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Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy
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New Biotechnology xxx (2016) xxx–xxx
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New Biotechnology journal homepage: www.elsevier.com/locate/nbt
Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy Valeria Anconaa , Anna Barra Caracciolob,* , Paola Grennib , Martina Di Lenolab , Claudia Campanalea , Angelantonio Calabresea , Vito Felice Uricchioa , Giuseppe Mascoloa , Angelo Massaccic a b c
Water Research Institute, National Research Council (IRSA-CNR), V.le F. De Blasio 5, 70132 Bari, Italy Water Research Institute, National Research Council (IRSA-CNR), Via Salaria km 29.300, 00015 Monterotondo Rome, Italy Institute of Agro-Environment and Forest Biology, National Research Council (IBAF-CNR), Via Salaria km 29.300, 00015 Monterotondo Rome, Italy
A R T I C L E I N F O
Article history: Received 22 November 2015 Received in revised form 6 September 2016 Accepted 23 September 2016 Available online xxx Keywords: Phyto-assisted bioremediation Poplar Rhizosphere Polychlorinated biphenyls Heavy metals Microbial activity
A B S T R A C T
A plant-assisted bioremediation strategy was applied in an area located in Southern Italy, close to the city of Taranto, historically contaminated by polychlorinated biphenyls (PCBs) and heavy metals. A specific poplar clone (Monviso) was selected for its ability to promote organic pollutant degradation in the rhizosphere, as demonstrated elsewhere. Chemical and microbiological analyses were performed at the time of poplar planting in selected plots at different distances from the trunk (0.25–1 m) and at different soil depths (0–20 and 20–40 cm), at day 420. A significant decrease in PCB congeners and a reduction in all heavy metals was observed where the poplar trees were present. No evidence of PCB and heavy metal reduction was observed in the non poplar-vegetated soil. Microbial analyses (dehydrogenase activity, cell viability, microbial abundance) of the autochthonous microbial community showed an improvement in soil quality. In particular, microbial activity generally increased in the poplar-rhizosphere and a positive effect was observed in some cases at up to 1 m distance from the trunk and up to 40 cm depth. The Monviso clone was effective in promoting both a general decrease in contaminant occurrence and an increase in microbial activity in the chronically polluted area a little more than one year after planting. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Polychlorinated biphenyls (PCBs) are persistent and ubiquitous organic contaminants widely used in industrial applications. Although their production has been banned since the 1970s their high recalcitrance and toxicity have caused the contamination of lakes, sediments and soils and their removal from contaminated ecosystems continues to present a challenge [1,2]. Among remediation strategies, the use of biological systems represents an effective, cost-competitive and environmentally friendly alternative to the thermal and physico-chemical technologies more traditionally used [3]. Microbial metabolism is potentially capable of degrading persistent organic pollutants, including polychlorinated biphenyls (PCBs) [4]. Laboratory studies have identified several bacterial strains able to aerobically transform lower chlorinated congeners through metabolic and co-metabolic
Abbreviations: PCB, Polychlorinated biphenyl. * Corresponding author. E-mail address: [email protected] (A. Barra Caracciolo).
pathways, and anaerobically highly chlorinated ones by using them as electron acceptors [5–8]. The role of specific microbial species in promoting PCB de-halogenation has been evaluated in several reports [9–11]. However, the complete degradative pathways and the environmental parameters involved in PCB transformation need to be better clarified [12]. Consequently, the next major focus of research into PCBs should be to maximise the potential of these natural degraders in order to accelerate environmental degradation processes [13]. Natural microbial communities in contaminated soils can be stimulated to degrade PCBs by providing them with specific nutrients [14] or the degradation can be promoted through the addition of specific degradative microbial populations [11]. Plant-microorganism association can improve PCB degradation in the rhizosphere thanks to synergic interactions between roots and the natural soil microbial community [15–17,12,13]. Plants can help natural microbial communities to transform, remove and contain contaminants in soil in so-called plant-assisted bioremediation [18]. Many plant species are capable of thriving in PCB contaminated soils and can stimulate indigenous soil populations with a degradative capability [3,19–21]. Plant roots tend to transfer
http://dx.doi.org/10.1016/j.nbt.2016.09.006 1871-6784/ã 2016 Elsevier B.V. All rights reserved.
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contaminants from the bulk soil to the rhizosphere where microbial activity and toxic compound degradation are promoted by root exudates. Some exudates may contain plant secondary metabolites, which can act as chemical signals promoting or inducing the bacterial enzymes involved in PCB degradation [12,13,22–25]. In return, degrading bacteria can produce plant growth stimulators or can suppress pathogens through competition and antibiotic production [26,27]. A wide range of plant genera has been shown to enhance the dissipation of PCBs in soil, from trees such as Populus and Salix to different forages, both grasses and legumes [28–30,15]. In particular poplar, owing to its fast growth rate and deep and wide-spreading root system, plus its ability to grow in nutrient poor soil and to resist high metal concentrations [31–34] it has been used successfully to stimulate the biodegradation of xenobiotic compounds. both in field [35–37] and microcosm studies [13,38]. Poplar was also found to be effective in heavy metal phytoremediation experiments [39]. However, only a few investigations with large-scale trials have been attempted and were often not completed [16,40]. No evidence of PCB degradation in the field has been found so far in historically and multi-contaminated soils, where PCB molecules are strongly bound to soil particles and therefore less available for biodegradation processes. Consequently, carrying out field experiments in degraded sites is a challenge whose implementation makes it possible to select new candidate plant and microbe species and/or identify bioremediation strategies for recovery of multi-contaminated soils. In the present study an area in Southern Italy historically contaminated by PCBs and heavy metals was planted with a selected poplar clone in order to test its ability to promote PCB bioremediation. The overall results of the chemical and microbiological analyses performed 420 days after poplar planting showed not only a significant decrease in all PCB congeners detected, but also a considerable heavy metal phyto-containment and a general increase in the microbial activity of the autochthonous microbial community. 2. Materials and methods 2.1. Experimental site and soil characterisation The study site was located in Southern Italy (40 280 04.9200 N, 17180 12.6800 E) close to the city of Taranto (Apulia Region). The northern part was next to a small power station with electrical transformers. Uncontrolled spilling and improper disposal of dielectric fluids have resulted in PCB pollution over a 30-year period. Moreover, since the same site was used as an uncontrolled waste dump, different waste layers (e.g. construction rubbish, polluted sediments originating from the “Mar Piccolo” facing it) accumulated above the original limestone soil; soil texture analysis confirmed the presence of inhomogeneous materials in the original topsoil due to the accumulation of different kinds of waste. The area was also characterised by a non-homogeneous natural vegetation cover with various widespread non-vegetated zones. A previous analysis by the Environmental Agency of the Apulia Region found PCB contamination at several soil sample points, exceeding the national legislative limit of 60 ng/g for gardens, parks and residential areas (Italian D. Lgs. 152/06), with the highest value being 220 ng/g at a particular point referred to here as PCBcontaminated plot zone P1. A preliminary sampling of the experimental site (more than 30 points in about 3.400 m2) was performed for a characterisation of the soil quality in terms of pH, main nutrients (N, P), organic carbon content and contaminant occurrence (PCB and heavy metals).
Waste and stone removal, ploughing, scarifying, hand weeding and soil milling were carried out in both the planting and nonplanting areas. 2.2. Poplar planting design The hybrid poplar genotype Monviso (Populus generosa Populus nigra) was selected for its ability, tested in a previous bioremediation study, to promote hexachlorocyclohexane degradation [37,41]. In April 2013 about 650 poplar cuttings were planted in a site sub-area of 785 m2 including the PCB and heavy metal contaminated plot zone (P1) and the heavy metal contaminated plot zones P2, P3 and P4. The cuttings were placed in 8 rows (each 2 m from the other). Inside each row the cuttings were at a distance of 0.5 m from each other. Before planting, a suitable irrigation system was set up (a drip tube for each row) in order to provide water every 0.2 m. Mulching was also performed, in order to control weeds in the early stages of poplar growth. The experimental area was supervised weekly to assess plant growth and health. Plant growth was well documented by taking a photographic record over time. 2.3. Sampling of soil, roots and leaves A soil sampling was performed before the poplar planting (t = day 0, March 2013) for the chemical (PCBs and heavy metals) and microbiological analyses (total microbial abundance, cell viability, microbial activity). Another sampling was performed 14 months (June 2014) after planting in order to assess chemical (PCBs and heavy metals both in soil and plant tissue) and microbiological (total microbial abundance, cell viability, microbial activity) parameters. Four tree-planted contaminated plot zones (P1, P2, P3 andP4) and one untreated plot (control contaminated plot) were selected. The area of each plot was 1 m2, with the plant at its centre (Fig. 1). Using a spiral auger (2.5 cm diameter) soil samples were collected both at 0 cm from the trunk (at 0–30 cm depth, up to where the rhizosphere was developed) and at different distances from the trunk and from the surface. In particular, four samples were collected at a 0.25 m distance from the trunk in the surface soil (0–20 cm) and another four at 20–40 cm depth. Similarly, four samples were collected at 1 m distance from the trunk in the surface soil (0–20 cm) and another four at 20–40 cm depth (Fig. 1). Composite soil samples were made using the four samples from the same distance and depth and designated A (0.25 m, 0–20 cm), B (0.25 m, 20–40 cm), C (1 m, 0–20 cm), and D (1 m, 20–40 cm), Fig. 1. Roots were found and sampled at 0 m from the poplar trunk, at 0–30 cm of depth. Leaf samples were collected from the outer canopy located in the upper third of each tree, the area in which it is reported that leaves tend to accumulate more mineral elements than inner ones due to their higher transpiration rates [42,43]. For each target plant, leaves were sampled by cutting four branches (one for each cardinal point in order to consider all possible sun exposure) and placed in polyethylene bags sealed with a nonmetallic closure. Equal amounts of leaves, detached from the four branches, were then mixed to obtain a single representative sample. After sampling the vegetal material was kept at 4 C until processing in the lab. Before all chemical analyses, all leaves were carefully washed with tap water and rinsed with deionised water to remove any soil and surface dust [44]. In order to eliminate soil particles adhering to roots, and to ensure that only the PCBs and heavy metals absorbed by the plant, and not those adsorbed on the radical wall, were measured, the washing step was performed according to Barillot et al. [45]. Firstly, each root system was manually stirred for 10 min, then washed in a saline solution (0.9%
Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006
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Fig. 1. Soil sampling design for each poplar-treated plot (P1, P2, P3 and P4).
NaCl) for another 10 min and finally rinsed quickly under running water on a sieve. Plant tissues were dried at room temperature for five days, oven-dried at 40 C for 24 h, triturated in a ceramic mortar to obtain a fine powder and stored in a drier until the extraction and mineralisation steps were carried out. To evaluate the moisture content of the vegetable matrices, fresh leaves (10 g) and roots (1 g) were weighed; subsequently they were oven-dried at 60 C for 3 d and immediately reweighed at intervals of 3 h until a constant weight was achieved. 2.3.1. Soil organic carbon, pH and water content Each soil sample (about 500 g) was homogenised, air dried at room temperature for 2 weeks and then crushed and pulverised to pass through a 2 mm sieve. Each sieved sample was ground manually using a mortar and a pestle and some aliquots were used to determine dry weight (60 C, 3 days), pH and organic carbon content. Organic carbon was determined following the Walkley & Black Method [46]. The pH value was measured using a portable pH meter (HI 9124, Hanna Instruments) in a 1:2.5 soil-water suspension [47].
2.4. Chemical analyses 2.4.1. PCB extraction and analytical procedures PCBs were extracted from plant and soil samples with an Accelerated Solvent Extractor (ASE 300 DIONEX). Samples were purified on-line, in an ASE extraction cell, using Silica Gel activated for one night at 200 C and then mixed for 4 h with concentrated sulphuric acid (3:2) in accordance with 210 DIONEX Technical notes. The extraction solvent used was n-hexane operating at 100 C and 1500 psi. The extract was then concentrated to 1 mL using a TurboVap (Caliper Life Sciences TurboVap II Concentration Workstation). Analyses were performed by gas chromatography (GC) with a mass spectrometric detector on a Trace GC ultra, equipped with a 30-m 0.25-mm 0.25-mm FactorFour VF–5 ms capillary column, connected to a MS Polaris Q (Thermo Scientific) operated in the electron impact ionization (EI) mode. Two ions were monitored for each PCB homologous group. The injector and transfer line temperatures were 280 and 250 C, respectively. The oven temperature was held at 80 C for 2 min, increased at first to 160 C at a rate of 25 C/min, then increased to 210 C at a rate of 4 C/min, further increased to 280 C at a rate of 10 C/min, and finally increased to 310 C at a rate of 30 C/min held for 4 min.
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Helium was employed as the carrier gas with a constant flow of 1.1 mL/min. For quantitative analysis, a PCB mixture standard containing 17 PCB congeners was used. Quantification was based on a five-point calibration curve and data were reported as ng of PCBs per g of dry sample. Average values of at least two replicates were taken for each analysis. 2.4.2. Heavy metal analysis Four trace elements (V, Cr, Sn, Pb) were measured by ICP-MS (inductively coupled plasma-mass spectrometry) in digested samples of soil, aerial parts and roots. Firstly, the dried material was milled to a fine powder in a mortar and then accurately weighed and mineralised. Mineralisation was performed by treating 500 mg of powdered samples with 9 mL of concentrated HCl and 3 mL of HNO3 [42] and by heating (Ethos Touch Control, Milestone, Microwave Laboratory Systems) in a two-step procedure: 10 min to reach 200 C followed by 15 min at 200 C. After cooling, the mineralised samples were transferred into vials, brought to 50 mL volume with MilliQ water and diluted 25 times before ICP-MS analysis, in order to have a maximum content of 5% of acids and of 0.2% dissolved solids. Quantification of all mineral elements was performed by mass spectrometry with an inductively coupled plasma source (ICP-MS) equipped with 7700x Agilent (Agilent Technologies, Japan). Standard Reference Materials (Multi-element calibration standards 2A, Agilent) were used for precision, quality assurance and checking the measurements. The average values of three replicates were taken for each analysis. 2.5. Microbiological analyses 2.5.1. Total microbial abundance, cell viability, dehydrogenase activity Total microbial abundance (N. cell/g soil) was measured by the epifluorescence direct count method, using DAPI (40 ,6-diamidino2-phenylindole) as the DNA fluorescent dye. The DAPI method is able to detect all the microbial cells in a sample whatever their physiological state and metabolic activity and for this reason is suitable for total microbial counts [48]. At day 420 three soil (rhizosphere) or composite subsamples (1 g each) were analysed for each experimental plot considered in the study (Rhizosphere, A, B, C, D soil). At day 0, topsoil (0–20 cm) only was used for microbiological analyses. All samples were immediately transferred into a fixed solution containing 1.5% formaldehyde and 0.5% Tween 20. After shaking, the suspension was left for 24 h to allow larger particles to settle out. DAPI was then added to an aliquot of the supernatant (30 min in the dark at 4 C) and subsequently the solution was filtered through a 0.2 mm polycarbonate filter. The microbial cells were counted under a Leica DM 4000 B fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) [48]. Similarly, in order to estimate cell viability (% live cells/ live + dead), three sub-sample replicates (1 g each) of fresh soil were analysed for each plot considered in the study (Topsoil for t = 0 days; Rhizosphere, A, B, C and D soil for t = 420 days). Two fluorescent dyes, SYBR Green II and propidium iodide (Sigma– Aldrich, Germany), were used to distinguish viable (green) and dead (red) cells under the fluorescence microscope (Leica DM 4000B Leica Microsystems GmbH, Wetzlar, Germany), as reported previously [49–51]. This method makes it possible to assess microorganism viability because the red propidium iodide can only enter cells that are dead or damaged (which could be detected by DAPI stain because DNA has a relatively high stability). Finally, three other soil sub-samples (5 g each) were collected from each plot to analyse microbial dehydrogenase activity (Topsoil for t = 0 days; Rhizosphere, A, B, C and D soil for t = 420 days). Dehydrogenase activity is a good indicator for evaluating the status and functioning of soil microbial communities, as it reflects microbial respiration rate, which provides
information on the active portion of the soil microbial community [49,52]. This method is based on extraction and colorimetric determination of the intensely coloured 2,3,5-triphenyl formazan (TPF) produced from the reduction of colourless 2,3,5-triphenyltetrazoliumchloride (TTC) in soil samples 24 h after an incubation at 37 C in the dark [49,52]. Soil dehydrogenase activity was expressed as mg TPF/g dry soil. 2.6. Statistical analysis Analysis of variance (one-way analysis of variance) was used to assess the significant differences in PCB and heavy metal concentration, dehydrogenase activity, total microbial abundance and organic carbon among the soil samples before and after the poplar planting. The PC Program used was SIGMASTAT 3.1 software (Systat Software Inc., Point Richmond, USA). 3. Results 3.1. Poplar growth Over 99% of the poplar cuttings took root, the vegetative growth of cuttings being rapid and luxuriant. During the summer (two months after planting), an attack by the parasitic wasp Messa hortulana was observed on some of the poplar leaves; however, the plants displayed an optimal resistance and the damage was limited and no longer visible after a short time. The Monviso clone proved able to grow luxuriantly in this area chronically polluted by PCBs and heavy metals and 420 days (14 months) after their planting, the trees were 5 m high. 3.2. Soil physico-chemical parameters The pH average value was 8 0.25, the organic carbon content (OC) was quite low (15.02 3.21 g/kg) and very low values of soil mineral nutrients (N = 0.17 0.07 g/kg, P = 8.12 1.81 mg/kg) were also found. These values are indicative of a reduced content of nutrients in the soil [53]. In the P2, P3 and P4 contaminated plot zones, the soil organic carbon content (OC) increased 420 days after poplar planting as compared to its initial average values, and this increase was particularly evident in the corresponding rhizosphere (p < 0.05) soil samples. On the contrary, in the P1 plot the OC values generally decreased and in the rhizosphere they halved. The average pH values remained similar to the initial ones (pH = 8 0.5). In line with the watering of the trees and rhizosphere formation, soil water content values were generally higher at day 420 than day 0, and the highest values were found in the rhizosphere soil samples. 3.3. PCB disappearance in soil Chemical analyses were performed on the original topsoil collected at the survey site before planting the poplars (t = 0 d topsoil) and on soil and plant tissue (leaves and roots) samples collected in the selected contaminated plot zones (P1, P2, P3, P4) of the poplar area at day 420. Similarly, control soil samples were collected from the non-planted control plot at days 0 and 420, respectively. Chemical analysis showed a high occurrence of PCBs (values exceeding the Italian legal limit by fivefold) and heavy metals (V, Cr, Sn and Pb) in four different sub-areas of the site (here referred to as P1, P2, P3 and P4 contaminated plot zones) included in the planting area. For this study, an additional PCB contaminated plot was identified outside the poplar planting area at a distance of 105 m from P1 and was considered the control.
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Table 1 PCB congeners concentrations (average values expressed as ng/g standard deviation) detected in: (i) soil samples at day 0 from poplar planting (topsoil: 0–20 cm depth) and at day 420 (Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth); (ii) plant tissues at day 420 from the poplar planting.
Control soil sample
P1
PCB (ng/g)
28
52
101
118
153
105
138
180
TOT
t=0 t = 420 d
1.82 0.09 7.46 1.83
8.75 1.20 26.08 3.61
27.25 1.13 33.57 0.78
24.87 0.35 39.77 0.22
106.5 4.06 86.33 2.03
12.50 0.57 20.79 0.35
67.75 0.46 77.43 0.22
111.5 7.7 79.3 6.78
360.95 15.66 370.73 15.84
topsoil t = 0
7.25 0.07
11.90 0.42
10.35 0.77
25.75 0.35
59.75 1.76
15.55 1.34
73.25 3.18
42.15 0.77
245.95 8.69
Rhizosphere A B C D leaves roots
0.25 0.00 0.41 0.23 0.25 0.00 0.96 0.35 0.41 0.09 2.44 1.26 0.25 0.00
0.56 0.43 0.73 0.13 0.54 0.41 1.08 0.35 0.88 0.15 1.66 0.75 3.21 1.04
1.05 0.77 1.08 0.10 0.82 0.41 1.53 0.02 1.99 2.10 1.38 2.09 4.00 1.23
1.55 0.50 1.60 0.12 1.49 0.21 1.98 0.10 1.52 0.19 3.58 1.06 10.76 0.07
4.04 2.41 3.88 0.61 2.90 1.37 5.73 1.05 14.11 3.49 6.58 2.33 12.23 3.14
1.07 0.81 1.45 0.01 1.35 0.07 1.61 0.48 1.43 0.09 2.23 2.34 0.25 0.00
3.74 2.31 3.24 0.00 2.51 0.97 4.11 0.67 7.09 1.45 5.57 1.75 10.49 2.53
5.84 2.92 5.44 0.83 4.07 2.36 7.44 1.78 20.90 4.69 6.54 2.84 9.42 2.15
18.12 11.27 17.86 2.03 13.95 5.8 24.46 4.8 48.35 12.28 30.01 11.88 50.63 10.16
In particular, at day 0, 17 different PCBs (28, 52, 77, 81, 101, 105, 114, 118, 123, 126, 138, 153, 157, 167, 169, 180, and 189) were searched for and PCB contamination was found in only the P1 plot zone. Eight PCB congeners (28, 52, 101, 105, 118, 138, 153 and 180) were detected in all P1 samples collected at different distances from the trunk and depths and the sum of their concentration was 246 ng/g; PCB-138 alone exceeded the legal concentration limit of 60 ng/g. Interestingly, the lower chlorinated congeners 28 and 52 were present in the original soil with%ages of just 2.95% and 4.84% of the total PCBs found. In the control plot soil at day 0, the sum of PCB concentration was comparable to that of plot 1 (360 ng/g) and similarly the most abundant congeners were 180, 153 and 138. At day 420 a significant decrease (p < 0.01) in total PCB concentration was observed both in the rhizosphere soil and at difference distances from the trunk and depths (A–D) in the P1 plot (Table 1). All the congener values were significantly below the legal limit. The highest residual PCB values were found for the congeners 101, 153, 180 in the soils sampled at 20–40 cm depth and at 1 m distance from the poplar trunk and rhizosphere (D soil, Fig. 2).
In all the soil samples (rhizosphere soil, A–D composite soils) the congeners 52, 118 and 138 decreased by more than 90% of their initial concentration. The highest%age removal of all congeners investigated was found in the rhizosphere and in both soils sampled at 0.25 m of distance from the trunk (A and B, respectively). 3.4. PCB concentration in roots and leaves Chemical analysis of the plant tissues sampled in the P1 contaminated plot zone revealed that total PCB concentration was higher than that observed in each soil sample analysed (rhizosphere, A, B, C, D soils), and equal to 51 and 31 ng/g in root and leave samples, respectively (Table 1). PCB-28, the lowest chlorinated congener analysed (3 Cl), was found in a higher concentration in leaves than in root samples and PCB-52 (4 Cl) was present more in roots than in leaves. The latter result is in accordance with Liu et al. [35], who report that owing to its higher Kow value, it can bind strongly to root tissue, which consequently hinders entry into the transpiration stream. The transportation of PCBs from soil to
Fig. 2. Residual percentages (average values standard deviation, %) of PCB congeners in soils sampled at different distances and depths from the trunk (Rhizosphere, A, B, C, D), 420 days after the poplar planting. Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth.
Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006
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Fig. 3. Bioaccumulation factor (BAF, average values standard deviation) of each single PCB congener calculated for leaves and roots 420 days after the poplar planting.
poplar roots or leaves was evaluated by calculating the bioaccumulation factors (BAFs, where BAF plant part = [PCB]plantpart/ [PCB]soil). Root and leaf BAFs, calculated using the soil PCB concentration before (t = 0 d topsoil P1) and 420 days after poplar planting in the rhizosphere soil were 0.22 and 0.38, respectively. These values are in the range (0.14–0.45) of those found by others [54,55] and, given that they are <1, they show that the poplar plant was not substantially able to phyto-extract PCB. The BAF value calculated for each single congener shows a value higher than 0.5 only for the tri-chlorinated congener 28 in leaves and for the tetrachlorinated 52 and penta-chlorinated 101 and 118 in roots (Fig. 3). 3.5. Heavy metal concentrations in soil The soil concentrations of the heavy metals (V, Cr, Sn and Pb) decreased strongly (p < 0.01) 14 months after poplar planting (Table 2) and in most cases was above the Italian legal limit (D.Lgs 152/06) at all points sampled in each plot (Rhizosphere, A–D soil): Cr in P1, P2, P3, P4; V in the P1 and P4; Pb in P1, P2 and P4. Although V and Pb values were still above the legal limits of 90 mg/kg and 100 mg/kg, respectively, their concentrations (except for the D sample) in the P3 plot halved when compared to their values at 0 d (Table 2). The highest decrease in heavy metal concentrations was found in topsoil at 25 cm from the poplar trunk (A) and in the rhizosphere soil. In the soil samples collected from the nonplanted area (control) at day 420 no significant decrease in heavy metal concentrations was observed (Table 2). 3.6. Heavy metal concentrations in roots and leaves Heavy metal concentrations in plant tissues (leaves and roots) were generally higher in roots than in leaves, except in the case of Pb and Sn in P1 and of Pb in P2, (Table 2). Consequently, the translocation factors (TF), calculated as the ratio of metal concentration in leaves to that in roots [56], were >1 only in the latter cases (Table 2).
3.7. Microbiological analysis The results of the microbiological analysis performed on soil sampled before the poplar planting (t = 0) and at day 420 in the rhizosphere and at different distances and depths from the poplar trunk soils (A–D) are shown in Table 3. The values of total microbial abundance and cell viability at day 0 were not directly related (ANOVA test not significant) either to OC content or to dehydrogenase activity (DHA). Although DHA is generally influenced positively by organic matter, because it stimulates microbial activity [49,52], the lowest values were found in the P1 (10.89 mg TPF/g) and control (19.05 mg TPF/g) plots, where the OC values were the highest (Table 3). Such low values of dehydrogenase activity indicate that the microbial community was not in a good condition [49,52,57] and are in line with the low quality state of the soil owing to the joint presence of PCBs and heavy metals, which presumably had a detrimental effect on microbial activity. At day 420 in the control, nonplanted soil microbiological parameters remained unvaried, demonstrating that the contamination persisted, while in the P1 rhizosphere a significant increase (p < 0.01) in DHA in line with a decrease in PCBs and heavy metals was observed. At the same time, the decrease in total microbial abundance and organic carbon content observed in the P1 rhizosphere may be ascribable to the stimulation of specific bacterial populations within the microbial community, that activated and transformed PCBs. Finally, the decrease in soil organic carbon is in line with its probable use as an electron donor for PCB reductive dehalogenation [58]. At day 420 a significant increase in dehydrogenase activity (DHA) was also found in the P3 and P4 plots, where a significant decrease in heavy metal pollution was observed. In these plots poplar planting also favoured an increase in soil organic carbon content. Finally, when plotting DHA values vs OC ones at day 420, a positive correlation was found (p < 0.01), suggesting a significant improvement in soil quality.
Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006
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V. Ancona et al. / New Biotechnology xxx (2016) xxx–xxx Table 2 Heavy metal concentrations (average values standard deviation) in soils and plant tissues sampled (leaves, roots; TF: translocation factors) at day 0 (t = 0) and 420 days after poplar-planting in different plots (P1, P2, P3 and P4). The metal concentrations above the National legal limit in D.Lgs 152/06 are in bold (V: 90 mg/kg; Cr: 150 mg/ kg; Sn: 1 mg/kg; Pb: 100 mg/kg). V
Cr
Sn
Pb
P1
t = 0 topsoil rhizosphere A B C D leaves roots TF
127.4 7.8 43.8 8.3 37.2 3.9 43.7 7.8 46.0 6.6 45.7 3.6 0.04 0.02 0.86 0.03 0.04
179.5 10.6 82.0 8.0 67.6 7.7 79.3 9.2 57.5 3.6 79.4 7.4 11.3 6.58 31.5 2.44 0.35
5.9 2.1 2.4 1.3 2.8 2.0 2.1 0.4 2.9 0.4 2.5 0.9 0.99 1.20 0.41 0.19 2.41
74.5 7.7 14.1 1.0 15.0 0.4 16.0 3.5 23.4 9.5 14.8 1.9 12.7 2.9 6.3 7.1 2.00
P2
t = 0 topsoil rhizosphere A B C D leaves roots TF
66.6 4.1 43.8 2.7 40.0 0.4 41.6 5.7 41.8 2.8 40.6 6.3 0.05 0.01 0.40 0.05 0.12
60.3 15.4 48.9 6.1 47.4 3.0 46.0 3.6 41.9 6.3 45.1 8.3 9.7 5.0 20.4 7.5 0.48
60.8 7.6 3.5 0.2 3.0 1.0 4.1 2.3 3.1 0.6 5.9 1.6 <0.6 0.97 0.8 –
193.8 5.2 41.6 7.4 40.0 6.6 48.8 8.9 39.2 6.1 58.1 8.8 10.7 3.7 4.2 0.5 2.51
P3
t = 0 topsoil rhizosphere A B C D leaves roots TF
275.7 15.7 117.9 3.3 105.3 12.2 344.8 6.9 141.7 7.1 129.6 3.0 0.38 0.54 1.02 0.08 0.37
283.7 1.79 83.7 2.7 67.8 5.6 69.8 3.6 83.2 3.9 141.9 4.9 8.1 2.8 22.0 4.0 0.36
15.3 5.72 4.2 1.3 5.2 1.1 4.9 0.7 7.0 1.3 15.6 3.4 0.6 0.54 3.0 3.84 0.19
289.1 13.03 102.4 8.3 93.1 11.4 131.6 5.7 136.0 5.0 90.6 3.1 0.6 0.54 20.0 3.95 0.03
P4
t = 0 topsoil rhizosphere A B C D leaves roots TF
336.4 7.5 51.6 4.3 90.4 6.4 50.9 3.6 65.8 7.2 48.7 1.6 0.3 0.56 1.0 0.15 0.35
217.0 3.4 43.5 8.7 56.9 8.3 39.5 0.8 41.5 2.9 37.9 1.6 16.3 4.2 24.3 11.4 0.67
10.5 3.2 6.2 2.8 3.6 1.5 4.5 0.6 4.7 1.2 4.6 1.5 1.0 0.79 2.0 1.01 0.51
199.0 11.23 41.4 7.8 74.4 5.2 38.6 3.8 58.6 5.0 62.5 7.7 12.0 2.0 15.5 2.9 0.77
61.4 1.6
60.3 1.6
10.5 0.6
144.1 11.1
63.7 1.8
62.9 1.3
12.3 1.6
147.3 9.6
Control Topsoil t = 0 d Topsoil 420 d
Topsoil: 0–20 cm depth; Rhizosphere: soil attached and surrounding roots, 0– 30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0– 20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth
4. Discussion The significant decrease in all PCB congeners, to less than the Italian environmental law limit (60 ng/g), in the poplar-planted plot shows the effectiveness of the Monviso clone at promoting the degradation of these persistent compounds. Confirming this result is the fact that in the control plot soil, outside the poplar-planting area, PCBs had not decreased by day 420 (Table 1). As can be seen in Fig. 2, a decreasing trend in the percentage of PCB removal is observable from the soil points at higher distances (C and D) to those closer to the rhizosphere (A and B), showing a clear role in chemical removal. The overall low BAF values in leaves being just higher than 0.5 for the congener 28 pointed to there being a negligible transportation of PCBs into poplar tissue. Owing to their high hydrophobicity, PCBs are not expected to enter the transpiration stream. Nevertheless, in such cases transportation to within plants
