RESEARCH ARTICLE

Low impact of phenanthrene dissipation on the bacterialcommunity in grassland soil

Maïté Niepceron & Jérémie Beguet & Florence Portet-Koltalo &

Fabrice Martin-Laurent & Laurent Quillet & Josselin Bodilis

Received: 4 June 2013 /Accepted: 17 October 2013 /Published online: 30 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The effect of phenanthrene on the bacterialcommunity was studied on permanent grassland soilhistorically presenting low contamination (i.e. less than1 mg kg−1) by polycyclic aromatic hydrocarbons (PAHs).Microcosms of soil were spiked with phenanthrene at300 mg kg−1. After 30 days of incubation, the phenanthreneconcentration decreased rapidly until its total dissipation within90 days. During this incubation period, significant changes ofthe total bacterial community diversity were observed, asassessed by automated-ribosomal intergenic spacer analysisfingerprinting. In order to get a deeper view of the effect ofphenanthrene on the bacterial community, the abundances of

ten phyla and classes (Actinobacteria, Acidobacteria,Bacteroidetes, Alphaproteobacteria, Betaproteobacteria,Gammaproteobacteria, Firmicutes, Verrucomicrobiales,Gemmatimonadetes, and Planctomycetes) were monitored byquantitative polymerase chain reaction performed on soil DNAextracts. Interestingly, abundances of some bacterial taxasignificantly changed as compared with controls. Moreover,among these bacterial groups impacted by phenanthrenespiking, some of them presented the potential of phenanthrenedegradation, as assessed by PAH-ring hydroxylatingdioxygenase (PAH-RHDα) gene detection. However, neitherthe abundance nor the diversity of the PAH-RHDα genes wassignificantly impacted by phenanthrene spiking, highlightingthe low impact of this organic contaminant on the functionalbacterial diversities in grassland soil.

Keywords Polycyclic aromatic hydrocarbon .Microcosm .

Phenanthrene . A-RISA . qPCR . Dioxygenase

Introduction

The Seine watershed covers approximately 78,650 km2 andrepresents 40 % of the French economic activity and 33 % ofall French oil refining. It is a highly urbanised and industrialisedwatershed but also a highly productive agricultural area, due tofavourable climatic conditions and to the natural richness oftheir deep silty soils. Consequently, the agricultural soils of theSeine basin are exposed to organic micro-pollutants includingpolycyclic aromatic hydrocarbons (PAHs). Contamination ofagricultural soils by PAH may represent a human health riskdue to the consumption of contaminated meat or vegetables(Wild and Jones 1992; Fismes et al. 2002; Grova et al. 2002;Martorell et al. 2010). PAHs are produced by incompletecombustion of wood and fossilised organic matter (Motelay-Massei et al. 2007; Wang et al. 2009). They are semi-volatile

Responsible editor: Robert Duran

Electronic supplementary material The online version of this article(doi:10.1007/s11356-013-2258-9) contains supplementary material,which is available to authorized users.

M. Niepceron : L. Quillet : J. BodilisLaboratoire de Microbiologie Signaux et Microenvironnement,Université de Rouen, EA 4312, 76821 Mont Saint Aignan, France

M. NiepceronLaboratoire M2C, UMR CNRS 6143, Université de Rouen,76821 Mont Saint Aignan, France

F. Portet-KoltaloLaboratoire COBRA, UMR CNRS 6014, Université de Rouen,27000 Evreux, France

J. Beguet : F. Martin-LaurentINRA, UMR 1347 Agroécologie, BP 86510, 21065 Dijon, France

F. Martin-LaurentINRA, Service de Séquençage et de Génotypage, SSG, BP 86510,21065 Dijon, France

J. Bodilis (*)LMSM, Bâtiment IRESEB, UFR des Sciences, Université de Rouen,76821 Mont Saint Aignan, Francee-mail: josselin.bodilis@univ-rouen.fr

Environ Sci Pollut Res (2014) 21:2977–2987DOI 10.1007/s11356-013-2258-9

compounds emitted in the atmosphere and dispersed in theenvironment where they are considered as ubiquitouscontaminants. Because of their chemical stability andhydrophobicity, these contaminants accumulate progressivelyin soils, where they persist for many years. PAHs can betransferred from soil to surface water by surface runoff(Motelay-Massei et al. 2007), which is a highly variableprocess depending on several parameters including land useor meteorological conditions (Motelay-Massei et al. 2004;Mouhri et al. 2008).

Although the fate of PAHs in soil is partly driven by abioticprocesses such as adsorption, volatilisation, photolysis andchemical degradation, microbial degradation is recognised asthe main driver of PAH dissipation (Park et al. 1990). Indeed,microorganisms can use PAHs as an energy or substratesource for their growth, by converting PAHs to carbondioxide (Peng et al. 2008; Seo et al. 2009). Manybacterial isolates are known to effectively degrade PAHsin soil among which are Pseudomonas spp., Mycobacteriumspp.,Haemophilus spp., Rhodococcus spp. and Paenibacillusspp (Cerniglia 1992; Juhasz and Naidu 2000; Niepceronet al. 2010).

Contamination with PAHs of agricultural soils alreadyimpacted by agricultural practices may affect soil microbialcommunities which are key players in soil ecosystem services.The impact of PAH contamination on the bacterial communityis dependent on the type and history of the soil (Wilson andJones 1993), but few studies have been conducted tocharacterise the impact of PAHs on agricultural soils,particularly in the pedoclimatic context of North West France(Johnsen and Karlson 2005). Although (punctual or diffuse)PAH contamination results generally in contamination byvarious PAHs (Motelay-Massei et al. 2004), most of thestudies conducted in soil microcosms under laboratoryconditions use phenanthrene as a PAH model because it ismore bioavailable to soil-borne organisms, and its degradationpathways are well-known (Johnsen et al. 2002; De Menezeset al. 2012).

This study proposes to evaluate the effect of PAHcontamination (simulating an accidental contamination) onthe bacterial community of agricultural soil historicallycontaminated with low amount of PAHs (i.e. less than1 mg kg−1) collected in the Seine basin. Microcosms ofpermanent grassland soil were spiked with 300 mg kg−1 ofphenanthrene. The dissipation of phenanthrene in the soilmicrocosms was monitored. The impact of this PAH on theabundance and structure of the bacterial community wasestimated by molecular approaches relying on direct soilDNA extraction and further analyses by PCR-basedapproaches. The abundance and structure of the PAH-degrading bacterial populations were also characterisedby targeting the PAH-ring hydroxylating dioxygenase(PAH-RHDα) genes.

Materials and methods

Soil samples

Soil was sampled in spring 2010 from a long-term grassland (arye-grass clover monoculture with permanent cover and notillage for 25 years) located in Yvetot (Upper-Normandy,France). The Upper-Normandy region is dominated by anoceanic and temperate climate characterised by mildtemperatures. The soil, containing 15 % clay, 65 % silt, and20 % sand, is representative of the Paris Basin, and classifiedas silty (e.g. loess) soil. From the surface horizon (0–15 cm) ofthis site, 20 samples were collected and combined into a bulkcomposite sample (15 kg). After sieving to 2-mm particle size,field-moist soils were stored at 20 °C for 1 week (underhumidity control). In order to obtain a final concentration inmicrocosms at 300 mg kg−1, part of the soil (10 %) wassterilised by autoclaving and supplemented with phenanthreneat 3,000 mg kg−1.

Microcosm design

Microcosms were established in hermetically closed 500-mLsterile glass flasks containing 90 g of dry weight equivalent soil(24.75 % vol/wt of water, corresponding to 70 % of water-holding capacity) and 10 g of dry sterilised soil spiked withphenanthrene (at 3,000 mg kg−1). The initial phenanthrenesolution used to spike the soil was prepared in acetone, mixedto dry sterilised soil, and incubated for 2 days under a fumehood to evaporate the acetone and homogenise thephenanthrene distribution in soil aggregates. Microcosmsspiked with 300 mg kg−1 (equivalent to about 5,000-fold theamount of phenanthrene found in the native soil) wereobtained by mixing the phenanthrene spiked soil with thewet native soil and homogenised. Microcosms spiked withphenanthrene or not (control) were established in triplicate(n =3, per treatment). The flasks were incubated in a dark roomat 20 °C, in a static mode, and opened once a week for 15 minto ensure aerobic conditions in the flasks. Analyses werecarried out after 8, 30, 60 and 90 days of incubation, usingsacrificial batches (27 batches in total, including three batchesfor studying the diversity of the initial bacterial community).

PAH extraction from soil and quantification by GC/MS

Phenanthrene remaining in the spiked soil, after the differentperiods of incubation, was analyzed by gas chromatography-mass spectrometry (GC-MS). Three aliquots of 1.5–2 g of soilwere taken from each of three microcosms and were driedovernight by incubating at 35 °C. After crushing andweighting,1 g aliquot of each of the nine soil samples were extractedsimultaneously by microwave-assisted extraction (MAE)(MARS X, CEM Corporation, Matthews, USA). Each aliquot

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was mixed with 20 mL of toluene/acetone 50/50 (v/v) and thenheated at 140 °C during 30min, with power set at 1,200W. Theextracting solvent was filtered with a Phenex Teflon PTFE filter(0.45 μm) (Phenomenex, Le Pecq, France) and added withperdeuterated phenanthrene as an internal standard(100 mg L−1) One microliter of the extract was injected(splitless injection at 280 °C) in the gas chromatographer(6850 model from Agilent, Santa Clara, USA). The MSdetector (5975C model from Agilent) operated at 70 eV withan electron voltage of 1,600 V in positive ion mode(temperature of the transfer line, 300 °C). Separation wasperformed using a 50 m×0.25 mm i.d. DB5-MS capillarycolumn (0.25 μm film thickness, coated with a (5 % phenyl)methyl polysiloxane stationary phase) from J & W Scientific(Folsom, CA, USA), with helium as a carrier gas(1.2 mL min−1). Oven temperature was programmed at 55 °Cfor 1.2 min, increased to 180 °C (at 40 °C min−1) and then to300 °C at 4 °C perminute. Quantificationwas based on selectedion monitoring for better sensitivity. The detection thresholds inSIM mode, calculated as three times the standard deviation of

blank sample noise, was 1.5 μg L−1 for phenanthrene, and thelimit of quantification was 5 μg L−1.

