ENVIRONMENTAL BIOTECHNOLOGY

Enhancement of the microbial community biomassand diversity during air sparging bioremediation of a soilhighly contaminated with kerosene and BTEX

Nadja Kabelitz & Jirina Machackova & Gwenaël Imfeld &

Maria Brennerova & Dietmar H. Pieper &

Hermann J. Heipieper & Howard Junca

Received: 6 October 2008 /Revised: 9 January 2009 /Accepted: 10 January 2009# Springer-Verlag 2009

Abstract In order to obtain insights in complexity shiftstaking place in natural microbial communities under strongselective pressure, soils from a former air force base in theCzech Republic, highly contaminated with jet fuel and atdifferent stages of a bioremediation air sparging treatment,were analyzed. By tracking phospholipid fatty acids and16S rRNA genes, a detailed monitoring of the changes in

quantities and composition of the microbial communitiesdeveloped at different stages of the bioventing treatmentprogress was performed. Depending on the length of the airsparging treatment that led to a significant reduction in thecontamination level, we observed a clear shift in the soilmicrobial community being dominated by Pseudomonadsunder the harsh conditions of high aromatic contaminationto a status of low aromatic concentrations, increasedbiomass content, and a complex composition with diversebacterial taxonomical branches.

Keywords BTEX . Air sparging . Bioremediation .

Biodiversity .Microbiota

Introduction

The spillage of organic compounds represents one of thebiggest problems of contamination in soils and ground-water, especially in eastern European countries. Militaryareas particularly represent a major problem due to theirhigh pollutant concentration. Therefore, massive attemptsare being carried out to remediate such sites, commonlyhighly polluted with alkanes and benzene, toluene, ethyl-benzene, and xylene (BTEX) compounds. One of the in situbioremediation technologies directed toward volatile hydro-carbons, mainly BTEX and gasoline relying on the aerobicstimulation of the catabolic capabilities of the autochtho-nous bacteria, is air sparging (Marley et al. 1992; Reddy etal. 1995; Bass et al. 2000; Hall et al. 2000; Heron et al.2002; Yang et al. 2005). However, despite the wideapplication of this technique to enhance the bioremediationof nonchlorinated aromatic contamination in situ, there isstill a scarcity of knowledge on the biocatalysts being

Appl Microbiol BiotechnolDOI 10.1007/s00253-009-1868-0

Electronic supplementary material The online version of this article(doi:10.1007/s00253-009-1868-0) contains supplementary material,which is available to authorized users.

N. Kabelitz :H. J. Heipieper (*)Department of Bioremediation,Helmholtz Centre for Environmental Research (UFZ),Permoserstr. 15,04318 Leipzig, Germanye-mail: hermann.heipieper@ufz.de

J. MachackovaEarth Tech CZ s.r.o.,Trojská 92,171 00 Prague 7, Czech Republic

G. ImfeldDepartment of Isotope Biogeochemistry,Helmholtz Centre for Environmental Research (UFZ),Permoserstr. 15,04318 Leipzig, Germany

M. BrennerovaInstitute of Microbiology (IMIC), Czech Academy of Sciences,Videnska 1083,142 20 Prague 4-Krc, Czech Republic

D. H. Pieper :H. JuncaBiodegradation Research Group,Helmholtz Centre for Infection Research (HZI),Inhoffenstrasse 7,38124 Braunschweig, Germany

stimulated and the overall microbiological characteristics ofthe process. In contrast, bioremediation studies often usedto be restricted to follow the disappearance of hazardouspollutants (Frostegard et al. 1993a; Frostegard et al. 1996)and still regard this system as a black box. As it is knownthat traditional culture-dependent methods are highly biasedwhen analyzing environmental samples (Amann et al.1995), culture-independent methods have been appliedsince two decades in order to characterize microbialcommunity structures and their shifts under changingenvironmental conditions. Lipid biomarker-based techni-ques (Guckert et al. 1991; White 1993; Frostegard et al.1996; White et al. 1996; Zelles 1997; MacNaughton et al.1999) provide culture-independent insights into severalimportant characteristics of microbial communities such asviable biomass, community structure, nutritional status, orphysiological stress responses of the bacteria (Guckert et al.1991; Heipieper et al. 1996; Pennanen et al. 1996;MacNaughton et al. 1999). However, the insight gainedfrom lipid biomarker analysis primarily concerns nutritionalor physiological status with little differentiation amongbacterial species. Complementary genetic methods targetingand discerning the sequence complexity of 16S rRNAgenes as a bacterial taxonomical biomarker allow themonitoring of taxonomical shifts in microbial communitystructure at greater details (Janssen 2006).

