water research 45 (2011) 1213e1228

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Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (, )

Laurence Haller a, Mauro Tonolla b,d, Jakob Zopfi c, Raffaele Peduzzi b, Walter Wildi a, John Pote´ a,* a University of Geneva, Institute F.A. Forel, 10 route de Suisse, CP 416, CH-1290 Versoix, Switzerland b Microbial Ecology, Microbiology Unit, Plant Biology Department, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva, Switzerland c University of , Institute of Geology and Paleontology, Laboratory of Biogeosciences, CH-1015 Lausanne, Switzerland d Cantonal Institute of Microbiology, Via Mirasole 22A, CH-6500 Bellinzona, Switzerland article info abstract

Article history: The aim of this study was to compare the composition of bacterial and archaeal Received 31 August 2010 communities in contaminated sediments ( Bay) with uncontaminated sediments Received in revised form ( area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were 8 November 2010 analysed for physicochemical characteristics including porewater composition, organic Accepted 14 November 2010 carbon, and heavy metals. Results show high concentrations of contaminants in sediments Available online 20 November 2010 from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense mineralisation, which was dominated by sulphate-reduction and methanogenesis. The Keywords: bacterial diversity in Vidy sediments was significantly different from the communities in Lake Geneva the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of Sediment pollution clones in Vidy sediments related to Dechloromonas sp., a group of dech- Heavy metal lorinating and contaminant degrading . Deltaproteobacteria, including clones related Microbial diversity to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more 16S rRNA abundant in the contaminated sediments. The archaeal communities consisted essentially Clone library of methanogenic Euryarchaeota, mainly found in the contaminated sediments rich in organic matter. Multiple factor analysis revealed that the microbial community composi- tion and the environmental variables were correlated at the two sites, which suggests that in addition to environmental parameters, pollution may be one of the factors affecting microbial community structure. ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction compounds including heavy metals (HMs) and hydrophobic organic compounds, such as polycyclic aromatic hydrocar- Untreated or only partly treated wastewaters including bons (PAHs), polychlorinated biphenyls (PCBs) and organo- industrial, agricultural and domestic effluents constitute the chlorine pesticides (OCPs) (e.g. Koelmans et al., 2001; Eggleton main contamination sources in aquatic environments. Sedi- and Thomas, 2004). The increasing contamination of sedi- ment contamination is usually due to inorganic and organic ments by inorganic and organic micro-pollutants is a big

* Corresponding author. Tel.: þ41 22 379 03 21; fax: þ 41 22 379 03 29. E-mail address: [email protected] (J. Pote´). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.11.018 1214 water research 45 (2011) 1213e1228

