bacterial community structure of sediments of the bizerte lagoon (tunisia), a southern mediterranean...

12

Click here to load reader

Upload: olfa-ben-said

Post on 10-Jul-2016

215 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

ENVIRONMENTAL MICROBIOLOGY

Bacterial Community Structure of Sediments of the BizerteLagoon (Tunisia), a Southern Mediterranean CoastalAnthropized Lagoon

Olfa Ben Said & Marisol Goñi-Urriza & Monia El Bour &

Patricia Aissa & Robert Duran

Received: 11 April 2009 /Accepted: 27 August 2009 /Published online: 30 September 2009# Springer Science + Business Media, LLC 2009

Abstract In order to estimate how pollution affects thebacterial community structure and composition of sedi-ments, chemical and molecular approaches were combinedto investigate eight stations around the Bizerte lagoon.Terminal restriction fragment length polymorphism(T-RFLP) analysis of PCR-amplified 16S rRNA genesrevealed that each station was characterized by a specificbacterial community structure. The combination of this datawith those of chemical analysis showed a correlationbetween the bacterial fingerprint and the pollutant content,principally with hydrocarbon pollution. The composition ofthe bacterial community of two contrasted stations relatedto the pollution revealed sequences affiliated to α, β, γ, δ,ε subclass of the Proteobacteria, Actinobacteria, andAcidobacteria in both stations although in different extent.Gamma and delta subclass of the Proteobacteria were

dominant and represent 70% of clones in the heavy-metal-contaminated station and 47% in the polyaromatic hydro-carbon (PAH)-contaminated. Nevertheless, most of thesequences found were unaffiliated to cultured bacteria.The adaptation of the bacterial community mainly to PAHcompounds demonstrated here and the fact that thesebacterial communities are mainly unknown suggest thatthe Bizerte lagoon is an interesting environment tounderstand the capacity of bacteria to cope with somepollutants.

Introduction

Organic and inorganic pollution of coastal zones is a majorubiquitous environmental problem since they can accumu-late in sediments [13]. Nevertheless, the contaminant can betransformed in this environment, principally by bacteria,since they are the most abundant organisms in the sediment[13]. On the opposite, the structure and composition ofmicrobial communities are affected by many differentabiotic and biotic parameters including pollutants compo-nents [35, 44]. Recent studies have demonstrated thatnatural attenuation and bioremediation of organic contam-inants and heavy metals cannot be effectively applied atmany sites until we have a better understanding of thephysiology, ecology, and phylogeny of microbial commu-nities at contaminated sites [42]. In light of these data, weneed to increase our understanding of how microbialcommunities are affected by and interact with thesecompounds in contaminated sites. Recently, fundamentalviews of the capacities of the bacterial communitiesinhabiting marine sediments to cope with pollutants havebeen given [6, 7, 21, 22, 29, 38], but relatively little is

Electronic supplementary material The online version of this article(doi:10.1007/s00248-009-9585-x) contains supplementary material,which is available to authorized users.

O. Ben Said :M. Goñi-Urriza : R. Duran (*)Equipe Environnement et Microbiologie-IPREM UMR5254-IBEAS, Université de Pau et des Pays de l’Adour,Avenue de l’Université, BP 1155, 64013 Pau cedex, Francee-mail: [email protected]

O. Ben Said :M. El BourLaboratoire de Bactériologie–Pathologie, Institut Nationaldes Sciences et Technologies de la Mer INSTM,Salammbô, Tunisia

O. Ben Said : P. AissaFaculté des Sciences de Bizerte, Laboratoire de Biosurveillancede l’Environnement,Zarzouna, Tunisia

Microb Ecol (2010) 59:445–456DOI 10.1007/s00248-009-9585-x

Page 2: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

known about bacterial communities in contaminated sedi-ments [1, 11, 12, 18]. Moreover, the combined effect ofdifferent pollutants as polyaromatic hydrocarbons (PAH)and heavy metals on microbial activities and communitycomposition is still unclear since few studies haveaddressed this point [10].

Costal urbanized and industrialized zones have oftenbeen shown to be characterized by the concomitantpresence of organic and inorganic pollutants [11]. Thesecoastal zones provide a good study site for the investigationof microbial communities inhabiting mixed contaminatedsediments. The Bizerte lagoon (Tunisia) is an example ofsuch ecosystem; it is located in an urbanized andindustrialized area subjected to the input of variouspollutants. The discharges of untreated domestic effluentsand wastewater from industries generate heavily pollutedsediments characterized by the concomitant presence ofPAHs, heavy metals, and drugs such as antibiotics [27]. Wepreviously characterized the aerobic PAH-degrading bacte-ria isolated from sediments of different stations locatedwithin the Bizerte lagoon [4]. The present work combineschemical and microbial molecular approaches in order toinvestigate the influence of PAHs and heavy metals

concentrations on microbial community structure andcomposition. First, bacterial T-RFLP fingerprints werecorrelated with pollutant concentrations, and second, thebacterial composition of both the most PAH-polluted andthe most heavy metal-polluted stations were determined by16S rRNA gene libraries analyses.

Materials and Methods

Site Description

Bizerte lagoon (Southern Mediterranean), a canalizedlagoon system located in the Northern of Tunisia (Fig. 1),has been exploited for fishing activities since severalcenturies and for mussel farming since 1964. This areaextends over 150 km2 and constitutes a receptor of severalindustrial sewages, aquaculture wastes, fertilizers, andpesticides through runoff and soil erosion, wastewatersfrom towns implanted around. At least, five differentcontamination sources can be identified in the Bizertelagoon: Menzel Bourguiba-Tinja and Menzel Bourguibalandfill; Bizerte, Menzel Abderahmen, and Menzel Jmil

Mediterranean Sea

Jarzouna

Urban zone

37°16’NUrban zone

Bizerte

Industrial zone

1

2

0

Menzel AbderahmenUrban zone Menzel Jmil

Urban zone

6 Industrialzone

8Bizerte Lagoon

Agricultural zone

9

37°08’N

09°56’E5 Km

10Industrial

zone

09°47’E

Tinja12

Industrial zone

Landfill

Menzel BourguibaUrban zone

TU

NISIA

Figure 1 Map of the Bizerte lagoon indicating the location of the sampling stations (dots). Urban zones (dashed circles), industrial zones (solidcircles), and agricultural zones (little dashed circles) are indicated. The arrows indicate streams

446 O. B. Said et al.

Page 3: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

industrial zones; and Southeastern agricultural zone [45].Furthermore, this lagoon is connected to Mediterranean Seathrough of a narrow channel that is exposed to intensivemaritime traffic and indirectly to several pollutants comingfrom oil and steel factories.

Sediment Collection and Field Measurements

Undisturbed surface sediments (0–5 cm depth) werecollected in May 2004 with a Van Veen Grab at eightstations (station nos. 0, 1, 2, 6, 8, 9, 10, and 12) locatedaround the Bizerte lagoon and the bay (Fig. 1). Samplingsites were selected because of the extent of anthropogeniccontamination is different [45]. Water column temperature,pH, and salinity were determined in the field with ahandheld multi-parameter system WTW Multi-197i. Thewater temperature and level of dissolved oxygen weredetermined with a multiparameter probe (YSI GRANT3800). Sediments were sampled, quickly homogenized, andfrozen in liquid nitrogen for both chemical and molecularanalyses. Samples were stored at −80°C until analysis.

Chemical Analysis

Chemical analyses, namely, ammonium (NH4), nitrates(NO3), nitrites (NO2), orthophosphate (PO4), and totalphosphorus (Pt), were performed using standard methods[32]. Chlorophyll a concentrations were obtained byspectrophotometry described in [37].

Heavy metal composition analysis (cadmium, cobalt,copper, lead, manganese, nickel, zinc, chromium) wereperformed according to the NF EN ISO 11885 standardsissued in March 1998 [34]. Sediment samples wereacidified using nitric acid (pH<2), then assays were carriedout with an inductively coupled plasma atomic emissionspectrometer. Heterogeneity of samples was determinedduring the calibration checks of the technique. Thedetection limit was estimated lower than 0.005 mg kg−1

for Cd lower than 0.0002 mg kg−1 for Hg and lower than0.050 mg kg−1 for the rest of heave metals analyzed. Thereliability of the quantitative measurements was checked byanalyzing the Standard Reference sediment Sed.IAEA, 405.The deviation of the measure was below to 10%.

Polycyclic aromatic hydrocarbon analyses in the sedi-ments were conducted by an automated extraction withASE 200 (Accelerated Solvent Extractor-Dionex), extractpurification, and gas chromatography-mass spectrometry.Approximately 30 mg of the samples were purified throughlow-pressure liquid chromatography on an open silica-alumina column. The GC was an HP 6890 N (Hewlett-Packard, Palo Alto, CA, USA) equipped with a split/splitless injector (pulsed splitless time: 1 min, flow50 mL min−1). The injector temperature was maintained at

270°C. The interface temperature was 290°C and the GCtemperature programmed from 50°C (1 min) to 300°C(20 min) at 5°C/min. The carrier gas was Helium at aconstant flow of 1 mL min−1. The capillary column usedwas an HP 5 MS (Hewlett-Packard, Palo Alto, CA, USA)=60 m×0.25 mm ID×0.25 μm film thickness. The GC wascoupled to an HP 5973 mass selective detector (ElectronicImpact: 70 eV, voltage: 1,200 V). Quantification wasperformed using Single Ion Monitoring mode with themolecular ion of each compound at 1.4 cycles s−1.

The reliability of the quantitative measurements waschecked by analyzing the Standard Reference Material1941b “Organics in Marine Sediment” (NIST, Gaithers-burg, Maryland, USA). Perdeuterated PAHs were obtainedfrom LGC Standards (Molsheim, France). Calibration curveswere established from n-alkane and PAH mixtures obtainedfrom LGC Standards (Molsheim, France). These mixturescontain n-alkanes from nC8 to nC32, and with regard toPAHs, all the parent PAHs mentioned in result table.

T-RFLP Analysis

Mixed community DNAwas extracted directly from sedimentsamples using an UltraClean soil DNA isolation Kit (MoBioLaboratories, CA) by following the manufacturer’s protocolwith minor modifications as previously described [36]. Genesencoding 16S rRNA were PCR amplified from extractedsamples using fluorescent-labeled bacterial primers 8F HEX(5-Hexa-chloro-fluorescein; 5′-AGAGTTTGATCCTGGCTCAG-3′, [26]) and 1489R TET (5-Tetrachloro-fluorescein;5′-TACCTTGTTACGACTTCA-3′, [43]). The PCR amplifi-cation mixture contained 12.5 µL Hot Start Taq polymerasemaster mix (Qiagen), 0.5 µL of each primer (20 µM), and10 ng of DNA template. A final volume of 50 µL wasadjusted with distilled water. 16S rRNA gene amplificationreactions were cycled in a PTC200 thermocycler (MJResearch) with a hot start step at 94°C for 15 min, followedby 35 cycles of 94°C for 1 min, 52°C for 1.5 min, and 72°Cfor 1 min, with a final extension step at 72°C for 10 min.

PCR products were purified with the GFX PCR DNApurification kit (Amersham-Pharmacia). Purified PCRproducts (600 to 700 ng) were digested with three units ofenzyme HaeIII (New England Biolabs). The length ofterminal fluorescent-labeled fragments from the digestedPCR products was determined by capillary electrophoresison ABI prism 310, (Applied Biosystems) as previouslydescribed [14]. Briefly, about 50 ng of the digested DNAfrom each sample was mixed with 18.5 µL of deionizedformamide and 0.5 µL of TAMRA size standard (AppliedBiosystems) and then denatured at 94°C for 2 min andimmediately chilled on ice prior to electrophoresis. After aninjection step of 10 s, electrophoresis was carried out for upto 30 min applying a voltage of 15 KV.

Bacterial Communities in Bizerte Lagoon-Polluted Sediments 447

Page 4: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

The T-RFLP profiles (T-RFs) were analyzed usingGene Scan Software version 3.1 (Applied Biosystem).Dominant T-RFs were selected by comparison of nu-merical values and electropherograms. Only the T-RFsrepresenting more than 1% of the total fluorescence wereconsidered [19].

T-RFLP profiles were compared by canonical correspon-dence analysis (CCA) using MVSP software (multivariatestatistical package 3.12d, Kovach Computing Services,1985–2001, UK). This test is based on the linear correlationbetween community data (abundance of each T-RF) andenvironmental parameters in the sediments. Linear regres-sion analysis between some T-RFs, and chemical data wasperformed in order to avoid overinterpretation of thecorrelation.

16S rRNA Gene Libraries Analyses and 16S rRNA GeneSequencing and Sequences Analysis

Bacterial composition of the stations 1 and 2 was furtheranalyzed by cloning PCR amplified 16S rRNA genes inEscherichia coli. PCR amplifications from these sampleswere carried out with unlabeled 8F and 1489R primers asdescribed previously [7]. PCR products were purified withthe GFX PCR DNA purification kit (Amersham-Pharma-cia) then cloned in E. coli TOP10 using the Topo-TAcloning kit (Invitrogen). Seventy-three clones from eachlibrary were selected randomly, and inserts were amplifiedusing the primers M13F (5′-CTGGCCGTCGTTTTAC-3′)and M13R (5′-GGTCATAAGCTGTTTCCTG-3′).

Partial sequences of the 16S rRNA gene weredetermined by the dideoxy nucleotide chain-terminationmethod using the BigDye cycle sequencing kit (AppliedBiosystems) on an ABI PRISM 310 205 Geneticanalyzer (Applied Biosystems) at the Génotypage-Séquençage de Bordeaux (France) using the primersM13F and M13R.

DNA sequences were compared to those present in thedatabank via the NCBI server (http://www.ncbi.nlm.nih.gov) using the basic local alignment search tool (BLAST)[2]. Sequence data were checked using the CHECKCHIMERA program (http://rdp8.cme.msu.edu/html/) todetermine the presence of hybrid sequences [30]. Nucleo-tide sequences were initially aligned with the ribosomaldatabase project (RDP) database [30] by means of theautomatic alignment function of the RDP phylogenyinference package interface, after sequences were manuallyaligned using ClustalW [41]. The phylogenetic tree wasconstructed with the MEGA software version 3.0 [25] usingNeighbor-Joining method [39]; the distance was calculatedon the basis of Kimura’s two-parameter algorithm [24]; 100bootstrap resamplings were performed to estimate thereproducibility of the tree.