The present study shows the monitoring of a former airforce base in the Czech Republic highly contaminated withjet fuel that is currently under bioremediation by the airsparging technique (Bass et al. 2000; Hall et al. 2000). Thesite is a part of the Bohemian Cretaceous Basin, the mostimportant resource of high-quality groundwater in theCzech Republic (Masak et al. 2003; Machackova et al.2005). The endangered aquifer is the only source ofdrinking water in the region and the presence of extensivecontamination limits future use and revitalization of the

site. Several principal source zones of petroleum pollutionwere identified at the site which has a size of 28.3 ha—three storage areas and the jet-fuelling depot. The pollutantsmigrated significant down-gradient distances due to morethan 20 years of massive fuel leakages in source areas andhigh permeability of sandstones. The amount of total petrolhydrocarbon (TPH) released into soil and groundwater until1997 is estimated as 7,150 t. At the start of the treatment,light nonaqueous-phase liquid (LNAPL) phase was fre-quently present in the wells with a thickness >0.5 m. Thepollution consisted mainly of jet fuel (70%) with admixtureof gasoline and diesel.

Figure 1 shows a scheme of the Hradčany site and theclean-up procedure carried out since 1997, when in situtechnologies have been gradually applied. LNAPL soilvapour extraction (SVE) and air sparging (AS) withapplication of nutrient solutions (N, P, and K) have beenapplied to the site (Masak et al. 2003). The first clean-upphase focused on maximum removal of LNAPL by vacuumextraction, whereas the second phase aimed at creatingfavorable conditions enabling aerobic degradation in theentire contaminated profile by AS and SVE. In the timeframe of 1997–2006, 3,667 t of TPH were removed fromthe site and it was estimated that biodegradation accountsfor 93%, vacuum extraction of LNAPL for 5%, and SVE/AS for 2% of the TPH amounts eliminated (Machackova etal. 2005). In this study, the development of microbialcommunities in samples taken from three locations of thatsite representing different stages of the treatment progresswas studied using microbial community analyses byphospholipid fatty acid (PLFA) profiling and 16S rRNAgene library analysis.

The results presented in this polyphasic approach showlinks between the depletion of contaminants (in this case, astrong selector) in natural setups due to oxygen amendmentand an increase of the abundance and complexity of the

Fig. 1 Schematic representationof the clean-up procedurecarried out since 1997 atHradčany site (AS air sparging,VE venting, GWT groundwater table)

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autochthonous microbial soil community. Most probably, theobserved changes in the microbial community are relatedand associated with the successful remediation of the soil.

Materials and methods

Sampling

All samples were taken from a site of high kerosenecontamination, located in the Czech Republic, referred hereas the Hradčany site. Since the Second World War until1990, the site was used as a military airport, and themilitary activities resulted in an extensive contamination ofthe soil and groundwater by petroleum products (mainly byjet fuel). The upper layers of the site (0.5–3 m) are formedby quaternary river sediments (sands, gravels); the aquiferis composed of middle- to fine-grained Middle Turoniansandstone with a thickness of 67 to 75 m. The base of theaquifer consists of Lower Turonian siltstones and marliteswith a thickness of about 75 m. The groundwater tabledepth varies from 3 to 8 m below the surface. In 1997, afull-scale clean-up was initiated (Masak et al. 2003;Machackova et al. 2005). Soil samples were taken usingspiral auger drilling technique. The actual level of thegroundwater table (GWT) was measured prior to drilling inthe adjacent monitoring point for preliminary setting ofsampling depth. The three sampling sites are located withinthe Hradcãny area (approximately 30 ha) with a reasonabledistance of several hundred meters between each other(HRB-3: highest contamination, beginning of clean-up;HRB-2: 2.5 years of treatment; HRB-1: 5 years oftreatment). Samples were taken in the depth of 0.5 mabove–1 m under the actual GWT level from the 0.2-mlayer of maximum contamination. All samples representvery similar soils, both from the geological (soil scientific)and hydrogeological aspects. From each of the three sites,approximately 2 kg of soil were taken. The soil of each sitewas then homogenized in a sterile bucket and then packedinto glass jars and stored at 4°C under aerobic conditions.Sampling for petroleum hydrocarbon quantification wasperformed prior to soil mixing as petroleum contaminationquickly volatilizes during homogenization. Two splitsamples for contamination content analyses were takenfrom the sampled interval. Content of TPH was measuredby standard gas chromatography and infrared detection(ISO TR 11046 and ISO TR11046[2]); BTEX was analyzedby standard gas chromatographic methods (EPA 601).

Fatty acid extraction and separation

The extraction of total fatty acids was performed with thesoil samples (five split samples of each site) that were

previously lyophilized for 24 h and was carried out usingaccelerated solvent extraction in an ASE 200 apparatus(Dionex), allowing an efficient extraction of lipids fromsoils under high temperatures and pressures. Methanol,chloroform, and buffer were applied in ratios described byBligh and Dyer (1959).

