Close association of active nitrifiers with Beggiatoa mats covering deep-sea hydrothermal sediments Matthias Winkel, 1 Dirk de Beer, 1 Gaute Lavik, 1 Jörg Peplies 2 and Marc Mußmann 1 * 1 Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany. 2 Ribocon GmbH, Fahrenheitstrasse 1, 28359 Bremen, Germany. Summary Hydrothermal sediments in the Guaymas Basin are covered by microbial mats that are dominated by nitrate-respiring and sulphide-oxidizing Beggiatoa. The presence of these mats strongly correlates with sulphide- and ammonium-rich fluids venting from the subsurface. Because ammonium and oxygen form opposed gradients at the sediment surface, we hypothesized that nitrification is an active process in these Beggiatoa mats. Using biogeochemical and molecular methods, we measured nitrification and determined the diversity and abundance of nitrifiers. Nitrification rates ranged from 74 to 605 μmol N l −1 mat day −1 , which exceeded those previously meas- ured in hydrothermal plumes and other deep-sea habitats. Diversity and abundance analyses of archaeal and bacterial ammonia monooxygenase subunit A genes, archaeal 16S ribosomal RNA pyrotags and fluorescence in situ hybridization con- firmed that ammonia- and nitrite-oxidizing microor- ganisms were associated with Beggiatoa mats. Intriguingly, we observed cells of bacterial and poten- tial thaumarchaeotal ammonia oxidizers attached to narrow, Beggiatoa-like filaments. Such a close spatial coupling of nitrification and nitrate respiration in mats of large sulphur bacteria is novel and may facilitate mat-internal cycling of nitrogen, thereby reducing loss of bioavailable nitrogen in deep-sea sediments. Introduction Deep-sea hydrothermal fluids usually contain reduced electron donors such as sulphide, hydrogen and metal ions that fuel microbial chemoautotrophy (Jannasch and Mottl, 1985). In contrast, the significance of ammonium for chemoautotrophy at hydrothermal vent systems is largely unknown. Ammonium concentration in end-member fluids range from 0.07 to 7 mM in some basaltic and ultramafic (Lilley et al., 1993; Orcutt et al., 2011; Bourbonnais et al., 2012a), and up to 16 mM in sedimented hydrothermal vent systems (Von Damm et al., 1985; Nunoura et al., 2010). When mixing with sea water at the seafloor, ammo- nium ascends the water column in buoyant hydrothermal plumes and stimulates the aerobic oxidation of ammonia by bacteria (Lam et al., 2004; 2008) or archaea (Baker et al., 2012; Lesniewski et al., 2012). Because hydrother- mal fluids can contain significant amounts of ammonium, it has been proposed to be an important energy source not only in hydrothermal plumes, but also in the direct vicinity of hydrothermal vents (Nakagawa and Takai, 2008). However, nitrification, the oxidation of ammonia to nitrite and further to nitrate, is largely unexplored at hydro- thermal vents. Isotope composition in hydrothermal fluids from the Juan de Fuca Ridge suggested that nitrate could be regenerated from nitrification by subsurface microbial communities (Bourbonnais et al., 2012b). Furthermore, the detection of genes from bacterial nitrifiers in hydro- thermally influenced sediments and chimneys (Davis et al., 2009; Kato et al., 2009; Wang et al., 2009; Nunoura et al., 2010) indicated a genetic potential for nitrification. Nevertheless, there is no study that determined the nitri- fication potential and the involved microorganisms in benthic compartments of hydrothermal systems. The end-member fluids of the sediment-covered hydro- thermal system of the Guaymas Basin, Gulf of California, contain up to 16 mM ammonium (Von Damm et al., 1985). These fluids mix with sea water below the sediment surface and cool down before entering the water column, which allows the formation of conspicuous microbial mats up to several centimetres thick. These mats are domi- nated by filamentous, sulphide-oxidizing Beggiatoa. Their horizontal distribution is tightly coupled to subsurface pro- cesses (Lloyd et al., 2010) and indicates intense venting of sulphide- and ammonium-rich fluids in the Guaymas Basin system (Jannasch et al., 1989; Gundersen et al., 1992; Magenheim and Gieskes, 1992; McKay et al., 2012). Mats of large sulphur bacteria are generally considered as hot spots of nitrogen cycling (Prokopenko Received 16 May, 2013; accepted 20 October, 2013. *For correspondence. E-mail mmussman@mpi-bremen.de; Tel. (+49) 421202 8936; Fax (+49) 421202 8790. Environmental Microbiology (2014) 16(6), 1612–1626 doi:10.1111/1462-2920.12316 © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd