Environmental Pollution 180 (2013) 199e205
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GammaProteobacteria as a potential bioindicator of a multiple contamination by polycyclic aromatic hydrocarbons (PAHs) in agricultural soils Maïté Niepceron a, b, Fabrice Martin-Laurent c, d, Marc Crampon a, e, Florence Portet-Koltalo e, Marthe Akpa-Vinceslas f, Marc Legras g, David Bru d, Fabrice Bureau f, Josselin Bodilis a, * a Université de Rouen, Laboratoire de Microbiologie Signaux et Microenvironnement, EA 4312, 76821 Mont Saint Aignan, France b Université de Rouen, Laboratoire M2C, UMR CNRS 6143, 76821 Mont Saint Aignan, France c INRA, UMR 1347 Agroécologie, BP 86510, 21065 Dijon, France d INRA, Service de Séquençage et de Génotypage (SSG), BP 86510, 21065 Dijon, France e Université de Rouen, Laboratoire COBRA, UMR CNRS 6014, 27000 Evreux, France f Université de Rouen, Laboratoire ECODIV, 76821 Mont Saint Aignan, France g Esitpa, Unité Agri’Terr, Laboratoire Biosol, 76134 Mont Saint Aignan, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2012 Received in revised form 22 May 2013 Accepted 23 May 2013
The impact of a multiple contamination by polycyclic aromatic hydrocarbons (PAHs) was studied on permanent grassland soil, historically presenting low contamination (i.e. less than 1 mg kg1). Soil microcosms were spiked at 300 mg kg1 with either single or a mixture of seven PAHs. While total dissipation of the phenanthrene was reached in under 90 days, only 60% of the PAH mixture were dissipated after 90 days. Interestingly, after 30 days, the abundance of the GammaProteobacteria class (assessed by qPCR) become significantly higher in microcosms spiked with the PAH mixture. In addition, the specific abundance of the cultivable Pseudomonas spp., which belong to the GammaProteobacteria class, increased earlier and transiently (after 8 days) in the microcosms spiked with the PAH mixture. Consequently, we propose to use the GammaProteobacteria as a bioindicator to detect the impact on the bacterial community of a multiple contamination by PAHs in agricultural soils. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Polycyclic aromatic hydrocarbon Bioindicator Agricultural soil qPCR Pseudomonas
1. Introduction Due to the conjunction of favorable climatic conditions and the natural richness of the deep silty soils of northern France, the Seine watershed is a highly productive agricultural area. However, the Seine basin is also highly urbanized and industrialized, and is consequently exposed to a range of pollutants, including polycyclic aromatic hydrocarbons (PAHs) (Blanchard et al., 2007). PAHs are hydrophobic aromatic compounds containing two or more fused phenyl and/or pentacyclic rings in linear, angular or cluster arrangements (Cerniglia, 1992). They are known to be carcinogenic, mutagenic and genotoxic to both aquatic and terrestrial organisms (Phillips and Grover, 1994; Cachot et al.,
* Corresponding author. E-mail address:
[email protected] (J. Bodilis). 0269-7491/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2013.05.040
2006). PAHs are ubiquitous, semi-volatile pollutants produced by incomplete combustion of organic material, fossil fuel, petroleum product spillage, and also come partly from natural sources such as forest fires and volcanic eruptions (Motelay-Massei et al., 2007; Wang et al., 2009). Because of their hydrophobicity and chemical stability, these compounds accumulate progressively in soils, where they persist for many years. PAH contamination of agricultural soils may contribute to food chain contamination and therefore represents a human health risk due to the consumption of contaminated meat or vegetables (Wild and Jones, 1992; Fismes et al., 2002; Grova et al., 2002; Martorell et al., 2010). Moreover, PAH contamination of agricultural soils may also alter soil microbial functioning which is the key player for soil ecosystem services. Soil contaminations resulting from chronic atmospheric PAH deposition are characterized by a multiple contamination including several different PAHs (Motelay-Massei et al., 2004, 2007). However, most of the studies assessing the impact of a PAH
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2. Materials and methods
overnight at 35 C. Then, they were crushed and weighted to 1 g, and the nine soil samples were extracted simultaneously by MAE, in 20 mL of a mixture of acetone/ toluene 50/50 (v/v) at 140 C, for 30 min, at a power of 1200 W. Then, the extracts were filtered using Teflon PTFE filters (0.45 mm) purchased from Phenomenex (Le Pecq, France). After an optimization step of the MAE process, mean recovery yields of the seven PAHs were obtained from six samples, in the best conditions of extraction; extractions were performed on the Yvetot soil, previously sterilized, spiked and aged for 40 days. Recovery yields were evaluated at 85.4 6.9% for phenanthrene, 77.8 5.2% for fluoranthene, 76.9 6.3% for pyrene, 83.9 10.5% for benzo[b]fluoranthene, 85.2 9.1% for benzo[k]fluoranthene, 76.9 8.5% for benz[a]pyrene and 81.7 10.2% for benzo[g,h,i]perylene. For the next step of quantification, 10 mL of an internal standard (perdeuterated phenanthrene at 100 mg L1) were added to 990 mL of each extract prior to analysis. Analyses were performed with a 6850 gas chromatograph from Agilent (Santa Clara, USA) fitted with a mass spectrometer detector (model 5975C from Agilent). The carrier gas (helium) was set at a constant flow rate of 1.2 mL min1. The injector was maintained at 280 C and the injection volume was 1 mL (pulsed splitless mode). The temperature was set at 55 C for 1.2 min, then it was increased to 180 C at 40 C min1, then to 300 C at 4 C min1 and finally maintained at 300 C for 7 min. The DB5-MS capillary column used (50 m 0.25-mm inner diameter, film thickness 0.25 mm; J & W Scientific, Folsom, CA, USA) was coated with a (5% phenyl) methyl polysiloxane stationary phase. The mass detector (electron impact ionization 70 eV, electron multiplier voltage 1600 V) operated in the selected ion monitoring (SIM) mode for better sensitivity. The temperature of the transfer line was 300 C. The detection and quantification thresholds in SIM mode, calculated respectively as three and ten times the standard deviation of blank sample noise, were respectively 1.5 mg L1 and 5 mg L1 for phenanthrene, 2.5 mg L1 and 8.5 mg L1 for fluoranthene, pyrene, benzo[b]fluoranthene and benzo[k]fluoranthene, 3.5 mg L1 and 11.5 mg L1 for benz[a]pyrene and 5 mg L1 and 16.5 mg L1 for benzo[g,h,i]perylene.
2.1. Sample site and sample collection
2.4. Organic carbon mineralization
Soil samples were collected in spring 2010 from an experimental field site of long-term grassland (a rye-grass clover monoculture with permanent cover and no tillage for 25 years) located in Yvetot (Upper-Normandy, France) between the industrial port of Le Havre (49 300 N 0 060 E, 245 000 inhabitants), representative of a very industrialized area with an electric power plant (using coal), refineries and petrochemical industries, and Rouen (49 270 N 10640 E, 465 000 inhabitants), typical of an urban site with high volumes of road traffic and domestic heating. The UpperNormandy region is dominated by an oceanic and temperate climate characterized by mild temperatures, rainfall with a mean of 800e900 mm yr1 and narrow seasonal ranges. The soil, representative of the Paris Basin, is classified as silty (e.g. loess) soil containing 15% clay, 65% silt, and 20% sand. Twenty samples were collected from the surface horizon (0e15 cm) of the long term experimental grassland site and combined into a bulk composite sample (15 kg). Field-moist soils were sieved to 2-mm particle size and stored at 20 C for one week (under humidity control). Part of the soil (10%) was sterilized by autoclaving (in order to avoid PAH biodegradation during the microcosm establishment) and supplemented with phenanthrene, pyrene or a PAH mixture at 3000 mg kg1 (in order to obtain a final concentration at 300 mg kg1).
The mineralization of organic carbon by soil microbial communities was assessed by measurements of the CO2 release using the conductimetry method (Heemsbergen et al., 2004). A NaOH (20 mL at 0.5 mol L1 or 0.2 mol L1) trap was placed in each microcosm. The carbon mineralization was defined as the quantity of carbon (mg) released per kilogram of dry soil and was calculated by comparing initial and final soda conductivity after a given incubation period. CO2 measurements were performed every seven days during the 90 days of the experiment and cumulative curves were drawn to compare the carbon mineralization kinetics induced by the presence of PAHs.