7
may occur, owing to plant root exudates, which can act as surfactants forming a more hydrophilic complex with PCBs, making their transportation into plants possible [59,60]. Heavy metals were found in the poplar tissue, but their concentrations were generally higher in roots than in leaves (Table 2). The low values for translocation factors indicate that transport to upper parts of the plant was quite limited [56]. The translocation of metals can be toxic for photosynthetic activity, chlorophyll synthesis and antioxidant enzymes [61] and for this reason some authors report the presence of exclusion mechanisms in some plant species [62]. The Monviso clone was therefore not a hyperaccumulator plant, but merely able to bioconcentrate heavy metals, avoiding their translocation; consequently it was shown to be useful for heavy metal phyto-stabilisation [63]. This process exploits the ability of plant roots to change environmental conditions via root exudates. Plants can immobilise heavy metals through absorption and accumulation by roots, adsorption onto roots or precipitation within the rhizosphere. This process reduces the mobility of the metals and leaching into ground water, as well as their bioavailability in the food chain. One advantage of this strategy is that the disposal of the metal-laden plant material is not required [63]. However, there needs to be research into the turnover of nutritive roots and the potential release of metals from decomposing roots [64]. The initially low values for dehydrogenase activity at all sampled points indicated that the microbial community was not in a good condition. DHA is a good indicator of soil quality state, because it is affected by different factors such as organic matter content and its quality [57], oxygen and contamination. Heavy metals can reduce microbial activity because they interact with enzyme-substrate complexes and can influence the synthesis of bacterial cellular enzymes [65]. Moreover, soil aeration may also influence this activity, being an oxidative process. In the soil investigated, many initial factors therefore co-existed, such as PCBs, heavy metals and the absence of both a pedologically evolved soil and a homogeneous soil texture. The overall increase in dehydrogenase activity 420 days after the poplar planting in the P1, P2, P3 and P4 plots is therefore ascribable to rhizosphere formation, which is known to favour microbial activity [66,51]. Moreover, the simultaneous occurrence of the increase in microbial activity and the PCB degradation suggests that the presence of poplar directly favoured some natural bacterial populations in their degradation and demonstrates a synergistic microbial-plant action in soil quality recovery. The use of poplar, for plant-assisted bioremediation of sites polluted by PCBs and heavy metals, has been tested in other experiments [67,42,13]. Although the Monviso clone selected has already been used in Central Italy for promoting hexachlorocyclohexane degradation [41,37], this is the first time that the same clone has been successfully applied in a multi-contaminated area in Southern Italy. Poplar-assisted bioremediation can be considered an economical, environmentally sustainable and landscapeenhancing technology, complying with green remediation, as it has a low impact on climate change and on the environment, in line with environmental sustainability as reported by the US EPA [68]. 5. Conclusions The Monviso clone was applied to a historically contaminated soil to promote PCB biodegradation through its root system. At about 1 year after planting, the overall results of the chemical analyses (PCBs and heavy metals) showed the effectiveness of this green technology at recovering degraded soil not only from organic, but also from inorganic contamination (phyto-containment, [69]). The soil remediation strategy is still being applied and the planting of the Monviso clone in other contaminated parts of
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Table 3 Average values (standard error, s.e.) of Total microbial abundance, Cell viability, Dehydrogenase activity and Organic carbon content in soils sampled at day 0 (t = 0) and 420 days after poplar-planting in different plots (P1, P2, P3 and P4). Total microbial abundance N. cells/g s.e.
Cell viability % s.e.
Dehydrogenase activity mg TPF/g s.e.
Organic carbon g/kg s.e.
P1
topsoil t = 0 Rhizosphere A B C D
2.11 * 107 2.4 * 105 4.66 * 106 5.1 * 105 1.38 * 107 1.1 * 106 2.17 * 107 1.1 * 106 1.17 * 107 1.1 * 106 1.74 * 107 1.2 * 106
59.7 6.2 36.6 5.1 20.2 3.2 17.9 4.5 20.2 1.5 46.1 5.5
8.96 3.5 31.9 1.2 37.3 4.5 28.9 11.8 32.9 0.9 27.9 2.0
10.9 1.2 4.8 0.6 7.8 0.4 6.0 0.5 6.4 1.0 5.9 0.2
P2
topsoil t = 0 Rhizosphere A B C D
9.84 * 106 1.8 * 105 1.33 * 107 1.4 * 106 1.57 * 107 9.7 * 105 1.52 * 107 4.1 * 105 9.84 * 106 4.0 * 105 1.03 * 107 1.9 * 106
57.5 7.0 19.7 2.0 20.0 1.2 32.3 6.2 15.7 1.8 38.6 5.2
84.5 7.2 82.4 5.6 47.27 1.1 44.8 5.7 37.5 1.3 35.06 0.7
9.5 0.9 12.5 0.5 8.5 0.6 8.4 0.5 9.5 0.4 8.6 0.4
P3
topsoil t = 0 Rhizosphere A B C D
1.40 * 107 1.5 * 106 3.89 * 106 2.6 * 105 7.47 * 106 1.0 * 106 2.31 * 107 2.1 * 106 1.19 * 107 9.2 * 105 7.85 * 106 9.6 * 105
43.2 5.3 19.5 2.2 27.3 3.5 20.2 3.2 52.1 4.2 20.2 1.6
125.2 10.0 213.0 12.1 143.4 19.0 140.5 49.3 197.9 11.9 143.0 19.8
10.7 0.4 12.7 0.2 12.4 0.1 11.4 0.2 12.4 0.1 11.5 0.2
P4
topsoil t = 0 Rhizosphere A B C D t=0 d t = 420 d
5.37 * 106 6.4 * 105 9.12 * 106 4.6 * 105 1.63 * 107 8.7 * 105 1.17 * 107 9.8 * 105 7.71 * 106 3.1 * 105 4.74 * 106 3.8 * 105 4.88 * 106 3.9 * 105 4.00 * 106 4.2 * 105
46.5 3.7 53.5 6.1 20.2 2.6 20.2 1.8 20.2 1.8 29.8 1.9 53.2 7.5 51.5 6.1
74.1 9.4 192.9 13.0 131.2 9.5 132.6 8.7 168.6 4.2 113.1 4.4 27.8 3.5 28.2 3.6
11.3 0.5 14.7 0.9 15.0 1.2 15.6 1.2 16.3 0.8 14.0 1.1 19.0 2.1 18.7 1.8
Control soil
Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth.
the same area has been performed. An annual sampling assessment of contaminants, microbial activity and plant biomass production will be carried out for at least the next 3–4 years. Currently, further analysis of the microbial structure and the composition of the microbial populations directly involved in the process of bioremediation is in progress. Acknowledgments The authors thank Giuseppe Bagnuolo (CNR-IRSA) for his technical support in the chemical analyses of the pollutants, Guido Del Moro, Andrea Decembrino and Vito Nicola Palmisano (CNRIRSA) for their useful assistance in the poplar planting, and Francesca Falconi (CNR-IRSA) for the microbiological analysis. The authors also thank Daniele Bianconi (CNR-IBAF) for his scientific support in the poplar plantation design and the Reverend Nicola Preziuso and Michele Arcangelo D’Alessandro (CEM of Taranto) and Pasquale Carmigiano (CODITA, Taranto) for their technical assistance during the field activities in the study area. Moreover, the authors thank CISA S.p.A. (Massafra, Italy) which partially funded this research within the Research Project “Applicazione di tecniche di fitorimedio a basso costo in località excampo Cimino-Manganecchia a Taranto” Prot. IRSA-CNR N. 0005159, 04/12/2012 . References [1] Furukawa K, Fujihara H. Microbial degradation of polychlorinated biphenyls: biochemical and molecular features. J Biosci Bioeng 2008;105:433–49. [2] Robertson LW, Hansen LG. PCBs recent advances in environmental toxicology and health effects. Lexington: University Press of Kentucky; 2015.
