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Environmental Pollution 177 (2013) 98e105

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Experimental increase in availability of a PAH complex organic


contamination from an aged contaminated soil: Consequences on
biodegradation
Aurlie Cbron a, b, *, Pierre Faure a, c, Catherine Lorgeoux a, c, Stphanie Ouvrard a, d,
Corinne Leyval a, b
a

Universit de Lorraine, Facult des sciences, bd des Aiguillettes, 54506 Vandoeuvre-ls-Nancy, France
CNRS, LIMOS UMR7137, BP70239, Facult des sciences, 54506 Vandoeuvre-ls-Nancy, France
CNRS, G2R UMR7566, BP70239, Facult des sciences, 54506 Vandoeuvre-ls-Nancy, France
d
INPL-INRA (ENSAIA), LSE UMR1120, BP 172, 54505 Vandoeuvre-ls-Nancy, France
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 October 2012
Received in revised form
25 January 2013
Accepted 30 January 2013

Although high PAH content and detection of PAH-degraders, the PAH biodegradation is limited in agedcontaminated soils due to low PAH availability (i.e., 1%). Here, we tried to experimentally increase the soil
PAH availability by keeping both soil properties and contamination composition. Organic extract was rst
removed and then re-incorporated in the raw soil as fresh contaminants. Though drastic, this procedure
only allowed a 6-time increase in the PAH availability suggesting that the organic constituents more than
ageing were responsible for low availability. In the re-contaminated soil, the mineralization rate was
twice more important, the proportion of 5e6 cycles PAH was higher indicating a preferential degradation
of lower molecular weight PAH. The extraction treatment induced bacterial and fungal community
structures modications, Pseudomonas and Fusarium solani species were favoured, and the relative
quantity of fungi increased. In re-contaminated soil the percentage of PAH-dioxygenase gene increased,
with 10 times more Gram negative representatives.
2013 Elsevier Ltd. All rights reserved.

Keywords:
PAH
Bioavailability
Availability
Complex organic contamination
Aged-contaminated soil
Microorganisms

1. Introduction
Polycyclic aromatic hydrocarbons (PAH) are common contaminants of industrial wasteland soils where they are associated
with a wide range of organic and metallic pollutants. In agedcontaminated soils the PAH level can be relatively elevated, but
major part of these organic contaminants are sequestrated within
soil constituents (Northcott and Jone, 2001). During ageing process, adsorption to soil organic matter (SOM) (Cornelissen et al.,
2005) or clay mineral (Mller et al., 2007) reduces PAH availability, explaining their recalcitrance to biodegradation. Microbial
biodegradation by bacterial and fungal strains is largely studied
and reviewed, authors use mainly pure cultures and few PAHcompounds as model compounds, either in simplied liquid
medium or in articially contaminated soils (Johnsen et al., 2005;
Haritash and Kaushik, 2009; Lu et al., 2011). In spite these studies
allow to understand the PAH-degradation kinetics and pathways

* Corresponding author.
E-mail address: aurelie.cebron@univ-lorraine.fr (A. Cbron).
0269-7491/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.envpol.2013.01.043

as well as the microbial physiologies and metabolisms, they do


not contribute to understand the fate of PAH in real agedcontaminated soils.
The multicontaminated soil from the Neuves-Maisons wasteland (Meurthe-et-Moselle, France) has been largely studied. In a
long term in situ experiment of natural attenuation, the PAH concentration was shown to evolve slowly during time (Ouvrard et al.,
2011) although a functional bacterial population with biodegradation potential was detected (Cbron et al., 2009). Lack of PAHbiodegradation could be explained either by the occurrence of
toxic compounds inhibiting bacterial activity or by the really low
availability of aged-PAH in this aged soil. Indeed, Tenax-available
PAH represented less than 1% of the total PAH concentration, as
estimated using Tenax resin extraction (Ouvrard et al., 2011). Other
procedures to evaluate availability of PAH, based on the measurement of chemical oxidation efciency (Cuypers et al., 2000) were
carried out on the same soil, using sodium persulfate (Usman et al.,
2012a) and Fenton like oxidations (Usman et al., 2012b) and
conrm the PAH unavailability (no PAH degradation was observed
whatever the oxidant). Moreover, using the same soil, freshly
spiked and easily available phenanthrene, was efciently degraded