Paleontological Statistics v1.60 software from http://folk.uio.no/ohammer/past/ website was used to perform rarefac-tion analysis and calculate diversity indices for each clonelibrary with clone phenotype identity defined at 97%. Inorder to determine the significance of differences betweenthe clone libraries, LIBSHUFF method was applied [40].

Nucleotide Sequence Accession Numbers

The sequences determined in this study have beensubmitted to the GenBank database and assigned AccessionNos. AM889144 to AM889146, AM889150, AM889157 toAM889198, AM889201 to AM889204, FM211753 toFM211808, and FM211815 to FM211816.

Results

Physicochemical Parameters and Metals and PAHConcentrations in Bizerte Lagoon’s Sediments

The physical and chemical parameters observed in thesampling stations are summarized in Table 1. The channelstations 0, 1, 2 and, in less extent, 6, are submitted to themarine influence with lower temperature and highersalinities. Channel station showed lower O2 concentrations.

Total heavy metal contents varied from 222 (station 9) to1,709 mg kg−1 of dry weight sediment (station 10; Table 1).Whatever the station investigated, magnesium and zincwere dominant (data not shown). Station 2, located in thechannel, showed the second high score for metals contents,mainly contaminated with magnesium, zinc, and chrome(respectively, 397, 322, and 163 mg kg−1 of dry weightsediment).

Total PAH concentrations in the coastal sedimentsranged from 24 (station 8, Southeast zone) to 877 (station1, channel) µg kg−1 dry weight sediment (Fig. 2a andTable 1). Two different PAH concentration profiles weredistinguished: stations 1, 2, and 6 were more contaminatedby high molecular weight PAHs (H PAH, mainly fluoran-thene, pyrene, and benzo(a)pyrene for stations 1 and 2 andfive aromatic rings PAHs for station 6) whereas station 12,10, 8, 9, and 0 where mainly contaminated by lowmolecular weight PAHs (L PAH) such as naphthalene (datanot shown). The unexpected high L PAH/H PAH ratiofound in station 8 is due to a hardly absence of PAHs ofhigh molecular weight.

Bacterial Community Structures

The T-RFLP profiles contained 25±7.5 T-RFs defined asribotypes. The lowest ribotype number (14) was observedin station 0 which is located at the channel entrance with

448 O. B. Said et al.

Page 5: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

the Mediterranean Sea subjected to both marine and lagooninfluences. In contrast, the highest ribotype number (37)was recorded in station 12. No clear relationship could beestablished between the number of ribotypes and thepollution level, even if the higher number of ribotypeswere found in the stations showing low level of contam-ination (stations 12 and 8, Table 1, Fig. 2a).

The T-RFs of 73, 192, 204, and 206 bp were detected in allthe stations (Fig. 2b). Whereas most of them showed a lowrelative abundance, the T-RF 204 bp is the more abundant inall the stations except for station 1. It represents up to 60% oftotal fluorescence in station 9. In contrast, some T-RFs werestation-specific such as T-RFs of 36 to 37 bp (station 1),49 bp (station 2), 56 bp (station 0), 114, 120, 127, 186,209 bp (station 8), 110, 111, 215, 259, 388, 399 bp (station12), 227, 290, 291 bp (station 10). Except for T-RF 36 bp instation 1 which is dominant, all of them were low abundant.Some T-RFs (such as 204, 256 bp, and others) have alsobeen detected (after in silico digestion of their 16S rRNAgenes) in strains isolated from the stations. These strains,belonging to the Pseudomonas or Acinetobacter genera,have been described as hydrocarbonoclastics [4].

CCA of the T-RFLP fingerprints correlated with somephysical and chemical parameters (Fig. 3) showing that thebacterial community in station 1 was highly influenced byPAHs contents. Stations 9 and 6 were influenced by NO3

concentrations while station 12 was the least impacted bythis pollutant. Station 2 was mainly influenced by NO3,PO4, and heavy metals. Distribution of T-RFs according tothese parameters is shown in Supplementary Data (Fig. S1).

The T-RFLP fingerprints correlated by CCA with thedifferent PAH molecules (Fig. 4) showed that the sedimentbacterial community of station 1 was influenced by all thePAHs analyzed (Fig. 4, insert). To observe the influence ofthe PAH content on the bacterial community structure ofthe other stations, data concerning the station 1 wasexcluded. In this condition, the first two axes of the CCAdescribed 48.2% of the variation (Fig. 4; axis 1, 25.5% andaxis 2, 22.7%). The bacterial community structures ofstations 6 and 9 were mostly influenced by fluorine,dibenzanthracene, and benzoperylene while those of sta-tions 0, 2, and 10 by fluoranthene and pyrene (Fig. 4). Incontrast, bacterial community structures of stations 8 and12 were not influenced by PAH contents (Fig. 4). Ribotypes73 and 74 bp were positively correlated with the totalcontents of PAH (respectively, R2 of 0.72 and 0.93),whereas ribotype 204 bp was negatively correlated. Also,the ribotype 256 bp, detected in stations 6 and 9, wasmostly influenced by fluorine, dibenzanthracene, andbenzoperylene (see Supplementary Data, Fig. S2).

The impact of heavy metals in the bacterial communitystructure could be observed in stations 8 and 10, whichwere mainly influenced by Mn and Co, and stations 1 andT

able

1Phy

sicochem

ical

parametersof

thestations

locatedarou

ndtheBizerte

lago

on

Statio

nLocation

Depth

(m)

T(°C)

S(psu)

SM

(gm

−3)

O2(gm

−3)

NO2(m

gm−3)

NO3(m

gm−3)

NH4(m

gm−3)

Nt(m

gm−3)

PO4(m

gm−3)

Pt(m

gm−3)

Chla(m

gm−3)

PAHt(µgg

−1)

Metalst(m

gkg−

1)

037°15.452′

N11

21.6

3620

5.5

11.69

308

180

235

4.4

30554

9°51.821

′E

137°14.600′

N11

21.8

36.3

185.5

13.13

26.96

11.94

208.36

11.19

47.21

4.12

877

425

9°50.420

′E

237°14.000′N

1121.9

3611.89

5.5

3.43

45.34

14.01

219.73

18.84

41.57

5.00

340

1065

9°49.400

′E

637°12.460′

N7.5

22.3

35.7

208.3

5.81

37.33

6.77

178.01

1.05

35.83

4.56

91453

9°55.790

′E

837°11.040′

N1.5

23,3

34.2

809.1

7,35

13.67

9.42

227.31

1241,85

4.95

24649

9°55.790

′E

937°09.770′

N3.9

2334.5

807

13.5

359

135

1230

5.1

38222

9°54.893

′E

1037°08.480′

N3

23.1

34.6

656.5

1040

18135

1237

5.4

110

1709

9°49.400

′E

1237°11.042′

N3

22.2

33.7

187.1

6.21

4.49

8.31

200.77

12.4

41.57

5.23

54395

9°47.313

′E

T,S,SM,O2,NO2,NO3,NH4,Nt,PO4,Pt,Chla,

PAHt,andMetalstweredeterm

ined

ineigh

tsitesof

Bizerte

lago

on(M

ay20

04)

Ttemperature,Ssalin

ity,SM

suspendedmatter,O2dissolvedox

ygen,NO2nitrites,NO3nitrates,NH4am

mon

ium,Nttotalnitrog

en,PO4orthop

hosphate,Pttotalph

osph

orus,Chlachloroph

yll,

PAHttotalPA

H,Metalsttotalheavymetals

Bacterial Communities in Bizerte Lagoon-Polluted Sediments 449

Page 6: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

0, influenced, respectively, by Cd and Pb (data not shown).Despite the high concentrations of heavy metals in station2, no clear influence of a specific metal in the communitystructure of this station could be characterized. Further-more; no OTU could be associated by the regressionanalysis with a particular heavy metal, neither with thetotal heavy metal content (data not shown).

