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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
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
99
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
100
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.
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.
101
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.
102
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.
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.
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
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).
105