A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

by bacteria (Cbron et al., 2011). However, in this experiment 30%


of the freshly added carbon from phenanthrene was also rapidly
(5 days) sequestrated (not extractable with organic solvent) in the
soil matrix (Cbron et al., 2011). The input of a PAH complex organic
contamination in the original organic and mineral matrixes of the
soil, would better mimic the complex interactions and parameters
that inuence the PAH-bioavailability level in real soils. However
such approach has never been tested.
The aim of this study was to check whether the limitation of PAH
biodegradation was due to low PAH bioavailability in the soil. The
originality of our approach is that, PAH availability was articially
increased by extracting the solvent extractable organic matter
(EOM) from the soil and then re-incorporating it in the residual soil.
Such procedure was selected as it was already carried out on the
same soil to evaluate its consequence on the sodium persulfate and
Fenton like oxidation efciencies. This extraction and re-incorporation allows to increase the oxidation efciency (50% of PAH were
degraded) suggesting that this procedure increases the availability
of the complex organic contamination and especially PAH (Usman
et al., 2012a, 2012b). In this present work, re-contaminated soil
and raw soil were then incubated for two months to compare the
PAH biodegradation efciency of the freshly added organic
contamination in re-contaminated soil to the non-extracted soil.
PAH Tenax-availability along with the total organic compound
concentrations (including 15 PAH from the US-EPA list) were
measured at the beginning and at the end of the experiment.
Moreover, microbial activity was monitored through mineralization measurement, the bacterial and fungal community structures
were studied and microorganisms potentially capable of organic
compounds and PAH-degradation were quantied.
2. Material and methods
2.1. Soil sample
Experiments were performed using an aged-PAH and heavy metal (HM)
contaminated soil (NM soil) from a former coking plant industry located in NeuvesMaisons (Meurthe-et-Moselle, France). This soil has been extensively studied and its
properties and physical characteristics have been previously described (Biache et al.,
2008; Cbron et al., 2009; Ouvrard et al., 2011). Briey, NM soil was characterized by,
66% sand, 22% silt and 12% clay particles, a C/N ratio of 25 with 65 g kg1 of organic
carbon, and a pH of 7.4. The 16 US-EPA PAH concentration was ca. 1214 mg kg1, and
heavy metals as zinc, lead or cadmium were found in high concentrations (2250,
490, 2.7 mg kg1, respectively). Soil sample was air dried and sieved at 2 mm.

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2.4. Carbon mineralization


A 3 ml portion of the ask atmosphere was sampled with a syringe and analysed
for CO2 content through infrared spectrophotometer (Binos 1004) (Quantin et al.,
2005). The produced CO2 was expressed as carbon mass per gram of dry soil
(mg C g soil dw1). After mineralization measurements, asks were opened for 1 h
under a fume hood for aeration, humidity was adjusted with sterile deionized water,
and then asks were sealed and incubated again. Measurements were performed
after 1, 2, 4 days of incubation and then twice a week.
2.5. Analyses of PAH and PPAC
The freeze samples were rst lyophilized. The organic extraction of each dry
replicate was carried out on 1 g of soil, with an automated extractor Dionex ASE
350 (Accelerated Solvent Extractor, using dichloromethane, 130  C, 100 bars).
Copper was added to the samples prior to the extraction to eliminate the molecular sulphur and Na2SO4. Then, the organic extracts were diluted with
dichloromethane to 20 ml and an aliquot (5 ml) was sampled and dried under
nitrogen to determine the content of solvent extractable organic matter (EOM)
yield.
The 16 PAH including in the US-EPA list and 7 polar polycyclic aromatic compounds (PPAC) including 5 oxygenated PPAC (dibenzofuran, 9H-uorenone,
anthraquinone, benzanthrone and benzoantracendione) and 2 nitrogenous PPAC
(acridine and carbazole) were quantied in each organic extract. This quantication was carried out using a gas chromatographyemass spectrometry (GCeMS)
with a GC 2010 plus (Shimadzu) instrument equipped with a DB 5-MS column
(60  0.25 mm, J&W) coupled to a QP2010 Ultra (Shimadzu) mass spectrometer
operated in full scan mode. Samples were injected in split mode (split ratio 1:5) at
300  C. The GC oven temperature was programmed from 70  C (held 2 min) to
130  C at 15  C/min, then from 130  C to 315  C (held 2 min) at 4  C/min. Quantitative analyses were performed using added internal standards. Five perdeuteriated PAHs (naphthalene D8, acenaphtene D10, chrysene D12, perylene D12) were
added to organic extract prior the injection in the GCeMS. The GCeMS was previously calibrated with mixtures of each molecule to be quantied (16 PAHs and 7
PPAC).
2.6. Analyses of available PAH
Available PAH were estimated on air dried soil samples. The method used
involved a solid phase extraction using Tenax resin with a protocol adapted from
Cornelissen et al. (1997) to represent the aged pollution behaviour observed in
contaminated soils (Barnier, 2009). Briey, two grams of soil were agitated during
30 h with 2 g of Tenax and 300 ml of a 0.01 M CaCl2 and 200 mg l1 NaN3 solution.
The Tenax beads were then recovered, air dried and extracted twice for 60 min with
50 ml of a 1:1 acetone hexane mix in a sonication bath. The extract was evaporated
under nitrogen ux (TurboVap LV, Caliper LifeSciences) and recovered in 5 ml
acetonitrile. The acetonitrile extract was analysed for the 16 US EPA PAH (except for
acenapthylene) with an HPLC line equipped with photodiode array detector and
uorescence detector (Varian). Separation was performed on a Pursuit 3PAH (Varian) column with a water:acetonitrile gradient.
2.7. DNA extraction