contamination on the bacterial community of an agricultural soil constructed microcosms spiked with only one PAH, often the phenanthrene (Johnsen et al., 2002; De Menezes et al., 2012). Only a few studies have dealt with multiple contaminations by PAHs in agricultural soils (Hamdi et al., 2007; Couling et al., 2010). The main objective of this study was to propose a microbial bioindicator in order to evaluate the impact of multiple contaminations by PAHs in agricultural soils. Soil microcosms prepared with permanent grassland soil were spiked with a single PAH or with a mixture of the seven different PAHs most often found in the native soil tested. Over a 90-day period, PAHs were regularly extracted from the soil by microwave-assisted extraction (MAE) and analyzed by GCeMS (gas chromatograph coupled with a mass detector) to determine dissipation kinetics. Concomitantly, the organic matter mineralization was measured to assess the microbial activity. Several qPCR analyses targeting the 16S RNA gene using universal or taxon-specific primer sets were performed to assess the impact of PAH contamination on soil microbial community structure. Finally, the abundance of culturable Pseudomonas isolates, known to be major PAH degraders in soil and water (Cerniglia, 1992; Juhasz and Naidu, 2000; Niepceron et al., 2010) was determined.
2.2. Microcosm setup and incubation conditions Incubations were performed in hermetically-closed 500 mL sterilized glass flasks. Microcosms consisted of 90 g of dry soil (24.75% vol/wt of water, corresponding to 70% of water-holding capacity) and 10 g of dry sterilized soil spiked with PAHs. The PAH solutions were prepared in acetone in order to obtain a final concentration in dry soil of 3000 mg kg1 for the single PAHs or the PAH mixture (i.e. seven PAHs at 430 mg kg1 each). The dry sterilized soil was mixed under an extractor hood for two days to homogenize the PAH distribution and evaporate the acetone. After a week of stabilization, dry sterilized soil spiked with PAHs was mixed with wet native soil (then the humidity was adjusted) and homogenized to obtain soil microcosms contaminated with 300 mg kg1, equivalent to about 500-fold the concentration of PAHs initially found in this grassland soil. Microcosms of permanent grassland soil were spiked with (i) phenanthrene, (ii) pyrene or (iii) a mixture of 7 PAHs (phenanthrene, pyrene, fluoranthene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, and benzo[g,h,i]perylene, each one being at 43 mg kg1 to obtain a total of 300 mg kg1), and were established in triplicate. Control microcosms not exposed to PAHs but only spiked with acetone were also prepared in triplicate, in order to control evaporation of the solvent (n ¼ 3, per treatment). The flasks were opened once a week for 15 min to oxygenate the medium, and were incubated at 20 C in static mode in a dark room. Analyses and experiments were carried out after 8, 30, 60 and 90 days of incubation. 2.3. PAH extraction from soil and quantification by GC/MS Extractions from the spiked soil were performed by using the Mars X microwave-accelerated extraction (MAE) system (CEM Corporation, Matthews, NC, USA). Three aliquots of 1.5 g of wet soil were taken from each microcosm and dried
2.5. Relative abundance of culturable Pseudomonas Soil microbial communities were extracted by shaking 5 g of soil in 45 mL of 0.85% NaCl solution. 10-fold serial dilutions in sterile 0.85% NaCl solution were prepared and plated on R2A medium to estimate the abundance of total culturable heterotrophic bacteria (Olsen and Bakken, 1987). The abundance of the culturable Pseudomonas was determined by inoculating in triplicate on CFC (Cetrimide Fucidin Cephaloridine) medium supplemented with Cefalotin. Plates were incubated at 20 C in the dark and bacterial colonies counted after 48 h. Data were expressed as Colony Forming Units (CFU) per gram of dry soil. The relative abundance of culturable Pseudomonas was calculated as the ratio of the culturable Pseudomonas to the total culturable heterotrophic bacteria abundances. 2.6. DNA extraction For each microcosm, nucleic acids (DNA) were directly extracted from 0.5 g of moist sieved soils (0.4 g dry weight equivalent) using the BIO101 Fast DNA spin Kit for Soil according to the manufacturer’s recommendations. DNA was re-suspended in 50 mL sterile de-ionized water and quantified on agarose gel stained with ethidium bromide (0.5 mg mL1) with Software Quantum-CaptÒ on Quantum ST4 Image Acquisition SystemÒ (Vilber LourmatÔ). DNA extracts were stored at 20 C. 2.7. 16S rRNA genes qPCR using universal or phylum-specific primer sets The copy number of 16S rRNA genes in soil DNA samples was estimated by quantitative PCR (qPCR) using universal or taxon-specific primers as was previously described (Philippot et al., 2010). Amplification reactions were carried out in a StepOnePlusÒ Real-Time PCR Systems (Applied BiosystemsÔ). Reaction mixtures contained 7.5 mL SYBR GreenÒ PCR Master Mix (Absolute QPCR SYBR Green Rox AbgeneÔ), 250 ng of T4 gp 32 (QBiogeneÔ), 4 ng of soil DNA in a final volume of 15 mL. Fluorescence acquisition was performed at 80 C to avoid interference by unspecific products. For each of the eleven 16S rRNA targets, a standard curve was established using serial dilutions of linearized plasmid pGEM-T (102e107 copies) containing a relevant 16S rRNA gene. Melting curves were generated after amplification by increasing the temperature from 80 C to 95 C. qPCR results are averages of two replicates, and are expressed as copy numbers per gram of dry soil. The relative abundance of each taxon was calculated as the ratio of the copy number of
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this taxon to the total number of 16S rRNA sequences, determined by using universal primers.