concern in aquatic ecosystems (Fo¨ rstner and Wittmann, 1979; biogeochemical, and ecotoxicological studies. This research Pardos et al., 2004; Schwarzenbach et al., 2006). Sediment represents the first assessment of Bacteria and Archaea in contamination by HMs and other micro-pollutants might contaminated and uncontaminated sediments of Lake Geneva cause potential adverse effects to ecosystems and also pose and serves as important background information for these human health risks (Salomons and Fo¨ rstner, 1984; Verweij studies. For a better understanding of the microbial commu- et al., 2004; Wang et al., 2004). The main environmental risk nity structures, molecular analyses were complemented by is remobilization of the contaminants and their return to the a detailed physicochemical characterisation of the sediments. hydrosphere either by sediment re-suspension or by infiltra- tion into the groundwater (Wildi et al., 2004). Therefore, the removal of HMs from wastewater or their accumulation in 2. Materials and methods sediments should be examined extensively (e.g. Wang et al., 2004; Wildi et al., 2004). Microbial communities may be 2.1. Study site description and sampling procedure sensitive indicators for pollution in aquatic ecosystems and may be applied for biomonitoring and assessment purposes In August 2005, sediment was collected at two locations in (Pronk et al., 2009). Heavy metal contamination, for example, Lake Geneva (Fig. 1): (i) within the Bay of Vidy near the outlet may lead to a reduction of bacterial diversity (Sandaa et al., pipe of the WWTP of Lausanne (Swiss coordinates X: 534682, 1999). However, other studies observed either an increase in Y: 151410) and (ii) near the Ouchy area (Swiss coordinates X: microbial diversity along with heavy metal contamination 537985, Y: 150390). Sampling was done from R/V “La Licorne” (Sorci et al., 1999) or no significant variation (Gillan et al., 2005). using a core sampler (Benthos Inc, USA). Three cores (6.7 cm Other environmental factors as well as the time of exposure i.d., 1.5 m length) were retrieved from each site at a depth of might explain these differences. 40 m. For microbiological analyses, the cores were opened Lake Geneva is the largest freshwater reservoir of Western longitudinally and sliced into 2 cm thick sections until 10 cm Europe with a volume of 89 km3 and a maximum depth of depth. The sediment samples were placed into sterile plastic 309 m. It is a monomictic temperate lake, with early spring containers, stored in an icebox and treated in the laboratory overturn not occurring every year. The lake was considered within 24 h. For chemical analysis, the intact sediment cores eutrophic in 1970s and 1980s, but has become mesotrophic were transported to the laboratory and stored vertically in after drastic reduction of phosphorus inputs (Dorioz et al., a cold-room at 4 C until analysis. 1998). Approximately 700,000 people are supplied with water from Lake Geneva. The city of Lausanne, located on the 2.2. Chemical analysis northern shore, discharges the largest volume of treated wastewater into the nearby Bay of Vidy. The wastewater Two cores per site were used for the chemical analyses: one treatment plant (WWTP) of the city treats nowadays approx- was used to measure organic matter, nutrients and HMs imately 220,000 equivalent-inhabitants of wastewater. The contents and the other one was used to determine sulphur WWTP effluent is released into the Bay of Vidy 700 m from the and iron concentrations and the porewater constituents. shore at 30 m depth. As a consequence, Vidy Bay is the most Organic matter and nutrients: the cores were opened longitu- contaminated area of Lake Geneva. Published data document dinally and sliced every 2 cm down to a depth of 10 cm. Before the accumulation of contaminants close to a recreational area analysis, sediment samples were air-dried at ambient room as well as the related ecological impacts and health risks temperature. The particle grain size was measured with a laser (Loizeau et al., 2004; Pardos et al., 2004; Wildi et al., 2004). Coulter LS-100 diffractometer (Beckman Coulter, Fullerton, CA, Several studies focused on the quality of bottom sediments in USA), after a 5-min ultrasonic dispersal in deionised water the Bay of Vidy and on the spatial distribution of organic according to the method described by Loizeau et al. (1994).The matter, faecal indicator bacteria, heavy metals, and hydro- proportions of three major size classes (clay < 2 mm; silt 2e63 mm; phobic organic compounds (Pote´ et al., 2008; Haller et al., and sand > 63 mm) were determined from size distributions. 2009). However, there is still a paucity of information Total organic matter content in sediments was estimated by loss regarding sedimentary microbial communities in Lake Geneva on ignition (LOI) at 550 C for 1 h in a Salvis oven (Salvis AG and particularly around the WWTP discharge outlet. Emmenbru¨ cke, Luzern, Switzerland) on 5 g of dried sediments. Sediments are complex habitats densely colonised by Total organic carbon (TOC) was determined by titrimetry diverse groups of microorganisms, which play key roles in following acid oxidation on 5 g of dried sediments. Total biogeochemical cycling, aquatic food webs and the remobili- nitrogen (TN) was determined according to Kjeldahl (APHA, zation of heavy metals (Nealson, 1997; Lors et al., 2004; Ye 1985) on 2 g of dried sediments. Total phosphorus (TP) and its et al., 2009). Many studies have been performed to examine different forms were measured on 150 mg of dried sediments the composition and variability of microbial communities in with a spectrophotometer (Helios Gamma UVeVis, Thermo extreme or complex aquatic ecosystems. However, only a few Scientific, USA) at 850 nm, following the fractionation scheme of studies compared microbial community structures in Williams et al. (1976) as modified by Burrus et al. (1990).The contaminated and uncontaminated sediments (Powell et al., results are expressed in mg kg 1 dry weight sediment (ppm). 2003; Zhang et al., 2008). The aim of the present study was Solid phases: sulphur and iron contents were determined to compare the composition of the sediment-associated every centimeter down to 10 cm depth. The sediment was microbial communities in the contaminated Bay of Vidy with sliced at room temperature in a N2-filled glove bag and fixed in a closeby less polluted site in the Ouchy area. The Bay of Vidy 50 mL tubes containing 10 mL zinc acetate solution (10%). is currently used as a model system for several limnological, Sulphide was extracted as acid volatile sulphur [AVS; water research 45 (2011) 1213e1228 1215

Fig. 1 e Study area with the two sampling locations in the Bay of Vidy and the Ouchy area. The upper left corner of the map represents 463200000 N and 63300000 E and the lower right corner represents 462900000 N and 63800000 E.