2.2. Preparation of soil organic matter extract


Soxhlet extraction was performed on 200 g of raw soil with chloroform during
48 h. The organic extract recovered was reduced to 200 ml by evaporation steps
(rotavapor Buchi). The extracted soil organic matter (SOM) residues were air dried
under a fume hood to remove residual organic solvent.
2.3. Experimental set up
A batch experiment was set up in 125 ml glass asks. Three conditions performed in 6 replicates were studied: 1) 20 g of raw soil (NM-raw), 2) mix of half raw
soil and half extracted soil (NM-extracted), and 3) mix of half raw soil and half
extracted soil where organic extract was re-incorporated (NM-ORGextract). For the
2 later conditions 10 g of previously chloroform extracted soil were distributed in 12
asks, 10 ml of chloroform were added to the 6 asks dedicated to the NM-extracted
condition and 10 ml of organic extract dissolved in chloroform were added to the 6
asks dedicated to the NM-ORGextract. After complete evaporation of the chloroform under a fume hood, 10 g of raw soil were added to the 12 asks to provide half
of the soil with the native microorganism community. Water (4.13 ml) was added to
the 18 asks to reach 60% of the water holding capacity, and asks were sealed
hermetically. Three replicates of each condition were harvested immediately after
setting up and the 3 other replicates were incubated at 24  C during 2 months (8
weeks). After 2 months, asks were harvested, humidity was estimated (soil was
weight before and after air drying), 10 g air dried soil were kept for Tenax
extraction, 3 g were frozen at 80  C before lyophilization and total PAH extraction,
and 2 g were frozen at 20  C for further DNA extraction.

Genomic DNA was extracted from 0.5 g of soil using Fast DNA spin Kit for Soil
(MP Biomedicals, France) following manufacturer instruction. DNA concentration
was estimated spectrouorimetrically (Shimadzu) using a TrayCell adaptor
(Hellma). DNA was stored at 20  C until further analyses.
2.8. Real-time PCR quantication of bacteria, fungi and functional bacteria
The genomic DNA was used to quantify the total bacteria and fungi using
968F/1401R (Felske et al., 1998) and Fung5f/FF390r (Smit et al., 1999; Vainio and
Hantula, 2000) primers targeting bacterial 16S rRNA gene and fungal 18S rRNA
gene, respectively. Functional genes, i.e., PAH-ring hydroxylating dioxygenase
genes from gram negative and positive bacteria, catechol-1,2-dioxygenase genes
and catechol-2,3-dioxygenase genes, were quantied using previously published
primers, i.e., PAH-RHDa GN F/R, PAH-RHDa GP F/R (Cbron et al., 2008), CATA-F/
CATA-R (El Azhari et al., 2010) and C23O-F/C23O-R (Sei et al., 1999), respectively. Real-time PCR quantications were performed as described in Cbron et al.
(2008) using an iCycler iQ apparatus (Bio-Rad), associated with iCycler optical
system interface software (version 2.3; Bio-Rad) for data collection and subsequent melting curve analysis. Amplication reactions were carried out in a volume
of 20 ml containing 1X iQ SYBR green Supermix (Bio-Rad, France), 0.8 ml of each
primer (at 0.4 mM for 16S rRNA, 18S rRNA, PAH-RHDa, and C23O primers and 2 mM
for CATA primers), 15 mg of bovine serum albumin, 0.25 ml of dimethyl sulfoxide,
0.1 ml of T4 bacteriophage gene 32 product (MPBiomedicals, France), and 1 ml of
the template DNA (genomic DNA or standard 10 times dilution series, from 108 to
102 gene copies.ml1) or 1 ml of distilled water (negative control). The amplications were carried out with the following temperature proles: step 1 consisted of

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A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

heating to 95  C (5 min), followed by four steps of 45 cycles, 30 s at 95  C, 30 s at


the primer-specic annealing temperatures (56  C for 16S rRNA, 50  C for 18S
rRNA, 57  C for PAH-RHDa GN, 54  C for PAH-RHDa GP, and 58  C for CATA and
C23O primers), 30 s at 72  C, and 10 s at 82  C to dissociate the primers dimers
and capture the uorescence intensity of the SYBR green. At the end, a melting
curve analysis was performed from 50  C to 95  C, with a temperature increase of
0.5  C every 10 s.
2.9. Bacterial and fungal community structure and diversity
Bacterial 16S rRNA gene and fungal 18S rRNA gene fragments were amplied
using the same primer sets described above but with addition of GC clamps on the
forward and reverse primer, respectively. PCR was performed using an iCycler (BioRad) in a 50 ml volume with Taq DNA polymerase (Invitrogen). A touchdown temperature prole was chosen according to Cbron et al. (2009) for 16S rRNA gene
amplication and a traditional three-step program with 35 cycles with hybridization
at 50  C was used for 18S rRNA gene amplication. PCR products were loaded onto a
temporal thermal gradient gel electrophoresis (TTGE) DCode system (Bio-Rad). TTGE
was performed as previously described (i) in Cbron et al. (2009) for 16S rRNA gene
(7 M urea, 6% acrylamide/bisacrylamide, run at 130 V, with temperature gradient
from 57  C to 67  C and an increment of 2  C h1) and (ii) in Thion et al. (2012b) for
18S rRNA gene (5.5 M urea, 6% acrylamide/bisacrylamide, run at 130 V, with temperature gradient from 51.5  C to 60.5  C and an increment of 1.5  C h1). After
migration, gels were stained with SYBRGold (1/10,000 nal, Molecular Probes) and
analysed on a GelDoc transilluminator (bio-rad). QuantityOne 4.0.1 software (BioRad) was used for image and band pattern analysis. Bands were detected and their
relative abundance was calculated to generate a Pearsons correlation matrix. This
correlation matrix was analysed through Principal Component Analysis (PCA) using
XLStat 2011 software (Addinsoft). Some bands (dominant or newly appearing ones)
were excised, dissolved over night in 30 ml sterile water, and re-amplied using the
same PCR primers without GC-clamp. After purication (High Pure PCR product
purication Kit, Roche), PCR products were sequenced (MWG-Eurons). Sequences
were phylogenetically afliated using GenBank (BlastN) and RDP databases. Partial
16S and 18S rDNA sequences were deposited in the GenBank database under the
following accession numbers: KC507782 to KC507788.
2.10. Statistical analyses
Statistical analyses were performed using XLStat 2011 software (Addinsoft) with
p < 0.05. One- and two-ways analyses of variance (ANOVA) considering treatment
and time as independent factors, followed by NewmaneKeuls multiple comparison
tests were performed to detect the effects of treatment and incubation period on the
CO2 production data and on the microbial and organic compounds parameters,
respectively. Student t test was performed on band intensity matrix generated from
TTGE data.