DNA extraction

For each sacrificial batch, soil was carefully homogenised, andDNAwas extracted from 0.4 g dry weight equivalent of sievedsoils using the BIO101 Fast DNA spin Kit for Soil. DNAextracts were resuspended in 50 μL sterile deionised water.They were quantified on agarose gel stained with ethidiumbromide (0.5 μg ml−1) and stored at −20 °C until their use.

16S rRNA genes qPCR using universal or phylum-specificprimer sets

In order to estimate the copy number of 16S rRNA genes inDNA samples, quantitative polymerase chain reaction (qPCR)were carried out using universal or taxon-specific primers(Table 1). qPCR were performed in a StepOnePlus® Real-Time PCR Systems (Applied Biosystems™). Each 15-μL

Table 1 Primer sets used in our study

Gene targeted Primer name Sequence (5′→3′) Reference

16S rRNA (qPCR)

Total bacteria 341 F CCTACG GGA GGC AGC AG Lopez-Gutierrez et al. 2004

534R ATTACC GCG GCT GCT GGC A Lopez-Gutierrez et al. 2004

Acidobacteria Acid31 GAT CCT GGC TCA GAATC Fierer et al. 2005

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

Actinobacteria Actino235 CGC GGC CTATCA GCT TGT TG Fierer et al. 2005

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

Bacteroidetes Cfb319 GTA CTG AGA CAC GGA CCA Fierer et al. 2005

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

Firmicutes Lgc353 GCA GTA GGG AAT CTT CCG Fierer et al. 2005

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

Gemmatimonadetes Gem440 TTC GGR KTG TAA ACC ACT GT Philippot et al. 2009

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

Planctomycetes Plancto352f GGC TGC AGT CGA GRATCT Muhling et al. 2008

Plancto920r TGT GTG AGC CCC CGT CAA Muhling et al. 2008

Alphaproteobacteria Eub338 ACT CCTACG GGA GGC AGC AG Fierer et al. 2005

Alfa685 TCTACG RAT TTC ACC YC TAC Fierer et al. 2005

Betaproteobacteria Eub338 ACT CCTACG GGA GGC AGC AG Fierer et al. 2005

Bet680 TCA CTG CTA CAC GYG Fierer et al. 2005

Gammaproteobacteria Gamma395f CMATGC CGC GTG TGT GAA Muhling et al. 2008

Gamma 871r ACT CCC CAG GCG GTC DAC TTA Muhling et al. 2008

Verrucomicrobia Verr349 GYG GCA SCA GKC GMG AAW Philippot et al. 2009

Eub518 ATTACC GCG GCT GCT GG Fierer et al. 2005

16S–23S Intergenic spacer (ARISA) ARISA-132r CCG GGT TTC CCC ATT CGC Ranjard et al. 2001

ARISA-1552f TCG GGC TGG ATG ACC TCC TT Ranjard et al. 2001

PAH-RHDα genes (qPCR) PAH-RHDα-396 F ATT GCG CTTAYC AYG GBT GG Ding et al. 2010

PAH-RHDα-696R ATA GGT GTC TCC AAC RAA RTT Ding et al. 2010

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reaction mixture contained 7.5 μL SYBR Green® PCR MasterMix (Absolute QPCR SYBR Green Rox Abgene™), 250 ng ofT4 gp 32 (QBiogene™), and 4 ng of DNA. Annealingtemperature varied between 55 °C and 60 °C, in accordancewith the primer pair used (for more details see references inTable 1). In order to avoid interference by unspecific products,fluorescence acquisition was carried out just after the annealingcycle at 80 °C. A standard curve was established using serialdilutions of linearised plasmid pGEM-T (102–107 copies)containing a relevant 16S rRNA gene. After amplification,melting curves were obtained by increasing the temperaturefrom 80 °C to 95 °C. For each DNA sample, average of tworeplicates was determined as copy number per gram of dry soil.The relative abundance of each taxon was estimated as the ratioof the copy number of this taxon to the total number of 16SrRNA genes, generated by using universal primers.

Bacterial community fingerprinting by automated-ribosomalintergenic spacer analysis (A-RISA)

Automated-ribosomal intergenic spacer analysis (A-RISA) wasapplied to estimate the effect of PAHs on the genetic structure ofthe bacterial community in the soil samples (Ranjard et al.2001). To amplify the 16S–23S intergenic spacer of the bacterialrRNA, PCR reactions were carried out in a final volume of25μL containing 0.5μMof universal primers ARISA-132r andARISA-1552f (Table 1) labelled at position 5′with IRD 800 dyefluorochrome (MWG SA Biotech, Ebersberg, Germany) and,2.5 U of Taq DNA polymerase (Appligene Oncor, France) and0.1 ng of template DNA per microlitre of PCR mixture. PCRreactions were done in a gradient thermocycler Venti™(AppliedBiosystems, USA) using the following program:94 °C for 5 min, 35 cycles of 1 min at 94 °C, 1 min at 55 °Cand 2 min at 72 °C, plus an additional 15 min at 72 °C. Theamount of A-RISA amplicons was evaluated afterelectrophoresis on 2 % agarose gel loaded with 5 μL of SmartLadder DNA marker (Eurogentec, Belgium) usingImageQuaNT software (Molecular Dynamics, Evry, France).A-RISA amplicon concentrations were normalised in order toload 40 ng of amplicons per lane on A-RISA acrylamide gels.After denaturation at 92 °C for 3min, amplicons were loaded ona 3.7 % polyacrylamide gel (66 cm in length) and run on aLiCor 4300DNAAnalysis System (Biosciences, USA) for 15 hat 1,500 V/80W. A-RISA fingerprints were analyzed using 1-DScan (ScienceTec, France) in order to estimate the size of eachband (in base pairs) as comparedwith DNA ladder ranging from200 to 1,200 bp aswell as the height of each peak correspondingto the relative abundance of each peak.

PAH-RHDα qPCR

The copy number of the PAH-RHDα sequences in DNAsampleswas determined by qPCR using the 20-mer degenerated

forward PAH-RHDα-396 F primer and the 21-mer degeneratedreverse PAH-RHDα-696R primer (Table 1) targeting a 320 bpsequence found in the 40 PAH-RHDα genes previouslydescribed (Ding et al. 2010). The PCR mix contained 12.5 μLSYBR Green® PCR Master Mix (MESA Blue qPCR Master-mix for SYBRGreen, Eurogentec™), 12.5 μg of BSA (MP™),2 ng of soil DNA in a final volume of 25 μL. A standard curvewas carried out using serial dilutions of plasmid pGEM-T (101–106 copies) containing a PAH-RHDα gene. Amplificationreactions were carried out in a 96-well optical plate with aChromo 4 Real Time PCR Detector (Bio-Rad™), as follows:denaturation for 5 min at 94 °C, 5 pre-amplification cycles with1 min denaturation at 94 °C, annealing at 46 °C for 2 min,extension at 72 °C for 1 min, followed by 30 cycles of 1 min at95 °C, 30 sec at 58.5 °C and 1 min at 72 °C, and then a 10 minextension at 72 °C. Melting curves were generated afteramplification by increasing the temperature from 30 °C to100 °C. For each DNA sample, average of two PCR replicateswas determined as copy number per gram of dry soil. Therelative abundance of PAH-RHD genes was estimatedas the ratio of the copy numbers of this gene to thetotal number of 16S rRNA genes.

Construction of PAH-RHDα gene libraries and phylogeneticanalysis

DNA extracted after 60 days of incubation from control orphenanthrene-spiked microcosms was used as a template toconstruct PAH-RHDα gene libraries. PCR amplification wasperformed using the same primers as for the real-time PCRquantification. The PCR mixtures (50 μL) contained 0.4 μMof each primer, 100 mM of each dNTP (Eurogentec™),0.25μg L-1 of BSA (MP™), IVD-cGMPTaqDNAPolymerasebuffer, 1.25 mM of MgCl2, 2.5 U of IVD-cGMP Taq DNAPolymerase with red loading dye (Eurogentec™), and onebacterial colony as template. Amplification was performed ina GeneAmp PCR system 9700 Thermocycler (AppliedBiosystems™) using the same program as for real-time PCRquantification of PAH-RHD genes. PCR products were easilyligated into pGEM-T using a TA cloning kit according to themanufacturer’s instructions (Promega™). About 100 cloneswere selected randomly and checked for correct insert size viaPCR and agarose gel electrophoresis and sequenced using theT7 primer of the plasmid [GenBank: JX861270–JX861369].The reference sequences used for the phylogenetic analysiswere retrieved from GenBank. The amino acid alignment wascarried out using the Clustal algorithm implemented in theSeaview software (Thompson et al. 1997; Gouy et al. 2010),then optimised visually, and all ambiguous positions andpositions with gaps were removed. The unrooted dendrogramwas generated from amino acid sequences, using neighbor-joining analysis (with Poisson correction) with the MEGAv5.0 software (Tamura et al. 2011). The degree of statistical

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support for the branches was determined with 1,000 bootstrapreplicates.

Statistical analyses

From qPCR results, the significant differences between themicrocosm conditions was tested with the non-parametricKruskal–Wallis test (p<0.05) using XLStat v. 6.0 (Addinsoft,Brooklyn, USA). According to the test requirements, all datapoints were independent from each other, and sample sizeswere equal with three data points analysed for each treatment.A-RISA fingerprints were analysed using PrepRISA (http://pbil.univ-lyon1.fr/ADE-4/microb/) and R package (Veganlibrary). Using PrepRISA, the data from the 1D-Scan(ScienceTec, France) were converted into a Bray-Curtissimilarity matrix summarising the bands’ presence (i.e. peaks)and intensity (i.e. peak heights). Non-metric multi-dimensional scaling (NMDS) was performed for eachmicrocosm (stress=0.16 that corresponds to a quite goodordination with no prospect for misleading interpretations).From A-RISA fingerprint, the significant of differencesbetween the microcosm conditions was tested using the two-way crossed ANOSIM of Bray-Curtis similarities.

