wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8

Avai lab le a t www.sc iencedi rec t .com

journa l homepage : www.e lsev ie r . com/ loca te /wat res

Composition of bacterial and archaeal communities infreshwater sediments with different contaminationlevels (Lake Geneva, Switzerland)

Laurence Haller a, Mauro Tonolla b,d, Jakob Zopfi c, Raffaele Peduzzi b,Walter Wildi a, John Pote a,*aUniversity of Geneva, Institute F.A. Forel, 10 route de Suisse, CP 416, CH-1290 Versoix, SwitzerlandbMicrobial Ecology, Microbiology Unit, Plant Biology Department, University of Geneva, 30, Quai Ernest-Ansermet,

CH-1211 Geneva, SwitzerlandcUniversity of Lausanne, Institute of Geology and Paleontology, Laboratory of Biogeosciences, CH-1015 Lausanne, SwitzerlanddCantonal Institute of Microbiology, Via Mirasole 22A, CH-6500 Bellinzona, Switzerland

a r t i c l e i n f o

Article history:

Received 31 August 2010

Received in revised form

8 November 2010

Accepted 14 November 2010

Available online 20 November 2010

Keywords:

Lake Geneva

Sediment pollution

Heavy metal

Microbial diversity

16S rRNA

Clone library

* Corresponding author. Tel.: þ41 22 379 03 2E-mail address: john.pote@unige.ch (J. Po

0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.11.018

a b s t r a c t

The aim of this study was to compare the composition of bacterial and archaeal

communities in contaminated sediments (Vidy Bay) with uncontaminated sediments

(Ouchy area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were

analysed for physicochemical characteristics including porewater composition, organic

carbon, and heavy metals. Results show high concentrations of contaminants in sediments

from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense

mineralisation, which was dominated by sulphate-reduction and methanogenesis. The

bacterial diversity in Vidy sediments was significantly different from the communities in

the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of

Betaproteobacteria clones in Vidy sediments related to Dechloromonas sp., a group of dech-

lorinating and contaminant degrading bacteria. Deltaproteobacteria, including clones related

to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more

abundant in the contaminated sediments. The archaeal communities consisted essentially

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

Untreated or only partly treated wastewaters including

industrial, agricultural and domestic effluents constitute the

main contamination sources in aquatic environments. Sedi-

ment contamination is usually due to inorganic and organic

1; fax: þ 41 22 379 03 29.te).ier Ltd. All rights reserved

organic compounds, such as polycyclic aromatic hydrocar-

bons (PAHs), polychlorinated biphenyls (PCBs) and organo-

chlorine pesticides (OCPs) (e.g. Koelmans et al., 2001; Eggleton

and Thomas, 2004). The increasing contamination of sedi-

ments by inorganic and organic micro-pollutants is a big

.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81214

concern in aquatic ecosystems (Forstner andWittmann, 1979;

Pardos et al., 2004; Schwarzenbach et al., 2006). Sediment

contamination by HMs and other micro-pollutants might

cause potential adverse effects to ecosystems and also pose

human health risks (Salomons and Forstner, 1984; Verweij

et al., 2004; Wang et al., 2004). The main environmental risk

is remobilization of the contaminants and their return to the

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

sediments should be examined extensively (e.g. Wang et al.,

2004; Wildi et al., 2004). Microbial communities may be

sensitive indicators for pollution in aquatic ecosystems and

may be applied for biomonitoring and assessment purposes

(Pronk et al., 2009). Heavy metal contamination, for example,

may lead to a reduction of bacterial diversity (Sandaa et al.,

1999). However, other studies observed either an increase in

microbial diversity along with heavy metal contamination

(Sorci et al., 1999) or no significant variation (Gillan et al., 2005).

Other environmental factors as well as the time of exposure

might explain these differences.

Lake Geneva is the largest freshwater reservoir of Western

Europe with a volume of 89 km3 and a maximum depth of

309 m. It is a monomictic temperate lake, with early spring

overturn not occurring every year. The lake was considered

eutrophic in 1970s and 1980s, but has become mesotrophic

after drastic reduction of phosphorus inputs (Dorioz et al.,

1998). Approximately 700,000 people are supplied with water

from Lake Geneva. The city of Lausanne, located on the

northern shore, discharges the largest volume of treated

wastewater into the nearby Bay of Vidy. The wastewater

treatment plant (WWTP) of the city treats nowadays approx-

imately 220,000 equivalent-inhabitants of wastewater. The

WWTP effluent is released into the Bay of Vidy 700m from the

shore at 30 m depth. As a consequence, Vidy Bay is the most

contaminated area of Lake Geneva. Published data document

the accumulation of contaminants close to a recreational area

as well as the related ecological impacts and health risks

(Loizeau et al., 2004; Pardos et al., 2004; Wildi et al., 2004).

Several studies focused on the quality of bottom sediments in

the Bay of Vidy and on the spatial distribution of organic

matter, faecal indicator bacteria, heavy metals, and hydro-

phobic organic compounds (Pote et al., 2008; Haller et al.,

2009). However, there is still a paucity of information

regarding sedimentarymicrobial communities in Lake Geneva

and particularly around the WWTP discharge outlet.

Sediments are complex habitats densely colonised by

diverse groups of microorganisms, which play key roles in

biogeochemical cycling, aquatic food webs and the remobili-

zation of heavy metals (Nealson, 1997; Lors et al., 2004; Ye

et al., 2009). Many studies have been performed to examine

the composition and variability of microbial communities in

extreme or complex aquatic ecosystems. However, only a few

studies compared microbial community structures in

contaminated and uncontaminated sediments (Powell et al.,

2003; Zhang et al., 2008). The aim of the present study was

to compare the composition of the sediment-associated

microbial communities in the contaminated Bay of Vidy with

a closeby less polluted site in the Ouchy area. The Bay of Vidy

is currently used as a model system for several limnological,

biogeochemical, and ecotoxicological studies. This research

represents the first assessment of Bacteria and Archaea in

contaminated and uncontaminated sediments of Lake Geneva

and serves as important background information for these

studies. For a better understanding of the microbial commu-

nity structures, molecular analyses were complemented by

a detailed physicochemical characterisation of the sediments.

2. Materials and methods

2.1. Study site description and sampling procedure

In August 2005, sediment was collected at two locations in

Lake Geneva (Fig. 1): (i) within the Bay of Vidy near the outlet

pipe of the WWTP of Lausanne (Swiss coordinates X: 534682,

Y: 151410) and (ii) near the Ouchy area (Swiss coordinates X:

537985, Y: 150390). Sampling was done from R/V “La Licorne”

using a core sampler (Benthos Inc, USA). Three cores (6.7 cm

i.d., 1.5 m length) were retrieved from each site at a depth of

40 m. For microbiological analyses, the cores were opened

longitudinally and sliced into 2 cm thick sections until 10 cm

depth. The sediment samples were placed into sterile plastic

containers, stored in an icebox and treated in the laboratory

within 24 h. For chemical analysis, the intact sediment cores

were transported to the laboratory and stored vertically in

a cold-room at 4 �C until analysis.

2.2. Chemical analysis

Two cores per site were used for the chemical analyses: one

was used to measure organic matter, nutrients and HMs

contents and the other one was used to determine sulphur

and iron concentrations and the porewater constituents.

Organic matter and nutrients: the cores were opened longitu-

dinally and sliced every 2 cm down to a depth of 10 cm. Before

analysis, sediment samples were air-dried at ambient room

temperature. The particle grain size wasmeasured with a laser

Coulter� LS-100diffractometer (BeckmanCoulter, Fullerton,CA,

USA), after a 5-min ultrasonic dispersal in deionised water

according to the method described by Loizeau et al. (1994). The

proportionsof threemajorsizeclasses (clay<2mm;silt 2e63mm;

and sand > 63 mm) were determined from size distributions.

Totalorganicmattercontent insedimentswasestimatedby loss

on ignition (LOI) at 550 �C for 1 h in a Salvis oven (Salvis AG

Emmenbrucke, Luzern, Switzerland) on 5 g of dried sediments.

Total organic carbon (TOC) was determined by titrimetry

following acid oxidation on 5 g of dried sediments. Total

nitrogen (TN) was determined according to Kjeldahl (APHA,

1985) on 2 g of dried sediments. Total phosphorus (TP) and its

different forms were measured on 150 mg of dried sediments

with a spectrophotometer (Helios Gamma UVeVis, Thermo

Scientific,USA)at 850nm, following the fractionationschemeof

Williams et al. (1976) as modified by Burrus et al. (1990). The

results are expressed inmg kg�1 dry weight sediment (ppm).

Solid phases: sulphur and iron contents were determined

every centimeter down to 10 cm depth. The sediment was

sliced at room temperature in a N2-filled glove bag and fixed in

50 mL tubes containing 10 mL zinc acetate solution (10%).

Sulphide was extracted as acid volatile sulphur [AVS;

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 46�3200000 N and 6�3300000 E and the lower right corner represents 46�2900000 N and 6�3800000 E.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1215

dissolved sulphide (H2S) and iron sulphides (FeS)] and chro-

mium reducible sulphur [CRS; primarily pyrite (FeS2),

elemental sulphur (S0), and some organic sulphur]. AVS and

CRS were measured by a two step distillation process with

cold 6 N HCl followed by boiling 1 M acidic CrCl2 solution

(Fossing and Jørgensen, 1989; Zopfi et al., 2008). Poorly cris-

talline Fe(III)oxides were extracted with 0.5M HCl according to

Thamdrup et al. (1994).

Total contents of Cu, Cd, Cr, Zn, and Pbwere determined by

quadrupole-based Inductively Coupled PlasmaeMass Spec-

trometry (ICPeMS) (HP 4500, Agilent) following the digestion

of 1 g of dried sediment in analytical grade 2 M HNO3 (Pardos

et al., 2004). Total Hg was quantified by atomic absorption

spectrophotometry (Advanced Mercury Analyser; AMA 254,

Altec, Czech Rep.) according to Hall and Pelchat (1997) and

Ross-Barraclough et al. (2002). Themethod is based on sample

combustion, gold amalgamation and atomic absorption

spectrometry (AAS). Average values of triplicate measure-

ments are expressed in mg kg�1 dry weight sediment (ppm).

Porewater constituents: porewater was harvested by centri-

fugation (4000 rpm) under an N2 atmosphere to avoid Fe2þ and

H2S oxidation. Samples for dissolved Fe2þ were acidified with

0.5 M HCl and analysed by the photometric Ferrozine method

(Thamdrup et al., 1994). Dissolved sulphidewas determined on

Zn-acetate fixed samples using the colorimetric methylene-

blue method (Cline, 1969; Zopfi et al., 2008). The major anions

Cl�, SO42�, NO3

�, and PO43� were determined by ion-chromatog-

raphy on a DIONEX DX-120 system using an IonPac� AS14A

anion exchange column, Na2CO3/NaHCO3 (8 mM/1 mM) as

eluent, an Anion Self-Regenerating Suppressor (ASRS� 300,

4 mm) module and a conductivity detector.

2.3. DNA extraction

The samples from 0 to 2 cm and 4e6 cm depth were used for

microbiological analyses. Total DNA was extracted from

250 mg of sediment using the PowerSoil� DNA Isolation Kit

(Mo-Bio Laboratories, Carlsbad, CA, USA), according to the

manufacturer’s instructions. The concentration of extracted

DNA was measured spectrophotometrically (OD260) and

aliquots were used to estimate DNA quality by electrophoresis

on 0.8%agarose gel stainedwith ethidiumbromide (10 mgml�1,

SigmaeAldrich Chemie GmbH, Buchs, Switzerland). DNA

extractswere stored at�20 �C until used for PCR amplification.

2.4. PCR amplification

Nearly complete bacterial 16S rRNA genes were amplified

using the primers 26f (50-AGAGTTTGATCATGGCTCA-30) and

1392r (50-GTGTGACGGGCGGTGTGTA-30) (Brosius et al., 1981;

Lane, 1991). Archaeal 16S rRNA genes were amplified using

the primers 109f (50-ACKGCTCAGTAACACGT-30) and 915r (50-GTGCTCCCCCGCCAATTCCT-30) (Grobkopf et al., 1998; Stahl

and Amann, 1991).

Both archaeal and bacterial PCR amplifications were per-

formed separately using the Taq PCR Master Mix Kit (Qiagen,

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81216

Basel, Switzerland). Each PCR reaction was carried out in

a volume of 50 mL, containing 1� PCR buffer, 1.5 mM of MgCl2,200 mM of each dNTP, 0.3 mM of each primer, 2.5 units of Taq

DNA polymerase, 2.5 mg ml�1 of bovine serum albumin

(Invitrogen, Basel, Switzerland) and 2 mL of DNA (about 100 ng)

of each sample. The following conditions were used for PCR

amplification: initial denaturation at 94 �C for 5 min; 35 cycles

of denaturation (94 �C for 30 s), annealing (52 �C for 30 s),

extension (72 �C for 1min) and a final extension step of 10min

at 72 �C. PCR products (10 mL) were separated by electropho-

resis on 0.8% agarose gels and visualised by ethidium bromide

staining and UV illumination.

2.5. Clone library construction and DNA sequencing

PCR products were purified using the NucleoSpin� Extract II

Kit (MachereyeNagel, Oensingen, Switzerland) according to

themanufacturer’s instructions. The amplified DNAwas then

quantified using the PicoGreen� dsDNA Quantitation Reagent

(Molecular Probes Inc., Eugene, OR, USA) and a TD-700 Fluo-

rometer (Turner Designs Sunnyvale, CA, USA). Approximately

20e30 ng of amplified 16S rDNA were cloned into competent

Escherichia coli cells using the TOPO TA cloning kit (Invitrogen,

Basel, Switzerland) following the manufacturer’s recommen-

dations. The transformed cells were plated on LB medium

containing 50 mg L�1 ampicillin, 60 mg L�1 of IPTG (isopropyl-

b-D-thiogalactopyranoside), and 100mg L�1 of X-gal (5-bromo-

4-chloro-3-indolyl-b-D-galactopyranoside), and incubated

overnight at 37 �C.White recombinants were transferred to LB

medium plates for 24 h. 80 and 40 recombinants were picked

from samples taken at 0e2 cm and 4e6 cm, respectively, in

each location, to constitute the Bacteria clone libraries. For the

Archaea, only 2 recombinants (1 per site) were found at 0e2 cm

and 34 colonies were picked from 4 to 6 cm. The insert size of

all picked colonies, was determined by direct PCR using M13

forward and reverse primers included in the cloning kit. The

products were subsequently purified with the NucleoSpin�

Extract II Kit and sequenced using the BigDye� Terminator

Cycle Sequencing Ready reaction kit (Applied Biosystems

International, Inc., Rotkreuz, Switzerland). Sequences were

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

bacterial clones (Lane, 1991).

2.6. Phylogenetic analysis

The obtained sequences were checked and edited using the

programEditSeq� (DNAStar Inc.,Madison,WI,USA).A chimera

Table 1 e General characterisation of the contaminated (Vidy)clone library construction.

Sediment section OM (%) TOC (%) TN (mg kg�1) NH4-

Vidy 0e2 cm 18.7 10.8 11.6

Vidy 4e6 cm 23.7 13.7 13.5

Ouchy 0e2 cm 5 2.9 2.2

Ouchy 4e6 cm 4.3 2.5 1.7

OM: Organic matter; TOC: Total organic carbon; TN: Total nitrogen; NH4-

detectionprogramwasused to exclude chimeras (Maidak et al.,

1999). NCBI BLAST (http://www.ncbi.nih.gov) was used to

identify themost closely related 16S rRNA gene sequences. The

partial 16S rRNA gene sequences were then aligned in the

Clustal W implementation of MEGA 3.0 (Kumar et al., 2004).

The same program was used to produce neighbour-joining

phylogenetic trees (Kimura-2 correction; bootstrap values for

500 replicates). The sequences were identified using the ribo-

somal database project classifier (Wang et al., 2007). Sequences

from this study have been deposited with the EMBL database

under accession numbers FN679050eFN679294.

The sequences were assigned to individual OTUs based on

the 97% sequence similarity criterion. The number of OTUs

and the rarefaction curves were estimated from the sequence

data using Mothur v.1.12.3 (Schloss et al., 2009).

Comparison between the clone libraries from the two sites

was done on the basis of genetic diversity by means of the

parsimony test using Mothur v.1.12.3 (Schloss et al., 2009) and

the the FST-test using the program Arlequin, v.2.0 (Schneider

et al., 2000).

A Mantel test using Spearman correlation (Mantel, 1967)

was applied to relate the microbial community compositions

from the two sites with environmental variables. Multiple

factor analysis (MFA)was done to obtain an integrative picture

of the relationship between the bacterial community struc-

tures and the environmental factors across the 2 sites. The

environmentalparameterswereseparated into2matrices, one

including organic matter contents and nutrient variables and

one with the heavymetal concentrations. The MFA allows the

simultaneous ordination of a composite table obtained by the

juxtaposition of the species and environmental datasets after

weighting thedifferentmatrices (Escofier andPages, 1994). The

final ordinationplot showsglobal points indicating the relative

positions of the objects described by the combination of data-

sets. Each global point is surrounded by partial points indi-

cating the relative positions of the datasets taken separately

(Escofier and Pages, 1994; Becue-Bertaut and Pages, 2008).

These statistical analyses were carried out in “R”, a free soft-

ware environment for statistical computing and graphics,

using the Vegan library (R Development Core Team, 2005).

3. Results

3.1. Chemical analysis

Some general sediment characteristics including particle

grain size, organicmatter and nutrient contents are presented

in Table 1. These sediments were mostly composed of silts,

and non-contaminated (Ouchy) sediment sections used for

N (mg kg�1) TP (mg kg�1) Clay/silt/sand proportion (%)

1.6 6783.8 0/64/35

1.6 9650.9 0/65/34

0.5 862.1 0.9/78/21

0.4 759.5 0.6/80/19

N: Ammonium; TP: Total phosphorus.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1217

approximately 65% and 80% for Vidy and Ouchy, respectively.

Average organic matter contents in Vidy Bay sediments were

much higher (21%) than in Ouchy sediments (4.5%). Average

nutrient concentrations such as total nitrogen, ammonium

and total phosphorus were also higher in Vidy sediments

(12.6 ppm, 1.6 ppm and 8217 ppm, respectively) while they

were considered low in the sediments from Ouchy (2 ppm,

0.5 ppmand 810 ppm, respectively). However, at both stations,

no substantial differences in the nutrient contents between

samples collected at 0e2 cm and 4e6 cm were observed.

Porewater concentrations of major ions, including Fe2þ,H2S (¼H2S þ HS� þ S2�) and SO4

2� are shown in Fig. 2. High

concentrations of porewater PO43�, reaching about 270 mM

were only detected in the uppermost cm of Vidy Bay sedi-

ments (not shown). Nitrate was not detected in either one of

the two cores. Porewater sulphate in the Ouchy sediment

decreased from about 550 mM at the surface to 0 mM at 7 cm

depth, whereas it was essentially depleted in the Vidy Bay

sediment. Dissolved Fe2þ concentrations were only high in

Vidy sediment where they reached 260 mM. It is unlikely that

under such Fe2þ-rich conditions free sulphide exists in pore-

water as it rapidly reacts with iron to form FeS. The measured

porewater sulphide in the Vidy core represents thus mainly

soluble FeS complexes or colloidal FeS phases. Minor amounts

of poorly crystalline Fe(III)oxides were only detected in the

uppermost section of the Ouchy sediment core. Below 1 cm

depth, and in the whole core from the Vidy Bay, HCl extract-

able Fe(III) was absent (Fig. 2). Differences in the iron-sulphur

geochemistry exist between the two sites as indicated also by

AVS and CRS data (Fig. 3). Unlike the Vidy sediments, solid

phase sulphur species in the Ouchy sediments accumulated

gradually with depth and reached a plateau at around 3 cm.

Less reduced sulphur species such as S0, FeS2, Fe3S4 may play

a role as indicated by higher CRS contents. Iron contents in the

Vidy sediments were about 50% higher than in the Ouchy site

and a constant Fe/S ratio of 1 along the whole core suggests

FeS as dominant phase.

The contents of heavy metals (in mg kg�1) are reported in

Table 2. All measured metal concentrations, except for Cr,

were higher in the Vidy sediments, where peak concentra-

tions reached 2.8 mg kg�1 for Cd, 181.4 mg kg�1 for Cu,

164.7 mg kg�1 for Pb, and 2.3 mg kg�1 for Hg.

3.2. Bacterial 16S rRNA gene clone libraries

Ateach site, sediment samples from0 to2 cmand4e6 cmdepth

were used for clone library construction of bacterial 16S rRNA

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-

tifiedinthesediments fromVidyandOuchy, respectively (Fig. 4).

The dominant groups (Beta-, Gamma-, Delta-proteobacteria and

Bacteroidetes) were found at both sites and all depths. Most

sequences in the Bay of Vidy were affiliated with Betaproteobac-

teria (37%), Deltaproteobacteria (15%), Gammaproteobacteria (15%),

and Bacteroidetes (23%). Also in the Ouchy sediments, most

sequences were related to Betaproteobacteria (22%), Deltaproteo-

bacteria (6%), Gammaproteobacteria (24%), and Bacteroidetes (12%).

The sequences were assigned to individual OTUs based on

their phylogenetic positions and the 97% sequence similarity

criterion. The sequences (n ¼ 208) were grouped into 132

OTUs: 40, 26, 53, and 32 for Vidy 0e2 cm, Vidy 4e6 cm, Ouchy

0e2 cm, and Ouchy 4e6 cm, respectively (Table 3). The

number of unique OTUs was higher in Ouchy than in Vidy

sediments, 38% and 54% of the OTUs were exclusively found

in Vidy and Ouchy, respectively. Only 8% of all OTUs were

shared between the libraries from both sites. The OTU rich-

ness estimates based on rarefaction curves suggests a higher

bacterial diversity for the sediments from Ouchy than from

Vidy (Fig. 5).

By means of the parsimony test (P-Test) and the FST-test,

a significant genetic differentiation was observed between the

sediment bacterial communities of the two sites at all depths

(0e2 and 4e6 cm), as well as between the two samples from

Ouchy taken at different depths (Table 4). Significance for both

tests signals less genetic diversity within each community

than for two communities combined and that the different

communities harbour distinct phylogenetic lineages (Martin,

2002).

Neighbour-joining phylogenetic trees of Betaproteobacteria

(Fig. 6), Deltaproteobacteria (Fig. 7), and Gammaproteobacteria

(Fig. 8) are presented.

The microbial communities of both sites were correlated

with the environmental variables, as indicated by the Mantel

test result (r¼ 0.9429, p¼ 0.044). Amore detailed picture of the

relationship between bacterial community structures and

environmental factors at the two sites was obtained by MFA

(Fig. 10). Results show that (i) the sampling sites Ouchy and

Vidy were clearly different in terms of their microbial

communities and environmental factors; (ii) the two sampling

depths in Vidy were not significantly different in terms of

microbial communities, OM, nutrients and heavy metals; (iii)

the similarity among the datasets from Ouchy is much lower

than from Vidy.

3.3. Archaeal 16S rRNA gene clone libraries

All Archaea found in both sites fell into the Euryarchaeota

division and most of them were from 4 to 6 cm of depth,

except for 2 clones, which were retrieved from surface sedi-

ments: one from Vidy and one from Ouchy. The 36 obtained

sequences were grouped into 18 OTUs: 1 OTU for Vidy 0e2 cm,

5 OTUs for Vidy 4e6 cm, 1 OTU for Ouchy 0e2 cm, and 12 for

Ouchy 4e6 cm. A large proportion of these Euryarchaeota

phylotypes, mostly originating from Vidy, were assigned to

the methanogenic families Methanosaetaceae and Meth-

anomicrobiaceae (Fig. 9).

4. Discussion

Sediments were sampled in the area of Ouchy, a site close to

the city of Lausanne but without known impact of contami-

nated water, and in the Bay of Vidy near the WWTP outlet

pipe. The Bay of Vidy, contaminated with heavy metals,

hydrophobic organic compounds and faecal bacteria, is

currently the most polluted area of Lake Geneva (Pardos et al.,

2004; Pote et al., 2008; Haller et al., 2009). Organic matter

contents ranged from 18.7% (0e2 cm) to 23.7% (4e6 cm) and

were much higher than in the Ouchy area (max. 5%) or

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.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81218

Fig. 3 e Depth profiles of acid volatile sulphur (AVS [ H2S D FeS), chromium reducible sulphur (CRS [ S0 D FeS2 D Fe3S4),

and total reducible sulphur (TRS [ AVS D CRS) in the sediments from Vidy and Ouchy.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1219

elsewhere in Lake Geneva (max. 5e8%) (Pote et al., 2008). The

intense mineralisation of organic matter, indicated by high

porewater phosphate, led to strongly reducing conditions in

the Vidy sediments. Porewater sulphate was used up by

sulphate-reducing bacteria and iron was essentially present

as FeS. Due to the absence of any other significant oxidant,

methanogenesis is suspected to be the major process for

organic matter degradation at this site. The conditions were

less reducing in the Ouchy sediments, where iron-reduction

may have prevailed in the uppermost 1e2 cm of the sediment

profile. Underneath, at 2.5 cm depth, the sediment was sul-

phidic due to the activity of sulphate-reducing bacteria. Below

7 cm depth, where sulphate was depleted, methanogenic

conditions prevailed (Canfield and Thamdrup, 2009).

Heavymetal concentrations in theBayofVidywereup tosix

times higher than in Ouchy. According to the Canadian Sedi-

ment Quality Guidelines for the Protection of Aquatic Life

(Conseil Canadien desMinistres de l’Environnement, 1999) the

heavymetal concentrations in Vidy Baywere 2e8 times higher

than reported PELs (probable effect levels). These results are in

agreement with previous data from the same sampling site

Table 2 e Depth variation of heavy metal contents (mg kgL1 d

Depth Cu Zn Cd

Ouchy Vidy Ouchy Vidy Ouchy Vid

0e2 cm 54.7 142.5 126.8 341.3 0.5 1.

2e4 cm 68.3 135.9 155.1 327.8 0.7 1.

4e6 cm 86.8 133.6 218.1 344.9 1.2 1.

6e8 cm 135.5 181.4 305.5 446.8 1.7 2.

8e10 cm 136.5 161.4 242.8 518.2 1.2 2.

a Total variation coefficients for triplicate measurements were <15% for

(Pote et al., 2008). In that survey, hydrophobic organic

compounds, such as PAHs, PCBs and OCPs were investigated

and concentrations of up to 2, 156 and 45 mg kg�1 were deter-

mined, respectively. These values are considered high and

above average levels for Lake Geneva (Corvi et al., 1986). It

appears clearly that the sediments around the WWTP outlet

pipe in the Bay of Vidy are heavily contaminated with many

kinds of pollutants, possibly representing a significant source

of toxicity for microbial communities and benthic organisms.

Results from this study indicate that the dominant phylo-

genetic groups at both sites were the Beta-, Gamma- and Delta-

proteobacteria and Bacteroidetes, which is in agreementwith 16S

rRNA analyses of lake bacterioplankton (Zwart et al., 2002;

Glockner et al., 2000; Hiorns et al., 1997). Nevertheless, some

differences between the two sites were observed in the rela-

tive proportion of the different Proteobacteria subdivisions.

Proteobacteria accounted for 64% of the clones in Vidy and

55% of the clones in Ouchy. Proportions of Beta- and Deltap-

roteobacteria were much higher in Vidy Bay that in Ouchy

sediments. In contrast, the Gammaproteobacteria were more

abundant in Ouchy sediments (Fig. 4).

ry weight sediment)a in Vidy Bay and Ouchy sediments.

Pb Cr Hg

y Ouchy Vidy Ouchy Vidy Ouchy Vidy

6 38.8 89.4 60.3 74.2 0.2 1.2

5 49.7 89.3 63 68.3 0.6 1

7 75 100.7 69.7 67.9 1 2.3

3 106.5 135.9 88.4 77.2 1.3 2.3

8 91 164.7 81.4 88.9 1.7 2.2

all elements.

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

taxonomic units were defined with a 97% sequence

similarity cut-off.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81220

Betaproteobacteria appear to be one of the major divisions

present in freshwater systems (Zwart et al., 2002; Hahn, 2006).

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-

cyclaceae, which are phenotypically and ecologically very

diverse. Twenty clones of this family were affiliated to Dech-

loromonas, a genus containing many heterotrophic and facul-

tative anaerobic chlorate-, perchlorate- or nitrate respiring

bacteria (Wolterink et al., 2005). Members of this genus such as

Dechloromonas aromatica are found in aquatic habitats and are

capable of oxidising aromatic compounds such as toluene,

benzoate, and chlorobenzoate. D. aromatica is currently the

only pure culture being able to degrade anaerobically benzene

with nitrate as electron acceptor (Coates et al., 2001) A few

clones, also found only in Vidy sediments, were related to

Methylophilus (Methylophilaceae) a group of methylotrophic

organisms. However, while methanol is oxidised as the sole

carbon and energy source, some species may grow also on

a limited range of other carbon compounds such as methyl-

amines, formate, glucose, and fructose (Jenkins et al., 1987).

Similar Methylophilaceae sequences were found in wastewater

treatment pools in China (AY863077) and in freshwater

calcareous mats (EF580978). The remaining Betaproteobacteria

clones were found in both sites and included various genera.

Several clones were affiliated to Propionivibrio (Rhodocyclaceae),

which are aerotolerant or obligate anaerobic chemo-

organotrophic bacteria. These bacteria are typical inhabitants

Table 3 e Distribution of Bacteria phylotypes in clonelibraries from Vidy and Ouchy sediments (Lake Geneva).“A” stands for the 0e2 cm sediment section and “B” forthe 4e6 cm sediment section.

Vidy A Vidy B Ouchy A Ouchy B

Total no of sequences 68 41 60 40

Total no of OTUs 40 26 53 32

Percentage of unique OTUs 17 10 30 18

of anaerobic, muddy freshwater sediments (Tanaka et al.,

2003). Two further clones were related to Thiobacillus (Hydro-

genophilaceae), which are ubiquitous in soils and sediments,

and may grow on reduced sulphur compounds.

Compared to Betaproteobacteria, the Deltaproteobacteria

subdivision (Fig. 7) was less abundant (15% and 6% of the

clones in Vidy and Ouchy sediments, respectively). Most

Deltaproteobacteria are sulphate-, iron- or proton-reducing

(syntrophic) bacteria that play major roles in anoxic settings

like meromictic lakes and sediments (Lehours et al., 2007;

Karr et al., 2005). The large number of clones related to

sulphate-reducing bacteria (Desulfobacteraceae) in Vidy Bay

was not unexpected since sulphate consumption in the

sediment was obvious from the porewater data. Moreover,

the pool of total reduced sulphur (TRS) was quite high,

especially in Vidy sediments. Several additional clones, only

found in Vidy sediments, were related to Geobacter sp.

(Geobacteraceae). These anaerobic bacteria have been isolated

from freshwater sediments, soils and subsurface environ-

ments. They are traditionally considered chemo-

organotrophic Fe(III)-reducing bacteria. However, most

Table 4 e Summary of FST and P-test results for thecomparison of microbial communities between Vidy andOuchy sediments. “A” stands for the 0e2 cm sedimentsection and “B” for the 4e6 cm sediment section.

P valuea

Group FST-test P test

Vidy A vs Vidy B NS NS

Ouchy A vs Ouchy B < 0.00 0.001

Vidy A vs Ouchy A 0.001 0.001

Vidy B vs Ouchy B 0.005 0.001

a NS, not significant.

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.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1221

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.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81222

species utilise a wide range of alternative electron acceptors,

including NO3�, S0, and other sulphur compounds (Coates

et al., 1998). Similar sequences related to Geobacter sp. were

retrieved from anthropogenically impacted urban creek

sediment (EU284415) and from an aquifer where Fe(III)

reduction was associated with aromatic hydrocarbon

degradation (Nevin et al., 2005; AY653549). Another group of

clones exclusively found in Vidy sediments belonged to the

Synthrophaceae, with one clone related to Smithella sp. Similar

sequences were identified as core microorganisms involved

in the anaerobic digestion of sludge (Riviere et al., 2009;

CU922073).

The Gammaproteobacteria were more abundant in Ouchy

sediments than in Vidy and were phylogenetically diverse

(Fig. 8). Clones from both sites were affiliated to Methylobacter

sp. (Methylococcaceae), which are characterised by their

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.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1223

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.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81224

Fig. 10 e The Multiple factor analysis (MFA) is a PCA-based

technique allowing the simultaneous ordination of

a composite table obtained by the juxtaposition of the

species and the two environmental datasets, after

weighting the different matrices The superimposed

representation shows one global point for each site, Vidy

and Ouchy, at each depth (“A” stands for the 0e2 cm

sediment section and “B” for the 4e6 cm sediment section).

The three associated partial points correspond to the three

datasets (microbial composition, organic matter and

nutrients and heavy metals). The values on the axes

indicate the percentage of total variation.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1225

specialisedmetabolism restricted to the oxidation ofmethane

or methanol. Similar sequences were retrieved from

a permafrost soil in Siberia (Liebner et al., 2009; EU124843) and

an Arctic wetland soil (Wartiainen et al., 2006; AJ414655).

Surprisingly, a few clones from Vidy sediments were related

to phototrophic purple sulphur bacteria (Chromatiaceae)

despite the fact that there is not much light to be expected at

30 m depth. However, some species can also grow under

chemotrophic conditions in the dark, either autotrophically or

heterotrophically using oxygen as terminal electron acceptor.

Sequences similar to ours were retrieved from an anaerobic

digestor (Riviere et al., 2009) and the sediment surface of

Fayetteville Green Lake, USA (FJ437977). Several groups of

Gammaproteobacteria clones remained unclassified but with

similar sequences found in bacterioplankton communities of

Lake Michigan (Mueller-Spitz et al., 2009. EU640647), river

sediments (Li et al., 2008. EF590053), mangroves (EF125457)

and agricultural soils (FJ444695).

A large number of sequences affiliated with the division

Bacteroidetes (CytophagaeFlexibactereBacteroidetes) were found

in both sites, particularly in Vidy sediments. Bacteroidetes

constitute the second largest group in Vidy sediments after

the Betaproteobacteria, and the third largest group in Ouchy

sediments (Fig. 4). Bacteroidetes phylotypes were diverse and

their closest relatives were sequences found in freshwater

lakes (Mueller-Spitz et al., 2009), anthropogenically impacted

sediments, tundra soils (Liebner et al., 2008) and aquifers.

Most of the sequences found in the 2 investigated sites

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

fromanaerobicsewagedigestorsorsewagesludge (Zinderetal.,

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

theanoxic zoneof ahydropowerplant 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

wouldbedifferent and reflect thedifferences in environmental

conditions. Fig. 4 and Figs. 6e9 clearly show the diverse

bacterial and archaeal lineages detected in the two sites. The

results of the genetic diversity tests confirm the significant

genetic differentiation of the sediment bacterial communities

between the two sites at all depths. Seven and twelve

divisions were identified in the sediments from Vidy and

Ouchy, respectively. Among them Nitropsira, Planctomycetes,

Verrucomicrobia and Gemmatimonadetes were only detected in

Ouchy sediments. The apparent lower bacterial diversity in

Vidy sedimentsmay be explained by the high levels of a broad

range of pollutants, which may induce adverse biological

effects on microbial communities. The bacterial community

composition changed with depth in the uncontaminated

sediment. Conversely, no statistically significant variations

were observed for the two sediment layers (0e2 and 4e6 cm) in

the Bay of Vidy, which is explained by the high sedimentation

rates and the non-consolidated nature of the sediment,

permitting mobilisation and vertical mixing.

The microbial composition of both sites was correlated

with the environmental variables, as shown by the Mantel

correlation test (r¼ 0.9429, p¼ 0.044). This result suggests that

the diversity of microbial communities may be affected by

nutrients, organic matter as well as the degree of pollution.

Many environmental variables are implicated, which is

a situation inherent to all field studies. The Bay of Vidy is

contaminated by all kinds of contaminants but also with high

quantities of organic matter and nutrients. Under these

conditions it is difficult to separate out the influence of the

different environmental variables on microbial diversity and

community composition. To learn more about the relative

importance of individual environmental factors, microcosm

studies will be required.

The integrative picture of the relationship between bacte-

rial community structures and environmental factors at the

two sites (Fig. 10) indicated that the sampling sites Ouchy and

Vidy were clearly different with respect to both. Previous

results already showed that the bacterial diversity in compa-

rable contaminated and uncontaminated environments may

differ significantly. The difference may be explained by the

nature of pollution and a wide diversity of organic carbon

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81226

sources (Sandaa et al., 1999; Sorci et al., 1999; Zhang et al.,

2008). The polluted environment of Vidy Bay may have

selected, among the dispersed microbes in sediments, certain

functional bacterial groups which adapted to these conditions

and became more dominant in that particular environment.

5. Conclusion

This is the first study reporting on the microbial community

structures of Bacteria and Archaea in contaminated and

uncontaminated sediments of Lake Geneva. Results show that

the sediments of the two sites differed clearly in their organic

matter and nutrient contents. Intense mineralisation of

organic matter under sulphate-reducing and methanogenic

conditions was indicated for the sediments from Vidy Bay.

Furthermore, results confirmdata of previous studies showing

that the area around the WWTP outlet pipe in the Vidy Bay is

heavily contaminated with various organic and inorganic

pollutants. Phylogenetic analysis of sedimentary prokaryotes

revealed that (i) archaeal and bacterial communities differed

significantly between the contaminated and the non-

contaminated sediments. (ii) For both sites, a correlation was

observed between the microbial community structure and

environmental variables suggesting that microbial diversity

may be affected by nutrients, organic matter content and by

thedegreeofpollution. (iii)Betaproteobacteriawas thedominant

bacterial group, representingmore than30%of the clones from

surface sediments at both sites. (iv) A large proportion

of Betaproteobacteria clones, mostly from Vidy sediments,

were related to the reductively dechlorinating Dechloromonas

sp. (iv) Consistent with geochemical data, Deltaproteobacteria

including clones related to iron- (Geobacter sp.) and sulphate-

reducing bacteria, were relatively more abundant in the

contaminated sediments. (v) The archaeal communities were

dominated by methanogenic Euryarchaeota, particularly in the

organic matter-rich Vidy Bay sediments.

This study suggests that each site harbours a specific sedi-

ment microbial community. The apparent lower bacterial

diversity inVidy sedimentsmaybeexplainedby the significant

concentrations of contaminants, which may induce adverse

biological effects on benthic metazoa andmicrobes. However,

given the long history of pollution in the bay, specific bacterial

and archaeal communities may well have adapted to these

particular conditions. Hence, more research on microbial

community composition and specific activities of microor-

ganisms inhabiting similar environments should be per-

formed, in order to improve the understanding how pollution

and eutrophication may affect microbial communities.

Acknowledgements

We thank Nadia Ruggeri-Bernardi (Cantonal Institute of

Microbiology, Bellinzona, Switzerland) for assisting with the

molecular analysis, Dr. Pierre Rossi and Noam Shani (EPFL-IIE-

LBE, Switzerland) and Dr. Mathias Currat (University of

Geneva, Switzerland) for helping with the statistical analyses.

Philippe Arpagaus (Institute FA Forel) is acknowledged for

navigating R/V “La Licorne” and the Municipality of Lausanne

for financial support.

r e f e r e n c e s

APHA, 1985. Standard Methods for the Examination of Water andWastewater. American Public Health Association,Washington. 408e410.

Becue-Bertaut, M., Pages, J., 2008. Multiple factor analysis andclustering of a mixture of quantitative, categorical andfrequency data. Computational Statistics & Data Analysis 52,3255e3268.

Brosius, J., Dull, T.J., Sleeter, D.D., Noller, H.F., 1981. Geneorganization and primary structure of ribosomal RNA operonfrom Escherichia coli. Journal of Molecular Biology 148, 107e127.

Burrus, D., Thomas, R.L., Dominik, B., Vernet, J.P., Dominik, J.,1990. Characteristics of suspended sediment in the UpperRhone River, Switzerland, including the particulate forms ofphosphorus. Hydrological Processes 4, 85e98.

Canfield, D.E., Thamdrup, B., 2009. Towards a consistentclassification schemefor geochemical environments, or,whywewish the term “suboxic” would go away. Geobiology 7, 385e392.

Cline, J.D., 1969. Spectrophotometric determination of hydrogensulfide in natural waters. Limnology & Oceanography 14,454e458.

Coates, J.D., Chakraborty, R., Lack, J.G., O’Connor, S.M., Cole, K.A.,Bender, K.S., Achenbach, L.A., 2001. Anaerobic benzeneoxidation coupled to nitrate reduction in pure culture by twostrains of Dechloromonas. Nature 411, 1039e1043.

Coates, J.D., Ellis, D.J., Blunt-Harris, E.L., Roden, E., Gaw, C.,Lovley, D.R., 1998. Recovery and identification of humic-reducing bacteria from a diversity of environments. Appliedand Environmental Microbiology 64, 1504e1509.

Conseil Canadien des Ministres de l’Environnement, 1999.Introduction, dans Recommandations canadiennes pour laqualite de l’environnement. Winnipeg, le Conseil.

Corvi, C., Majeux, C., Vogel, J., 1986. Les polychlorobiphenyles et leDDE dans les sediments superficiels du Leman et de sesaffluents. Rapport de la Commission Internationale pour laprotection des eaux du Leman contre pollution. Campagne1985, 205e216.

Dorioz, J.M., Pelletier, J.P., Benoit, P., 1998. Physico-chemicalproperties and bioavailability of particulate phosphorus ofvarious origins in a watershed of Lake Geneva (France). WaterResearch 32, 275e286.