3. Results 3.1. Dissipation rates of PAHs As consequence of a chronic and diffuse exposure to PAH contamination from urban and industrial atmospheric depositions, the grassland soil of Yvetot was contaminated with about 0.6 mg kg1 of PAHs (representing the sum of the 16 priority PAHs, as defined by the US-EPA), including 0.055 0.022 mg kg1 of phenanthrene, 0.063 0.021 mg kg1 of pyrene, 0.074 0.023 mg kg1 of fluoranthene, 0.065 0.027 mg kg1 of benzo[b]fluoranthene, 0.050 0.034 mg kg1 of benzo[k]fluoranthene, 0.043 0.016 mg kg1 of benzo[g,h,i]perylene and 0.039 0.018 mg kg1 of benzo [a]pyrene (these values were obtained from 5 independent soil samples). In order to simulate an accidental pollution, this soil was spiked with 300 mg kg1 of phenanthrene, pyrene or a mixture of seven PAHs (phenanthrene, pyrene, fluoranthene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[g,h,i]perylene). The seven PAHs of the mixture we used were the seven most representative PAHs in the native soil. This PAH spiking was 500-fold the PAHs content of the native soil. PAH dissipation kinetics was monitored after extraction of PAHs by MAE and identification/quantification by GCeMS over 90 days (Fig. 1). After a 30-day lag phase, a rapid decrease in the phenanthrene concentration was observed in the microcosms spiked with the phenanthrene alone (Fig. 1A), as well as in those spiked with the mixture of PAHs (Fig. 1B). DT50 (Dissipation Time 50%) was reached after 50 days of incubation. After 90 days of incubation, phenanthrene was no longer detectable (i.e. below the detection limit). Interestingly, the lag phase of the pyrene and the fluoranthene dissipations was longer than that of the phenanthrene dissipation, i.e. 60 days (Fig. 1). After 90 days, only about 60% of the pyrene was dissipated in microcosms spiked with pyrene alone (Fig. 1A), whereas more than 95% of the pyrene and the fluoranthene were dissipated in microcosms spiked with the PAH mixture (Fig. 1B). Finally, while less than 20% of the benzo[b]fluoranthene, the benzo[k]fluoranthene and the benzo[g,h,i]perylene was dissipated after 90 days, a rapid decrease in the benzo[a]pyrene concentration was observed in under 8 days (reaching up to 75% of dissipation) followed by a stabilization of the PAH concentration (Fig. 1B). 3.2. Mineralization rates in the microcosms The organic carbon mineralization rates were measured in all the microcosms (Fig. 2). During the first 20 days of incubation, the mineralization rates were high in all the microcosms (17 mg CeCO2 kg1 dry soil day1) and then decreased and stabilized at 9 mg Ce CO2 kg1 dry soil day1. While the rates of mineralization in both microcosms spiked with pyrene and those spiked with the PAH mixture were identical to that of controls, a slight increase was observed in microcosms spiked with phenanthrene (Fig. 2). After 90 days of incubation, the cumulative mineralization of organic carbon was significantly higher (P < 0.025; ManneWhitney test) in the microcosms spiked with phenanthrene, compared to controls. 3.3. Abundance of the total bacterial community During the PAH dissipation kinetics, DNA was directly extracted from soil samples at each sampling time and for each treatment, and qPCR assays were performed to monitor the evolution of the abundance of the total bacterial community. This abundance ranged between 5 108 and 1.9 109 copies of 16S rRNA gene per
Fig. 1. (A) PAH dissipation kinetics (milligram of PAHs per kilogram of dry soil) at 300 mg kg1 in permanent grassland of Yvetot (PGY) incubated at 20 C for 90 days (n ¼ 9). Microcosm spiked with phenanthrene ; microcosm spiked with pyrene ; microcosm spiked with the PAH mixture . (B) PAH dissipation kinetics (milligram of PAHs per kilogram of dry soil) in microcosms enriched with the PAH mixture (300 mg S 7 PAHs kg1 of dry soil): phenanthrene, fluoranthene; pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo[g,h,i] perylene.
