Ann Microbiol (2014) 64:1691–1705 DOI 10.1007/s13213-014-0813-3 ORIGINAL ARTICLE Diversity and abundance of Bacteria and nirS-encoding denitrifiers associated with the Juan de Fuca Ridge hydrothermal system Annie Bourbonnais & S. Kim Juniper & David A. Butterfield & Rika E. Anderson & Moritz F. Lehmann Received: 9 August 2013 /Accepted: 10 January 2014 /Published online: 4 February 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Denitrification, which results in the loss of bioavail- communities were diverse and dominated by members of the able nitrogen—an essential macronutrient for all living organ- ε-andγ-proteobacteria, including taxonomic groups contain- isms—may potentially affect chemosynthetic primary produc- ing known denitrifiers. Assemblages of denitrifiers that could tion in hydrothermal vent ecosystems where sub-oxic condi- be evaluated by nirS gene sequence comparisons showed low tions favorable to denitrification are common. Here we de- diversity. The single nirS sequence shared by the two loca- scribe the diversity and abundance of denitrifying bacteria in tions, affiliated with a γ-proteobacteria isolated from estuarine the subsurface biosphere at Axial Volcano and the Endeavour sediments (Pseudomonas sp. BA2), represented more than Segment on the Juan de Fuca Ridge using a combination of half of all sequences recovered when clustered at 97 % iden- quantitative polymerase chain reaction assays, and small sub- tity. All other nirS sequences clustered into different taxonom- unit ribosomal RNA (SSU or 16S rRNA) pyrotag and nitrite ic groups, indicating important differences in denitrifier com- reductase (nirS) clone library sequencing methods. Bacterial munity membership between the two sites. Total nirS gene abundance was at least two orders of magnitude lower than 16S rRNA abundance. Overall, our results demonstrate that Electronic supplementary material The online version of this article (doi:10.1007/s13213-014-0813-3) contains supplementary material, the diversity and abundance of the nirS gene-containing bac- which is available to authorized users. terial community are rather low, as might be expected under A. Bourbonnais : S. K. Juniper the extreme conditions encountered in the subsurface bio- School of Earth and Ocean Sciences, University of Victoria, Victoria, sphere of hydrothermal vent systems, and do not correlate BC V8P 5C2, Canada clearly with any environmental variables investigated (i.e., − + pH, temperature, and H2S, NO3 ,NH4 concentrations). D. A. Butterfield Joint Institute for the Study of the Atmosphere and Ocean, University of Washington, Seattle, WA 98105-5672, USA Keywords nirS genes . 16S rRNA genes . Denitrifying bacteria . Diffuse hydrothermal vent fluids . Juan de Fuca D. A. Butterfield Pacific Marine Environmental Laboratory, National Oceanic and Ridge Atmospheric Administration, Seattle, WA 98115, USA R. E. Anderson School of Oceanography and Astrobiology, University of Introduction Washington, Seattle, WA 98195-7940, USA Nitrogen (N) is an essential macronutrient for all organisms, M. F. Lehmann and its availability often limits primary productivity in marine Department of Environmental Sciences, University of Basel, 4056 Basel, Switzerland environments (Mulholland and Lomas 2008). At seafloor hydrothermal vents and beneath the seafloor in the subsurface Present Address: biosphere, denitrification, which results in the loss of bioavail- * A. Bourbonnais ( ) able N [e.g., nitrate (NO −)], has the potential to affect at least School for Marine Science and Technology (SMAST), University of 3 Massachusetts Dartmouth, New Bedford, MA 02744-1221, USA local (chemosynthetic) primary productivity. For example, e-mail: [email protected] Lee and Childress (1994) showed that S-oxidizing bacteria 1692 Ann Microbiol (2014) 64:1691–1705 living in symbiosis with the HV tubeworm Riftia pachyptila Knowledge of the diversity and abundance of denitrifiers in the − exclusively assimilate NO3 , even in the presence of ammo- subsurface biosphere of hydrothermal vents is needed to better + nium (NH4 ). In these settings, bioavailable N is supplied understand nutrient cycling and mass balance in these systems. − + mainly by NO3 -rich seawater that is entrained and reduced Previous studies have reported large differences in NH4 con- + to NH4 during hydrothermal circulation and possibly by centrations in hydrothermal fields on the JFR, including fluids autochthonous N2 fixation (e.g., Mehta and Baross 2006). from diffuse flow vents at Axial Volcano and the Endeavour The process of denitrification involves the stepwise reduc- Segment (Lilley et al. 1993; Bourbonnais et al. 2012b). The − + tion of NO3 through a series of intermediates including nitrite higher NH4 concentrations