dissolved sulphide (H2S) and iron sulphides (FeS)] and chro- anion exchange column, Na2CO3/NaHCO3 (8 mM/1 mM) as mium reducible sulphur [CRS; primarily pyrite (FeS2), eluent, an Anion Self-Regenerating Suppressor (ASRS 300, elemental sulphur (S0), and some organic sulphur]. AVS and 4 mm) module and a conductivity detector. CRS were measured by a two step distillation process with cold 6 N HCl followed by boiling 1 M acidic CrCl2 solution 2.3. DNA extraction (Fossing and Jørgensen, 1989; Zopfi et al., 2008). Poorly cris- talline Fe(III)oxides were extracted with 0.5M HCl according to The samples from 0 to 2 cm and 4e6 cm depth were used for Thamdrup et al. (1994). microbiological analyses. Total DNA was extracted from Total contents of Cu, Cd, Cr, Zn, and Pb were determined by 250 mg of sediment using the PowerSoil DNA Isolation Kit quadrupole-based Inductively Coupled PlasmaeMass Spec- (Mo-Bio Laboratories, Carlsbad, CA, USA), according to the trometry (ICPeMS) (HP 4500, Agilent) following the digestion manufacturer’s instructions. The concentration of extracted of 1 g of dried sediment in analytical grade 2 M HNO3 (Pardos DNA was measured spectrophotometrically (OD260) and et al., 2004). Total Hg was quantified by atomic absorption aliquots were used to estimate DNA quality by electrophoresis spectrophotometry (Advanced Mercury Analyser; AMA 254, on 0.8% agarose gel stained with ethidium bromide (10 mgml 1, Altec, Czech Rep.) according to Hall and Pelchat (1997) and SigmaeAldrich Chemie GmbH, Buchs, Switzerland). DNA Ross-Barraclough et al. (2002). The method is based on sample extracts were stored at 20 C until used for PCR amplification. combustion, gold amalgamation and atomic absorption spectrometry (AAS). Average values of triplicate measure- 2.4. PCR amplification ments are expressed in mg kg 1 dry weight sediment (ppm). Porewater constituents: porewater was harvested by centri- Nearly complete bacterial 16S rRNA genes were amplified 2þ 0 0 fugation (4000 rpm) under an N2 atmosphere to avoid Fe and using the primers 26f (5 -AGAGTTTGATCATGGCTCA-3 ) and 2þ 0 0 H2S oxidation. Samples for dissolved Fe were acidified with 1392r (5 -GTGTGACGGGCGGTGTGTA-3 )(Brosius et al., 1981; 0.5 M HCl and analysed by the photometric Ferrozine method Lane, 1991). Archaeal 16S rRNA genes were amplified using (Thamdrup et al., 1994). Dissolved sulphide was determined on the primers 109f (50-ACKGCTCAGTAACACGT-30) and 915r (50- Zn-acetate fixed samples using the colorimetric methylene- GTGCTCCCCCGCCAATTCCT-30)(Grobkopf et al., 1998; Stahl blue method (Cline, 1969; Zopfi et al., 2008). The major anions and Amann, 1991). 2 3 Cl ,SO4 ,NO3 , and PO4 were determined by ion-chromatog- Both archaeal and bacterial PCR amplifications were per- raphy on a DIONEX DX-120 system using an IonPac AS14A formed separately using the Taq PCR Master Mix Kit (Qiagen, 1216 water research 45 (2011) 1213e1228

Basel, Switzerland). Each PCR reaction was carried out in detection program was used to exclude chimeras (Maidak et al., m a volume of 50 L, containing 1 PCR buffer, 1.5 mM of MgCl2, 1999). NCBI BLAST (http://www.ncbi.nih.gov) was used to 200 mM of each dNTP, 0.3 mM of each primer, 2.5 units of Taq identify the most closely related 16S rRNA gene sequences. The DNA polymerase, 2.5 mg ml 1 of bovine serum albumin partial 16S rRNA gene sequences were then aligned in the (Invitrogen, Basel, Switzerland) and 2 mL of DNA (about 100 ng) Clustal W implementation of MEGA 3.0 (Kumar et al., 2004). of each sample. The following conditions were used for PCR The same program was used to produce neighbour-joining amplification: initial denaturation at 94 C for 5 min; 35 cycles phylogenetic trees (Kimura-2 correction; bootstrap values for of denaturation (94 C for 30 s), annealing (52 C for 30 s), 500 replicates). The sequences were identified using the ribo- extension (72 C for 1 min) and a final extension step of 10 min somal database project classifier (Wang et al., 2007). Sequences at 72 C. PCR products (10 mL) were separated by electropho- from this study have been deposited with the EMBL database resis on 0.8% agarose gels and visualised by ethidium bromide under accession numbers FN679050eFN679294. staining and UV illumination. The sequences were assigned to individual OTUs based on the 97% sequence similarity criterion. The number of OTUs 2.5. Clone library construction and DNA sequencing and the rarefaction curves were estimated from the sequence data using Mothur v.1.12.3 (Schloss et al., 2009). PCR products were purified using the NucleoSpin Extract II Comparison between the clone libraries from the two sites Kit (MachereyeNagel, Oensingen, Switzerland) according to was done on the basis of genetic diversity by means of the the manufacturer’s instructions. The amplified DNA was then parsimony test using Mothur v.1.12.3 (Schloss et al., 2009) and quantified using the PicoGreen dsDNA Quantitation Reagent the the FST-test using the program Arlequin, v.2.0 (Schneider (Molecular Probes Inc., Eugene, OR, USA) and a TD-700 Fluo- et al., 2000). rometer (Turner Designs Sunnyvale, CA, USA). Approximately A Mantel test using Spearman correlation (Mantel, 1967) 20e30 ng of amplified 16S rDNA were cloned into competent was applied to relate the microbial community compositions Escherichia coli cells using the TOPO TA cloning kit (Invitrogen, from the two sites with environmental variables. Multiple Basel, Switzerland) following the manufacturer’s recommen- factor analysis (MFA) was done to obtain an integrative picture dations. The transformed cells were plated on LB medium of the relationship between the bacterial community struc- containing 50 mg L 1 ampicillin, 60 mg L 1 of IPTG (isopropyl- tures and the environmental factors across the 2 sites. The 1 b-D-thiogalactopyranoside), and 100 mg L of X-gal (5-bromo- environmental parameters were separated into 2 matrices, one 4-chloro-3-indolyl-b-D-galactopyranoside), and incubated including organic matter contents and nutrient variables and overnight at 37 C. White recombinants were transferred to LB one with the heavy metal concentrations. The MFA allows the medium plates for 24 h. 80 and 40 recombinants were picked simultaneous ordination of a composite table obtained by the from samples taken at 0e2 cm and 4e6 cm, respectively, in juxtaposition of the species and environmental datasets after each location, to constitute the Bacteria clone libraries. For the weighting the different matrices (Escofier and Pages, 1994). The Archaea, only 2 recombinants (1 per site) were found at 0e2cm final ordination plot shows global points indicating the relative and 34 colonies were picked from 4 to 6 cm. The insert size of positions of the objects described by the combination of data- all picked colonies, was determined by direct PCR using M13 sets. Each global point is surrounded by partial points indi- forward and reverse primers included in the cloning kit. The cating the relative positions of the datasets taken separately products were subsequently purified with the NucleoSpin (Escofier and Pages, 1994; Becue-Bertaut and Pages, 2008). Extract II Kit and sequenced using the BigDye Terminator These statistical analyses were carried out in “R”, a free soft- Cycle Sequencing Ready reaction kit (Applied Biosystems ware environment for statistical computing and graphics, International, Inc., Rotkreuz, Switzerland). Sequences were using the Vegan library (R Development Core Team, 2005). obtained on an ABI PRISM 310 Genetic Analyser, (Perkin Elmer, Schwerzenbach, Switzerland) using either the sequencing primer M13 for archaeal clones or primer 27f for 3. Results bacterial clones (Lane, 1991). 3.1. Chemical analysis 2.6. Phylogenetic analysis Some general sediment characteristics including particle The obtained sequences were checked and edited using the grain size, organic matter and nutrient contents are presented program EditSeq (DNAStar Inc., Madison, WI, USA). A chimera in Table 1. These sediments were mostly composed of silts,