gram of dry soil (Fig. 3). Whatever the treatment considered, the bacterial abundance remained stable throughout the experiment, with a slight and transient (but not significant) increase after 30 days of incubation. It therefore seems that PAH spiking did not affect the abundance of total bacterial community. 3.4. Relative abundance of different taxonomical groups In order to get a deeper view of the impact of PAHs on the soil bacterial community, the abundances of ten phyla and classes (Actinobacteria, Acidobacteria, Firmicutes, AlphaProteobacteria, Bacteroidetes, Gemmatimonadetes, Verrucomicrobiales, BetaProteobacteria, Planctomycetes, and GammaProteobacteria) were monitored by qPCR (Philippot et al., 2010). The relative abundance of each bacterial group was calculated at each sampling time and for each treatment (Fig. 4; Fig. S1, Supplementary data). Some significant differences could be observed between the controls and the microcosms spiked with a single PAH and/or with the PAH mixture. However, most of these differences were only transient
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water (Cerniglia, 1992; Juhasz and Naidu, 2000; Singleton et al., 2005; Niepceron et al., 2010). However, it is important to note that culturable bacteria represent only a fraction of the total bacterial diversity (estimated to about 1% in our studied soil). In addition, several studies have highlighted a role of uncultured members of GammaProteobacteria in the degradation of PAH (Singleton et al., 2006; Martin et al., 2012). Nevertheless, the relative abundance of culturable Pseudomonas was about 1% at the beginning of the experiment and transiently increased in all the microcosms (including the controls), with a maximum after 8 days of incubation (Fig. 5). Interestingly, only the microcosms spiked with either phenanthrene or the PAH mixture showed a significantly (P < 0.05; ManneWhitney test) higher abundance of Pseudomonas than controls (Fig. 5). Specifically, the relative abundance of culturable Pseudomonas was significantly higher in the microcosms spiked with the PAH mixture from 8 to 60 days of incubation. Fig. 2. Kinetic of organic carbon mineralization in CeCO2 (mg C kg1 of dry soil) in permanent grassland of Yvetot (PGY) incubated at 20 C for 90 days (n ¼ 3). Control ; microcosm enriched with phenanthrene ; microcosm microcosm enriched with pyrene ; microcosm enriched with the PAH mixture .
(Fig. 4). At a given incubation time, the relative abundances of some bacterial groups decreased (e.g. the Acidobacteria phylum), while those of other bacterial groups increased, as compared to controls (e.g. the BetaProteobacteria class). In complex ways, for the same treatment, the relative abundances of some bacterial groups increased significantly at a given incubation time, and then decreased significantly at another incubation time (e.g. the Bacteroïdetes phylum). Interestingly, the impact of the PAH mixture on the GammaProteobacteria was both early and sustainable (Fig. 4). The relative abundance of this bacterial group in microcosms spiked with the PAH mixture was significantly higher than in controls, from 30 days of incubation to, at least, the end of our experiment (i.e. 90 days of incubation). 3.5. Abundance and structure of the culturable Pseudomonas In order to give another angle of analysis, the relative abundance of the culturable Pseudomonas was monitored during the kinetics of PAH dissipation (Fig. 5). Pseudomonas are known to be PAH degraders of small PAHs (Naphthalene and Phenanthrene) in soil and
Fig. 3. 16S rRNA gene abundance (per gram of dry soil) in the different microcosms as determined by real-time PCR (n ¼ 3). Control microcosms ; phenanthrenespiked microcosms ; pyrene-spiked microcosms ; PAH-mixture-spiked microcosms .