Results

Phenanthrene dissipation rate

The grassland soil of Yvetot has been exposed for severaldecades to chronic and diffuse contamination of PAHoriginating from urban and industrial atmospheric deposition.As a consequence, it contained about 0.6 mg kg−1 of PAHs(total amount of the 16 priority PAHs as defined by theEnvironmental Protection Agency of the USA), including0.055±0.022 mg kg−1 of phenanthrene, 0.074±0.023 mg kg−1

of fluoranthene, 0.063±0.021 mg kg−1 of pyrene and 0.039±0.018 mg kg−1 of benzo[a]pyrene. In order to simulate anaccidental pollution, microcosms of soils, collected from theYvetot permanent grasslands, were spiked with 300 mg kg−1 ofphenanthrene. This artificial contamination represents 500-foldthe total PAH content of the native soil and 5,000-fold thephenanthrene content of the native soil.

Phenanthrene dissipation kinetics was monitored by GC-MS during the 90-day period of incubation (Fig. 1). Althoughthe soil was spiked with a target concentration of 300 mg kg−1

of phenanthrene, only about 250 mg kg−1 could be retrieved inthe soil at T0 (i.e. a few minutes after the preparation of themicrocosms). This discrepancy between theoretical andmeasured values was reproducible (measured from nine soilsamples) and probably results essentially from lossesoccurring during MAE extraction. Indeed, during theoptimisation step of MAE, the observed recovery yield of

phenanthrene was approaching 85.4±6.9 % in Yvetot soil.Slight losses may also result from both the evaporation ofacetone from soil and the adsorption on the glass walls of the500-mL flasks.

In the microcosms, the phenanthrene concentrationdecreased rapidly after a 30-day lag phase, phenanthrenebeing no longer detectable after 90 days of incubation (i.e.detection limit of GC-MS estimated to 0.1 mg kg−1).

Abundance and structure of the total bacterial community

During the phenanthrene dissipation, changes of theabundance of the bacterial community were monitored byqPCR assay targeting the 16S rRNA gene from soil DNA.The abundance of the bacterial community ranged between5.83×108 and 1.65×109 copies of 16S rRNA gene per gramof soil (dried weight equivalent) (Fig. 2) and was not

Ph

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Fig. 1 Phenanthrene dissipation kinetic (milligrams of phenanthrene perkilogram of dry soil) at 300 mg kg−1 in permanent grassland of Yvetotincubated at 20 °C during 90 days (n =9). Each plot corresponds to meanvalues based on nine replicates±standard errors

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Fig. 2 Number of 16S rRNA gene copies per gram of dry soil frommicrocosms spiked with phenanthrene at 300 mg kg−1 of soil (blacksquare) and acetone control microcosms (black triangle), as determinedby real-time PCR (n=3). Each plot corresponds to mean values based onthree replicates± standard errors. All the differences betweenphenanthrene-spiked and control microcosms were not significant (P >0.05; Mann–Whitney U test)

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significantly affected by the phenanthrene enrichment (P >0.05; Kruskal–Wallis test), whatever the incubation time.

In order to further address the impact of phenanthrene, thetotal bacterial structure was monitored all along the dissipationkinetics (Fig. S1, Electronic supplementary data). A-RISAfingerprints were relatively complex, beingmade of more than100 bands. Visual observation of the gel revealed thatreplicates were similar, indicating a good reproducibility ofthe overall experimental procedure (Fig. S1, supplementarydata). Interestingly, while most of the bands presented thesame intensity over time and whatever the microcosmconditions (i.e. with or without phenanthrene), a few numberof bands showed significantly higher intensities in thephenanthrene-spiked microcosms as compared with control(Fig. S1, Electronic supplementary data). Unfortunately, thepopulations corresponding to these particular bands could notbe identified because of technical difficulties in extractingbands from 66 cm acrylamide gel and also because of only afew numbers of 16S-23S ISR sequences available in thedatabases. To further estimate the impact of phenanthreneenrichment by comparison to the control microcosm, A-RISA fingerprints were analysed to determine bands’presence (i.e. peaks) and intensity (i.e. peak heights) in orderto build up a Bray-Curtis similarity matrix which wasanalysed by NMDS (Fig. S1, Electronic supplementary data).

Visual observation revealed an important time effect duringthe experiment that was confirmed by an ANOSIM test (P <0.0001, R =0.762; ANOSIM test). Visual observationrevealed also slight differences between the microcosmsspiked with phenanthrene and the controls, starting to beobservable after 60 days of incubation and being moremarkedafter 90 days of incubation (Fig. S1, Electronic supplementarydata). These differences in the structure of bacterialcommunity in response to phenanthrene exposure wasstatistically significant according to ANOSIM test (P =0.004, R =0.407; ANOSIM test).

