Subground Animalcule Retrieval (SUGAR) Project, Extremobiosphere Research Center (XBR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan,¹ Research Center for Deep Geological Environments, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba 305-8567, Japan,² Department of Earth Science, University of Bergen, Allégaten 41, 5007 Bergen, Norway,³ Department of Marine Sciences, University of North Carolina at Chapel Hill, 12-7 Venable Hall CB #3300, Chapel Hill, North Carolina 27599,⁴ Institut für Chemie und Biologie Des Meeres (ICBM), Carl von Ossietzky Universitát Oldenburg, Carl von Ossietzky Strasse 9-11, 26111 Oldenburg, Germany,⁵ Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan,⁶ Global Undersea Research Unit, University of Alaska Fairbanks, P.O. Box 475, Moss Landing, California 95039⁷

Received 30 May 2006/ Accepted 8 August 2006

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.

Published ahead of print on 21 August 2006.

Supplemental material for this article may be found at http://aem.asm.org/.