For the extraction, from each of the samples, 30 g of soilwere lyophilized and filled in an extraction cell (volume=22 mL) together with the mix of solvents, heated for 5 min,and pressurized to 120 bar. The temperature and pressurewere kept constant for 10 min (static extraction, twocycles). The total amount of solvent used for each cellwas about 25 mL. The extracts were collected andseparated by addition of appropriate volumes of distilledwater and chloroform. The chloroform phase, whichcontained the total fatty acids, was isolated and dried overanhydrous sodium sulfate. The PLFA fraction was separat-ed by liquid chromatography using silica gel columns(Bakerbond spe, Baker). By subsequent elution withchloroform, acetone, and methanol, neutral glycolipidsand phospholipids were collected separately according toZelles (1997). The methanol fraction containing the PLFAwas transesterified to the respective fatty acid methyl esters(FAMEs) with trimethylchlorosilane in methanol (1:9, v/v)at 60°C for 2 h. The solvent was evaporated under a gentlestream of nitrogen, and residues were resuspended inhexane.

Analysis of fatty acid composition by GC-MS and GC-FID

Analysis of FAMEs in hexane was performed using aquadruple GC system (HP8690, Hewlett & Packard, PaloAlto, USA) equipped with a split/splitless injector. A BPX-5 capillary column (SGE, Darmstadt Germany; length,30 m; inner diameter, 0.32 mm; 0.25 μm film) was usedfor separation where the injector temperature was held at240°C. The injection was splitless and He was used ascarrier gas at a flow of 2 mL/min. The temperature programwas: 40°C, 2 min isothermal; 4°C/min to 230°C; 5 minisothermal at 230°C. The pressure was held constant at7,57 psi. Additionally, a GC system with flame ionizationdetector was used (Agilent 6890N) with a special FAMEcolumn (CP-Sil88 Varian Chromopack; length, 50 m; innerdiameter, 0.25 mm; 0.2 μL film) to reach better separation.The pressure program was as follows: start, 27,64 psi for2 min; increase, 0.82 psi/min up to 45.7 psi; isobaric for5.5 min. The temperature program started at 40°C (2 min),increased 8°C/min up to 220°C, and was held there for5 min. Injector temperature was 240°C, detector temperature270°C.

The peak areas of the carboxylic acids in total ionchromatograms (TIC) were used to determine their relativeamounts. The fatty acids were identified by their mass

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spectra and retention time compared to coinjections ofauthentic reference compounds obtained from Supelco(Bellefonte, USA).

Statistical analysis

Principal component analyses (PCA) were applied on thebasis of numerical data matrices converted using theprogram R (R: Copyright 2005, The R Foundation forStatistical Computing Version 2.1.1). The relative amountsof PLFA data were subjected to PCA to investigate theinterrelationships between the soil samples and to deter-mine the predominant PLFA species in the samples. In thefirst attempt, the investigated soil samples corresponded tothe object represented in the multidimensional space andthe PLFA values to the descriptors of the multivariateanalysis. In a second PCA, reciprocal analysis was carriedout with the soil samples corresponding to the descriptorsof the analysis.

DNA extraction

For DNA extraction, fractions of the soil samples werefrozen with solid carbon dioxide at the time of samplingand maintained in this condition during transportation.Later on, samples were stored at −70°C until furtherprocessing. DNA was extracted with the FastDNA Spinkit for soil (QBiogene, Carlsbad, CA, USA) from 800 mgof soil per reaction tube, according to the instructions of themanufacturer with the only exception that the final elutionof DNA from the filter was with 75 μL of Tris–HCl buffer3.33 mM pH 8.0. Five DNA extractions, equivalent to 4 gof soil, were performed for each soil sample, and extractedDNA were pooled together in a single reaction tube. TheDNA was dried and the final volume adjusted to 40 μLwith MilliQ water. DNA concentrations were quantifiedusing the Quant-iT PicoGreen dsDNA quantitation kit(Invitrogen—Molecular Probes Europe BV, Leiden, TheNetherlands).

PCR amplification, cloning, sequencing, and analyses

The pooled DNA extracts were used as template inpolymerase chain reaction (PCR) amplifications withprimers targeting two highly conserved regions identifiedon bacterial 16S rRNA genes (Marchesi et al. 1998) [63F:5′-CAG GCC TAA CAC ATG CAA GTC-3′ and 1387R:5′-GGG CGG WGT GTA CAA GGC-3′]. The finalamounts or concentrations of the reagents for PCR in avolume of 50 μL were: 1X colorless GoTaq reaction buffer(Promega, Madison, WI, USA), 5 U of GoTaq polymerase(Promega, Madison, WI, USA), 200 μM of dNTPs (MBIFermentas, Germany), and 10 pmol of each primer