4. Discussion After a 30-day lag phase, the phenanthrene (3-ring PAH) in our soil microcosms was entirely dissipated before 90 days of incubation, both in microcosms spiked with phenanthrene alone and in microcosms spiked with the PAH mixture. Dissipation of PAHs is known to result from the combination of abiotic (adsorption to mineral or organic particles) and biotic (mineralization and/or biomass formation) processes. The shape of the phenanthrene dissipation observed here is in favor of the second process, since adsorption contribution is expected to be rapid, which is in contradiction with the 30-day lag phase observed. The lag phase recorded prior to dissipation is known to be the time required for the growth of degrading populations (Soulas, 1993; Amellal et al., 2006). Concerning the pyrene and the fluoranthene (4-ring PAHs), the lagphase was longer than for the phenanthrene dissipation (60 days), but the high dissipation rate of these PAHs between 60 and 90 days suggests that these PAHs were also partially biodegraded (Fig. 1). For the pyrene, the different percentages of dissipation observed between microcosms containing only the pyrene and those containing the PAH mixture (60% and 95%, respectively) could be explained either by the initial lower amount of pyrene or by a co-metabolism in the microcosms spiked with the PAH mixture (Wilson and Jones, 1993; Bouchez et al., 1999; Johnsen et al., 2005). For three of the high molecular weight PAHs spiked (the benzo [b]fluoranthene, 5-ring PAH; the benzo[k]fluoranthene, 5-ring PAH; and the benzo[g,h,i]perylene, 6-ring PAH), a little dissipation could be observed after 90 days, and the biodegradation was probably negligible. Indeed, their recovery yields in soil were around 80%, which corresponds to the error interval of the analytical process (see Materials and methods). Surprisingly, more than 75% of the benzo[a]pyrene (5-ring PAH) dissipated very quickly during the first eight days, then no further dissipation was observed. Because of its speed, particularly compared to the phenanthrene dissipation, this benzo[a]pyrene dissipation seemed at first sight to result essentially from abiotic processes, i.e. adsorption to mineral or organic particles (Kottler and Alexander, 2001). However, it is difficult to explain why the benzo[a]pyrene and the three other high molecular weight PAHs (with similar hydrophobic properties) would follow different abiotic dissipation shapes. Moreover, additional analyses using the same soil, but sterilized by autoclaving, showed no significant dissipation of the benzo[a]pyrene over at least 90 days (data not shown). So, it is difficult to conclude about the processes (biotic or abiotic) involved in the benzo[a]pyrene dissipation. The initial high mineralization rates in all the microcosms (19 mg CeCO2 kg1 dry soil day1) could be explained by both the better initial oxygenation and the priming effect resulting from the
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Fig. 4. Relative abundances of phylum-specific 16S rRNA genes in soil DNA during incubations with PAHs. DNA was extracted from soil ( ) before microcosm establishment (T0) and from the control ( ), phenanthrene ( ), pyrene ( ) and PAH mixture ( ) microcosms at T8, T30, T60 and T90. Stars highlight significant differences compared to control DNA at the same incubation time (P < 0.05, ManneWhitney test): (A) Acidobacteria; (B) BetaProteobacteria; (C) Bacteroidetes; (D) GammaProteobacteria.
Fig. 5. Pseudomonas proportion among chemo-organo-heterotrophic bacteria soil community during incubation with PAHs. Control microcosms ( ); microcosms spiked with phenanthrene ( ); microcosms spiked with pyrene ( ); microcosms spiked with the PAH mixture ( ).