Eggleton, J., Thomas, K.V., 2004. A review of factors affecting therelease and bioavailability of contaminants during sedimentdisturbance events. Environment International 30, 973e980.

Escofier, B., Pages, J., 1994. Multiple factor-analysis (AfmultPackage). Computational Statistics & Data Analysis 18,121e140.

Forstner, U., Wittmann, G.T.W., 1979. Metal Pollution in theAquatic Environment. SpringereVerlag, Berlin, 486 pp.

Fossing, H., Jørgensen, B.B., 1989. Measurement of bacterialsulphate reduction in sediments: evaluation of a single-stepchromium reduction method. Biogeochemistry 8, 205e222.

Gillan, C.D., Danis, B., Pernet, P., Joly, G., Dubois, P., 2005.Structure of sediment-associated microbial communitiesalong a heavy-metal contamination gradient in the marineenvironment. Applied and Environmental Microbiology 71,679e690.

Glockner, F.O., Zaichikov, E., Belkova, N., Denissova, L.,Pernthaler, J., Pernthaler, A., Amann, R., 2000. Comparative16S rRNA analysis of lakebacterioplankton reveals globallydistributed phylogenetic clusters including an abundant group

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 8 1227

of Actinobacteria. Applied and Environmental Microbiology 66,5053e5065.

Grobkopf, R., Janssen, P.H., Liesack, W., 1998. Diversity andstructure of the methanogenic community in anoxic ricepaddy soil microcosms as examined by cultivation and direct16S rRNA gene sequence retrieval. Applied and EnvironmentalMicrobiology 64, 960e969.

Hahn, M.W., 2006. The microbial diversity of inland waters.Current Opinion in Biotechnology 17, 256e261.

Hall, G.E.M., Pelchat, P., 1997. Evaluation of a direct solid samplingatomic absorption spectrometer for the trace determination ofmercury in geological samples. The Analyst 122, 921e924.

Haller, L., Pote, J., Loizeau, J.-L., Wildi, W., 2009. Distribution andsurvival of faecal indicator bacteria in the sediments of theBay of Vidy, Lake Geneva, Switzerland. Ecological Indicators 9,540e547.

Hiorns, W.D., Methe, B.A., Nierzwicki-Bauer, S.A., Zehr, J.P., 1997.Bacterial diversity in Adirondack mountain lakes as revealedby 16S rRNA Gene sequences. Applied and EnvironmentalMicrobiology 63, 2957e2960.

Huser, B.A., Wuhrmann, K., Zehnder, A.J.B., 1982. Methanothrixsoehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogen-oxidizing methane bacterium. Archive ofMicrobiology 132, 1e9.

Jenkins, O., Byrom, D., Jones, D., 1987. Methylophilus: a new genusof methanol-utilizing bacteria. International Journal ofSystematic and Evolution Microbiology 37, 446e448.

Karr, E.A., Sattley, W.M., Rice, M.R., Jung, D.O., Madigan, M.T.,Achenbach, L.A., 2005. Diversity and distribution of sulphate-reducing bacteria in permanently frozen Lake Fryxell,McMurdo Dry Valleys, Antarctica. Applied and EnvironmentalMicrobiology 71, 6353e6359.

Koelmans, A.A., Van der Heijde, A., Knijff, L.M., Alderink, R.H.,2001. Integrated modelling of eutrophication and organiccontaminant fate & effects in aquatic ecosystems. A review.Water Research 15, 3517e3536.

Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated softwarefor Molecular Evolutionary Genetics Analysis and sequencealignment. Brief Bioinform 5, 150e163.

Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E.,Goodfellow, M. (Eds.), Nucleic Acid Techniques in BacterialSystematics. John Wiley & Sons, Inc., New York, pp.115e175.

Lehours, A.C., Evans, P., Bardot, C., Joblin, K., Fonty, G., 2007.Phylogenetic diversity of archaea and bacteria in the anoxiczone of a meromictic lake (Lake Pavin, France). Applied andEnvironmental Microbiology 73, 2016e2019.

Li, D., Yang, M., Li, Z., Qi, R., He, J., Liu, H., 2008. Change ofbacterial communities in sediments along Songhua River inNortheastern China after a nitrobenzene pollution event.FEMS Microbiology Ecology 65, 494e503.

Liebner, S., Harder, J., Wagner, D., 2008. Bacterial diversity andcommunity structure in polygonal tundra soils from SamoylovIsland, Lena Delta, Siberia. International Microbiology 11,195e202.

Liebner, S., Rublack, K., Stuehrmann, T., Wagner, D., 2009.Diversity of aerobic methanotrophic bacteria in a permafrostactive layer soil of the Lena Delta, Siberia. Microbial Ecology57, 25e35.

Loizeau, J.-L., Arbouillle, D., Santiago, S., Vernet, J.-P., 1994.Evaluation of a wide range laser diffraction grain size analyserfor use with sediments. Sedimentology 41, 353e361.

Loizeau, J.-L., Pardos, M., Monna, F., Peytremann, C., Haller, L.,Dominik, J., 2004. The impact of a sewage treatment plant’seffluent on sediment quality in a small bay in Lake Geneva(Switzerland-France). Part 2: Temporal evolution of heavymetals. Lakes & Reservoirs: Research and Management 9,53e63.

Lors, C., Tiffreau, C., Laboudigue, A., 2004. Effects of bacterialactivities on the release of heavy metals from contaminateddredged sediments. Chemosphere 56, 619e630.

Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J.,Woese, C.R., 1999. A new version of the RDP (RibosomalDatabase Project). Nucleic Acids Research 27, 171e173.

Mantel, N., 1967. The detection of disease clustering anda generalized regression approach. Cancer Research 27,209e220.

Martin, A.P., 2002. Phylogenetic approaches for describing andcomparing the diversity of microbial communities. Appliedand Environmental Microbiology 68, 3673e3682.

Mueller-Spitz, S.R., Goetz, G.W., McLellan, S.L., 2009. Temporaland spatial variability in nearshore bacterioplanktoncommunities of Lake Michigan. FEMS Microbiology Ecology 67,511e522.

Nealson, K.H., 1997. Sediment bacteria: who’s there, what arethey doing, and what’s new? Articles in Annual Review ofEarth and Planetary Sciences 25, 403e434.

Nevin, K.P., Holmes, D.E., Woodard, T.L., Hinlein, E.S.,Ostendorf, D.W., Lovley, D.R., 2005. Geobacter bemidjiensis sp.nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates. International Journal ofSystematic and Evolutionary Microbiology 55, 1667e1674.

Pardos, M., Benninghoff, C., de Alencastro, L.P., Wildi, W., 2004.The impact of a sewage treatment plant’s effluent onsediment quality in a small bay in Lake Geneva (Switzerland-France). Part 1: spatial distribution of contaminants and thepotential for biological impacts. Lakes & Reservoirs: Researchand Management 9, 41e52.

Pote, J., Haller, L., Loizeau, J.-L., Garcia Bravo, A., Sastre, V.,Wildi, W., 2008. Effects of a sewage treatment plant outlet pipeextension on the distribution of contaminants in thesediments of the Bay of Vidy, Lake Geneva, Switzerland.Bioresource Technology 99, 7122e7131.