(synthesized by Invitrogen, Karlsruhe, Germany). Forthermal cycling, a Hybaid PCR Express Thermocycler(Thermo Electron, Waltham, MA, USA) was used asfollows: initial denaturation at 94°C for 4 min, 35 cyclesof 95°C for 45 s, 55°C for 45 s, and 72°C for 1.5 min.These cycles were followed by one elongation step at 72°Cfor 7 min. PCR products were purified by using theQIAquick PCR purification kit (Qiagen, Hilden, Germany)and cloned in pGEM-T easy vector system (Promega,Madison, WI, USA). Plasmid inserts were amplified byPCR with vector-specific M13 forward and reverse primers(Sambrook et al. 1989) on transformant colonies dissolvedin water and previously incubated at 95°C for 10 min.Amplified ribosomal DNA restriction analysis (ARDRA)was performed as previously described (Junca and Pieper2004). The purified PCR products were used as DNAtemplates in independent sequencing reactions of bothstrands using the BigDye terminator v1.1 cycle sequencingkit (Applied Biosystems, Foster City, CA, USA) using M13primers and primers annealing at four different conservedregions in two directions inside the 16S rRNA genesequences as described previously (Lane 1991). Sequencingreactions were analyzed in an Applied Biosystems 3130xlGenetic Analyzer (Applied Biosystems, Foster City, CA,USA) and sequence contigs were assembled usingSequencher version 4.0.5 (Genes Codes, Ann Arbor, MI,USA). The sequences were cleaned of vector sequencesusing VecScreen Blast program (NCBI, USA) and orientedin 5′–3′ of the 16S rRNA genes using OrientationChecker(Bioinformatics Toolkit, Cardiff School of Biosciences,UK). Sequences were analyzed for potential chimericsequences with the service available at the RibosomalDatabase Project II (Cole et al. 2003). Additional potentialchimeras were assessed with the program MALLARD(Ashelford et al. 2006). The final datasets were alignedwith the multiple sequence alignment method MUSCLE(Edgar 2004). A block of sequence alignments was selectedwith GeneDoc multiple sequence alignment editor software(Nicolas 1997). A collection of the nearest neighbors to thesequences obtained against the 16S rRNA gene sequencesreported and classified in the Ribosomal Database Project IIwere found using Seqmatch (Cole et al. 2003). Neighbor-joining trees were calculated from the composite align-ments together with calculated bootstrapped values of 1,000trials using the functions implemented inside Clustal W(Thompson et al. 1994). Tree files were graphicallydisplayed with MEGA 3.1 software (Kumar et al. 2004).For calculation of rarefaction curves and Shannon diversityindexes, the program DOTUR was used (Schloss andHandelsman 2005) using the distance matrices computedwith Dnadist program (Felsenstein 1989) from the nucleo-tide sequence alignments of the sequence libraries obtainedin this study.

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Microbiological culture techniques

Fractions of the soil samples were kept at 4°C aftercollection. Colony forming units were determined in R2Aagar (Difco, Livonia, MI, USA) in triplicates after platingof the appropriate dilution that were carried out onphosphate buffer (50 mM, pH 7.0).

Results

Biomass development in the Hradčany soil

Soil samples from the Hradčany site representative fordifferent steps of the air sparging bioremediation processwere investigated for pollutant content. In general, the threesites can be characterized as follows: HRB-3, soil from anuntreated site contains high organic contamination (concen-trations of TPH=6,400 mg/kg and BTEX=4,400 mg/kg dryweight); HRB-2, soil from a 3-year clean-up site exhibits amoderate organic contamination (TPH=3,900 mg/kg andBTEX=190 mg/kg dry weight); and HRB-1, soil from a5.5-year clean-up site contains low organic contamination(TPH=1,500 mg/kg and BTEX=9 mg/kg dry weight). Fromeach site, five samples were investigated which were taken inthe same drilling campaign. As the geological and hydro-geological specificities of all three sites were similar, thedifferences in microbiota are most likely due to thedifference in pollution level and cannot be explained bygeological or other aspects.

In order to compare the abundance and complexity ofmicrobial biomass of the samples subject to different timesof air sparging treatment, the overall abundance of PLFAwas analyzed (Fig. 2a). On the other hand, in the nontreatedsoil, which contains very high toxic concentrations ofBTEX compounds, PLFAs were only present in very lowamounts; this content increased by more than two orders ofmagnitude in the air sparging treated soils. As PLFA areonly present in living (micro)organisms (MacNaughton etal. 1999; Kindler et al. 2006), this is a clear indication thatthis bioremediation treatment leads to a significant increasein overall microbial biomass. The increase in biomass wasalso reflected in strong differences between the soil samplesregarding quantities of heterotrophic bacteria as quantifiedby the number of colony forming units per gram of soil(CFU/g) (Fig. 2a) with HRB-3 exhibiting CFUs/g twoorders of magnitude lower than HRB-1. Analysis of DNAconcentrations by fluorescence quantification (see the“Materials and methods” section) revealed a concentrationof 40 ng dsDNA per gram of HRB-1 soil, whereas DNAfrom HRB-2 was observable after gel electrophoresis butbelow the concentration of 0.5 ng/μL dsDNA which couldbe accurately quantified. Amounts of DNA extractable

from HRB-3 were even lower and only detectable afterPCR amplification. Thus, compatible results were obtainedwhen comparing, as biomarkers, total DNA extract con-centrations, CFUs, or PLFA concentration, which all pointto an increase in living microbial biomass.

