View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library FEMS Microbiology Ecology 43 (2003) 89^98 www.fems-microbiology.org Spatio-temporal distribution of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland) Mauro Tonolla a, Sandro Peduzzi a;b, Dittmar Hahn b;Ã, Ra¡aele Peduzzi a a Cantonal Institute of Microbiology, Microbial Ecology (University of Geneva), Via Giuseppe Bu⁄ 6, CH-6904 Lugano, Switzerland b Department of Chemical Engineering, New Jersey Institute of Technology (NJIT), and Department of Biological Sciences, Rutgers University, 101 Warren Street, Smith Hall 135, Newark, NJ 07102-1811, USA Received 24 May 2002; received in revised form 26 July 2002; accepted 30 July 2002 First published online 4 September 2002 Abstract In situ hybridization was used to study the spatio-temporal distribution of phototrophic sulfur bacteria in the permanent chemocline of meromictic Lake Cadagno, Switzerland. At all four sampling times during the year the numerically most important phototrophic sulfur bacteria in the chemocline were small-celled purple sulfur bacteria of two yet uncultured populations designated Dand F. Other small- celled purple sulfur bacteria (Amoebobacter purpureus and Lamprocystis roseopersicina) were found in numbers about one order of magnitude lower. These numbers were similar to those of large-celled purple sulfur bacteria (Chromatium okenii) and green sulfur bacteria that almost entirely consisted of Chlorobium phaeobacteroides. In March and June when low light intensities reached the chemocline, cell densities of all populations, with the exception of L. roseopersicina, were about one order of magnitude lower than in August and October when light intensities were much higher. Most populations were evenly distributed throughout the whole chemocline during March and June, while in August and October a microstratification of populations was detected suggesting specific eco-physiological adaptations of different populations of phototrophic sulfur bacteria to the steep physico-chemical gradients in the chemocline of Lake Cadagno. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. 1. Introduction mixolimnion, a narrow chemocline and the anoxic moni- molimnion. The chemocline is permanent and stabilized by Studies on the ecology of microorganisms are often density di¡erences of salt-rich water constantly supplied complicated by the heterogeneous and dynamic nature of by subaquatic springs to the monimolimnion and of elec- their habitat together with the small and discontinuous trolyte-poor surface water feeding the mixolimnion [5]. size distribution of microhabitats. In this regard, water High concentrations of sulfate and steep gradients of sul- columns of strati¢ed lakes o¡er de¢ned physico-chemical ¢de in the chemocline [6,7] support the growth of elevated conditions or unidirectional gradients in depth intervals numbers of bacteria (up to 107 cells ml31) indicating that a ranging from cm to m [1]. As such, meromictic lakes are bacterial community making use of these gradients is interesting model systems for research on bacterioplank- present [8,9]. Molecular techniques that permit analysis ton because a number of di¡erent physiological groups of of microbial community structure una¡ected by the limi- bacteria substitute each other along the vertical gradient tations of culturability showed that almost all bacteria of light, oxygen and sul¢de [2^4]. Lake Cadagno in Swit- belonged to the Proteobacteria [8,9] with numbers for zerland represents such a model system. The water body of the K-, L-, Q- and N-subdivisions of Proteobacteria, respec- this lake is structured in three distinct layers, the oxic tively, accounting for 23, 17, 45 and 15% of the total number of bacteria [8,9]. Purple sulfur bacteria were most prominent numerically with on average 33% of all bacteria [10,11]. All large-celled purple sulfur bacteria were identi¢ed as Chromatium okenii, while small-celled purple sulfur bacteria consisted of four major populations * Corresponding author. Tel.: +1 (973) 353 5235; forming a tight cluster with Amoebobacter purpureus (re- Fax: +1 (973) 353 5518. cently reclassi¢ed as Lamprocystis purpurea [12]) and E-mail address: [email protected] (D. Hahn). Lamprocystis roseopersicina [9]. These small-celled purple 0168-6496 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0168-6496(02)00354-9 FEMSEC 1415 16-1-03 90 M. Tonolla et al. / FEMS Microbiology Ecology 43 (2003) 89^98 sulfur bacteria were usually found in aggregates, together 2.2. Chemical analysis with sulfate-reducing bacteria of the family Desulfovibrio- naceae [8,9]. From these samples, 11-ml subsamples were immedi- The populations of small-celled purple sulfur bacteria ately transferred to screw-capped tubes containing 0.8 ml displayed di¡erent distribution pro¢les in the chemocline of a 4% zinc acetate solution. These were stored on ice and of Lake Cadagno indicating di¡erent eco-physiological used