consumption of labile organic matter released after the establishment of the microcosms (see Materials and methods; Grosser et al., 1991). The mineralization rate, significantly higher for the microcosms spiked with phenanthrene, was consistent with a probable complete biodegradation of this PAH in less than 90 days. However, we cannot be sure that the excess of emitted CO2 resulted only from the mineralized phenanthrene. To prove this last point, the use of phenanthrene labeled with 13C or 14C would be necessary. In the case of the other microcosms containing pyrene alone or the mixture of 7 PAHs, even if the biodegradation of fluoranthene and pyrene did actually occur, it was certainly not as complete as the biodegradation of phenanthrene after 90 days. Indeed, because intermediary oxygenated metabolites are formed during the biodegradation process by successive ring cleavages, pyrene and fluoranthene were certainly degraded to various degrees, but not completely transformed to the final CO2. Nevertheless, it is noteworthy that a relatively strong spiking of a PAH mixture (i.e. 500fold the initial content) had no detectable impact on the bacterial activity (i.e. carbon mineralization) in an agricultural soil. Although polycyclic aromatic hydrocarbons are of special concern because they are persistent and accumulate along the trophic chain, their impact on the structure and abundance of soil bacterial communities remains a matter of debate (Peng et al., 2010). In our study, although we found changes in the specific abundance of several bacterial classes or phyla, neither the abundance of the total bacterial community nor the mineralization rate was significantly affected in response to spiking with a PAH
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mixture. Keeping in mind that PAH exposure leads to the selection of PAH-degrading populations, which represent less than 1% of the overall microbial populations (Cebron et al., 2008; Martin et al., 2012), one might expect that they would not be seen by studying the overall bacterial community. In order to further study the impact of PAHs on soil microorganisms, the relative abundances of 10 bacterial groups were estimated in each microcosm all through the incubation period (Fig. 4; Fig. S1, Supplementary data). We found that the relative abundance of some taxa either increased or decreased transiently in microcosms spiked with single or multiple PAHs (Fig. 4). We hypothesize that the decrease of a given relative abundance probably resulted from an ecotoxicological effect of the PAHs (e.g. for the Acidobacteria phylum), when an increase resulted either from a better resistance to PAH toxicity and/or from the use of PAHs as carbon and energy sources. The mean objective of our study was to identify some bacterial groups sensitive to a multiple contamination by PAHs, thus representing a possible bioindicator of PAH contamination. Among the bacterial groups for which the abundance increased in response to the addition of phenanthrene, pyrene and/or the PAH mix, the BetaProteobacteria presented a significantly higher relative abundance, compared to controls (Fig. 4). Members of this group have been described as major bacterial degraders of phenanthrene and pyrene in soil (Jeon et al., 2003; Singleton et al., 2005; Martin et al., 2012). However, the increase in their abundance was late (after 60 days of incubation) and only transient which makes their use as a bioindicator difficult. Interestingly, starting from 30 days the relative abundance of the GammaProteobacteria significantly increased in response to spiking with the PAH mixture. In addition, between 8 and 60 days of incubation, the microcosms spiked with only phenanthrene or with the PAH mixture showed a relative abundance of culturable Pseudomonas (affiliated to the GammaProteobacteria) that was significantly higher than in controls. However, because the increase of the Pseudomonas abundance was earlier, compared to the kinetic of the GammaProteobacteria abundance, these two phenomena could not be directly linked. Indeed, uncultured members of GammaProteobacteria may play a major role of in the phenanthrene degradation in soil (Singleton et al., 2006; Martin et al., 2012). In this present study, even though the phenanthrene was also present in the PAH mixture (with 6 other PAHs), we think that the selection of phenanthrene-degraders was not sufficient to explain, alone, both the selection of the GammaProteobacteria and culturable Pseudomonas in microcosms spiked with the PAH mixture. On the one hand, the initial phenanthrene concentration was 7-fold less in the microcosms spiked with the PAH mixture than in those spiked with only the phenanthrene. On the other hand, after only 30 days of incubation, when no phenanthrene dissipation had yet been observed, the relative abundance of the GammaProteobacteria was already significantly higher in microcosms spiked with the PAH mixtures, compared to controls. Thus, the increase in the relative abundance of the GammaProteobacteria in microcosms spiked with PAHs resulted probably from both a better PAH-degrading capacity and a better resistance of this bacterial group to the ecotoxicologic impact of the PAHs. Interestingly, previous studies have already shown that the GammaProteobacteria may represent a good bioindicator for the potential biodegradation of 2-, 3- and 4-ring PAHs in a coal-tarcontaminated soil (Lors et al., 2010, 2012). In these studies, the authors found that the GammaProteobacteria were specifically selected at the beginning of their experiment, when the PAH concentration was about 3000 mg kg1, and then other bacterial groups, such as the BetaProteobacteria, appeared over the course of time, when the PAH concentration was low enough to strongly