commonly found in Endeavour − (NO2 ), nitric oxide (NO), and nitrous oxide (N2O), ultimately Segment vent fluids have the potential to influence the + resulting in dinitrogen (N2) production. Nitrite conversion to activity and diversity of denitrifying bacteria, with NH4 − gaseous N oxides (NOx) and N2 results in N loss, and in the being a potential source of NO3 for denitrifiers through case of N2O, greenhouse gas production. Two structurally nitrification at the oxic end of the redox gradient in different, but functionally equivalent enzymes can catalyze subseafloor mixing zones. − NO2 reduction: the homotrimeric copper-containing enzyme Here we use 16S rRNA and nirS genes to investigate encoded by the nirK gene, and the homodimeric cytochrome the diversity of potential denitrifying bacteria in relation − − − cd1-NO2 reductase encoded by nirS. The NO2 reductase to environmental variables (temperature, pH, H2S, NO3 + gene has been used widely as a functional marker to investi- and NH4 concentrations) in diffuse-flow fluids at dif- gate the diversity and abundance of denitrifying bacteria in ferent hydrothermal vent sites at Axial Volcano and the terrestrial (e.g., Henry et al. 2004;Chonetal.2009), estuarine Endeavour Segment on the JFR. We also expand on and marine environments (e.g., Braker et al. 2000, 2001; sequencing results using quantitative polymerase chain Nogales et al. 2002; Liu et al. 2003; Jayakumar et al. 2004, reaction (qPCR) to quantify 16S rRNA and nirS gene 2009; Castro-González et al. 2005; Santoro et al. 2006;Falk abundances at 15 different vent sites at Axial Volcano et al. 2007; Oakley et al. 2007; Smith et al. 2007). Most and the Endeavour Segment. studies have used the nirS gene because of reported difficulties with existing nirK primers, especially for marine samples (e.g., Braker et al. 2000; Jayakumar et al. 2004). Materials and methods Evidence for denitrification has been reported for both seafloor and subseafloor hydrothermal habitats. Previous Site description and water sampling studies, using GeoChip and metagenomic approaches to in- vestigate microbial communities inhabiting hydrothermal Fluid samples were collected for geochemical and microbio- vent chimneys on the Juan de Fuca Ridge (JFR), detected logical analysis at hydrothermal vents of the Endeavour functional gene repertoires mediating the denitrification pro- Segment (∼48°N, 129°W, ∼2,200 m depth) and Axial − − cess, including NO3 reductase (nar), NO2 reductase (nir), Volcano (∼46°N, 130°W, ∼1,500 m depth) on the JFR, NO reductase (nor)andN2Oreductase(nos) (Wang et al. North-East Pacific Ocean. The samples were collected during 2009; Xie et al. 2010). More recently, Bourbonnais et al. two research cruises, in August 2007 and June 2009, onboard − (2012a, b) reported reduced NO3 concentrations (vs back- the R/V Thompson and the R/VAtlantis, respectively, as part ground seawater) in combination with enrichment of heavy 15 of the New Millenium Observatory (NEMO) and Endeavour- − 18 − N-NO3 and O-NO3 isotopes in low temperature (low-T) Axial Geochemistry and Ecology Research (EAGER) diffuse flow fluids on the JFR, observations that are consistent Projects (Fig. 1). The caldera of Axial Volcano rises 1,100 m − with isotope fractionation during active NO3 reduction. above the surrounding ocean floor, and has been the site of Potential denitrification rates up to 1,000 nmol L−1 day−1, two recent seafloor volcanic eruptions, in 1998 and 2011 measured in diffuse vent fluids at different sites on the JFR, (Chadwick et al. 2012). were always several fold higher than anammox The remotely operated vehicle (ROV) JASON and the (<5 nmol L−1 day−1) rates, indicating that, at least for deep submergence vehicle (DSV) Alvin were used to − JFR vents, denitrification is the dominant NO3 consuming collect all fluid samples. Samples for chemical analysis process within the subsurface biosphere, with unconstrained were collected with titanium gas-tight samplers, non-gas- feedback on chemosynthetic primary production. tight titanium syringe (“Major”) samplers, collapsible Multitrophic interactions and food web structure in hydro- Tedlar plastic bags with valves, or PVC piston samplers thermal vent ecosystems are typically sustained by with Teflon spring seals on the hydrothermal fluid and chemolithoautotrophic bacteria harnessing metabolic energy particle sampler (HFPS). Vent fluid sub-samples were from the oxidation of reduced sulfur species and molecular transferred from the collapsible bags using a syringe into − hydrogen, using dissolved O2 and NO3 as primary electron acid-washed and deionized-water-rinsed, 60-mL high- acceptors (e.g., Jannasch and Mottl 1985; Schrenk et al. 2010). density polyethylene (HDPE) brown bottles