Table 1 e General characterisation of the contaminated (Vidy) and non-contaminated (Ouchy) sediment sections used for clone library construction. Sediment section OM (%) TOC (%) TN (mg kg 1) NH4-N (mg kg 1)TP(mgkg1) Clay/silt/sand proportion (%)

Vidy 0e2 cm 18.7 10.8 11.6 1.6 6783.8 0/64/35 Vidy 4e6 cm 23.7 13.7 13.5 1.6 9650.9 0/65/34 Ouchy 0e2 cm 5 2.9 2.2 0.5 862.1 0.9/78/21 Ouchy 4e6 cm 4.3 2.5 1.7 0.4 759.5 0.6/80/19

OM: Organic matter; TOC: Total organic carbon; TN: Total nitrogen; NH4-N: Ammonium; TP: Total phosphorus. water research 45 (2011) 1213e1228 1217

approximately 65% and 80% for Vidy and Ouchy, respectively. criterion. The sequences (n ¼ 208) were grouped into 132 Average organic matter contents in Vidy Bay sediments were OTUs: 40, 26, 53, and 32 for Vidy 0e2 cm, Vidy 4e6 cm, Ouchy much higher (21%) than in Ouchy sediments (4.5%). Average 0e2 cm, and Ouchy 4e6 cm, respectively (Table 3). The nutrient concentrations such as total nitrogen, ammonium number of unique OTUs was higher in Ouchy than in Vidy and total phosphorus were also higher in Vidy sediments sediments, 38% and 54% of the OTUs were exclusively found (12.6 ppm, 1.6 ppm and 8217 ppm, respectively) while they in Vidy and Ouchy, respectively. Only 8% of all OTUs were were considered low in the sediments from Ouchy (2 ppm, shared between the libraries from both sites. The OTU rich- 0.5 ppm and 810 ppm, respectively). However, at both stations, ness estimates based on rarefaction curves suggests a higher no substantial differences in the nutrient contents between bacterial diversity for the sediments from Ouchy than from samples collected at 0e2 cm and 4e6 cm were observed. Vidy (Fig. 5). 2þ Porewater concentrations of major ions, including Fe , By means of the parsimony test (P-Test) and the FST-test, ¼ þ þ 2 2 H2S( H2S HS S ) and SO4 are shown in Fig. 2. High a significant genetic differentiation was observed between the 3 m concentrations of porewater PO4 , reaching about 270 M sediment bacterial communities of the two sites at all depths were only detected in the uppermost cm of Vidy Bay sedi- (0e2 and 4e6 cm), as well as between the two samples from ments (not shown). Nitrate was not detected in either one of Ouchy taken at different depths (Table 4). Significance for both the two cores. Porewater sulphate in the Ouchy sediment tests signals less genetic diversity within each community decreased from about 550 mM at the surface to 0 mMat7cm than for two communities combined and that the different depth, whereas it was essentially depleted in the Vidy Bay communities harbour distinct phylogenetic lineages (Martin, þ sediment. Dissolved Fe2 concentrations were only high in 2002). Vidy sediment where they reached 260 mM. It is unlikely that Neighbour-joining phylogenetic trees of Betaproteobacteria þ under such Fe2 -rich conditions free sulphide exists in pore- (Fig. 6), Deltaproteobacteria (Fig. 7), and Gammaproteobacteria water as it rapidly reacts with iron to form FeS. The measured (Fig. 8) are presented. porewater sulphide in the Vidy core represents thus mainly The microbial communities of both sites were correlated soluble FeS complexes or colloidal FeS phases. Minor amounts with the environmental variables, as indicated by the Mantel of poorly crystalline Fe(III)oxides were only detected in the test result (r ¼ 0.9429, p ¼ 0.044). A more detailed picture of the uppermost section of the Ouchy sediment core. Below 1 cm relationship between bacterial community structures and depth, and in the whole core from the Vidy Bay, HCl extract- environmental factors at the two sites was obtained by MFA able Fe(III) was absent (Fig. 2). Differences in the iron-sulphur (Fig. 10). Results show that (i) the sampling sites Ouchy and geochemistry exist between the two sites as indicated also by Vidy were clearly different in terms of their microbial AVS and CRS data (Fig. 3). Unlike the Vidy sediments, solid communities and environmental factors; (ii) the two sampling phase sulphur species in the Ouchy sediments accumulated depths in Vidy were not significantly different in terms of gradually with depth and reached a plateau at around 3 cm. microbial communities, OM, nutrients and heavy metals; (iii) 0 Less reduced sulphur species such as S , FeS2,Fe3S4 may play the similarity among the datasets from Ouchy is much lower a role as indicated by higher CRS contents. Iron contents in the than from Vidy. Vidy sediments were about 50% higher than in the Ouchy site and a constant Fe/S ratio of 1 along the whole core suggests 3.3. Archaeal 16S rRNA gene clone libraries FeS as dominant phase. The contents of heavy metals (in mg kg 1) are reported in All Archaea found in both sites fell into the Euryarchaeota Table 2. All measured metal concentrations, except for Cr, division and most of them were from 4 to 6 cm of depth, were higher in the Vidy sediments, where peak concentra- except for 2 clones, which were retrieved from surface sedi- tions reached 2.8 mg kg 1 for Cd, 181.4 mg kg 1 for Cu, ments: one from Vidy and one from Ouchy. The 36 obtained 164.7 mg kg 1 for Pb, and 2.3 mg kg 1 for Hg. sequences were grouped into 18 OTUs: 1 OTU for Vidy 0e2cm, 5 OTUs for Vidy 4e6 cm, 1 OTU for Ouchy 0e2 cm, and 12 for 3.2. Bacterial 16S rRNA gene clone libraries Ouchy 4e6 cm. A large proportion of these Euryarchaeota phylotypes, mostly originating from Vidy, were assigned to At each site, sediment samples from 0 to 2 cm and 4e6cmdepth the methanogenic families Methanosaetaceae and Meth- were used for clone library construction of bacterial 16S rRNA anomicrobiaceae (Fig. 9). genes. Phylogenetic analysis showed that approximately 85% of the retrieved clones fell into known divisions with the rest remaining unclassified. Seven and twelve divisions were iden- 4. Discussion tifiedin the sediments from Vidy and Ouchy, respectively (Fig. 4). The dominant groups (Beta-, Gamma-, Delta- and Sediments were sampled in the area of Ouchy, a site close to Bacteroidetes) were found at both sites and all depths. Most the city of Lausanne but without known impact of contami- sequences in the Bay of Vidy were affiliated with Betaproteobac- nated water, and in the Bay of Vidy near the WWTP outlet teria (37%), Deltaproteobacteria (15%), Gammaproteobacteria (15%), pipe. The Bay of Vidy, contaminated with heavy metals, and Bacteroidetes (23%). Also in the Ouchy sediments, most hydrophobic organic compounds and faecal bacteria, is sequences were related to Betaproteobacteria (22%), Deltaproteo- currently the most polluted area of Lake Geneva (Pardos et al., bacteria (6%), Gammaproteobacteria (24%), and Bacteroidetes (12%). 2004; Pote´ et al., 2008; Haller et al., 2009). Organic matter The sequences were assigned to individual OTUs based on contents ranged from 18.7% (0e2 cm) to 23.7% (4e6 cm) and their phylogenetic positions and the 97% sequence similarity were much higher than in the Ouchy area (max. 5%) or 1218 water research 45 (2011) 1213e1228

Fig. 2 e Concentration profiles of porewater sulphate and sulphide in the sediments from Vidy and Ouchy (above). The concentrations of iron(II) in the porewater and contents of solid phase iron(III)oxides are shown below. water research 45 (2011) 1213e1228 1219

e [ D [ 0 D D Fig. 3 Depth profiles of acid volatile sulphur (AVS H2S FeS), chromium reducible sulphur (CRS S FeS2 Fe3S4), and total reducible sulphur (TRS [ AVS D CRS) in the sediments from Vidy and Ouchy.