Relative abundance of different taxonomical groups

In order to get a deeper view of the impact of PAHs on the soilbacterial community, the abundances of ten phyla and classes(Actinobacteria, Acidobacteria, Bacteroidetes, Alpha-proteobacteria, Betaproteobacteria, Gammaproteobacteria,Firmicutes, Verrucomicrobiales, Gemmatimonadetes andPlanctomycetes) were determined by qPCR according toPhilippot et al. (2010). The relative abundance of each bacterialtaxon was calculated for each treatment and at each samplingtime (Fig. 3; Fig. S2, Electronic supplementary data).Significant differences have been observed between themicrocosms spiked with phenanthrene and the controls. Most

Bet

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CFig. 3 Relative abundances ofphylum-specific 16S rRNA genesin soil microcosms spiked withphenanthrene at 300 mg kg−1 ofsoil (black square) and acetonecontrol microcosms (blacktriangle), as determined by real-time PCR (n =3). Each plotcorresponds to mean values basedon three replicates±standarderrors. Stars highlight significantdifferences compared withcontrol DNA at the sameincubation time (P<0.05, Mann–Whitney U test): aActinobacteria; bBetaproteobacteria; cGammaproteobacteria; dFirmicutes

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of these differences were observed transiently during PAHdissipation (Fig. 3; Fig. S2, Electronic supplementary data).Interestingly, after 60 days of incubation, when almost 50 %of the initial amount of phenantrene was dissipated, theabundances of Actinobacteria and Firmicutes phyla and ofBeta- and Gammaproteobacteria classes were higher in soilmicrocosms spiked with phenanthrene than in correspondingcontrols. These abundance increases were significant for theFirmicutes phylum and the Beta- and Gammaproteobacteriaclasses. It is noteworthy that the impact of the phenanthreneon the Gammaproteobacteria was observed at the beginning ofthe incubation period, and this impact was kept all along theincubation period, except a transient decrease after 30 days ofincubation (Fig. 3).

Abundance and structure of the PAH-RHDα gene

In order to further study the effect of phenanthrene on thebacterial community, we specifically monitored the abundanceand diversity of bacterial PAH-degrading community. Theabundance of the total PAH-degrading bacterial communitywas estimated by qPCR assay targeting the PAH-RHDα genesencoding the alpha subunit of PAH-ring hydroxylatingdioxygenases involved in the initial aerobic oxidation ofaromatic compounds. The specific abundance of PAHdegraders in the total bacterial community was estimated bycalculating the ratio PAH-RHDα gene to 16S rRNA gene.Surprisingly, even if the soil studied presented a historicallylow PAH contamination, the specific abundance of PAHdegraders was high and remained stable over time in the controlmicrocosms (about 10−3 of the total bacterial community)(Fig. 4). Moreover, the specific abundance of PAH degradersin phenanthrene-spiked microcosms was of the same order of

magnitude as that of the controls (Fig. 4). In fact, the onlysignificant differences between the phenanthrene-spiked andcontrol microcosms was observed after 30 and 90 days, thespecific abundance of PAH degraders being significantly higher(P <0.05; Mann–Whitney U test) in control microcosms.

To further investigate the dominant PAH-degrading, twoPAH-RHDα clone libraries were constructed from the control(TA60) and phenanthrene-spiked (PHE60) microcosmscollected after 60 days of incubation when about 50 % ofPAH added to soil microcosms were already dissipated. Atotal of 100 clones were sequenced (Fig. 5). The control andphenanthrene-spiked microcosms showed a low andcomparable diversity with the PAH-RHDα sequencesgathering in two major operational taxonomic units (OTU-Burkholderia and OTU-Mycobacterium ). Overall, retrievedPAH-RHDα sequences were closely related to either theBurkholderia genus (affiliated to the Betaproteobacteria class)or the Mycobacterium genus (affiliated to the Actinobacteriaphylum). The proportion of PAH-RHDα sequences affiliatedto the Actinobacteria phylum was slightly higher, but notsignificantly (P=0.425; z test), in the phenanthrene-spikedmicrocosms (65 %) than in the control microcosms (56 %).

Discussion

After a 30-day lag phase, the phenanthrene in our soilmicrocosms was rapidly and entirely dissipated after 90 daysof incubation. Both the combination of biotic (mineralisationand/or biomass formation) and abiotic (adsorption to mineral ororganic particles, and evaporation) processes are known as maindrivers of phenantrene dissipation in soils. Since adsorptioncontribution is expected to be rapid following PAH-application, the observation of a lag-phase followed by a rapiddissipation is in favour of biotic degradation. PAH-biodegradation by soil microbial community would be inagreement with the low proportion of phenanthrene sequestratedin soil particles (Jones et al. 2011). In addition, the lag phaserecorded prior to dissipation is known to be the time required forthe growth of degrading populations (Soulas 1993). Thisobservation is in agreement with several studies showing thatphenanthrene dissipation mainly relies on biodegradationprocesses (Cebron et al. 2011; Martin et al. 2012).

Although PAHs are of environmental concern because theyare persistent and accumulate along the trophic chain, theirimpact on the diversity of soil bacterial communities is not clear(Peng et al. 2010). On one hand, PAHs might have toxic effecton soil bacterial community (Lors et al. 2010; Lors et al. 2012)and on the other hand, PAH exposure can lead to the selection ofbacterial populations able to use it as carbon and energy sourcesfor its growth, favouring their emergence in a contaminatedenvironment. The combination of these two opposite effectscontributes to modify the structure and the abundance of

Rat

io (

PA

H-R

HD

α/16

S r

RN

A g

ene)

Time (days)

