The ISME Journal (2008) 2, 590–601 & 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Niche differentiation among sulfur-oxidizing bacterial populations in cave waters Jennifer L Macalady1, Sharmishtha Dattagupta1, Irene Schaperdoth1, Daniel S Jones1, Greg K Druschel2 and Danielle Eastman2 1Department of Geosciences, Pennsylvania State University, Pennsylvania, PA, USA and 2Department of Geology, University of Vermont, Burlington, VT, USA The sulfidic Frasassi cave system affords a unique opportunity to investigate niche relationships among sulfur-oxidizing bacteria, including epsilonproteobacterial clades with no cultivated representatives. Oxygen and sulfide concentrations in the cave waters range over more than two orders of magnitude as a result of seasonally and spatially variable dilution of the sulfidic groundwater. A full-cycle rRNA approach was used to quantify dominant populations in biofilms collected in both diluted and undiluted zones. Sulfide concentration profiles within biofilms were obtained in situ using microelectrode voltammetry. Populations in rock-attached streamers depended on the sulfide/oxygen supply ratio of bulk water (r ¼ 0.97; Po0.0001). Filamentous epsilonproteobacteria dominated at high sulfide to oxygen ratios (4150), whereas Thiothrix dominated at low ratios (o75). In contrast, Beggiatoa was the dominant group in biofilms at the sediment–water interface regardless of sulfide and oxygen concentrations or supply ratio. Our results highlight the versatility and ecological success of Beggiatoa in diffusion-controlled niches, and demonstrate that high sulfide/oxygen ratios in turbulent water are important for the growth of filamentous epsilonproteobacteria. The ISME Journal (2008) 2, 590–601; doi:10.1038/ismej.2008.25; published online 20 March 2008 Subject Category: microbial population and community ecology Keywords: Beggiatoa; epsilonproteobacteria; Frasassi cave; microelectrode voltammetry; Thiothrix; Thiovirga Introduction correspond with morphological differences in the biofilms. Our initial observations motivated a Sulfidic caves form in limestone (CaCO3) rocks systematic, multi-year study of the population where sulfide-rich groundwater interacts with oxy- structure of biofilms collected from cave waters gen at the water table. The Frasassi cave system with a wide range of hydrological and geochemical hosts a rich, sulfur-based lithoautotrophic microbial characteristics. The goal of this study was to identify ecosystem (Sarbu et al., 2000; Vlasceanu et al., 2000; the ecological niches (defined as the range of Macalady et al., 2006, 2007; Jones et al., 2008). environmental conditions that permit growth) of Previous studies of the geochemistry of the cave major biofilm-forming populations. waters have revealed that they are mixtures of Unraveling the effects of changing hydrologic slightly salty, sulfidic groundwater diluted 10–60% conditions on microbial growth within the cave by oxygen-rich, downward-percolating meteoric system is not trivial because dilution of the sulfidic water (Galdenzi et al., 2007). Initial observations of groundwater has multiple effects relevant to micro- the abundant biofilms in cave streams and pools bial metabolism. The input of meteoric water suggested that they respond dynamically to seasonal increases water depth and flow rates, dilutes and episodic hydrologic changes. In particular, we dissolved species in the sulfidic aquifer and adds noted that changes in specific conductivity (tracking dissolved oxygen. Water flow conditions that in- freshwater dilution) and water flow characteristics crease turbulence also increase sulfide degassing from the water and oxygen transport into the water from the oxygenated cave air. We investigated Correspondence: JL Macalady, Department of Geosciences, microbial populations using a full-cycle rRNA Pennsylvania State University, University Park, PA 16802, USA. E-mail: [email protected] approach. Environmental conditions were measured Received 18 October 2007; revised 14 February 2008; accepted 20 using microelectrode voltammetry and bulk water February 2008; published online 20 March 2008 geochemical analyses. We found that both Niches of sulfur-oxidizing cave bacteria JL Macalady et al 591 sulfide/oxygen ratios and physical water flow for fluorescence in situ hybridization (FISH) were characteristics are important for determining the fixed in 4% (w/v) paraformaldehyde and stored at distributions of sulfur-oxidizing groups. À20 1C. Samples for clone library construction were preserved in four parts RNAlater (Ambion/Applied Biosystems, Foster City, CA, USA) to one part Materials and methods sample (v/v). Water samples were filtered (0.2 mm) into acid-washed polypropylene