Bacterial Community Composition of Sedimentsfrom Stations 1 and 2

The bacterial community composition of the channelstations 1 and 2 were determined in order to evaluate theimpact of pollutants on the bacterial diversity. Thesestations were chosen because (1) they are both channel

0

0,5

1

1,5

2

2,5

3

3,5

4

9

0

100

200

300

400

500

600

700

800

900

1000

10 9 12 8 6 2 1 00

10

20

30

40

50

Total PAHs

0

500

1000

1500

2000

0

0,5

1

1,5

2

2,5

3

3,5

4

9

Tot

al P

AH

s (µ

g. k

g-1 dr

y w

eigh

t)0

100

200

300

400

500

600

700

800

900

1000

10 9 12 8 6 2 1 00

10

20

30

40

50

0

10

20

30

40

50

L.PAHs/H.PAHs Ribotype number Total HM

0

500

1000

1500

2000

0

500

1000

1500

2000T

otal Heavy M

etals (mg. kg

-1 dry weight)

Ribotype num

ber

Low

PA

Hs

/Hig

h PA

Hs

Outdistance Mediterranean Sea

ChannelLagoon

A

B

Figure 2 Bacterial communitystructure analysis of Bizerte la-goon sediments. A Number ofribotypes in each station, totalPAH contents (µg kg−1 dw),total heavy metal contents(mg kg−1 dw), and low molecu-lar weight PAHs/high molecularweight PAHs ratio (LPAHs/HPAHs) are also indicated. BRelative abundance of T-RFs foreach station from T-RFLP pat-terns obtained by HaeIII diges-tion of 16S rRNA-amplifiedfragment

31 % 0

1

2

6

8

9

10

12

-0.6

-1.3

-1.9

-2.5

0.6

1.3

1.9

2.5

3.2

-0.6-1.3 -1.9 -2.5 0.6 1.3 1.9 2.5 3.2

Total Heavy Metals

Total PAHs

SalinitySM O 2

PO4

NO 3

21 %

31 % 0

1

2

6

8

9

10

12

-0.6

-1.3

-1.9

-2.5

0.6

1.3

1.9

2.5

3.2

-0.6-1.3 -1.9 -2.5 0.6 1.3 1.9 2.5 3.2

Total Heavy Metals

Total PAHs

SalinitySM O 2

PO4

NO 3

21 %

Figure 3 CCA betweensediment bacterial communitiescharacterized by T-RFLP fin-gerprints and some physical–chemical parameters: suspendedmatter (SM), dissolved oxygen(O2), nitrates (NO3), phosphates(PO4), Salinity, Total PAHs, andTotal Heavy Metals

450 O. B. Said et al.

Page 7: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

stations and their sediments have relatively similar physi-cochemical parameters (Table 1); (2) they are under theinfluence of PAH at different levels, station 1 being muchmore contaminated (Fig. 2a); and (3) the station 2 is thechannel station that is more contaminated with metals(Table 1). 16S rRNA gene library analysis of the stations 1and 2 was performed in order to determine the compositionof the bacterial communities. From the 73 clone sequencesanalyzed by library, 63 (47 singletons) and 69 (55 single-tons) phylotypes were found in library of stations 1 and 2,respectively. Shannon index was quite similar for bothlibraries (4.22 and 4.28 for library 1 and 2, respectively) andequitability was identical (0.97) indicating a high bacterialdiversity in both stations. No species (cutoff level of <97%identity) were found to be dominant as indicated by lowDominance indexes (1.73% for library 1 and 1.68% for library2). Although the libraries share some common sequences(28% and 41% for libraries 1 and 2, respectively, according tothe 97% identity cutoff), a comparison of the libraries with theLIBSHUFF method revealed that they were composed of

significantly different phylotypes (XY p=0.001; YX p=0.004, with a confidence of 99% (p=0.01)).

The phylogenetic analysis of the clone sequencesrevealed their distribution within six major taxonomicgroups of prokaryotic organisms (Fig. 5). Sequencesaffiliated to α, β, γ, δ, ε subclass of the Proteobacteria,to the phyla of Actinobacteria and Acidobacteria weredetected in both libraries, though in different extent.Sequences affiliated to the phyla of Nitrospirae andVerrucomicrobia were detected only in library 2. Neverthe-less, about 80% (library 1) and 90% (library 2) of the totalsequences could not be closely related to cultured organ-isms or already known sequences suggesting that they mayconstitute new taxa (Fig. 6). About 15% of the totalsequences in library 1 and 6% of the total sequences inlibrary 2 were closely related to 16S rRNA sequences ofunknown bacteria. The library 1 was dominated bysequences related to γ-Proteobacteria (31% of the sequen-ces), and 3% of these sequences were related to 16S rRNAsequences of identified species such as Pseudomonas sp.,

2

12

81

6

10

9 -1.06

5.31

-1.06 5.31

N

ANYF

PAFLPYC

BABBF+BkFBAP

INDBABPE

34.7 %

17.7 %

ANA

2

12

8

6

10

9

0

-1.06

5.31

-1.06 5.31ANYF

PAFLPYC

BABBF+BkFBAP

INDBABPE

34.7 %

17.7 %

ANA

2

12

81

6

10

9 -1.06

5.31

-1.06 5.31

N

ANYF

PAFLPYC

BABBF+BkFBAP

INDBABPE

34.7 %

17.7 %

ANA

2

12

8

6

10

9

0

-1.06

5.31

-1.06 5.31ANYF

PAFLPYC

BABBF+BkFBAP

INDBABPE

34.7 %

17.7 %

ANA

2

12 8

6

10

9

0

-0.4

-0.8

-1.1

0.4

0.8

1.1

1.5

1.9

-0.4-0.8-1.1 0.4 1.1 1.5 1.9

N

ANY

ANA

F

P

A

FL

PY

CBA

BBF+

BkFBAP

IN

DBA

BPE

22.7%

25.5 %

2

12 8

6

10

9

0

-0.4

-0.8

-1.1

0.4

0.8

1.1

1.5

1.9

-0.4-0.8-1.1 0.4 1.1 1.5 1.9

N

ANY

ANA

F

P

A

FL

PY

CBA

BBF+

BkFBAP

IN

DBA

BPE

22.7%

25.5 %

Figure 4 CCA between sedi-ment bacterial communitiescharacterized by T-RFLP fin-gerprints and individual PAHconcentrations. All stations arerepresented in the insert figure.The main figure represents allthe station, except station 1. NNaphthalene; ANY Acenaphty-lene; ANA Acenaphtene; FFluorine; P Phenanthrene; AAnthracene; FL Fluoranthene;PY Pyrene; C Chrysene; BABenzo(a)anthracene; BBF+BkFBenzo[b+k]fluoranthene;BAP Benzo(a)pyrene; IN Indeno(1,2,3-)pyrene; DBA Dibenzo(a,h)anthracène; BPE Benzo(g,h,i)perylene

Bacterial Communities in Bizerte Lagoon-Polluted Sediments 451

Page 8: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

Pseudomonas lanceolata, Legionella pneumophila, Acine-tobacter sp. (Fig. 6). Library 2 was characterized by the co-dominance of δ- and γ-Proteobacteria (38% and 37%,respectively; Fig. 5). Numerous sequences in the librarieswere related to sequences previously found in organic andinorganic contaminated sediment. Indeed, the main phylo-type, representing 6.1% and 12.4% of libraries 1 and 2,respectively, is affiliated within the γ-Proteobacteria andrelated to uncultured clones obtained from polluted envi-ronments, heavy metal-rich and/or organic-rich, originatedfrom harbor sediments, marine sediments, or fish farmsediments [5, 15, 46].