decrease the ecotoxicity of the soil, i.e. under 300 mg kg1 (Lors et al., 2010, 2012). In conclusion, probably both because of its PAH-degrading capacity and its better resistance to PAH toxicity, GammaProteobacteria could constitute a useful bioindicator to detect the impact on the bacterial community of a multiple contamination by PAHs in agricultural soils. In order to establish a baseline response of this putative bioindicator, further work will aim at estimating the influence of the soil type as well as the level and the age of the PAH contamination on the kinetic of the GammaProteobacteria. Acknowledgments This work was supported by grants from the Agence de l’Environnement et de la Maîtrise de l’Energie (ADEME) and the Région Haute Normandie (via RESSOLV and ALTERAGRO projects). The authors are grateful to Sylvaine Buquet for technical assistance and to Dilys Moscato for her critical reading of the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2013.05.040. References Amellal, S., Boivin, A., Ganier, C.P., Schiavon, M., 2006. High sorption of phenanthrene in agricultural soils. Journal Agronomy for Sustainable Development 26, 99e106. Blanchard, M., Teil, M.J., Guigon, E., Larcher-Tiphagne, K., Ollivon, D., Garban, B., Chevreuil, M., 2007. Persistent toxic substance inputs to the river Seine basin (France) via atmospheric deposition and urban sludge application. Science of the Total Environment 375, 232e243. Bouchez, M., Blanchet, D., Bardin, V., Haeseler, F., Vandecasteele, J.P., 1999. Efficiency of defined strains and of soil consortia in the biodegradation of polycyclic aromatic hydrocarbon (PAH) mixtures. Biodegradation 10, 429e435. Cachot, J., Geffard, O., Augagneur, S., Lacroix, S., Le Menach, K., Peluhet, L., Couteau, J., Denier, X., Devier, M.H., Pottier, D., Budzinski, H., 2006. Evidence of genotoxicity related to high PAH content of sediments in the upper part of the Seine estuary (Normandy, France). Aquatic Toxicology 79, 257e267. Cebron, A., Norini, M.P., Beguiristain, T., Leyval, C., 2008. Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDalpha) genes from Gram positive and Gram negative bacteria in soil and sediment samples. Journal of Microbiological Methods 73, 148e159. Cerniglia, C.E., 1992. Biodegradation of polycyclic aromatic hydrocarbons. In: Biodegradation, vol. 3. Springer, Netherlands, pp. 351e368. Couling, N.R., Towell, M.G., Semple, K.T., 2010. Biodegradation of PAHs in soil: influence of chemical structure, concentration and multiple amendment. Environmental Pollution 158, 3411e3420. De Menezes, A., Clipson, N., Doyle, E., 2012. Comparative metatranscriptomics reveals widespread community responses during phenanthrene degradation in soil. Environmental Microbiology 14, 2577e2588. Fismes, J., Perrin-Ganier, C., Empereur-Bissonnet, P., Morel, J.L., 2002. Soil-to-root transfer and translocation of polycyclic aromatic hydrocarbons by vegetables grown on industrial contaminated soils. Journal of Environmental Quality 31, 1649e1656. Grosser, R.J., Warshawsky, D., Vestal, J.R., 1991. Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils. Applied and Environmental Microbiology 57, 3462e3469. Grova, N., Feidt, C., Crepineau, C., Laurent, C., Lafargue, P.E., Hachimi, A., Rychen, G., 2002. Detection of polycyclic aromatic hydrocarbon levels in milk collected near potential contamination sources. Journal of Agricultural and Food Chemistry 50, 4640e4642. Hamdi, H., Benzarti, S., Manusadzianas, L., Aoyama, I., Jedidi, N., 2007. Solid-phase bioassays and soil microbial activities to evaluate PAH-spiked soil ecotoxicity after a long-term bioremediation process simulating landfarming. Chemosphere 70, 135e143. Heemsbergen, D.A., Berg, M.P., Loreau, M., van Hal, J.R., Faber, J.H., Verhoef, H.A., 2004. Biodiversity effects on soil processes explained by interspecific functional dissimilarity. Science 306, 1019e1020. Jeon, C.O., Park, W., Padmanabhan, P., DeRito, C., Snape, J.R., Madsen, E.L., 2003. Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proceedings of the National Academy of Sciences of the United States of America 100, 13591e13596. Johnsen, A.R., Wick, L.Y., Harms, H., 2005. Principles of microbial PAH-degradation in soil. Environmental Pollution 133, 71e84.
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