3. Results
The fate of the polycyclic aromatic compounds (PAH and PPAC),
the Tenax-available PAH and the microbial activity and community
structures were compared for three different conditions: the raw
NM soil (control that illustrates the functioning of the native soil), a
mix of half raw NM soil and half solvent extracted NM soil (control
that highlights the impact of the organic extraction on the soil
functioning) and a mix of half raw NM soil and half solvent
extracted NM soil with reintroduction of organic extract (test
condition to increase PAH availability and biodegradation). It was
necessary to have half of raw NM soil, to provide all batches for
alive microorganisms, adapted to the pollution and to the soil type.

At the beginning of the experiment (T0), extractable organic


matter (EOM), 16 PAH and 7 PPAC concentrations were not significantly different between NM-Raw and NM-ORGextract conditions
while, as expected, these parameters (excepted 7 PPAC concentration) were signicantly lower in the NM-extracted condition
(in theory we would expect half EOM and PAH concentration for
this condition; Table 1). Moreover, the percentage of Tenaxavailable PAH was 6-times higher in the NM-ORGextract than in
NM-raw soil (Table 1), conrming the hypothesis that extraction
and reintroduction of the organic extract allowed to experimentally
increase the PAH availability. However, with this method, available
PAH still represented only ca. 6.3% of the total PAH content.
Batches were incubated during 2 months with the monitoring of
mineralization through CO2 release measurement (Fig. 1A). Data
were transformed to estimate the CO2 production per week
(Fig. 1B). A lag of two days was observed at the beginning with low
mineralization rate for the three conditions. The mineralization in
the NM-raw batches stayed constant close to a linear kinetics of
2.4 mg CeCO2 produced g soil1 day1 during the rest of the incubation. In both NM-extracted and NM-ORGextract conditions, the
mineralization level was similar and signicantly higher than in the
NM-raw batches during the 10 rst days with a rate of 15.7 mg Ce
CO2 produced g soil1 day1. This corresponds to 125e130 mg Ce
CO2 produced g soil1 during the rst week. This similar rapid increase in mineralization intensity in both NM-extracted and NMORGextract, reect probably a close microbial activity. It could be
explained either by baring an easily degradable organic matter
previously inaccessible or by a priming effect due to the release of
carbon from dead biomass consequently to solvent extraction. Between the third and the sixth weeks, the mineralization kinetics in
the NM-extracted and NM-raw batches were similar, while in the
NM-ORGextract it was twice more important with a signicantly
higher rate of 3.8 mg CeCO2 produced g soil1 day1, that corresponded to an extra 92 mg C degraded g soil1 over the two months
period. During the last 2 weeks, the mineralization kinetics were
similar for the three conditions. However, whatever the condition
(NM-Raw, NM-ORGextract and NM-extracted), the percentage of
mineralized carbon is very low compared to the TOC and the EOM
values. For the NM-ORGextract condition, ca. 5.1& of soil total
organic carbon was mineralized after 2 months compare to 2.1&
for the NM-raw condition.
During the incubation period, 16 PAH content tended to
decrease for both NM-raw and NM-ORGextract conditions
(Table 1), while PAH content, mainly 4- to 6-cycles PAH, increased
in NM-extracted condition. The PAH concentration was heterogeneous in both solvent-extracted soil conditions (high standard
deviation). The PPAC concentration slightly decreased, though not
signicantly, in the three conditions. The quantity of Tenaxavailable PAH decreased in the NM-ORGextract batches and
reached a value similar to the NM-raw treatment. Furthermore, the
percentage of Tenax-available PAH for these two treatments after

Table 1
Organic extraction rate, total PAH concentration (sum of the 16 US-EPA PAH), concentration and percentage of available PAH extracted with Tenax (sum of the 16 US-EPA PAH),
and total PPAC (polar polycyclic aromatic compounds) concentration.
EOM (mg g
soil dw1)
NM-raw T0
NM-extracted T0
NM-ORGextract T0
NM-raw T2m
NM-extracted T2m
NM-ORGextract T2m

12.5
7.5
11.5
11.2
10.1
11.3








1.6A
2.6A
0.8A
1.6A
3.1A
1.2A

Sum of 16 PAH
(mg kg soil dw1)
1214
717
1036
1004
781
872








93A
235B
215AB
122AB
292AB
94AB

Sum of 15 Tenax-available
PAH (mg kg soil dw1)
12.0
16.1
61.2
20.3
15.6
22.9








1.1C
3.8a BC
4.6A
3.3B
8.3a C
3.3B

Percentage of
Tenax-available PAH
1.0
1.9
6.2
2.1
2.3
2.7








0.2B
1.4B
1.8A
0.6B
1.4B
0.6B

PPAC (mg kg
soil dw1)
34.3
30.1
28.3
25.6
26.4
26.1








1.7A
22.1A
8.7A
1.5A
6.0A
4.9A

Mean values (n 3) and standard deviations. Results from two-ways analyses of variance (ANOVA) followed by NewmaneKeuls comparison test (p < 0.05) are shown with
different uppercase letter indicating signicant differences at p < 0.05.
a
Data from only 2 replicates were kept for analysis.