Pouliot, J., Galand, P.E., Lovejoy, C., Vincent, W.F., 2009. Verticalstructure of archaeal communities and the distribution ofammonia monooxygenase A gene variants in two meromicticHigh Arctic lakes. Environmental Microbiology 11, 687e699.

Powell, S.M., Bowman, J.P., Snape, I., Stark, J.S., 2003. Microbialcommunity variation in pristine and polluted nearshoreAntartic sediments. FEMS Microbiology Ecology 45, 135e145.

Pronk, M., Goldscheider, N., Zopfi, J., 2009. Microbial communitiesin karst groundwater and their potential use forbiomonitoring. Hydrogeology Journal 17, 37e48.

R Development Core Team, 2005. R: A Language and Environmentfor Statistical Computing. R Foundation for StatisticalComputing, Vienna, Austria, ISBN 3-900051-07-0. http://www.R-project.org.

Riviere, D., Desvignes, V., Pelletier, E., Chaussonnerie, S.,Guermazi, S., Weissenbach, J., Li, T., Camacho, P., Sghir, A.,2009. Towards the definition of a core of microorganismsinvolved in anaerobic digestion of sludge. ISME Journal 3,700e714.

Ross-Barraclough, F., Givelet, N., Martinez-Cortizas, A.,Goodsite, M.E., Biester, H., Shotyk, W., 2002. An analyticalprotocol for the determination of total mercuryconcentrations in solid peat samples. Science of TotalEnvironment 292, 129e139.

Salomons, W., Forstner, U., 1984. Metals in the Hydrocycle.SpringereVerlag, Berlin, 349 pp.

Sandaa, R.A., Torsvik, V., Enger, O., Daae, F.L., Castberg, T.,Hahn, D., 1999. Analysis of bacterial communities in heavymetal-contaminated soils at different levels of resolution.FEMS Microbiology Ecology 30, 237e251.

Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M.,Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H.,Robinson, C.J., Sahl, J.W., Stres, B., Thallinger, G.G., Van

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 2 1 3e1 2 2 81228

Horn, D.J., Weber, C.F., 2009. Introducing mothur: open-source, platform-independent, community-supportedsoftware for describing and comparing microbialcommunities. Applied and Environmental Microbiology 75,7537e7541.

Schneider, S., Roessli, D., Excoffier, L., 2000. Arlequin Ver 2.0: ASoftware for Population Genetic Data Analysis. Genetics andBiometry Lab, University of Geneva, Geneva.

Schwarzenbach, R.P., Escher, B.I., Fenner, K., Hofstetter, T.B.,Johnson, C.A., vonGunten, U.,Wehrli, B., 2006. The challenge ofmicropollutants in aquatic systems. Science 313, 1072e1077.

Sorci, J.J., Paulauskis, J.D., Ford, T.E., 1999. 16 rRNA Restrictionfragment length polymorphism analysis of bacterial diversityas a biomarker of ecological health in polluted sediments fromNew Bedford Harbor, Massachusetts, USA. Marine PollutionBulletin 38, 663e675.

Stahl, D.A., Amann, R.I., 1991. Development and application ofnucleic acid probes. In: Stackebrandt, E., Goodfellow, M. (Eds.),Nucleic Acid Techniques in Bacterial Systematics. John Wiley& Sons, Inc., New York, pp. 205e248.

Tanaka, K., Nakamura, K., Mikami, E., 2003. Fermentation ofmaleate by a gram-negative strictly anaerobic non-spore-former, Propionivibrio dicarboxylicus gen.nov., sp.nov. Archiveof Microbiology 154, 323e328.

Thamdrup, B., Fossing, H., Jørgensen, B.B., 1994. Manganese, ironand sulfur cycling in a coastal marine sediment, Aarhus Bay,Denmark. Geochimica et Cosmochimica Acta 58, 5115e5129.

Verweij, F., Booij, K., Satumalay, K., Molen, N., Oost, R., 2004.Assessment of bioavailable PAH, PCB and OCP concentrationsin water, using semipermeable membrane devices (SPMDs),sediments and caged carp. Chemosphere 54, 1675e1689.

Wang, Q.G., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. NaıveBayesian classifier for rapid assignment of rRNA sequencesinto the new bacterial taxonomy. Applied EnvironmentalMicrobiology 73, 5261e5267.

Wang, Q., Kim, D., Dionysiou, D.D., Sorial, G.A., Timberlake, D.,2004. Sources and remediation for mercury contamination inaquatic systemsda literature review. Environmental Pollution131, 323e336.

Wartiainen, I., Hestnes, A.G., McDonald, I.R., Svenning, M.M.,2006. Methylobacter tundripaludum sp. nov., a methane-oxidizing bacterium from Arctic wetland soil on the Svalbardislands, Norway (78 degrees N). International Journal ofSystematic and Evolutionary Microbiology 56, 109e113.

Wildi, W., Dominik, J., Loizeau, J.-L., Thomas, R.L., Favarger, P.-Y.,Haller, L., Perroud, A., Peytremann, C., 2004. River, reservoirand lake sediment contamination by heavy metalsdownstream from urban areas of Switzerland. Lakes &Reservoirs: Research and Management 9, 75e87.

Williams, J.D.H., Jaquet, J.M., Thomas, R.L., 1976. Forms ofphosphorus in the superficial sediments of Lake Erie. Journalof the Fisheries Research Board of Canada 33, 413e429.

Wolterink, A., Kim, S., Muusse, M., Kim, I.S., Roholl, P.J.M., vanGinkel, C.G., Stams, A.J.M., Kengen, S.W.M., 2005.Dechloromonas hortensis sp. nov. and strain ASK-1, two novel(per)chlorate-reducing bacteria, and taxonomic description ofstrain GR-1. International Journal of Systematic andEvolutionary Microbiology 55, 2063e2068.

Ye, W., Liu, X., Lin, S., Tan, J., Pan, J., Li, D., Yang, H., 2009. Thevertical distribution of bacterial and archaeal communities inthe water and sediment of LakeTaihu. FEMS MicrobiologyEcology 70, 263e276.

Zhang, W., Ki, J.S., Qian, P.Y., 2008. Microbial diversity in pollutedharbor sediments I: bacterial community assessment basedon four clone libraries of 16S rDNA. Estuarine. Coastal andShelf Science 76, 668e681.

Zinder, S.H., Cardwell, S.C., Anguish, T., Lee, M., Koch, M., 1984.Methanogenesis in a thermophilic (58�C) anaerobic digestor:Methanothrix sp. as an important aceticlastic methanogen.Applied Environmental Microbiology 47, 796e807.

Zopfi, J., Bottcher, M.E., Jørgensen, B.B., 2008. Biogeochemistry ofsulfur and iron in Thioploca-colonized surface sediments in theupwelling area off central Chile. Geochimica et CosmochimicaActa 72, 827e843.

Zwart, G., Crump, B.C., Kamst-van Agterveld, M.P., Hagen, F.,Han, S.-K., 2002. Typical freshwater bacteria: an analysis ofavailable 16S rRNA gene sequences from plankton of lakesand rivers. Aquatic Microbial Ecology 28, 141e155.