Several 16S rRNA gene sequences could be relatedto T-RFLP’s ribotypes by predictive digestions (data notshown). Both molecular methods revealed the samedominant populations, i.e., clones sequences with an insilico digestion size of 204 bp, which is the dominantT-RF in T-RFLP analysis. BLAST analysis of thesequence of these clones having an in silico T-RF of204 affiliated them to γ-Proteobacteria, most of themuncultured strains.

Discussion

The impact of environmental and pollutant variables onsediment microbial communities was studied at eightdifferent stations located in a Southern Mediterraneanlagoon. Considering hydrological and trophic conditions,this ecosystem was divided into two zones: (1) the channel(stations 0, 1, and 2) and (2) the lagoon itself (stations 6, 8,9, 10, and 12). Such a division is attributed to the presenceof inflow streams among which, the Tinja wadi is the mostimportant [20]. The same division could also be observedconsidering the PAH contents in sediments (from 24 to877 ng/g dw), the channel stations being the most

contaminated. T-RFLP analysis revealed differences be-tween the microbial community structures. The lowestbacterial diversity was observed in the station closer to theMediterranean Sea (14 ribotypes, station 0) subjected toboth marine and lagoon influences and showing highestsalinity. In contrast, the highest bacterial diversity wasrecorded in station 12 (37 ribotypes) that shows lowestsalinity, low PAH-levels and that receives Tinja wadieffluent in wet season, when the samples were taken.Microbial diversity was shown to vary along gradients suchas salinity [16], pollution [20], and other parameters [11,14]. The bacterial community structure was correlated withthe PAH content allowing the identification of specificribotype. For example, ribotype of 256 bp wasfound specifically in fluorine-, dibenzanthracene-, andbenzoperylene-contaminated station. Interestingly, this ribo-type could correspond to Acinetobacter sp. and strainsbelonging to this genus (and having a predictive restrictionsize of 256 bp) have been previously isolated from Bizertelagoon sediment for their capacity to degrade PAHs [4].

The stations 1 and 2 showed similar physicochemicalparameters and different PAH and metal contaminationlevels. The comparison of their composition would revealthe effect of contaminants on the bacterial communitystructure. Comparison of 16S rRNA gene library analysisof these stations revealed clear differences between thebacterial community compositions with high diversity inbacterial community inhabiting superficial sediment ofstation 2. A considerable number of singletons in thecomposition of the two rRNA gene libraries were observed,and the majority of phylotypes detected were not closelyrelated to any cultivated representatives. These results are inaccordance with previous observations reported in marineenvironmental sequence analysis [20]. We observed mainlyphylotypes affiliated with α-, β-, γ-, δ-, ε-Proteobacteria.In addition, phylotypes related to the phyla of Actino-

24.6%

21.92%

13.7%

5.48%

12.33%

10.96%

2.74%

34.25%

34.25%

6.85%

1.37%

5.48%

4.11%

1.37% 1.37%

γ –Proteobacteria δ–Proteobacteria β-Proteobacteria ε– Proteobacteria

Acidobacteria Actinobacteria

α-Proteobacteria

Library 1 Library 2

Planctomycetacia Verrucomicribia

4.11%

6.85%

Unknown bacteria

8.22%

Figure 5 Repartition of clonesequences in representativephylogenetic groups detected on16S rRNA gene libraries ofstations 2 and 1 of Bizertelagoon sediment

452 O. B. Said et al.

Page 9: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

bacteria and Acidobacteria were also detected. Members ofthese phyla are commonly found in less permeable sedi-ments of Seas and estuaries [28]. Numerous sequences inthe libraries are related to sequences of bacteria that werepreviously found in heavy metal and hydrocarbon-contaminated sediments [5, 8, 17, 20]. The libraries weredominated by sequences related to γ- and δ-Proteobacteria;however, α-Proteobacteria-related sequences are less abun-dant. This is typically observed in marine microbialcommunities [9]. Previous research showed that themicrobial community structure in a long-term mixedwaste-contaminated site might reflect both metal andaromatic hydrocarbon concentrations [33]. The dominanceof γ-Proteobacteria-related bacteria in PAH-contaminated

sediments is not unexpected since rapid and strongselection for γ-Proteobacteria have been reported in oil-treated microcosms [7], in oil-contaminated marine sedi-ments [18, 35], and after oil-spill accident [23]. Moreover,75% of PAHs degrading strains isolated from the Bizertelagoon in a previous study were γ-Proteobacteria [4]. Bycloning and sequencing, we detected clones affiliated toAcinetobacter sp. Interestingly, we have isolated strainsaffiliated to the same genus capable of fluoranthene andpyrene mineralization [4]. They were also resistant to heavymetals (Zn, Pb, Co, Cr, and Ni) and to antibiotics (variousaminosides and β-lactam molecules) [4]. Their relativeabundance associated with their metabolic capacitiesindicates the possible role of strains of this genus in in situ

Sulfurimonas denitrificans (L40808)100

100

100

87

100

100

100

92

100

51

100

61

90

99

100

100

67

89

83

99

58

76

89

53

77

77

66

100

100

96

70

98

100

52

53

58

57

99

89

82

100

99

85

9073

86

83

53

50

59

51

0.02

1 (1.37%); 2 (1.37%)*Comamonas testosteroni (M11224)

Iodobacter fluviatilis (M22511)Achromobacter group

1 (1.37%)Burkholderia group

Nitrosomonas oligotropha (AF272422)Gallionella ferruginea (L07897)

Stenotrophomonas acidaminiphila (AF273080)1 (4.11%); 2 (2.74%)*

2 (1.37%)Beggiatoa alba (AF110274)

1 (8.22%); 2 (16.44%)* 1 (1.37%)

Pseudomonas groupEnterobacter group

Alteromonadales groupsMarinobacter group

Marinomonas mediterranea (AF063027)1 (1.37%)

Acinetobacter group2 (2.74%)

Oceanospirillum beijerinckii (AB006760)1 (1.37%); 2 (1.37%)

Halomonas nitritophilus (AJ309564)Alcanivorax borkumensis (Y12579)

1 (1.37%); 2 (5.48%)*Methylomicrobium pelagicum (X72775)

1 (1.37%); 2 (1.37%)*

Thiothrix nivea (L40993)1 (2.74%)