A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

Fig. 1. Carbon mineralization, estimated as CO2 measurement in the atmosphere of


batches, during the 2 months of incubation. A. Cumulated CO2 concentrations; B.
Estimation of the CO2 production per week. Mean values (n 3) and standard
deviations. Different letters within different times indicate signicant differences between treatments using one-way ANOVA followed by NewmaneKeuls comparison test
(p < 0.05).

two months of incubation was not signicantly different from the


value measured at the beginning (ca. 1e2%, Table 1).
Bacterial and fungal community structures in batch experiments
were studied through TTGE ngerprinting (Figs. 2A and 3A).
Organic extraction treatment did not affect drastically the bacterial
community structures, no shift in TTGE band patterns was observed
and all T0 samples were grouped together on the PCA analysis
(Fig. 2B). After 2 months of incubation, bacterial community
structure was different from the beginning of the experiment and

101

differed between conditions, i.e., PCA analysis revealed 2 groups


separating the NM-raw batches from the NM-extracted and NMORGextract conditions (Fig. 2). One replicate of the NM-extracted
condition differed from the two others. Although NM-extracted
and NM-ORGextract conditions were not separated on PCA analysis, some TTGE bands were signicantly more intense in the
NM-ORGextract samples based on band matrix statistical analyses
(student t test, p < 0.05). These bands were sequenced and afliated to three Gamma-Proteobacteria including 2 species belonging
to Pseudomonas genus.
At the beginning of the experiment (T0), the soil fungal community structure was dominated by one species (closest relatives in
GenBank with 95% sequence identity belonged to Basidiolobus
genus) and after the 2 months incubation this fungus still dominated the NM-raw batches (Fig. 3A). However, the fungal community structure completely shifted in the NM-extracted and NMORGextract conditions with a new dominant strain closely related
to Fusarium solani. This modication is highlighted on the PCA
analysis of TTGE band patterns (Fig. 3B), where NM-ORGextract and
NM-extracted samples after 2 months of incubation are spatially
separated from all the other samples on the rst axis which
explains most of the variability (30%). As previously observed for
the bacterial community structure, the third replicate of the NMextracted condition differed from the two others (Fig. 3A) and
this sample is separated from the others on the 2nd axis of the
PCA (Fig. 3B).
Microorganisms were quantied using real-time PCR tool
(Table 2). At the beginning of the experiment (T0), the quantication of all gene markers indicated that samples were not signicantly different. After 2 months of incubation, the bacterial and
fungal densities (i.e., 16S rRNA and 18S rRNA genes) increased in the
three conditions, and the percentage of fungi (percentage of 18S
rDNA relative to 16S rDNA) was signicantly higher in NMextracted and NM-ORGextract conditions than in NM-raw soil (7.1
and 3.7% compare to 1.3% respectively). Functional bacterial groups,
potentially implicated in the degradation of PAH and organic
compounds, were also quantied. PAH-degraders, quantied by
targeting the PAH-RHDa genes from Gram positive (GP) and Gram
negative (GN) bacteria, were signicantly more abundant after
2 months of incubation in NM-extracted and NM-ORGextract
batches. In NM-raw batches, only the Gram positive PAH-RHDa
genes were more abundant after 2 months. The percentages of both
Gram positive and Gram negative PAH-degraders were signicantly
more abundant in NM-ORGextract condition. The concentration of

Fig. 2. Bacterial community structure in the triplicate samples from the 3 batch conditions (NM-raw: raw soil without treatment, NM-ORGextract: soil with addition of organic
extract, NM-extracted: control extracted soil), at the beginning (T0) and after 2 months incubation (T2m). A. TTGE based on 16S rRNA gene fragment patterns, band labelled with an
arrow were sequenced and afliated using GenBank (BlastN) and RDP databases (1. and 2. Pseudomonas sp. (99%), 3. uncultured g-Proteobacterium); B. Principal Component
Analysis (PCA) based on TTGE banding patterns analysis.

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A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

Fig. 3. Fungal community structure in the triplicate samples from the 3 batch conditions (NM-raw: raw soil without treatment, NM-ORGextract: soil with addition of organic
extract, NM-extracted: control extracted soil), at the beginning (T0) and after 2 months incubation (T2m). A. TTGE based on 18S rRNA gene fragment patterns, band labelled with an
arrow were sequenced and afliated using BlastN (1. Uncultured fungus, 2. Geomyces sp. (99%), 3. Aspergillus sp. (99%), 4. Fusarium solani (100%)); B. Principal Component Analysis
(PCA) based on TTGE banding patterns analysis.