Phospholipid fatty acid composition of Hradčany soils

The PLFA composition of Hradcãny samples (Fig. 2b) showedsignificant differences depending on the time of air spargingtreatment. In the untreated samples, saturated fatty acids(16:0, 18:0) are predominant next to 18:1Δ9cis fatty acid.The major difference in the PLFA profiles between the threeinvestigated soil sampling sites was the significantly higherrelative abundance of 16:1Δ9cis and 18:1Δ11cis monoun-saturated fatty acids as well as cyclo19:0 cyclopropane fattyacid in the treated compared to the untreated samples.

A PCA of the PLFA profiles underlined the results givenabove. The first PCA (Fig. 3a) allowed to clearlydistinguish PLFA patterns associated with the soil fromthree sites differing in the level of BTEX and kerosenecontamination and treatment duration. The data of all thethree different sampling sites formed distinct clusters. ThisPCA showed a clear separation of the three conditions onthe biplot of the first two principal components, emphasiz-ing changes in the PLFA composition of the soils accordingto the length of treatment. A separation of the soil samplesfrom the lowest level of contamination to the highest one isoperating along the first principal component.

The second PCA stresses the dominant PLFAs that areassociated with the difference in the analyzed soil samples(Fig. 3b). The amount of variation explained by the firstand second principal components reached 86.3% of thetotal variation. This PCA relates the abundance of specificPLFAs (16:0; 18:0; 18:1cisΔ9) with the level of contam-ination (prior clean-up, HRB-3). On the other hand, otherPLFAs (18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase inresponse to the treatment time and were particularlyassociated with the 3 years treated (HRB-2) samples. Theother PLFAs were found in a close vicinity of the origin ofthe PCA plot, indicating that the relative amounts of thesePLFAs were not substantially affected by the level ofcontamination and the length of treatment.

Molecular biological analysis of the microbiotacomposition of Hradčany soil

To correlate shifts in lipid composition occurring at theinvestigated site with changes in bacterial taxonomicalcomplexity, the microbial community structures of the threesampling points were assessed by 16S rRNA gene libraries.PCR clone libraries of 16S rRNA gene were generated fromtotal pooled DNA extracts of the soils and initial screenings

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performed by ARDRA. For HRB-3, ARDRA screeningwith AluI on 96 clones showed identical patterns in 82 ofthe clones, suggesting the predominance of a singletaxonomic group in the library. A similar ARDRAscreening on HRB-1 and HRB-2 clone libraries did notgive evidence for any predominant pattern. Further screen-ing by random sequencing was performed on 79 clonesfrom HRB-1, 80 clones from HRB-2, and 28 clones fromHRB-3. The relationships of 187 assembled sequences,comprising a common 1-kb length block covering variableregions V2 to V6 of the 16S rRNA genes (Neefs et al.1993), corresponding to positions 103 to 1130 of Escher-ichia coli 16S rRNA gene (GenBank accession numberJ01695), are shown in Fig. 4a (expanded view and detailedlabeling of these results are available as Electronic

supplementary material). A global alignment of thesequences obtained together with the most closely related16S rRNA gene sequences from type strains and selectedsequences retrieved from public databases indicated thepresence of sequences related to diverse evolutionarybranches (Janssen 2006). The clones in the clone librarieswere assigned to operational taxonomic units (OTUs) using>99% (OTU0.01), >95% (OTU0.05), and >90% (OTU0.1)sequence identity as criteria, as sequences with greater thanthose identities are typically excluding differences based onoperon heterogeneity or are typically assigned to the samegenus or order, respectively (Acinas et al. 2004b; Schlossand Handelsman 2004).

Rarefaction analysis on each sequence library showedthat the higher number of clones sequenced from the HRB-

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Fig. 2 Effect of air spargingtreatment on PLFA abundanceand composition in Hradčanysoils. a Biomass development,given as the overall abundanceof PLFA (filled diamonds, rep-resented by area counts) andcolony forming units (opensquares) in the Hradčany sitecaused by the air sparging treat-ment. b PLFA patterns of soilsfrom the Hradčany site. Notreatment (HRB-3),total BTEX concentration=17,000 mg/kg; 3 years treatment(HRB-2), total BTEX concen-tration=960 mg/kg; 5.5 yearstreatment (HRB-1), total BTEXconcentration=70 mg/kg. Fromeach sampling point, fiveindependent soil samples wereextracted and analyzed for theirPLFA content. Standard devia-tion of these five independentmeasurements are given aserror bars

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Fig. 3 PCA analyses of PLFApatterns obtained fromHradčany soils. a Ordinationplot representing the relationshipbetween the contamination sitesand the PLFA patterns. Thecross indicates the origin ofcoordinates and values on theaxes indicate the percentage ofthe total explained variation. bPCA showing loading values forindividual PLFA. PLFAs foundon the right in the plot hadincreased in the no treatment(HRB-3) soils, whereas the onesfound in the lower part of theplot had increased the 5.5-yeartreatment (HRB-1) and 3-yeartreatment (HRB-2) soils

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1 and HRB-2 libraries were indeed necessary to obtaincoverage comparable to the one for the HRB-3 library (seeFig. 4).