1 (1.37%)Legionella pneumophila (M59157)

1 (1.37%); 2 (2.74%)Rhodospirillum rubrum (D30778)

Magnetite containing magnetic vibrio (L06455)Rickettsia prowazekii (M21789)

Sphingomonas group1 (2.74%)

Bradyrhizobium elkanii (U35000)1 (1.37%); 2 (2.74%)*

1 (1.37%); 2 (1.37%)Agrobacterium tumefaciens (DQ468100)

1 (2.74%)2 (1.37%)Rhizobiales groups

1 (2.74%); 2 (1.37%)

1 (2.74%); 2 (4.11%)Desulfonatronum lacustre (AF418171)

Desulfovibrio halophilus (U48243)1 (1.37%); 2 (2.74%)

Geobacter metallireducens (L07834)2 (1.37%)Pelobacter carbinolicus (X79413)

1 (4.11%); 2 (1.37%)*Desulfosarcina variabilis (M26632)

1 (2.74%)2 (1.37%)

Desulfococcus multivorans (AF418173)2 (1.37%)

Desulfobacterium anilini (AJ237601)2 (2.74%)

Desulfonema ishimotoei (U45992)Desulfobacca acetoxidans (AF002671)

1 (6.85%); 2 (5.48%)*Syntrophobacter sp. (X94911)

2 (2.74%)Desulfobulbus elongatus (X95180)

Desulfotalea arctica (AF099061)Desulfocapsa thiozymogenes (X95181)

Desulforhopalus vacuolatus (L42613)1 (4.11%); 2 (10.96%)*

1 (5.48%); 2 (2.74%)*Helicobacter baculiformis (EF070342)

Helicobacter nemestrinae (AF348617)1 (2.74%)

Campylobacter fetus subsp. fetus (DQ174128)2 (1.37%)

Candidatus Arcobacter sulfidicus (AY035822)Arcobacter cryaerophilus (L14624)

1 (1.37%)

Psychrobacter marincola (AY292940)

-Proteobacteria

-Proteobacteria

-Proteobacteria

-Proteobacteria

-Proteobacteria

Desulfobacter group

1 (1.37%)

Sulfurimonas denitrificans (L40808)100

100

100

87

100

100

100

92

100

51

100

61

90

99

100

100

67

89

83

99

58

76

89

53

77

77

66

100

100

96

70

98

100

52

53

58

57

99

89

82

100

99

85

9073

86

83

53

50

59

51

0.02

1 (1.37%); 2 (1.37%)*Comamonas testosteroni (M11224)

Iodobacter fluviatilis (M22511)Achromobacter group

1 (1.37%)Burkholderia group

Nitrosomonas oligotropha (AF272422)Gallionella ferruginea (L07897)

Stenotrophomonas acidaminiphila (AF273080)1 (4.11%); 2 (2.74%)*

2 (1.37%)Beggiatoa alba (AF110274)

1 (8.22%); 2 (16.44%)* 1 (1.37%)

Pseudomonas groupEnterobacter group

Alteromonadales groupsMarinobacter group

Marinomonas mediterranea (AF063027)1 (1.37%)

Acinetobacter group2 (2.74%)

Oceanospirillum beijerinckii (AB006760)1 (1.37%); 2 (1.37%)

Halomonas nitritophilus (AJ309564)Alcanivorax borkumensis (Y12579)

1 (1.37%); 2 (5.48%)*Methylomicrobium pelagicum (X72775)

1 (1.37%); 2 (1.37%)*

Thiothrix nivea (L40993)1 (2.74%)

1 (1.37%)Legionella pneumophila (M59157)

1 (1.37%); 2 (2.74%)Rhodospirillum rubrum (D30778)

Magnetite containing magnetic vibrio (L06455)Rickettsia prowazekii (M21789)

Sphingomonas group1 (2.74%)

Bradyrhizobium elkanii (U35000)1 (1.37%); 2 (2.74%)*

1 (1.37%); 2 (1.37%)Agrobacterium tumefaciens (DQ468100)

1 (2.74%)2 (1.37%)Rhizobiales groups

1 (2.74%); 2 (1.37%)

1 (2.74%); 2 (4.11%)Desulfonatronum lacustre (AF418171)

Desulfovibrio halophilus (U48243)1 (1.37%); 2 (2.74%)

Geobacter metallireducens (L07834)2 (1.37%)Pelobacter carbinolicus (X79413)

1 (4.11%); 2 (1.37%)*Desulfosarcina variabilis (M26632)

1 (2.74%)2 (1.37%)

Desulfococcus multivorans (AF418173)2 (1.37%)

Desulfobacterium anilini (AJ237601)2 (2.74%)

Desulfonema ishimotoei (U45992)Desulfobacca acetoxidans (AF002671)

1 (6.85%); 2 (5.48%)*Syntrophobacter sp. (X94911)

2 (2.74%)Desulfobulbus elongatus (X95180)

Desulfotalea arctica (AF099061)Desulfocapsa thiozymogenes (X95181)

Desulforhopalus vacuolatus (L42613)1 (4.11%); 2 (10.96%)*

1 (5.48%); 2 (2.74%)*Helicobacter baculiformis (EF070342)

Helicobacter nemestrinae (AF348617)1 (2.74%)

Campylobacter fetus subsp. fetus (DQ174128)2 (1.37%)

Candidatus Arcobacter sulfidicus (AY035822)Arcobacter cryaerophilus (L14624)

1 (1.37%)

Psychrobacter marincola (AY292940)

δ -Proteobacteria

ε -Proteobacteria

α -Proteobacteria

γ -Proteobacteria

β -Proteobacteria

Desulfobacter group

1 (1.37%)

aFigure 6 16S rRNA-basedphylogenetic reconstructionshowing the affiliation of Aproteobacteria clone sequencesand B nonproteobacteria clonesequences obtained from thesediments of stations 1 and 2 ofBizerte lagoon (in bold) withselected reference sequences.Percentages of 1,000 bootstrapresampling that supported thebranching orders in each analy-sis are shown above or near therelevant nodes (only values upto 50% are shown). The scalebar represents 2% estimatedsequence divergence. In brack-ets: percentage represented byeach sequence related to thetotal analyzed clones for eachlibrary. Asterisk phylotypes(sequences with similaritieshigher than 97%) presents inboth libraries. Accession numb-ers are A library 1:AM889157to AM889198; toAM889159; AM889201to AM889204; AM889144to AM889146; AM889150;FM211816 and FM211788.Library 2: FM211753 to FM211777; FM211779 toFM211808; FN424387FN424390; FN424394;AM889192. b Library 1:AM889146; AM889148;AM889149; AM889151to AM889156; AM889200;FN428746; FN424396. Library2: FN424385; FN424386;FN424388; FN424389;FN424391; FN424392;FN424395; FN424397;FN424398

Bacterial Communities in Bizerte Lagoon-Polluted Sediments 453

Page 10: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

biodegradation of these pollutants. In the previous study,we also isolated other hydrocarbonoclastic strains, mainlyaffiliated to the Pseudomonas genus [4] that we could notdetect in this study by library analysis but that we detect byT-RFLP analysis. These strains presented different capaci-ties of hydrocarbon degradation, most of them degradepreferentially the low molecular weight PAH, but somewere able to degrade pyrene more efficiently than phenan-threne, suggesting different ways of degradation. Some ofthem were resistant to the seven heavy metal tested andwere resistant to antibiotics [4].

Sediments of the most heavy-metal contaminated station(station 2) were dominated by δ-Proteobacteria-affiliatedsequences followed by γ-Proteobacteria. Numerous δ-Proteobacteria phylotypes were related to both sulfuroxidizers and sulfate reducers suggesting an active sulfurcycle in the sediments as usually found in sea sediments[20, 35]. Interestingly, δ-Proteobacteria-related sequenceshave been observed as predominant in sediments contam-inated with multiple pollutants (Hg, PAHs, and PCBs [12]).Nevertheless, Cordova-Kreylos and coworkers [11] foundthat metals had a greater effect on microbial communitycomposition than organic pollutants.

Sequences affiliated to β-Proteobacteria were detectedin both stations in low abundance (4% and 1%) as

reported in other studies of marine sediments [3, 8, 31].Since β-Proteobacteria-related phylotypes are knownto play a critical role in ecosystem function, Hunterand coworkers [20] hypothesized that the lack of β-Proteobacteria-related phylotypes is believed to be theresult of the lowest detection limits of these taxa ratherthan their absences.

In conclusion, our results revealed bacterial communitystructure differences along Bizerte lagoon sediments. Weshowed the influence of pollutants on bacterial communitystructure, which is clearly observed by the quantity and thetype of PAHs and to a lesser extent by some heavy metals.Strong relationships were observed between individual-PAH concentrations and some ribotypes. Clone librariesanalysis for two most contaminated PAHs and metalsediment stations have demonstrated significant differencesin bacterial community compositions, gamma proteobacte-rial phylotypes dominated the most PAH-polluted stationand delta and gamma proteobacterial phylotypes the mostmetal-polluted station. However, both libraries were dom-inated by sequences affiliated with uncultured bacteria.Future research might focus on in situ activity level of theγ-Proteobacteria, δ-Proteobacteria in order to determine ifthese groups play a role in the biotransformation of thepollutants.

Verrucomicrobia

Actinobacteria

Acidobacteria

Planctomycetacia

AB015546 Unidentified bacterium

100

60

61

41

95

79

73

91

56

90

74

43

39

97

100

95

52

94

29

31

97

87

82

51

55

38

59

26

33

33

24

43

6

11

39

0.02

X89560 Candidatus Microthrix

Z95733 Uncultured Acidobacteria bacterium X77215 Holophaga foetida DSM 6591T

AF050560 Uncultured eubacterium WCHB1-41

X94145 Nocardioides sp.M37200 Aeromicrobium erythreum

X77439 Rathayibacter rathayi

AF382139 Uncultured bacterium

AF424323 Uncultured bacterium MERTZ_21CM_220

Z95718 Holophaga/Acidobacterium phylum

X62910 Planctomyces marisL10942 Unidentified marine eubacterium

AJ241004 Uncultured Holophaga/Acidobacterium Sva0515

AF507898 Uncultured Verrucomicrobia bacterium clone ML623J-15

AY114335 Uncultured Verrucomicrobia bacterium clone LD1-PB3

AJ231183 Pirellula staleyi (strain DSM 6068T)X62912 Blastopirellula marina DSM 364

X54522 Gemmata obscuriglobus

1 (1.37%); 2 (1.37%)

1 (2.74%)1 (1.37%)

2 (1.37%)

2 (1.37%)1 (1.37%)

1 (1.37%); 2 (1.37%)1 (2.74%)

2 (1.37%)

2 (1.37%)

1 (1.37%)1 (2.74%); 2 (2.74%)

2 (1.37%)

1 (1.37%)

b

Fig. 6 continued.

454 O. B. Said et al.

Page 11: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

Acknowledgments This research was supported by the Tunisian“Ministère de la Recherche Scientifique, de la Technologie et duDeveloppement des Competences” (MRSTDC). We acknowledge thefinancial support of the Conseil Régional d’Aquitaine and the ConseilGénéral des Pyrénées Atlantiques. Sequencing experiments presentedin the present publication were performed at the Genotyping andSequencing facility of Bordeaux (grants from the Aquitaine RegionalGovernment Council no. 20030304002FA and 20040305003FA andfrom the European Union, FEDER no. 2003227).

References

1. Abulencia CB, Wyborski DL, Garcia JA, Podar M, Chen W,Chang SH, Chang HW, Watson D, Brodie EL, Hazen TC, KellerM (2006) Environmental whole-genome amplification to accessmicrobial populations in contaminated sediments. Appl EnvironMicrobiol 72:3291–3301

2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, MillerW, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic AcidsRes 25:3389–3402

3. Asami H, Aida M, Watanabe K (2005) Accelerated sulfur cycle incoastal marine sediment beneath areas of intensive shellfishaquaculture. Appl Environ Microbiol 71:2925–2933

4. Ben Said O, Goñi-Urriza MS, El Bour M, Dellali M, Aissa P,Duran R (2008) Characterization of aerobic polyaromatichydrocarbon-degrading bacteria from Bizerte lagoon sediments,Tunisia. J Appl Microbiol 104:987–997

5. Bissett A, Bowman J, Burke C (2006) Bacterial diversity inorganically-enriched fish farm sediments. FEMS Microbiol Ecol55:48–56

6. Bordenave S, Fourçans A, Blanchard S, Goñi-Urriza MS,Caumette P, Duran R (2004) Structure and fonctional analysesof bacterial communities changes in microbial mats followingpetroleum exposure. Ophelia 58:195–203

7. Bordenave S, Goñi-Urriza MS, Caumette P, Duran R (2007)Effects of heavy fuel oil on the bacterial community structure of apristine microbial mat. Appl Environ Microbiol 73:6089–6097

8. Bowman JP, Mccuaig RD (2003) Biodiversity, communitystructural shifts, and biogeography of prokaryotes within antarcticcontinental shelf sediment. Appl Environ Microbiol 69:2463–2483

9. Brown MV, Bowman JP (2001) A molecular phylogenetic surveyof sea-ice microbial communities (SIMCO). FEMS MicrobiolEcol 35:267–275

10. Cao Y, Cherr GN, Córdova-Kreylos AL, Fan TWM, Green PG,Higashi RM, LaMontagne MG, Scow KM, Vines CA, Yuan J,Holden PA (2006) Relationships between sediment microbialcommunities and pollutants in two California Salt Marshes. FEMSMicrobiol Ecol 52:619–633

11. Córdova-Kreylos AL, Cao Y, Green PG, Hwang HM, KuivilaKM, LaMontagne MG, Van De Werfhorst LC, Holden PA, ScowKM (2006) Diversity, composition, and geographical distributionof microbial communities in California Salt Marsh Sediments.Appl Environ Microbiol 72:3357–3366

12. Edlund A, Jansson JK (2006) Changes in active bacterialcommunities before and after dredging of highly polluted BalticSea sediments. Appl Environ Microbiol 72:6800–6807

13. Ford T, Ryan D (1995) Toxic metals in aquatic ecosystems: amicrobiological perspective. Environ Health Perspect 103:25–28

14. Fourçans A, Solé A, Diestra E, Ranchou-Peyruse A, Esteve I,Caumette P, Duran R (2006) Vertical migrations of phototrophicbacterial populations in a hypersaline microbial mat from

Salins-de-Giraud (Camargue, France). FEMS Microbiol Ecol57:367–377

15. Gillan DC, Pernet P (2007) Adherent bacteria in heavy metalcontaminated marine sediments. Biofouling 23:1–13

16. Goñi-Urriza MS, Point D, Amouroux D, Guyoneaud R, DonardOFX, Caumette P, Duran R (2007) Bacterial community structurealong the Adour estuary (French Atlantic coast): influence of salinitygradient versus metal contamination. Aquat Microb Ecol 49:47–56

17. Heijs SK, Damsté JS, Forney LJ (2005) Characterization of adeep-sea microbial mat from an active cold seep at the Milanomud volcano in the Eastern Mediterranean Sea. FEMS MicrobiolEcol 54:47–56

18. Hernández-Raquet G, Budzinski H, Caumette P, Dabert P, LeMénach K, Muyzer G, Duran R (2006) Molecular diversity studiesof bacterial communities of oil polluted microbial mats from theEtang de Berre (France). FEMS Microbiol Ecol 58:550–562

19. Hewson I, Fuhrman JA (2004) Richness and diveristy ofbacterioplankton along an estuarine gradient in Moreton Bay,Australia. Appl Environ Microbiol 70:3425–3433

20. Hunter EM, Mills HJ, Kostka JE (2006) Microbial communitydiversity associated with carbon and nitrogen cycling in perme-able shelf sediments. Appl Environ Microbiol 72:5689–5701

21. Inagaki F, Suzuki M, Takai K, Oida Sakamoto T, Aoki K, NealsonKH, Horikoshi K (2003) Microbial communities associated withgeological horizons in coastal subseafloor sediments from the seaof Okhotsk. Appl Environ Microbiol 69:7224–7235

22. Jiang SC, Paul JH (1996) Occurrence of lysogenic bacteria inmarine microbial communities as determined by prophageinduction. Mar Ecol Prog Ser 142:27–38

23. Kasai Y, Kishira H, Syutsubo K, Harayama S (2001) Moleculardetection of marine bacterial populations on beaches contaminated bythe Nakhodka tanker oil-spill accident. EnvironMicrobiol 3:246–255

24. Kimura M (1980) A simple method for estimating evolutionaryrates of base substitutions through comparative studies ofnucleotide sequences. J Mol Evol 16:111–120

25. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated softwarefor molecular evolutionary gnenetics analysis and sequencealignement. Brief Bioinform 5:1907–1919

26. Lane DJ (1991) rRNA sequencing. In: Stachenbradt E, Good-fellow M (eds) Nucleic acid 527 techniques in bacterialsystematics. Wiley, Chichester, pp 115–175

27. Li B, Zhang T, Xu Z, Fang HHP (2009) Rapid analysis of 21antibiotics of multiple classes in municipal wastewater using ultraperformance liquid chromatography-tandem mass spectrometry.Anal Chiml Acta 645(1–2):64–72

28. Lopez-Garcia P, Duperron S, Philippot P, Foriel J, Susini J,Moreira (2003) Bacterial diversity in hydrothermal sediment andepsilonproteobacterial dominance in experimental microcolonizersat the Mid-Atlantic Ridge. Environ Microbiol 5:961–976

29. Macnaughton SJ, Stephen JR, Venosa AD, Davis GA, Chang YJ,White DC (1999) Microbial population changes during bioreme-diation of an experimental oil spill. Appl Environ Microbiol65:95–101

30. Maidak BL, Cole JR, Lilburn TG, CT P Jr, Saxman PR, Farris RJ,Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM (2001) TheRDP-II (Ribosomal Database Project). Nucleic Acids Res 29:173–174

31. Mills HJ, Hodges C, Wilson K, MacDonald IR, Sobecky PA(2003) Microbial diversity in sediments associated with surface-breaching gas hydrate mounds in the Gulf of Mexico. FEMSMicrobiol Ecol 46:39–52

32. Murphy J, Riley JP (1962) A modified single solution method forthe determination of phosphate in naturel waters. Anal Chim Acta27:31–36

33. Nakatsu CH, Carmosini N, Baldwin B, Beasley F, Kourtev P,Konopka A (2005) Soil microbial community responses to

Bacterial Communities in Bizerte Lagoon-Polluted Sediments 455

Page 12: Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

additions of organic carbon substrates and heavy metals (Pb andCr). Appl Environ Microbiol 71:7679–7689

34. NF EN ISO 11885 (1998) Water quality-determination of 33elements by inductively coupled plasma atomic emission spec-troscopy. AFNOR, Paris

35. Païssé S, Coulon F, Goñi-Urriza MS, Peperzak LJ, McGenity T,Duran R (2008) Structure of bacterial communities along ahydrocarbon contamination gradient in a coastal sediment. FEMSMicrobiol Ecol 66:295–305

36. Precigou S, Goulas P, Duran R (2001) Rapid and specificidentification of nitrile hydratase (NHase)-encoding genes in soilsamples by polymerase chain reaction. FEMS Microbiol Lett204:155–161

37. Richards FA, Thompson TG (1952) The estimation and charac-terization of plankton populations by analysis. 2- a spectrophoto-metric method for the estimation of plankton pigment. J Mar Res11:156–172

38. Röling WFM, Milner MG, Jones DM, Lee K, Daniel F, SwannellRJP, Head IM (2002) Robust hydrocarbon degradation anddynamics of bacterial communities during nutrient-enhanced oilspill bioremediation. Appl Environ Microbiol 68:5537–5548

39. Saitou N, Nei M (1987) The neighbour-joining method: a newmethod for reconstructing phylogenetics trees. Mol Biol Evol4:406–425

40. Singleton DR, Furlong MA, Rathbun SL, Whitman WB (2001)Quantitative comparisons of 16S rDNA sequence libraries

from environmental samples. Appl Environ Microbiol 67:4373–4376

41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, HigginsDG (1997) The ClustalX-Windows interface: flexible strategiesfor multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 25:4876–4882

42. Vrionis HA, Anderson RT, Ortiz-Bernad I, O’Neill KR, Resch CT,Peacock AD, Dayvault R, White DC, Long PE, Lovley DR (2005)Microbiological and geochemical heterogeneity in an in situuranium bioremediation field site. Appl Environ Microbiol71:6308–6318

43. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16Sribosomal DNA amplification for phylogenetic study. J Bacteriol173:697–703

44. Yan T, Fields MW, Wu L, Zu Y, Tiedje JM, Zhou J (2003)Molecular diversity and characterization of nitrite reductase genefragments (nirK and nirS) from nitrate- and uranium-contaminatedgroundwater. Environ Microbiol 5:13–24

45. Yoshida M, Hamdi H, Abdulnasser I, Jedidi N (2002) Contam-ination of potentially toxic elements (PTEs) in Bizerte lagoonbottom sediments, surface sediment and sediment repository. In:Ghrabi A, Yoshida M (eds) Study on environmental pollution ofBizerte lagoon. INRST-JICA, Tunis, p 139

46. Zhang W, Ki JS, Qian PY (2008) Microbial diversity in pollutedharbor sediments I: bacterial community assessment based on fourclone libraries of 16S rDNA. Estuar Coast Shelf Sci 76:668–681

456 O. B. Said et al.