PAH-RHDa GN genes was ten times higher than PAH-RHDa GP


genes, representing 10.3% and 1.2% of PAH-RHDa GN and GP genes
relative to 16S rRNA genes, respectively. Catechol dioxygenase
genes were also quantied, catechol-1,2-dioxygenase (CATA) were
signicantly more abundant after 2 months of incubation but there
was no treatment impact on the abundance and relative proportion
of this functional group; on the other hand the abundance and
percentage of catechol-2,3-dioxygenase (C23O) was constant all
along the experiment.
Data were analysed together to highlight important variables for
qualitative sample discrimination. A PCA analysis was performed
based on normalized values of PAH, Tenax-available PAH, PPAC and
gene quantication data (Fig. 4). Most NM-raw and NM-extracted
samples, at T0 and after 2 months (T2m), were in the middle of
the PCA, except for 2 aberrant NM-extracted samples (i.e., one
replicate at T0 and one at T2m) linked probably to the important
heterogeneity of the coking plant soil, the aberrant NM-extracted
replicates presented a higher proportion of low-molecular weight
PAH (NAP, ANA, ANT, FLU, and PHE). NM-ORGextract samples, both
at T0 and T2m, were separated from all the others on the 2nd PCA
axis. At T0, NM-ORGextract samples were separated from other T0
samples mainly because of a higher proportion of Tenax-available
3e4 cycles PAH (i.e., ANT av, FLT av, PYR av, CHR av, and BaA av
according to correlation circle; Fig. 4). At T2m, NM-ORGextract
samples were separated from other T2m samples mainly because
of a higher proportion of 5e6 cycles PAH both total and Tenaxavailable concentration (i.e., DbPHE, IPY, and BPY; BbF av, BaP av,
DbA av, IPY av, and BPY av) and higher percentage of aromatic
compounds- and PAH-degradation genes (cata, PAH-RHDa GN
and GP).
4. Discussion
4.1. Availability of PAH in aged-contaminated soil
In industrial wasteland soils (coking plants, gas plants.), PAH
are generally released into the environment as byproducts of
incomplete combustion and pyrolysis process of coal. These pyrogenic PAH are usually emitted within diverse organic matrixes as
coal tar, pitch, or black carbon-like products such as coke or soot,
with strong sorption domains for PAH (Jonker and Koelmans, 2002;
Cornelissen et al., 2005; Khalil et al., 2006). Thus, due the
complexity of the organic constituents and to the ageing process,
major residual part of PAH is not available for microbial

biodegradation. Various methods have been developed to assess


available PAH concentration (Semple et al., 2003). Among others,
Tenax extraction method can provide a prediction of PAH concentration that is closer to what is really bioavailable than traditional
exhaustive solvent extractions (Cornelissen et al., 1997; Ten
Hulscher et al., 2003). In the studied soil, from the former coking
plant of Neuves-Maisons, the fraction of Tenax-available PAH was
found to be close to 1% of the total PAH content. Similar value was
previously measured on the same soil under in situ natural attenuation (Ouvrard et al., 2011). Our case study was quite extreme and
presented strikingly low availability values. Indeed, the percentage
of available PAH fraction, using a similar Tenax extraction method,
could represent a higher compartment, e.g. Schwab and Brack
(2007) found that 12e30% of PAH-pollutants were Tenaxavailable in sediment from the River Elbe downstream industrial
region. However, the available PAH fraction also depend on the
technique used for the measurement. On the same NM soil but
using a chemical oxidation technique with sodium persulfate,
previously used by Cuypers et al. (2000) to estimate bioavailability,
Usman et al. (2012a, 2012b) found also no PAH degradation conrming the weak availability of PAH in the Neuves-Maisons soil.
Many studies have showed that fresh contamination is more
bioavailable and biodegradable than aged-contaminants. As
example, the incubation of a freshly contaminated coking plant soil
by a 13C-phenanthrene reveals that the added model contaminant
was rapidly degraded (2 weeks) whereas the initial contamination
of the coking plant soil (including phenanthrene) is not affected
(Cbron et al., 2011), In another experiment, Khan et al. (2012) have
shown that still 40e50% of the freshly spiked phenanthrene was
still bioavailable even after a 150-days of ageing period. Differences
in ageing rate could depend on soil characteristics (Semple et al.,
2003). Allan et al. (2006) showed lower phenanthrene bioavailability (lesser than 10% extractable using cyclodextrin after 50 days)
in peat soil than in silt, sand and clay soils. Ageing is more marked
in soils rich in organic matter (Hatzinger and Alexander, 1995; Nam
et al., 1998), due (i) to diffusion and sequestration within nanopores
(Alexander, 2000) inaccessible to organisms and even to extracellular enzymes of microorganisms and (ii) to important sorption
between organic matter and lipophilic hydrocarbons (Allan et al.,
2006). Thus, the complexity in term of nature and abundance of
organic matter, that interacts with PAH, could largely inuence the
PAH availability.
After the treatment by organic solvent extraction and reintroduction of the EOM in the soil (NM-ORG-extract), the percentage of

0.1A
0.1A
0.1A
0.3A
 2.4A
0.1A
0.2A
1.1AB
0.1A
0.1A

Mean values (n 3) and standard deviations. Results from two-ways analyses of variance (ANOVA) followed by NewmaneKeuls comparison test (p < 0.05) are shown with different uppercase letter indicating signicant
differences at p < 0.05.
a
Only 2 replicates were considered because of the very divergent microbial population in the 3rd replicate (see Figs. 2 and 3).