Almost all the sequences obtained from HRB-3 weretightly clustering (>95% overall sequence similarity) insidethe genus Pseudomonas with the majority of these sequencesclosely related, but not identical (identities >1,022/1,030,99%), to those found in Pseudomonas cedrina or Pseudo-monas azotoformans type strains inside the Pseudomonasfluorescens group (Anzai et al. 2000). Such clusters ofsequence microdiversity in ribosomal genes are commonlyobserved in amplifications of environmental samples (Acinaset al. 2004a); however, its interpretation and significance isstill under discussion. Nevertheless, it is very likely that, dueto a strong selection caused by the hazardous environmentalconditions, only members of a bacterial genus tolerant tohigh solvent concentrations and possibly with the potential toaerobically degrade such compounds were observed. HRB-2exhibited a wider sequence diversification compared to thecontaminated nontreated state (HRB-3). A distinct Pseudo-monas intragenus microdiversity was evidenced in thissampling area with sequences highly similar to above-mentioned P. cedrina/P. azotoformans cluster still beingpredominant (22% of clones) but 5% of the clone sequencesbeing closely related (identities >1,016/1,030, 98%) to therecently described Pseudomonas rhizosphaerae type strain(Peix et al. 2003).

Discussion

The air sparging treatment of the Hradcãny site caused asignificant increase in the amount of biomass and, at leastpartially as a consequence, a decrease in organic contam-ination of the soil. The increase in living microbial biomasscould be shown by us; compatible results were obtainedwhen comparing, as biomarkers, total DNA extract con-centrations, CFUs, or PLFA concentration, which all pointto an increase in living microbial biomass.

The major difference in the PLFA profiles between thethree investigated soil sampling sites was the significantlyhigher relative abundance of 16:1Δ9cis and specifically18:1Δ11cis monounsaturated fatty acids in the treatedcompared to the untreated samples. As cis-vaccenic acid(18:1Δ11cis) is synthesized via the so-called anaerobicpathway of fatty acid synthesis that is exclusively present inseveral Gram-negative bacteria (Keweloh and Heipieper1996), this indicates the high abundance of Gram-negativebacteria in the treated samples. The predominance of Gram-negative bacteria in the treated samples is further supportedby the high abundance of Gram-negative-specific cyclo-propane fatty acids (cy17:0 and cy19:0) and the lowabundance of Gram-positive-specific iso- and anteiso-branched fatty acids (i15:0, a15:0, i16:0, and i17:0).However, inspection of discriminatory fatty acids showsthe presence of Gram-negative-specific cyclopropane fattyacids accounting for approximately 5% up to 40% of thetotal fatty acids, whereas abundance of Gram-positive-specific acids was negligible. This indicates also theuntreated site to be dominated by Gram-negative organ-isms, and treatment to exert a significant effect on thecomposition of the Gram-negative microbial communityfraction. In fact, taking the combined abundance of theGram-positive-specific fatty acids and the Gram-negative-specific fatty acids as a measure of the ratio between Gram-positive and Gram-negative bacteria (Margesin et al. 2007),the relative abundance of Gram-positive organisms washighest in the 5.5 years treated soil. The absence ofpolyunsaturated fatty acids shows that eukaryotes arepractically absent in these soils, which indicates that anormal soil microflora has not been completely establishedby the so far carried out bioremediation process.

The PCA carried out with data that stresses the dominantPLFAs associated with the difference in the analyzed soilsamples (Fig. 3b) clearly approves the tendencies visiblefrom the fatty acid profiles. The relation of specific PLFAs(16:0; 18:0; 18:1cisΔ9) with the highest level of contam-ination (prior clean-up, HRB-3) suggests that a highlyspecific microbiota is associated with these hazardousenvironmental conditions. On the other hand, other PLFAs(18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase in response tothe treatment time and were particularly associated with the

Fig. 4 Taxonomical distribution of the 16S rRNA gene sequencesretrieved from the contaminated soils DNA under different bioreme-diation treatments. a Neighbor-joining tree based on 16S rRNA genesequences obtained from HRB soil DNA amplifications. Circlesindicate sequences obtained by random screening of PCR clonelibraries of amplifications from DNA extracts of HRB-1 (blue), HRB-2 (green), and HRB-3 (orange) soils. Light purple trapezoids indicatesequences of closely related bacterial type strains or cultured strains.In cases where sequences with a similarity higher than 60% to anobserved HRB-derived sequence were not available from bacterialtype strains, sequences from uncultured bacteria were included fororientation (violet triangles). Rarefaction curves for different distancelevels (OTU0.01, OTU0.05, and OTU0.10) for each of the analyzedlibraries were calculated by DOTUR (Schloss and Handelsman 2005)and are given below the dendrogram. Coverages (C) at 95% distanceswere calculated according to Turing’s formula (Good 1953) where C=100 represents complete coverage. b Relative clone frequencies inmajor phylogenetic groups (Order–Class) of the clone librariesanalyzed. HRB-derived 16S rRNA gene sequences were assigned tobacterial classes using the RDP-naïve Bayesian classifier according tothe taxonomical hierarchy of Garrity and Lilburn (release 6.0) with thedefault confidence threshold of 80%. The colors used in the stackcolumn diagram correspond to bacterial Orders as defined to the rightof the columns. Orders were grouped as Classes as shown to the veryright of the figure, except for Bacillales and Clostridiales that, forsimplicity, were grouped in the higher rank (phylum) of Firmicutes.Sequences that could not be classified and that were retrieved only invery low amounts were collectively indicated as “Unclassifiedribosomal genes.” For additional details of the sequences obtainedand control sequences used in this figure, see the accompanyingElectronic supplementary material