elsewhere in Lake Geneva (max. 5e8%) (Pote´ et al., 2008). The (Pote´ et al., 2008). In that survey, hydrophobic organic intense mineralisation of organic matter, indicated by high compounds, such as PAHs, PCBs and OCPs were investigated porewater phosphate, led to strongly reducing conditions in and concentrations of up to 2, 156 and 45 mgkg 1 were deter- the Vidy sediments. Porewater sulphate was used up by mined, respectively. These values are considered high and sulphate-reducing bacteria and iron was essentially present above average levels for Lake Geneva (Corvi et al., 1986). It as FeS. Due to the absence of any other significant oxidant, appears clearly that the sediments around the WWTP outlet methanogenesis is suspected to be the major process for pipe in the Bay of Vidy are heavily contaminated with many organic matter degradation at this site. The conditions were kinds of pollutants, possibly representing a significant source less reducing in the Ouchy sediments, where iron-reduction of toxicity for microbial communities and benthic organisms. may have prevailed in the uppermost 1e2 cm of the sediment Results from this study indicate that the dominant phylo- profile. Underneath, at 2.5 cm depth, the sediment was sul- genetic groups at both sites were the Beta-, Gamma- and Delta- phidic due to the activity of sulphate-reducing bacteria. Below proteobacteria and Bacteroidetes, which is in agreement with 16S 7 cm depth, where sulphate was depleted, methanogenic rRNA analyses of lake bacterioplankton (Zwart et al., 2002; conditions prevailed (Canfield and Thamdrup, 2009). Glo¨ ckner et al., 2000; Hiorns et al., 1997). Nevertheless, some Heavy metal concentrations in the Bay of Vidy were up to six differences between the two sites were observed in the rela- times higher than in Ouchy. According to the Canadian Sedi- tive proportion of the different Proteobacteria subdivisions. ment Quality Guidelines for the Protection of Aquatic Life Proteobacteria accounted for 64% of the clones in Vidy and (Conseil Canadien des Ministres de l’Environnement, 1999) the 55% of the clones in Ouchy. Proportions of Beta- and Deltap- heavy metal concentrations in Vidy Bay were 2e8 times higher roteobacteria were much higher in Vidy Bay that in Ouchy than reported PELs (probable effect levels). These results are in sediments. In contrast, the Gammaproteobacteria were more agreement with previous data from the same sampling site abundant in Ouchy sediments (Fig. 4).

L Table 2 e Depth variation of heavy metal contents (mg kg 1 dry weight sediment)a in Vidy Bay and Ouchy sediments. Depth Cu Zn Cd Pb Cr Hg

Ouchy Vidy Ouchy Vidy Ouchy Vidy Ouchy Vidy Ouchy Vidy Ouchy Vidy

0e2 cm 54.7 142.5 126.8 341.3 0.5 1.6 38.8 89.4 60.3 74.2 0.2 1.2 2e4 cm 68.3 135.9 155.1 327.8 0.7 1.5 49.7 89.3 63 68.3 0.6 1 4e6 cm 86.8 133.6 218.1 344.9 1.2 1.7 75 100.7 69.7 67.9 1 2.3 6e8 cm 135.5 181.4 305.5 446.8 1.7 2.3 106.5 135.9 88.4 77.2 1.3 2.3 8e10 cm 136.5 161.4 242.8 518.2 1.2 2.8 91 164.7 81.4 88.9 1.7 2.2

a Total variation coefficients for triplicate measurements were <15% for all elements. 1220 water research 45 (2011) 1213e1228