0.1A
0.1A
0.4A
0.1A
0.1A
0.1A






0.6
0.9
0.7
0.3
0.3
0.4
0.1A
0.1A
0.2A
0.1A

0.5 
0.5 
0.4 
0.6 
0.7A
0.7 
0.1A
0.2A
0.1A
0.0A

3.9 
4.1 
4.1 
4.3 
4.1A
4.2 
0.1B
0.3B
0.0B
0.0A

3.8 
3.8 
3.7 
4.5 
4.48A
4.4 
0.1B
0.2B
0.1B
0.1B

0.5 
0.3 
0.3 
0.6 
0.4B
1.2 
0.1C
0.1C
0.1C
0.1C





0.1
0.2
0.3
0.2
7.7B
10.3
0.1C
0.2C
0.1C
0.1AB

3.8 
3.6 
3.5 
4.5 
4.3B
4.7 
0.2B
0.2B
0.0B
0.1B

3.3 
3.4 
3.6 
4.1 
5.4AB
5.6 
0.1B
0.2B
0.2B
0.3B

0.7 
0.4 
0.3 
1.3 
7.2A
3.7 
0.1B
0.1B
0.1B
0.1A

3.9 
3.7 
3.6 
4.8 
5.44A
5.2 
0.1B
0.3B
0.0B
0.0A

6.1 
6.1 
6.1 
6.7 
6.6A
6.6 
NM-raw T0
NM-extracted T0
NM-ORGextract T0
NM-raw T2m
NM-extracted T2ma
NM-ORGextract T2m

% C23O/16S
% CATA/16S
C23O (Log gene
copies g soil dw1)
CATA (Log gene
copies g soil dw1)
% PAH-RHDa
GP/16S
% PAH-RHDa
GN/16S
PAH-RHDaGP
(Log gene copies
g soil dw1)
PAH-RHDaGN
(Log gene copies
g soil dw1)
% 18S/16S
18S rDNA (Log gene
copies g soil dw1)
16S rDNA (Log gene
copies g soil dw1)

Table 2
Real-time PCR quantication of bacterial (16S rRNA genes) and fungal (18S rRNA genes) communities, and functional bacterial populations: PAH-degraders (PAH-RHDa genes from Gram negative GN and Gram positive GP
bacteria), and aromatic-hydrocarbon degraders (catechol-1,2-dioxygenase genes, CATA; catechol-2,3-dioxygenase genes, C23O) expressed as gene copies abundance or percentage relative to 16S rDNA quantication.

A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

103

Tenax-available PAH was only 6-times higher than in raw soil, and
represented 6.3% of the total PAH content. The same procedure
(extraction and reintroduction of EOM) on the same coking plant
soil was applied and followed by sodium persulfate (Usman et al.,
2012a) or Fenton-like (Usman et al., 2012b) oxidations. About 50%
of the 16 PAH were degraded whatever the oxidant suggesting an
important increase in the availability after extraction/reintroduction procedure. In theory, in our experiment, if most of the PAH
reintroduced with the organic extract would have been bioavailable, the mix with half raw soil would have resulted in about 50% of
bioavailable PAH. The far lower Tenax-available PAH fraction and
the contradiction with persulfate oxidation results suggest probably different availability levels: a Tenax-availability compatible
with bioavailability in our case study and a chemo-availability
deduced by persulfate or Fenton-like oxidations. This difference
can be linked to a sequestration/sorption of PAH within the soil
constituent (especially organic constituents) too high for biodegradation processes but accessible for chemical oxidants. Extractionreintroduction probably favours a good dispersion of EOM
(including PAH) on all the different mineral and organic constituents. Such dispersion increases the contact areas of organic contaminants but also favours the interaction (sorption/sequestration)
between soil particles and organic molecules. These two antagonistic phenomena after extraction-reintroduction can explain on
one hand the important increase in chemical oxidation efciency
(enlarge contact area) and on the other hand, the low biodegradation intensity (increase in interactions between PAH and soil
particles).
Following the solvent extraction procedure, the soil trapping
matrix was left free but though freshly added, PAH were still mostly
associated within the complex organic mixture that constitutes the
extractable organic matter (EOM). Contrarily to most of the previous studies performed using only fresh PAH compounds inputs, our
approach reveals the importance of the complexity of the organic
matter on the PAH bioavailability parameter. In EOM, the PAH are
associated with a complex mixture of other polycyclic aromatic
macromolecules, inherited from coal-tars that is an adsorbent
material with high sorption strength (Gosh et al., 2003). Moreover,
associated to coal tars, in industrial aged-contaminated soils, coke
and coal particles can be present in large quantities (Achten et al.,
2011), and high levels of PAH may occur associated to these solid
organic matrixes (Ghoshal and Luthy, 1996) acting as efcient
geosorbents (Achten and Hofmann, 2009). The behaviour of these
multi-component organic mixtures is complex and therefore
difcult to describe but better mimic the reality. Thereby, even
using a very drastic approach to increase PAH availability, it was not
highly efcient because chemical nature of the pollutant source and
surrounding rather than time seemed more important in controlling PAH availability. However, this small increase in available PAH
fraction was sufcient to highlight the biodegradation activity and
the PAH signature of NM-ORGextract samples before and after
microbial degradation.
4.2. Evidence of biodegradation consequently to the increase in
available PAH fraction
Although the increase in Tenax-availability was low, the recontaminated soil samples (NM-ORGextract) had a specic signature both at the beginning and after 2 months of incubation, since
they were clearly separated on PCA (Fig. 4). During the two-months
of batch incubation, the quantity of available PAH decreased.
Moreover, at T0 NM-ORGextract samples had a higher proportion
of Tenax-available PAH of 3e4 cycles while after 2 months these
samples had a higher proportion of high molecular weight PAH of
5e6 cycles, probably indicating a degradation of lower molecular