bottles and stored Field site, sample collection and geochemistry at 4 1C until analyzed. Conductivity, pH and tem- The Grotta Grande del Vento-Grotta del Fiume perature of the waters were measured in the field (Frasassi) cave system is actively forming in Jurassic using sensors attached to a 50i multimeter (WTW, limestone in the Appennine Mountains of the Weilheim, Germany). Dissolved sulfide and oxygen Marches Region, Central Italy. The waters of the concentrations were measured in the field using a cave system are near neutral (pH 6.9–7.4) and have portable spectrophotometer (Hach Co., Loveland, specific conductivities ranging from 1200 to CO, USA) using the methylene blue and indigo 3500 mScmÀ1, or roughly 4–5% of average marine carmine methods, respectively. Duplicate sulfide þ 2 þ À À salinity. The major ions are Na ,Ca ,Cl, HCO3 analyses were within 1% of each other. Replicate 2À and SO4 (Galdenzi et al., 2007). Concentrations of oxygen analyses were within 20% of each other. electron donors and acceptors other than sulfur Nitrate, nitrite, ammonium and sulfate were mea- species and oxygen are as follows: bicarbonate sured at the Osservatorio Geologico di Coldigioco (5.3–7.1 mM), ammonium (30–175 mM), methane Geomicrobiology Lab using a portable spectrophot- (1–20 mM), dissolved iron (o0.1 mM), dissolved man- ometer within 12 h of collection according to the ganese (o0.04 mM), nitrate (not detected, o0.7 mM) manufacturer’s instructions (Hach Co.). Light micro- and nitrite (not detected, o2.0 mM). Organic carbon scopy was performed on live samples within 8 h of concentrations range between 0.16 and 4.5 mg lÀ1. collection on a Zeiss Model 47-30-12-9901 optical Biofilms from cave springs and streams were microscope ( Â 1250) at the Osservatorio Geologico collected from 5 to 40 cm water depth at sample di Coldigioco Geomicrobiology Lab. locations shown in Figure 1 in May (wet season) and August (dry season) in 2005, 2006 and 2007. Biofilms were harvested using sterile plastic transfer Microelectrode voltammetry pipettes into sterile tubes, stored on ice and Voltammetric signals are produced when dissolved processed within 4–6 h of collection. Subsamples or colloidal species interact with the surface of a gold amalgam working electrode. Electron flow resulting from redox half-reactions at a 100 mm diameter tip is registered as a current that is proportional to concentration (Skoog et al., 1998; Taillefert and Rozan, 2002). The gradients associated with microbial metabolism in biofilms are thus readily measured using this technique. Aqueous and colloidal species that are electroactive at gold À amalgam electrode surfaces include: H2S, HS ,S8, 2À 2À À 2 þ 3 þ polysulfides, S2O3 ,S4O6 , HSO3 ,Fe ,Fe , 2 þ FeS(aq),Mn ,O2 and H2O2 (Luther et al., 1991, 2001; Xu et al., 1998; Taillefert et al., 2000; Druschel et al., 2003, 2004; Glazer et al., 2004, 2006). Voltammetric analyses of dissolved sulfur, iron and other species in the field were accomplished using a DLK-60 potentiostat powered with a 12 V battery and controlled with a GETAC ruggedized computer. To protect the electrochemical system from drip waters and high humidity, the potentiostat was contained inside a storm case containing dryrite humidity sponges and modified with rubber strip- ping to allow the communication ribbon cable and electrode cables to go outside the case while keeping the inside sealed. Electrodes were constructed as described in Brendel and Luther (1995). Figure 1 Map of the Frasassi cave system showing sample Voltammetric analyses in the cave system locations (open circles). The small inset shows the location of the involved placing working (gold amalgam) electro- Frasassi cave system in Italy. Major named caves in the Frasassi system are shown in different shades of gray. Topographic lines des into a narishege three-axis micromanipulator and elevations in meters refer to the surface topography. Base map with a two-arm magnetic base on a steel plate. At courtesy of the Gruppo Speleologico CAI di Fabriano. each sampling location, the reference and counter The ISME Journal Niches of sulfur-oxidizing cave bacteria JL Macalady et al 592 electrodes were placed in the flowing water near the Phred base calling using CodonCode Aligner v.1.2.4 biofilm. The working electrode was lowered to the (CodonCode Corp., Dedham, MA, USA) and manu- air–water interface, then to the biofilm–water inter- ally checked for ambiguities. The nearly full-length face and subsequently lowered in increments to gene sequences were compared against sequences in profile the biofilm. We used both cyclic voltammetry public databases using BLAST (Altschul et al., 1990) between À0.1 and À1.8 V (vs Ag/AgCl) at scan rates and submitted