Microbial Physiologies Associated with Hydrogenotrophic Metal/Metalloid Reduction from the Crustal Subseafloor of the Juan de Fuca Ridge Flank.

The crustal subseafloor biosphere is powered via chemosynthesis and has been estimated to inhabit the largest reservoir of microorganisms on Earth. Here we used anoxic subseafloor crustal fluids (65 °C, pH 7.0-7.3) from the Juan de Fuca Ridge (JdFR) flank to enrich and isolate for H2-oxidizing, metal/metalloid-reducing autotrophs. Selective enrichments using H2 as primary electron donor (PED), Fe(III) or ASO42- as terminal electron acceptors (TEA), and CO2 as the carbon (C) source were incubated at temperatures between 60 °C and 80 °C. Two successful Fe(III)-reducing cultures growing at 75 °C and 80 °C were obtained from three initial serial dilutions with Fe(OH)3 as TEA, followed by three serial dilutions with ferric citrate (C6H5FeO7) as TEA. Two successful ASO42—reducing cultures were obtained after six serial dilutions at 60 °C or 75 °C; these two cultures were also able to grow with C6H5FeO7 as TEA. Cellular morphologies and sizes associated with our cultures tend to vary with incubation times from small cocci to elongated bacilli, and/or filamentous cells. Microbial transfers made between two weeks and a month reflect doubling times between one and several days. Additional physiological, metabolic, and phylogenetic studies are ongoing in our efforts to characterize microorganisms capable of primary production in crustal subseafloor environments.