Fig. 4 e Relative abundance of bacterial taxonomic groups in the clone libraries established with sediments from the Bay of Vidy and the Ouchy area. Fig. 5 e Rarefaction curves for the Bacteria 16S rRNA gene sequences retrieved from Vidy and Ouchy sediment samples (depth intervals: 0e2 cm and 4e6 cm). Operational Betaproteobacteria appear to be one of the major divisions taxonomic units were defined with a 97% sequence present in freshwater systems (Zwart et al., 2002; Hahn, 2006). similarity cut-off. In this study they were also well represented in both locations (37% of clones in Vidy, 22% in Ouchy), particularly in surface sediments where they were dominant (Fig. 6). A prevalent group of bacteria in Vidy sediments, belonged to the Rhodo- of anaerobic, muddy freshwater sediments (Tanaka et al., cyclaceae, which are phenotypically and ecologically very 2003). Two further clones were related to Thiobacillus (Hydro- diverse. Twenty clones of this family were affiliated to Dech- genophilaceae), which are ubiquitous in soils and sediments, loromonas, a genus containing many heterotrophic and facul- and may grow on reduced sulphur compounds. tative anaerobic chlorate-, perchlorate- or nitrate respiring Compared to Betaproteobacteria, the Deltaproteobacteria bacteria (Wolterink et al., 2005). Members of this genus such as subdivision (Fig. 7) was less abundant (15% and 6% of the Dechloromonas aromatica are found in aquatic habitats and are clones in Vidy and Ouchy sediments, respectively). Most capable of oxidising aromatic compounds such as toluene, Deltaproteobacteria are sulphate-, iron- or proton-reducing benzoate, and chlorobenzoate. D. aromatica is currently the (syntrophic) bacteria that play major roles in anoxic settings only pure culture being able to degrade anaerobically benzene like meromictic lakes and sediments (Lehours et al., 2007; with nitrate as electron acceptor (Coates et al., 2001) A few Karr et al., 2005). The large number of clones related to clones, also found only in Vidy sediments, were related to sulphate-reducing bacteria (Desulfobacteraceae) in Vidy Bay Methylophilus (Methylophilaceae) a group of methylotrophic was not unexpected since sulphate consumption in the organisms. However, while methanol is oxidised as the sole sediment was obvious from the porewater data. Moreover, carbon and energy source, some species may grow also on the pool of total reduced sulphur (TRS) was quite high, a limited range of other carbon compounds such as methyl- especially in Vidy sediments. Several additional clones, only amines, formate, glucose, and fructose (Jenkins et al., 1987). found in Vidy sediments, were related to Geobacter sp. Similar Methylophilaceae sequences were found in wastewater (Geobacteraceae). These anaerobic bacteria have been isolated treatment pools in China (AY863077) and in freshwater from freshwater sediments, soils and subsurface environ- calcareous mats (EF580978). The remaining Betaproteobacteria ments. They are traditionally considered chemo- clones were found in both sites and included various genera. organotrophic Fe(III)-reducing bacteria. However, most Several clones were affiliated to Propionivibrio (), which are aerotolerant or obligate anaerobic chemo- organotrophic bacteria. These bacteria are typical inhabitants e P Table 4 Summary of FST and -test results for the comparison of microbial communities between Vidy and Ouchy sediments. “A” stands for the 0e2 cm sediment section and “B” for the 4e6 cm sediment section. Table 3 e Distribution of Bacteria phylotypes in clone P valuea libraries from Vidy and Ouchy sediments (Lake Geneva). “A” stands for the 0e2 cm sediment section and “B” for Group FST-test P test the 4e6 cm sediment section. Vidy A vs Vidy B NS NS Vidy A Vidy B Ouchy A Ouchy B Ouchy A vs Ouchy B < 0.00 0.001 Vidy A vs Ouchy A 0.001 0.001 Total no of sequences 68 41 60 40 Vidy B vs Ouchy B 0.005 0.001 Total no of OTUs 40 26 53 32 Percentage of unique OTUs 17 10 30 18 a NS, not significant. water research 45 (2011) 1213e1228 1221

Fig. 6 e Neighbour-joining phylogenetic tree of Betaproteobacteria 16S rRNA gene sequences retrieved from Vidy Bay and Ouchy sediments. “A” in clone names stands for the 0e2 cm sediment section and “B” for the 4e6 cm sediment section. Bootstrap values > 50% are shown (500 replicates). The scale bar represents 2% estimated sequence divergence. 1222 water research 45 (2011) 1213e1228

Fig. 7 e Neighbour-joining phylogenetic tree of Deltaproteobacteria 16S rRNA gene sequences from Vidy Bay and Ouchy sediments. “A” in clone names stands for the 0e2 cm sediment section and “B” for the 4e6 cm sediment section. Bootstrap values >50% are shown (500 replicates). The scale bar represents 2% estimated sequence divergence.

species utilise a wide range of alternative electron acceptors, Synthrophaceae, with one clone related to Smithella sp. Similar 0 including NO3 ,S, and other sulphur compounds (Coates sequences were identified as core microorganisms involved et al., 1998). Similar sequences related to Geobacter sp. were in the anaerobic digestion of sludge (Riviere et al., 2009; retrieved from anthropogenically impacted urban creek CU922073). sediment (EU284415) and from an aquifer where Fe(III) The Gammaproteobacteria were more abundant in Ouchy reduction was associated with aromatic hydrocarbon sediments than in Vidy and were phylogenetically diverse degradation (Nevin et al., 2005; AY653549). Another group of (Fig. 8). Clones from both sites were affiliated to Methylobacter clones exclusively found in Vidy sediments belonged to the sp. (Methylococcaceae), which are characterised by their water research 45 (2011) 1213e1228 1223

Fig. 8 e Neighbour-joining phylogenetic tree of Gammaproteobacteria 16S rRNA gene sequences from Vidy Bay and Ouchy sediments. “A” in clone names stands for the 0e2 cm sediment section and “B” for the 4e6 cm sediment section. Bootstrap values >50% are shown (500 replicates). The scale bar represents 2% estimated sequence divergence. 1224 water research 45 (2011) 1213e1228