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New Biotechnology xxx (2016) xxx–xxx
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Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy Valeria Anconaa , Anna Barra Caracciolob,* , Paola Grennib , Martina Di Lenolab , Claudia Campanalea , Angelantonio Calabresea , Vito Felice Uricchioa , Giuseppe Mascoloa , Angelo Massaccic a b c
Water Research Institute, National Research Council (IRSA-CNR), V.le F. De Blasio 5, 70132 Bari, Italy Water Research Institute, National Research Council (IRSA-CNR), Via Salaria km 29.300, 00015 Monterotondo Rome, Italy Institute of Agro-Environment and Forest Biology, National Research Council (IBAF-CNR), Via Salaria km 29.300, 00015 Monterotondo Rome, Italy
A R T I C L E I N F O
Article history: Received 22 November 2015 Received in revised form 6 September 2016 Accepted 23 September 2016 Available online xxx Keywords: Phyto-assisted bioremediation Poplar Rhizosphere Polychlorinated biphenyls Heavy metals Microbial activity
A B S T R A C T
A plant-assisted bioremediation strategy was applied in an area located in Southern Italy, close to the city of Taranto, historically contaminated by polychlorinated biphenyls (PCBs) and heavy metals. A specific poplar clone (Monviso) was selected for its ability to promote organic pollutant degradation in the rhizosphere, as demonstrated elsewhere. Chemical and microbiological analyses were performed at the time of poplar planting in selected plots at different distances from the trunk (0.25–1 m) and at different soil depths (0–20 and 20–40 cm), at day 420. A significant decrease in PCB congeners and a reduction in all heavy metals was observed where the poplar trees were present. No evidence of PCB and heavy metal reduction was observed in the non poplar-vegetated soil. Microbial analyses (dehydrogenase activity, cell viability, microbial abundance) of the autochthonous microbial community showed an improvement in soil quality. In particular, microbial activity generally increased in the poplar-rhizosphere and a positive effect was observed in some cases at up to 1 m distance from the trunk and up to 40 cm depth. The Monviso clone was effective in promoting both a general decrease in contaminant occurrence and an increase in microbial activity in the chronically polluted area a little more than one year after planting. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Polychlorinated biphenyls (PCBs) are persistent and ubiquitous organic contaminants widely used in industrial applications. Although their production has been banned since the 1970s their high recalcitrance and toxicity have caused the contamination of lakes, sediments and soils and their removal from contaminated ecosystems continues to present a challenge [1,2]. Among remediation strategies, the use of biological systems represents an effective, cost-competitive and environmentally friendly alternative to the thermal and physico-chemical technologies more traditionally used [3]. Microbial metabolism is potentially capable of degrading persistent organic pollutants, including polychlorinated biphenyls (PCBs) [4]. Laboratory studies have identified several bacterial strains able to aerobically transform lower chlorinated congeners through metabolic and co-metabolic
Abbreviations: PCB, Polychlorinated biphenyl. * Corresponding author. E-mail address: [email protected] (A. Barra Caracciolo).
pathways, and anaerobically highly chlorinated ones by using them as electron acceptors [5–8]. The role of specific microbial species in promoting PCB de-halogenation has been evaluated in several reports [9–11]. However, the complete degradative pathways and the environmental parameters involved in PCB transformation need to be better clarified [12]. Consequently, the next major focus of research into PCBs should be to maximise the potential of these natural degraders in order to accelerate environmental degradation processes [13]. Natural microbial communities in contaminated soils can be stimulated to degrade PCBs by providing them with specific nutrients [14] or the degradation can be promoted through the addition of specific degradative microbial populations [11]. Plant-microorganism association can improve PCB degradation in the rhizosphere thanks to synergic interactions between roots and the natural soil microbial community [15–17,12,13]. Plants can help natural microbial communities to transform, remove and contain contaminants in soil in so-called plant-assisted bioremediation [18]. Many plant species are capable of thriving in PCB contaminated soils and can stimulate indigenous soil populations with a degradative capability [3,19–21]. Plant roots tend to transfer
http://dx.doi.org/10.1016/j.nbt.2016.09.006 1871-6784/ã 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006
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contaminants from the bulk soil to the rhizosphere where microbial activity and toxic compound degradation are promoted by root exudates. Some exudates may contain plant secondary metabolites, which can act as chemical signals promoting or inducing the bacterial enzymes involved in PCB degradation [12,13,22–25]. In return, degrading bacteria can produce plant growth stimulators or can suppress pathogens through competition and antibiotic production [26,27]. A wide range of plant genera has been shown to enhance the dissipation of PCBs in soil, from trees such as Populus and Salix to different forages, both grasses and legumes [28–30,15]. In particular poplar, owing to its fast growth rate and deep and wide-spreading root system, plus its ability to grow in nutrient poor soil and to resist high metal concentrations [31–34] it has been used successfully to stimulate the biodegradation of xenobiotic compounds. both in field [35–37] and microcosm studies [13,38]. Poplar was also found to be effective in heavy metal phytoremediation experiments [39]. However, only a few investigations with large-scale trials have been attempted and were often not completed [16,40]. No evidence of PCB degradation in the field has been found so far in historically and multi-contaminated soils, where PCB molecules are strongly bound to soil particles and therefore less available for biodegradation processes. Consequently, carrying out field experiments in degraded sites is a challenge whose implementation makes it possible to select new candidate plant and microbe species and/or identify bioremediation strategies for recovery of multi-contaminated soils. In the present study an area in Southern Italy historically contaminated by PCBs and heavy metals was planted with a selected poplar clone in order to test its ability to promote PCB bioremediation. The overall results of the chemical and microbiological analyses performed 420 days after poplar planting showed not only a significant decrease in all PCB congeners detected, but also a considerable heavy metal phyto-containment and a general increase in the microbial activity of the autochthonous microbial community. 2. Materials and methods 2.1. Experimental site and soil characterisation The study site was located in Southern Italy (40 280 04.9200 N, 17180 12.6800 E) close to the city of Taranto (Apulia Region). The northern part was next to a small power station with electrical transformers. Uncontrolled spilling and improper disposal of dielectric fluids have resulted in PCB pollution over a 30-year period. Moreover, since the same site was used as an uncontrolled waste dump, different waste layers (e.g. construction rubbish, polluted sediments originating from the “Mar Piccolo” facing it) accumulated above the original limestone soil; soil texture analysis confirmed the presence of inhomogeneous materials in the original topsoil due to the accumulation of different kinds of waste. The area was also characterised by a non-homogeneous natural vegetation cover with various widespread non-vegetated zones. A previous analysis by the Environmental Agency of the Apulia Region found PCB contamination at several soil sample points, exceeding the national legislative limit of 60 ng/g for gardens, parks and residential areas (Italian D. Lgs. 152/06), with the highest value being 220 ng/g at a particular point referred to here as PCBcontaminated plot zone P1. A preliminary sampling of the experimental site (more than 30 points in about 3.400 m2) was performed for a characterisation of the soil quality in terms of pH, main nutrients (N, P), organic carbon content and contaminant occurrence (PCB and heavy metals).