to determine sul¢de concentrations by colorimetric adaptations [9,13,14]. Since most of these bacteria have analysis [22] using the Spectroquant0 kit of Merck (Swit- not been obtained in pure culture yet, the aim of this study zerland) [9,18]. Additional 10 ml was immediately ¢ltered was to gather information on their spatial and temporal through 0.22-Wm polyethersulfone membrane ¢lters distributions in the chemocline of Lake Cadagno and to (GyroDisc-PES25, Orange Scienti¢c, Waterloo, Belgium) analyze their interrelationships with environmental factors into plastic tubes containing 100 Wl of 65% nitric acid so- [13,14]. The analysis was based on in situ hybridization lution (Fluka, Buchs, Switzerland). These samples were using rRNA-targeted, Cy3-labeled oligonucleotide probes. further analyzed for dissolved iron by graphite furnace In addition to speci¢c populations of purple sulfur bacte- and air acetylene £ame atomic absorption with a Spec- ria (C. okenii, A. purpureus, L. roseopersicina, and two yet trAA-800 instrument (Varian, Melbourne, Australia). Am- uncultured and uncharacterized populations Dand F) [9], monium and sulfate concentrations were measured by iso- green sulfur bacteria were analyzed using a published [15] cratic ion chromatography with suppressed conductivity and a newly designed probe speci¢cally targeting a se- detection with a Dionex DX-500 ion chromatograph (Di- quence retrieved from a 16S rRNA gene clone library onex, Olten, Switzerland). For the determination of am- from the chemocline of Lake Cadagno [16]. monium, a CG-12 pre-column, a CS-12 column, a CSRS-1 suppressor, 20 mM methanesulfonic acid as an eluent at a £ow of 1.0 ml min31 were used, for that of sulfate an AG- 2. Materials and methods 14 pre-column, an AS-14 column, an ASRS Ultra suppres- sor, and a mixture of 3.5 mM Na-carbonate and 1.0 mM 2.1. Site description, physical analyses and sampling Na-bicarbonate as eluent were used at a £ow of 0.6 ml min31 [23]. Lake Cadagno is an alpine lake located 1923 m above sea level in the south of Switzerland (46‡33PN, 8‡43PE) in 2.3. Microbial analysis the catchment area of a dolomite vein rich in gypsum (Piora-Mulde). The lake has a surface area of 26U105 For the microbial analysis of the chemocline, 15-ml sub- m2 and a maximum depth of 21 m. Due to the in¢ltration samples of water obtained with the multi-syringe sampler of water through the dolomite vein, Lake Cadagno is a were ¢ltered immediately after sampling through 0.22-Wm meromictic lake characterized by a high salinity of the polycarbonate membrane ¢lters (25 mm diameter; Milli- monimolimnion and a permanent chemocline in a depth pore, Volketswil, Switzerland) [24]. Bacteria were ¢xed by between 9 and 14 m [5,17]. Samples were taken from the overlaying the ¢lters with 4% paraformaldehyde in phos- chemocline over the deepest site in the center of the lake phate-bu¡ered saline (PBS; 0.13 M NaCl, 7 mM Na2- (21 m) in October 1998, and in March, June and August HPO4, 3 mM NaH2PO4, pH 7.2) for 30 min at room 1999. The chemocline and the bacterial plume in Lake temperature [25]. The ¢lters were subsequently rinsed Cadagno were located at each sampling date using temper- twice with PBS by vacuum ¢ltration and transferred into ature, conductivity, pH, dissolved oxygen, turbidity and plastic bags with 1 ml of 50% ethanol in PBS. In sealed redox potential measurements with a YSI 6000 pro¢ler bags, the bacterioplankton was released from ¢lters and (Yellow Springs Inc., Yellow Springs, OH, USA) [9,18]. resuspended by slightly massing the ¢lter with thumb and In addition, PAR-light transmission conditions were de- fore¢nger [26]. The complete release of the bacteria from termined down to the chemocline in steps of 0.1 m using ¢lters was checked microscopically after DAPI staining. two LI-193SA spherical quantum sensors and a LI-COR Resuspended bacterial cells were than transferred into 1000 datalogger (LI-COR Ltd., Lincoln, NE, USA). The Eppendorf tubes and stored at 320‡C until further use latter measurements were used to calculate vertical attenu- [9,25]. ation coe⁄cients (Kd) of photosynthetically available radi- Aliquots (1 Wl) of the samples were spotted onto gelatin- ation [19] for the mixolimnion and the bacterial layer. The coated slides (0.1% gelatin, 0.01% KCr(SO4)2). The prep- YSI 6000 pro¢ler and LI-COR sensors were ¢xed at the arations were allowed to air-dry and subsequently dehy- lowest part of a thin-layer pneumatic multi-syringe sam- drated in 50, 80 and 96% of ethanol for 3 min each [25]. pler (University of Zurich, Institute of Microbiology, Swit- The analysis of purple and green sulfur bacteria in the zerland) that was used after detection of the chemocline to chemocline samples from Lake Cadagno was based on take 20 samples of 100 ml simultaneously each over a total in situ hybridization using rRNA-targeted, Cy3-labeled depth of 2 m, yielding in a depth resolution of 10 cm oligonucleotide probes (Table 1).