1,20. 10-3

1,00. 10-3

8,00. 10-4

6,00. 10-4

4,00. 10-4

2,00. 10-4

0

1,40. 10-3

0 20 40 60 80 100

*

*

Fig. 4 Proportion of phenanthrene degraders in the total bacterialcommunity (number of PAH-RHDα gene copies per number of 16SrRNA gene copies) from microcosms spiked with phenanthrene at300 mg kg−1 of soil (black square) and acetone control microcosms(black triangle ), as determined by real-time PCR (n =3). Each plotcorresponds to mean values based on three replicates±standard errors.Stars highlight significant differences compared with control DNA at thesame incubation time (P <0.05, Mann–Whitney U test)

Environ Sci Pollut Res (2014) 21:2977–2987 2983

bacterial populations. Keeping in mind that PAH-degradingpopulations represent less than 1 % of the overall microbialpopulations in soil adapted to PAH degradation (Martin et al.2012; Cebron et al. 2008), one might expect that they would notbeen seen on A-RISA fingerprints that are principally allowing

discriminating dominant phylotypes among the overall bacterialcommunity. This has already been suggested for differentpesticide-degrading populations which cannot be perceived inthe soil environment by monitoring changes in the structure ofthe total bacterial community (Piutti et al. 2002; Hussain et al.

NahAC(Pseudomonas-AAA25902)NahAC(Pseudomonas-AAO64274)

NahAC(Pseudomonas-AAL07262)PahAc(Pseudomonas-BAA20391)NagAC(Polaromonas-AAZ93388)NagAC(Ralstonia-AAD12610)PahAc(Comamonas-AF252550)NahAC(Pseudomonas-AAD02136)

PahA3(Pseudomonas-BAA12240)PhnAc(Alcaligenes-BAA76323)

ArhA1(Sphingomonas-BAE93949)PhnAc(Burkholderia-AAD09872)OTU-Burkholderia (23TA+14Phe)

PhnA1(Cycloclasticus-BAC81541)BphA1f(Novosphingobium-AAD03858)

BphA1a(Sphingomonas-CAG17576)BphA1f(Sphingobium-ABM91740)

AhdA1(Sphingomonas-BAC65446)Phe41

DbfA1(Terrabacter-BAB55886)DbfA1(Paenibacillus-BAE53401)RH-dioxygenase(Rhodobacteraceae-EBA03444)

TA187TA76TA67TA83

Phe91RH-dioxygenase(Mycobacterium-YP935836)

Phe101Phe106

CarAa(Sphingomonas-AAC38616)DxnA1(Sphingomonas-CAA51365)

CumA1(Pseudomonas-BAA07074)BphA(Pandoraea-AAC44526)

BphA1(Rhodococcus-CAA56346)BphA1(Rhodococcus-BAA06868)

TodC1(Pseudomonas-AAA26005)PdoA2(Mycobacterium-AAZ78216)PdoA2(Mycobacterium-CAD38643)TA53

PhdA(Nocardioides-BAA94708)NarAa(Rhodococcus-AF082663)

NarAa(Rhodococcus-AAR05114)NidA(Rhodococcus-AF121905)Phe143Phe184Phe136

Phe167NidA3(Mycobacterium-AAY85176)PdoA(Terrabacter-AAZ38356)

OTU-Mycobacterium (28TA+22Phe)PdoA1(Mycobacterium-AAQ12029)

NidA(Mycobacterium-AAQ12023)PdoA1(Mycobacterium-CAD38647)NidA(Mycobacterium-AAQ95206)NidA(Mycobacterium-BAD20297)NidA(Mycobacterium-AAN78312)NidA(Mycobacterium-AAN78316)

DitA(Pseudomonas-AAD21063)BenA(Acinetobacter-AAC46436)OhbB(Pseudomonas-AAD20006)

76

99

97

50

59

99

99

99

99

99

71

87

99

9999

99

67

53

59

95

99

90

99

89

90

94

51

83

87

67

85

76

PAH-RHDα (Gram negative bacteria)

PAH-RHDα(Gram positive bacteria)

A-RHDα

A-RHDα

0.1 substitution per site

Fig. 5 Ring hydroxylatingdioxygenase alpha subunit(RHDα) phylogeny frommicrocosms spiked (noted “Phe”)or not (noted “TA”) withphenanthrene. Clone librarysequences (n=100) andsequences of closest relatives areincluded in the analysis. Therepresentative sequences definethe different clusters highlighted.OTU-Burkholederia and OTU-Mycobacterium represent the twomajor groups of sequences withmore than 98 % identity. Theunrooted dendrogram wasgenerated from amino acidsequences, using neighbor-joining analysis (with Poissoncorrection). The scale barcorresponds to 0.1 substitutionsper nucleotide position. Numberson tree branches indicatebootstrap results (n =1,000) forthose branches having more than50 % support. Sequencesprovided in this study are in bold

2984 Environ Sci Pollut Res (2014) 21:2977–2987

2009). In our study, because the structure of the total bacterialdiversity was significantly affected in response to phenanthrenespiking, we suggest that these differences observed were mostlikely due to a toxic effect of PAHs.