Waste and stone removal, ploughing, scarifying, hand weeding and soil milling were carried out in both the planting and nonplanting areas. 2.2. Poplar planting design The hybrid poplar genotype Monviso (Populus generosa Populus nigra) was selected for its ability, tested in a previous bioremediation study, to promote hexachlorocyclohexane degradation [37,41]. In April 2013 about 650 poplar cuttings were planted in a site sub-area of 785 m2 including the PCB and heavy metal contaminated plot zone (P1) and the heavy metal contaminated plot zones P2, P3 and P4. The cuttings were placed in 8 rows (each 2 m from the other). Inside each row the cuttings were at a distance of 0.5 m from each other. Before planting, a suitable irrigation system was set up (a drip tube for each row) in order to provide water every 0.2 m. Mulching was also performed, in order to control weeds in the early stages of poplar growth. The experimental area was supervised weekly to assess plant growth and health. Plant growth was well documented by taking a photographic record over time. 2.3. Sampling of soil, roots and leaves A soil sampling was performed before the poplar planting (t = day 0, March 2013) for the chemical (PCBs and heavy metals) and microbiological analyses (total microbial abundance, cell viability, microbial activity). Another sampling was performed 14 months (June 2014) after planting in order to assess chemical (PCBs and heavy metals both in soil and plant tissue) and microbiological (total microbial abundance, cell viability, microbial activity) parameters. Four tree-planted contaminated plot zones (P1, P2, P3 andP4) and one untreated plot (control contaminated plot) were selected. The area of each plot was 1 m2, with the plant at its centre (Fig. 1). Using a spiral auger (2.5 cm diameter) soil samples were collected both at 0 cm from the trunk (at 0–30 cm depth, up to where the rhizosphere was developed) and at different distances from the trunk and from the surface. In particular, four samples were collected at a 0.25 m distance from the trunk in the surface soil (0–20 cm) and another four at 20–40 cm depth. Similarly, four samples were collected at 1 m distance from the trunk in the surface soil (0–20 cm) and another four at 20–40 cm depth (Fig. 1). Composite soil samples were made using the four samples from the same distance and depth and designated A (0.25 m, 0–20 cm), B (0.25 m, 20–40 cm), C (1 m, 0–20 cm), and D (1 m, 20–40 cm), Fig. 1. Roots were found and sampled at 0 m from the poplar trunk, at 0–30 cm of depth. Leaf samples were collected from the outer canopy located in the upper third of each tree, the area in which it is reported that leaves tend to accumulate more mineral elements than inner ones due to their higher transpiration rates [42,43]. For each target plant, leaves were sampled by cutting four branches (one for each cardinal point in order to consider all possible sun exposure) and placed in polyethylene bags sealed with a nonmetallic closure. Equal amounts of leaves, detached from the four branches, were then mixed to obtain a single representative sample. After sampling the vegetal material was kept at 4 C until processing in the lab. Before all chemical analyses, all leaves were carefully washed with tap water and rinsed with deionised water to remove any soil and surface dust [44]. In order to eliminate soil particles adhering to roots, and to ensure that only the PCBs and heavy metals absorbed by the plant, and not those adsorbed on the radical wall, were measured, the washing step was performed according to Barillot et al. [45]. Firstly, each root system was manually stirred for 10 min, then washed in a saline solution (0.9%
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Fig. 1. Soil sampling design for each poplar-treated plot (P1, P2, P3 and P4).
NaCl) for another 10 min and finally rinsed quickly under running water on a sieve. Plant tissues were dried at room temperature for five days, oven-dried at 40 C for 24 h, triturated in a ceramic mortar to obtain a fine powder and stored in a drier until the extraction and mineralisation steps were carried out. To evaluate the moisture content of the vegetable matrices, fresh leaves (10 g) and roots (1 g) were weighed; subsequently they were oven-dried at 60 C for 3 d and immediately reweighed at intervals of 3 h until a constant weight was achieved. 2.3.1. Soil organic carbon, pH and water content Each soil sample (about 500 g) was homogenised, air dried at room temperature for 2 weeks and then crushed and pulverised to pass through a 2 mm sieve. Each sieved sample was ground manually using a mortar and a pestle and some aliquots were used to determine dry weight (60 C, 3 days), pH and organic carbon content. Organic carbon was determined following the Walkley & Black Method [46]. The pH value was measured using a portable pH meter (HI 9124, Hanna Instruments) in a 1:2.5 soil-water suspension [47].
2.4. Chemical analyses 2.4.1. PCB extraction and analytical procedures PCBs were extracted from plant and soil samples with an Accelerated Solvent Extractor (ASE 300 DIONEX). Samples were purified on-line, in an ASE extraction cell, using Silica Gel activated for one night at 200 C and then mixed for 4 h with concentrated sulphuric acid (3:2) in accordance with 210 DIONEX Technical notes. The extraction solvent used was n-hexane operating at 100 C and 1500 psi. The extract was then concentrated to 1 mL using a TurboVap (Caliper Life Sciences TurboVap II Concentration Workstation). Analyses were performed by gas chromatography (GC) with a mass spectrometric detector on a Trace GC ultra, equipped with a 30-m 0.25-mm 0.25-mm FactorFour VF–5 ms capillary column, connected to a MS Polaris Q (Thermo Scientific) operated in the electron impact ionization (EI) mode. Two ions were monitored for each PCB homologous group. The injector and transfer line temperatures were 280 and 250 C, respectively. The oven temperature was held at 80 C for 2 min, increased at first to 160 C at a rate of 25 C/min, then increased to 210 C at a rate of 4 C/min, further increased to 280 C at a rate of 10 C/min, and finally increased to 310 C at a rate of 30 C/min held for 4 min.
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Helium was employed as the carrier gas with a constant flow of 1.1 mL/min. For quantitative analysis, a PCB mixture standard containing 17 PCB congeners was used. Quantification was based on a five-point calibration curve and data were reported as ng of PCBs per g of dry sample. Average values of at least two replicates were taken for each analysis. 2.4.2. Heavy metal analysis Four trace elements (V, Cr, Sn, Pb) were measured by ICP-MS (inductively coupled plasma-mass spectrometry) in digested samples of soil, aerial parts and roots. Firstly, the dried material was milled to a fine powder in a mortar and then accurately weighed and mineralised. Mineralisation was performed by treating 500 mg of powdered samples with 9 mL of concentrated HCl and 3 mL of HNO3 [42] and by heating (Ethos Touch Control, Milestone, Microwave Laboratory Systems) in a two-step procedure: 10 min to reach 200 C followed by 15 min at 200 C. After cooling, the mineralised samples were transferred into vials, brought to 50 mL volume with MilliQ water and diluted 25 times before ICP-MS analysis, in order to have a maximum content of 5% of acids and of 0.2% dissolved solids. Quantification of all mineral elements was performed by mass spectrometry with an inductively coupled plasma source (ICP-MS) equipped with 7700x Agilent (Agilent Technologies, Japan). Standard Reference Materials (Multi-element calibration standards 2A, Agilent) were used for precision, quality assurance and checking the measurements. The average values of three replicates were taken for each analysis. 2.5. Microbiological analyses 2.5.1. Total microbial abundance, cell viability, dehydrogenase activity Total microbial abundance (N. cell/g soil) was measured by the epifluorescence direct count method, using DAPI (40 ,6-diamidino2-phenylindole) as the DNA fluorescent dye. The DAPI method is able to detect all the microbial cells in a sample whatever their physiological state and metabolic activity and for this reason is suitable for total microbial counts [48]. At day 420 three soil (rhizosphere) or composite subsamples (1 g each) were analysed for each experimental plot considered in the study (Rhizosphere, A, B, C, D soil). At day 0, topsoil (0–20 cm) only was used for microbiological analyses. All samples were immediately transferred into a fixed solution containing 1.5% formaldehyde and 0.5% Tween 20. After shaking, the suspension was left for 24 h to allow larger particles to settle out. DAPI was then added to an aliquot of the supernatant (30 min in the dark at 4 C) and subsequently the solution was filtered through a 0.2 mm polycarbonate filter. The microbial cells were counted under a Leica DM 4000 B fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) [48]. Similarly, in order to estimate cell viability (% live cells/ live + dead), three sub-sample replicates (1 g each) of fresh soil were analysed for each plot considered in the study (Topsoil for t = 0 days; Rhizosphere, A, B, C and D soil for t = 420 days). Two fluorescent dyes, SYBR Green II and propidium iodide (Sigma– Aldrich, Germany), were used to distinguish viable (green) and dead (red) cells under the fluorescence microscope (Leica DM 4000B Leica Microsystems GmbH, Wetzlar, Germany), as reported previously [49–51]. This method makes it possible to assess microorganism viability because the red propidium iodide can only enter cells that are dead or damaged (which could be detected by DAPI stain because DNA has a relatively high stability). Finally, three other soil sub-samples (5 g each) were collected from each plot to analyse microbial dehydrogenase activity (Topsoil for t = 0 days; Rhizosphere, A, B, C and D soil for t = 420 days). Dehydrogenase activity is a good indicator for evaluating the status and functioning of soil microbial communities, as it reflects microbial respiration rate, which provides
information on the active portion of the soil microbial community [49,52]. This method is based on extraction and colorimetric determination of the intensely coloured 2,3,5-triphenyl formazan (TPF) produced from the reduction of colourless 2,3,5-triphenyltetrazoliumchloride (TTC) in soil samples 24 h after an incubation at 37 C in the dark [49,52]. Soil dehydrogenase activity was expressed as mg TPF/g dry soil. 2.6. Statistical analysis Analysis of variance (one-way analysis of variance) was used to assess the significant differences in PCB and heavy metal concentration, dehydrogenase activity, total microbial abundance and organic carbon among the soil samples before and after the poplar planting. The PC Program used was SIGMASTAT 3.1 software (Systat Software Inc., Point Richmond, USA). 3. Results 3.1. Poplar growth Over 99% of the poplar cuttings took root, the vegetative growth of cuttings being rapid and luxuriant. During the summer (two months after planting), an attack by the parasitic wasp Messa hortulana was observed on some of the poplar leaves; however, the plants displayed an optimal resistance and the damage was limited and no longer visible after a short time. The Monviso clone proved able to grow luxuriantly in this area chronically polluted by PCBs and heavy metals and 420 days (14 months) after their planting, the trees were 5 m high. 3.2. Soil physico-chemical parameters The pH average value was 8 0.25, the organic carbon content (OC) was quite low (15.02 3.21 g/kg) and very low values of soil mineral nutrients (N = 0.17 0.07 g/kg, P = 8.12 1.81 mg/kg) were also found. These values are indicative of a reduced content of nutrients in the soil [53]. In the P2, P3 and P4 contaminated plot zones, the soil organic carbon content (OC) increased 420 days after poplar planting as compared to its initial average values, and this increase was particularly evident in the corresponding rhizosphere (p < 0.05) soil samples. On the contrary, in the P1 plot the OC values generally decreased and in the rhizosphere they halved. The average pH values remained similar to the initial ones (pH = 8 0.5). In line with the watering of the trees and rhizosphere formation, soil water content values were generally higher at day 420 than day 0, and the highest values were found in the rhizosphere soil samples. 3.3. PCB disappearance in soil Chemical analyses were performed on the original topsoil collected at the survey site before planting the poplars (t = 0 d topsoil) and on soil and plant tissue (leaves and roots) samples collected in the selected contaminated plot zones (P1, P2, P3, P4) of the poplar area at day 420. Similarly, control soil samples were collected from the non-planted control plot at days 0 and 420, respectively. Chemical analysis showed a high occurrence of PCBs (values exceeding the Italian legal limit by fivefold) and heavy metals (V, Cr, Sn and Pb) in four different sub-areas of the site (here referred to as P1, P2, P3 and P4 contaminated plot zones) included in the planting area. For this study, an additional PCB contaminated plot was identified outside the poplar planting area at a distance of 105 m from P1 and was considered the control.
