid-ocean Ridges

Current interest in subsurface microbiology at hydrothermal vents has resulted in greater attention being given to the characterization of new microbial species and their feedback with the sub-seafloor environment. In response to a need for integrated biogeochemical studies, we have developed a new type of experimental apparatus to study microbial processes at in situ pressure (250 bars) and temperature (70° C). This high-pressure open-system apparatus, modified from HPLC technology, is capable of supplying fresh media, including dissolved gases, to the system, while removing metabolic waste products. Thus, we eliminate the need to artificially elevate certain components in the fluid chemistry and allow continuous growth of the culture, maximizing interaction between the bacteria and mineral surfaces. Preliminary pure culture experiments designed to monitor bacterial effects on mineral surfaces were completed using a new strain of denitrifying thermophilic bacteria, EX-H1. By monitoring aqueous and dissolved gas chemistry, we confirmed its use of the following energy-producing reaction during metabolism: 19H_2(aq) + 3NO₃^- + 5CO_2(aq) + 3H⁺ = N_2(aq) + C₅H₇O₂N + 17H_2(aq) where (C₅H₇O₂N) represents bacterial cell mass. Reaction rates at ambient pressure were on the order of 0.02 mmolal/hr in closed system. Abiotic control experiments at pressure confirmed that mineral dissolution/precipitation reactions were kinetically inhibited at 70° C over 18 hours. Biotic experiments were successful in showing attachment of cells to the quartz fiber substrate during cell growth in the reactor. Due to insufficient residence time of fluid in the reactor (50 minutes), however, metabolic changes in fluid chemistry were slight. Future experiments will incorporate longer residence times to allow comparison of the stoichiometry and rate of the denitrification reaction with theoretical estimates. Also, we will assess the effect of microbial growth on minerals in the reactor to quantify the link between cell growth, metabolism, and the broader geochemical environment.