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3 years treatment (HRB-2) indicates a specific Gram-negative bacterial community accumulating during the airsparging treatment.

However, next to community shifts, changes in themembrane fatty acid patterns of bacteria can also occur asadaptive response to pollutant toxicity and environmentalstress conditions (Frostegard et al. 1993b; Heipieper and deBont 1994; Heipieper et al. 1996). Therefore, it is necessaryto support the insights based on PLFA profiling also by othermethods such as, e.g., molecular biological techniques.Surprisingly, the expected trans–cis ratio of unsaturated fattyacids, a very useful parameter for stress monitoring inbacterial cultures (Guckert et al. 1986; Guckert et al. 1991;Heipieper et al. 1992; Heipieper et al. 1996), did not showsignificant changes in the samples analyzed (data notshown), probably due to its transient identity.

Although the PLFA analysis already demonstrated a shiftin the microbial community as well as an increase in livingbiomass, a detailed molecular biological analysis of themicrobiota was necessary. Here, a clear increase in themicrobial biodiversity of the site caused by the air spargingtreatment was visible. Whereas almost all the sequencesobtained from HRB-3 were clustering inside the genusPseudomonas, a tremendous increase in the identifiedbacterial diversity occurred in the samples taken from 3and 5.5 years of treatment.

While Pseudomonas is a genus defined as ubiquitousand of high environmental importance, these conclusionsare predominantly coming from observations using tradi-tional culture-dependent techniques (Moore et al. 2006)which are generally accepted to include a severe biastoward easy to culture microorganisms (Amann et al.1995). However, our study and some other recent reports(Duineveld et al. 2001; Kaplan and Kitts 2004; Gerdes et al.2005; Popp et al. 2006; Ferguson et al. 2007) are showingthat Pseudomonas may be defined as a predominantmember in communities of aerobic or microaerophilicecosystems where high concentrations of crude oil areacting as a strong selector.

However, whereas HRB-3 sequences affiliated withPseudomonas spp. comprise roughly 80% of all clones, only25% of HRB-2 clones were affiliated with that genus. Otherpredominant sequence types in HRB-2 were affiliated withthe classes of Actinobacteria, Acidobacteria, and Alphapro-teobacteria (predominantly members of the orders Rhizo-biales and Rhodospirillales). The higher diversity observedin HRB-2 (Fig. 4) was also reflected by a higher Shannondiversity index (H′, calculated for OTU0.05) of 2.67±0.25(95% confidence interval), compared to only 0.73±0.44 forHRB-3. An even slightly higher value compared to HRB-2was observed for HRB-1 (2.86±0.21), indicating diversityand balance of community composition to increase withbioremediation treatment time.

However, whereas there is only a small increase in theShannon diversity index from HRB-2 to HRB-1, both sitescomprise very different microbial community compositions.Most importantly, Pseudomonas spp. was barely detectable(one out of 79 sequences) in HRB-1. In contrast, sequencesaffiliated with Sphingomonadales, members of which hadbeen observed in various aromatic contaminated sites andwhich had been related to primary stages on polycyclicaromatic biodegradation (Leys et al. 2004, 2005), wereabundant only in HRB-1, comprising roughly 10% of therespective clone library, contrasting a single sequence in theHRB-2 clone library.

In addition, Betaproteobacteria-affiliated sequences, agroup only marginally detected (one sequence only) inHRB-2, are a significant fraction of the HRB-1 library,accounting for 20% of the total amount of sequences.Additional sequences exclusively observed in HRB-1constitute a new branch inside the family Xanthomonada-ceae (Gammaproteobacteria) with equal divergences (ap-proximately 15% of difference) against sequences fromstrains of the genera Frateuria and Rhodanobacter.Bacterial assemblages similar to that of HRB-1 andconsisting of Pseudomonas, Sphingomonas, Xanthomonas,Acidovorax, and Burkholderia sequences have been previ-ously observed, for instance, at anthropogenic hydrocarbon-contaminated coastal soils in Antarctica (Saul et al. 2005),while bacterial communities predominantly composed ofPseudomonas, Sphingomonas, and Acidobacteria had beenreported for instance in soil–groundwater ecosystems withpetroleum contamination (Popp et al. 2006).