104

A. Cbron et al. / Environmental Pollution 177 (2013) 98e105

Fig. 4. Principal Component Analysis (PCA) based on PAH, PPAC, available PAH, and gene quantication data. On the correlation circle, all variables are visible, abbreviations correspond
to: 16 PAHs (NAP, naphthalene; ANY, acenaphthylene; ANA, acenaphthene; FLU, uorene; PHE, phenanthrene; ANT, anthracene; FLT, uoranthene; PYR, pyrene; BaA, benz[a]
anthracene; CHR, chrysene; BbF, benzo[b]uoranthene; BkF, benzo[k]uoranthene; BaP, benzo[a]pyrene; IPY, indeno[1,2,3-c,d]pyrene; DbPHE, 1,2:7,8-dibenzophenanthrene; BPY,
benzo[ghi]perylene), for the available PAH data the abbreviated name is followed by av.; gene quantication data are expressed as the percentage of gene relative to 16S rRNA gene
quantity (18S/16S, ratio of 18S rRNA relative to 16S rRNA gene; PAH-RHD GN and GP, for PAH dioxygenases from Gram negative and Gram positive bacteria; cata, catechol-1,2dioxygenase; C23O, catechol-2,3-dioxygenase).

weight compounds. Concomitantly, the mineralization rate was


twice more important suggesting a higher biological activity. After
two months, the available PAH dissipation and the excess in
mineralization represented 38.7 mg of PAH kg soil1 and 92 mg Ce
CO2 produced kg soil1 respectively. These elements were evidences of the higher biodegradation of organic matter probably
both degradation of PAH and co-extracted organic compounds.
Contrarily to the rapid and easy degradation of freshly spiked
phenanthrene in the same soil (Cbron et al., 2011), the input of a
complex organic extract could partially inhibit the degradation of
PAH compounds due to molecular interactions. Although studies of
complex organic extract biodegradation are scarce, some authors
have showed processes of the degradation, solubilization or
transformation of coals by bacteria or fungi (Ahmed et al., 1999;
Silva-Stenico et al., 2007). In the re-contaminated soil samples
(NM-ORGextract) and in the soil that undergo solvent extraction
(NM-extracted), the percentage of fungi relative to bacteria was
higher than in raw soil. Moreover, the fungal community structure
completely shifted, and a new dominant strain closely related to
Fusarium solani, developed. Many studies have demonstrated the
ubiquity of Fusarium species in PAH-contaminated soils and sediments (Jacques et al., 2008; Mohsenzadeh et al., 2010) and Fusarium
solani was previously detected in the same soil under natural
attenuation in situ (Thion et al., 2012b). Many studies on Fusarium
spp., a saprophytic fungus, have shown its capability to degrade
high-molecular-weight organic compounds such as cellulose,
xylan, pectin, different hydrocarbons (Kang and Buchenauer, 2000)
as well as PAH (Chulalaksananukul et al., 2006; Thion et al., 2012a).
Furthermore, Fusarium sp. has been shown to be involved in coal
solubilization by increasing pH and production of non-enzymatic
agents (chelators) (Hlker et al., 1999). After two months of incubation, the bacterial community structure was also slightly
different in the condition with organic extract input. Two strains
belonging to Pseudomonas genus were favoured in this condition.
Pseudomonas species are well-known PAH-degraders and are
commonly isolated for their PAH-degradation efciency (Mueller
et al., 1997). Pseudomonas sp. are Gram negative gammaProteobacteria, having their catabolic genes on large plasmids, e.g.
Pseudomonas putida NCIB 9186-4 is a well-characterized bacterium
capable of utilizing naphthalene as sole carbon and energy source
(Simon et al., 1993). Both Gram positive and Gram negative PAH-

degraders were detected in higher abundance in the soil with


organic extract and PAH-RHDa GN genes were ten-times more
represented than PAH-RHDa GP genes (10.3% and 1.2%, respectively). Both these ndings suggest that Gram negative PAHdegraders, closely related to Pseudomonas sp., contributed to
PAH-degradation.
5. Conclusions
The extraction and re-contamination procedure was an original
approach to test the impact of PAH availability on the biodegradation activity. This approach only increase 6-times the availability
of PAH, thus demonstrating that ageing is not the main parameter
inuencing the PAH availability level, but that the organic constituents coexisting with PAH in soil also drive the PAH availability. In
the re-contaminated soil, the microbial mineralization activity
increased. The decrease in 2- to 4-cycles PAH proportion (available
and total) and the increase in the percentage of Gram negative and
Gram positive PAH-ring hydroxylating dioxygenase relative to 16S
rRNA genes, highlighted the stimulation of PAH biodegradation of
low molecular weight PAH by the re-contamination procedure.
Acknowledgements
This work was funded by the INSU-EC2CO-MicrobiEn Program.
We thank Romain Goudon for technical help.
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