Fig. 9 e Neighbour-joining phylogenetic tree showing 16S rRNA gene sequences of Archaea retrieved from Vidy Bay and Ouchy sediments. “A” in clone names stands for the 0e2 cm sediment section and “B” for the 4e6 cm sediment section. Bootstrap values >50% are shown (500 replicates). The scale bar represents 5% estimated sequence divergence. water research 45 (2011) 1213e1228 1225

specialised metabolism restricted to the oxidation of methane composition changed with depth in the uncontaminated or methanol. Similar sequences were retrieved from sediment. Conversely, no statistically significant variations a permafrost soil in Siberia (Liebner et al., 2009; EU124843) and were observed for the two sediment layers (0e2 and 4e6cm)in an Arctic wetland soil (Wartiainen et al., 2006; AJ414655). the Bay of Vidy, which is explained by the high sedimentation Surprisingly, a few clones from Vidy sediments were related rates and the non-consolidated nature of the sediment, to phototrophic purple sulphur bacteria (Chromatiaceae) permitting mobilisation and vertical mixing. despite the fact that there is not much light to be expected at The microbial composition of both sites was correlated 30 m depth. However, some species can also grow under with the environmental variables, as shown by the Mantel chemotrophic conditions in the dark, either autotrophically or correlation test (r ¼ 0.9429, p ¼ 0.044). This result suggests that heterotrophically using oxygen as terminal electron acceptor. the diversity of microbial communities may be affected by Sequences similar to ours were retrieved from an anaerobic nutrients, organic matter as well as the degree of pollution. digestor (Riviere et al., 2009) and the sediment surface of Many environmental variables are implicated, which is Fayetteville Green Lake, USA (FJ437977). Several groups of a situation inherent to all field studies. The Bay of Vidy is Gammaproteobacteria clones remained unclassified but with contaminated by all kinds of contaminants but also with high similar sequences found in bacterioplankton communities of quantities of organic matter and nutrients. Under these Lake Michigan (Mueller-Spitz et al., 2009. EU640647), river conditions it is difficult to separate out the influence of the sediments (Li et al., 2008. EF590053), mangroves (EF125457) different environmental variables on microbial diversity and and agricultural soils (FJ444695). community composition. To learn more about the relative A large number of sequences affiliated with the division importance of individual environmental factors, microcosm Bacteroidetes (CytophagaeFlexibactereBacteroidetes) were found studies will be required. in both sites, particularly in Vidy sediments. Bacteroidetes The integrative picture of the relationship between bacte- constitute the second largest group in Vidy sediments after rial community structures and environmental factors at the the Betaproteobacteria, and the third largest group in Ouchy two sites (Fig. 10) indicated that the sampling sites Ouchy and sediments (Fig. 4). Bacteroidetes phylotypes were diverse and Vidy were clearly different with respect to both. Previous their closest relatives were sequences found in freshwater results already showed that the bacterial diversity in compa- lakes (Mueller-Spitz et al., 2009), anthropogenically impacted rable contaminated and uncontaminated environments may sediments, tundra soils (Liebner et al., 2008) and aquifers. differ significantly. The difference may be explained by the Most of the sequences found in the 2 investigated sites nature of pollution and a wide diversity of organic carbon remained unclassified. One clone found in Vidy sediments was affiliated to the genus Cytophaga sp. and one clone from Ouchy was related to the genus Flavobacterium sp. A large proportion of the Euryarchaeota phylotypes, mostly retrieved from Vidy sediments, were related to methanogens like Methanosaeta sp. (Methanosaetaceae)andMethanomicrobiales (Fig. 9). A few species of Methanosaeta sp. have been isolated from anaerobic sewage digestors or sewage sludge (Zinder et al., 1984; Huser et al., 1982). Similar sequences to the clones found in this study, were retrieved from anaerobic sludge and a mer- omictic lake (Lehours et al., 2007). The rest of the archaeal sequences were only distantly related to any cultured species but similar to sequences retrieved from lake sediments (Pouliot et al., 2009; AY531743, EU782007), Arctic peat (AM712495) and the anoxic zone of a hydropower plant reservoir in the Brazilian Amazon (GU127420 and GU127500). The two investigated sites differ clearly in terms of sedi- ment chemical parameters and degree of pollution. It was therefore expected that the bacterial community composition would be different and reflect the differences in environmental conditions. Fig. 4 and Figs. 6e9 clearly show the diverse Fig. 10 e The Multiple factor analysis (MFA) is a PCA-based bacterial and archaeal lineages detected in the two sites. The technique allowing the simultaneous ordination of results of the genetic diversity tests confirm the significant a composite table obtained by the juxtaposition of the genetic differentiation of the sediment bacterial communities species and the two environmental datasets, after between the two sites at all depths. Seven and twelve weighting the different matrices The superimposed divisions were identified in the sediments from Vidy and representation shows one global point for each site, Vidy Ouchy, respectively. Among them Nitropsira, Planctomycetes, and Ouchy, at each depth (“A” stands for the 0e2cm Verrucomicrobia and Gemmatimonadetes were only detected in sediment section and “B” for the 4e6 cm sediment section). Ouchy sediments. The apparent lower bacterial diversity in The three associated partial points correspond to the three Vidy sediments may be explained by the high levels of a broad datasets (microbial composition, organic matter and range of pollutants, which may induce adverse biological nutrients and heavy metals). The values on the axes effects on microbial communities. The bacterial community indicate the percentage of total variation. 1226 water research 45 (2011) 1213e1228

sources (Sandaa et al., 1999; Sorci et al., 1999; Zhang et al., navigating R/V “La Licorne” and the Municipality of Lausanne 2008). The polluted environment of Vidy Bay may have for financial support. selected, among the dispersed microbes in sediments, certain functional bacterial groups which adapted to these conditions and became more dominant in that particular environment. references

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