Interestingly, by targeting separately the ten classes or phylathe most often found in the soil, we have been able to highlightthat, after 60 days of incubation, the abundance of the Beta- andGammaproteobacteria classes and of the Firmicutes phylumwere significantly higher than in corresponding control. Inaddition, a smaller (not significant) variation of theActinobacteria abundance was also observed. These increaseswere observed when the dissipation of phenantrene was themost active. In addition, one could notice that members of thesefour bacterial groups have been extensively described in theliterature as PAH degraders (Jeon et al. 2003; Singleton et al.2005; Ding et al. 2010; Jones et al. 2011; De Menezes et al.2012; Martin et al. 2012). In addition, some phenanthrenedegraders have also been highlighted among the Firmicutesphylum (Matlakowska and Sklodowska 2009; Cebron et al.,2011). Interestingly, the bacterial groups presenting lowabundances (especially the Gammaproteobacteria) were thosethat were the most affected by the spiking of soil microcosmswith phenanthrene. Moreover, it is noteworthy that theBacteroidetes phylum (the second most abundant bacterialgroup in our study, representing about 30 % of the totalBacteria) increased significantly after 60 days of incubation inthe control microcosms (Fig. S2, Electronic supplementarydata). Overall, this might explain why the impact ofphenanthrene was hardly observable when considering theabundance of total bacterial community. Indeed, the impact ofphenantrene on the abundances of minor groups was hidden byabundances of dominant bacterial groups. This highlights theimportance of having tools able to also target minor bacterialgroups to be able to quantify the impact of pollutants, such asPAHs on soil microbial diversity.

In order to further study the effect of phenanthrene on thebacterial community, we specifically monitored the bacterialPAH-degrading community by targeting the PAH-RHDαgenes. Surprisingly, the abundance of the PAH-degradingcommunity was found to be high in Yvetot soil (about 10−3 ofthe total bacterial community). It could be hypothesised that thecontinuous diffuse contamination with PAHs, characteristic ofthe highly urbanised and industrialised Seine watershed, led tothe adaptation of soil microbiota and the development of PAH-degrading populations (Motelay-Massei et al. 2007). Theabundance of this functional community was found to be stableall over the incubation of soil microcosms and not respondingto phenanthrene exposure. This result is in contradiction withprevious studies conducted on different soils, as well as on asimilar soil (Luvisol), reporting an increase in PAH-RHDαgene abundance of several orders of magnitude in response toenrichment with PAH (Ding et al. 2010; Peng et al. 2010). Thisdiscrepancy might be explained by the fact that the amount of

phenanthrene spiked was too low (e.g. compared with 2,000 mg kg−1 used in Ding et al. 2010) to significantly increasethe already high PAH-RHDα gene abundance.

It is noteworthy that the primers we used to amplify the fullPAH-RHDα gene diversity so far described (i.e. both fromGram-positive and Gram-negative bacteria) also amplifiedgenes encoding aromatic-ring hydroxylating dioxygenases(noted A-RHDα in Fig. 5), which are not involved in thedegradation of PAHs (Iwai et al. 2011). Therefore, one cannotexclude a small overestimation of the abundance of the PAH-RHDα sequences.

Retrieved PAH-RHDα sequences were closely related toeither the Burkholderia genus (affiliated to the Betapro-teobacteria class) or the Mycobacterium genus (affiliated tothe Actinobacteria phylum). Despite their significantabundance increases in response to phenanthrene spiking,the Gammaproteobacteria class and the Firmicutes phylumwere not represented among the potential phenanthrenedegraders, neither in the control nor in the phenanthrene-spiked soil microcosms. The rather low abundance of theGammaproteobacteria compared with the Betaproteobacteriaand Actinobacteria (10 and 100 times lower, respectively)probably explains the absence of PAH-RHDα sequencesaffiliated to this bacterial group in our clone libraries. To beable to get sequences affiliated to this group, either primerstargeting specifically this group should be used, or thesampling effort should be increased using next-generationsequencing. More generally, PAH degraders (especiallyamong these four bacterial groups) might possess otherfunctional genes involved in PAH degradation and nottargeted by our primers (Suenaga et al. 2009). Moreover,although the PAH-RHDα sequences from Gram-positiveand Gram-negative bacterial strains are evolutionary distant(Fig. 5), we cannot exclude that some horizontal genetransfers of PAH-RHDα genes between Gram-negativebacteria masked the existence of phenanthrene-degradingbacteria among the Gammaproteobacteria class oramong the Firmicutes phylum (Herrick et al. 1997;Wilson et al. 2003).

To conclude, our study showed that the microbiota of thegrassland soil of Yvetot historically exposed to diffusecontamination of PAHs harboured an abundant PAH-degrading population able to rapidly dissipate phenanthrene.In response to phenanthrene exposure, the abundance of thetotal Bacteria as well as the PAH-degrading populationwas found to be stable, when the overall bacterialcommunity structure varied slightly. In response tophenanthrene, only the abundance of minor bacterialgroups increased when dissipation rate was the highest.Altogether, these observations suggest that continuousexposure of grassland soil microbial community toPAHs led to the acquisition of tolerance to these atmosphericpollutants.

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Acknowledgements This work was supported by grants from theAgence de l’Environnement et de la Maîtrise de l’Energie (ADEME)and the Région Haute Normandie (via RESSOLV and ALTERAGROprojects). The authors are grateful to Sylvaine Buquet for technicalassistance, to Aymé Spor for statistical assistance and to Dilys Moscatofor her critical reading of the manuscript.

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