As shown in Fig. 4b, a comparison of communitycomposition at the level of bacterial classes, which is usedin many reports tracking shifts in microbial communities byFISH probes, T-RF sizes, or OTU definition (Pett-Ridgeand Firestone 2005; Yu et al. 2005; Watt et al. 2006; Allenet al. 2007; McGarvey et al. 2007) among others, woulddirect to misleading conclusions as it would suggest thepredominance and resilience of Gammaproteobacteria inthe sites independent of the treatment. However, whencomparisons are performed at the taxonomical scale Order,this shows to be an oversimplified assumption as strongshifts in composition inside the Order were observed. As anexample, among the Gammaproteobacterial sequences,only those affiliated with Pseudomonadales were predom-inant in the HRB-2 site but practically absent from HRB-1where Xanthomonadales and Unclassified Gammaproteo-bacteria are accounting for a relatively high amount of thetotal bacterial composition detected (>35%). This exampleshows how comparisons of taxonomical composition inbacterial communities should be at least at Order ranks.Lower resolution comparisons of Classes or even Phyla canbe misleading and fail to detect significant communitychanges.

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Integrating the information of overall microbial abun-dance (Fig. 2a) with the sequences obtained (Fig. 4), it isevident that even though the relative abundance ofPseudomonadales decreased in HRB-2 compared to HRB-3, the total number of Pseudomonadales cells per gramof soil in HRB-2 is very likely by at least one order ofmagnitude higher. Thus, the initial predominance ofPseudomonadales in HRB-3 may indicate a physiologicaladvantage. These cells not only survive under these harshconditions of low oxygen, high loads of aromatic carbonpollutants, and high solvent concentrations, but obviouslyhave been replicating under these conditions. This is inaccordance with culture-dependent studies on Pseudomo-nadales (Heipieper and de Bont 1994; Sikkema et al. 1995;Heipieper et al. 1996), which have shown members of thisgroup to be capable to replicate under harsh laboratoryconditions and high solvent stress. It can thus be proposedthat, at least at the site under study, Pseudomonads pavedthe road for other bacteria to be capable to replicate asshown by the increased diversity observed in HRB-2. Thus,for a certain time, Pseudomonas is sharing its habitat in abacterial community of increasing complexity, as lessrestricting conditions for other phylotypes are beinggenerated during the clean-up process, leading to thedecrease in community predominance of Pseudomonasand the increase of other, previously not detectablephylotypes. Particularly interesting is the increase in Acid-obacterial sequence types, which are supposed to beselected in low-nutrient soil or in soil with a high amountof recalcitrant substrates (Torsvik and Ovreas 2002). As,moreover, soils with a high content of nutrients showedpositive selection for Alphaproteobacteria and specificallyGammaproteobacteria (Amann et al. 1995), the ratiobetween the number of Proteobacteria and Acidobacteriumwas suggested to be indicative of the nutritional status ofsoils (Smit et al. 2001) with supposedly high ratios beingindicative for high input soil systems. In the soil systemsstudied here, Acidobacteria were absent from untreatedsoil, similar to the situation observed in other environmentshighly contaminated with petroleum hydrocarbons (Popp etal. 2006), and ratios reached values of 5.4 in HRB-2 and13.8 in HRB-1, indicating a significant recovery of thesystem. However, as ratios of up to 10 have been reportedin supposedly pristine soils (Kasai et al. 2005; Schloss andHandelsman 2006), obviously even such high values do notindicate the nutritional status.

The air sparging biodegradation technique applied in theHradcãny site led to a drastic decrease in pollutantconcentrations through the accelerated aerobic microbialactivity. Next to an increase in the amount of potentialdegraders, this reduction in the concentration of toxicorganic solvents also allows the reproduction of bacterialtypes sensitive to higher solvent concentrations that are not

necessarily able to degrade aromatics, but capable ofsurviving and growing in the cross-feeding mesh ofmetabolites excreted from the initial biomass of degraders.In accordance with decreased concentrations of aromatics(and thus lower solvent stress), a higher variety of bacterialtaxonomical types and higher biomass content was ob-served. This biodiversity restoration, which can be seen asan ecological succession, probably would not lead to thesame microbial composition of the soil as it was before thearomatic contamination occurred (Curtis et al. 2002).

Here, it is shown that the bacterial community underadaptation in these soils, concomitantly with the observeddegradation of the contaminants in situ, showed a dynamicsuccession of Gram-negative bacteria with the communitybeing initially restricted to Pseudomonadales at very lowdensities, developing an increased diversity comprising newproteobacterial types and Gram-positive bacteria. Compatibletrends were observed using ordination methods, whichshowed the clear separation of the different fatty acid clustersand indicated the predominance of Gram-negative bacteriaable to resist the solvent concentrations at untreatedcontaminated soils and the diversification in samples wherethe treatment is applied. Future studies will focus oncatabolic activities in these sites and the relationship withthe phylogenetic changes observed by means of culture-dependent and culture-independent studies.

Acknowledgments This work was supported by contract no.003998 (GOCE) of the European Commission within its SixthFramework Program project BIOTOOL. We would like to thank theexcellent technical assistance of Silke Kahl.

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