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Table 1 PCB congeners concentrations (average values expressed as ng/g standard deviation) detected in: (i) soil samples at day 0 from poplar planting (topsoil: 0–20 cm depth) and at day 420 (Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth); (ii) plant tissues at day 420 from the poplar planting.
Control soil sample
P1
PCB (ng/g)
28
52
101
118
153
105
138
180
TOT
t=0 t = 420 d
1.82 0.09 7.46 1.83
8.75 1.20 26.08 3.61
27.25 1.13 33.57 0.78
24.87 0.35 39.77 0.22
106.5 4.06 86.33 2.03
12.50 0.57 20.79 0.35
67.75 0.46 77.43 0.22
111.5 7.7 79.3 6.78
360.95 15.66 370.73 15.84
topsoil t = 0
7.25 0.07
11.90 0.42
10.35 0.77
25.75 0.35
59.75 1.76
15.55 1.34
73.25 3.18
42.15 0.77
245.95 8.69
Rhizosphere A B C D leaves roots
0.25 0.00 0.41 0.23 0.25 0.00 0.96 0.35 0.41 0.09 2.44 1.26 0.25 0.00
0.56 0.43 0.73 0.13 0.54 0.41 1.08 0.35 0.88 0.15 1.66 0.75 3.21 1.04
1.05 0.77 1.08 0.10 0.82 0.41 1.53 0.02 1.99 2.10 1.38 2.09 4.00 1.23
1.55 0.50 1.60 0.12 1.49 0.21 1.98 0.10 1.52 0.19 3.58 1.06 10.76 0.07
4.04 2.41 3.88 0.61 2.90 1.37 5.73 1.05 14.11 3.49 6.58 2.33 12.23 3.14
1.07 0.81 1.45 0.01 1.35 0.07 1.61 0.48 1.43 0.09 2.23 2.34 0.25 0.00
3.74 2.31 3.24 0.00 2.51 0.97 4.11 0.67 7.09 1.45 5.57 1.75 10.49 2.53
5.84 2.92 5.44 0.83 4.07 2.36 7.44 1.78 20.90 4.69 6.54 2.84 9.42 2.15
18.12 11.27 17.86 2.03 13.95 5.8 24.46 4.8 48.35 12.28 30.01 11.88 50.63 10.16
In particular, at day 0, 17 different PCBs (28, 52, 77, 81, 101, 105, 114, 118, 123, 126, 138, 153, 157, 167, 169, 180, and 189) were searched for and PCB contamination was found in only the P1 plot zone. Eight PCB congeners (28, 52, 101, 105, 118, 138, 153 and 180) were detected in all P1 samples collected at different distances from the trunk and depths and the sum of their concentration was 246 ng/g; PCB-138 alone exceeded the legal concentration limit of 60 ng/g. Interestingly, the lower chlorinated congeners 28 and 52 were present in the original soil with%ages of just 2.95% and 4.84% of the total PCBs found. In the control plot soil at day 0, the sum of PCB concentration was comparable to that of plot 1 (360 ng/g) and similarly the most abundant congeners were 180, 153 and 138. At day 420 a significant decrease (p < 0.01) in total PCB concentration was observed both in the rhizosphere soil and at difference distances from the trunk and depths (A–D) in the P1 plot (Table 1). All the congener values were significantly below the legal limit. The highest residual PCB values were found for the congeners 101, 153, 180 in the soils sampled at 20–40 cm depth and at 1 m distance from the poplar trunk and rhizosphere (D soil, Fig. 2).
In all the soil samples (rhizosphere soil, A–D composite soils) the congeners 52, 118 and 138 decreased by more than 90% of their initial concentration. The highest%age removal of all congeners investigated was found in the rhizosphere and in both soils sampled at 0.25 m of distance from the trunk (A and B, respectively). 3.4. PCB concentration in roots and leaves Chemical analysis of the plant tissues sampled in the P1 contaminated plot zone revealed that total PCB concentration was higher than that observed in each soil sample analysed (rhizosphere, A, B, C, D soils), and equal to 51 and 31 ng/g in root and leave samples, respectively (Table 1). PCB-28, the lowest chlorinated congener analysed (3 Cl), was found in a higher concentration in leaves than in root samples and PCB-52 (4 Cl) was present more in roots than in leaves. The latter result is in accordance with Liu et al. [35], who report that owing to its higher Kow value, it can bind strongly to root tissue, which consequently hinders entry into the transpiration stream. The transportation of PCBs from soil to
Fig. 2. Residual percentages (average values standard deviation, %) of PCB congeners in soils sampled at different distances and depths from the trunk (Rhizosphere, A, B, C, D), 420 days after the poplar planting. Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth.
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Fig. 3. Bioaccumulation factor (BAF, average values standard deviation) of each single PCB congener calculated for leaves and roots 420 days after the poplar planting.
poplar roots or leaves was evaluated by calculating the bioaccumulation factors (BAFs, where BAF plant part = [PCB]plantpart/ [PCB]soil). Root and leaf BAFs, calculated using the soil PCB concentration before (t = 0 d topsoil P1) and 420 days after poplar planting in the rhizosphere soil were 0.22 and 0.38, respectively. These values are in the range (0.14–0.45) of those found by others [54,55] and, given that they are <1, they show that the poplar plant was not substantially able to phyto-extract PCB. The BAF value calculated for each single congener shows a value higher than 0.5 only for the tri-chlorinated congener 28 in leaves and for the tetrachlorinated 52 and penta-chlorinated 101 and 118 in roots (Fig. 3). 3.5. Heavy metal concentrations in soil The soil concentrations of the heavy metals (V, Cr, Sn and Pb) decreased strongly (p < 0.01) 14 months after poplar planting (Table 2) and in most cases was above the Italian legal limit (D.Lgs 152/06) at all points sampled in each plot (Rhizosphere, A–D soil): Cr in P1, P2, P3, P4; V in the P1 and P4; Pb in P1, P2 and P4. Although V and Pb values were still above the legal limits of 90 mg/kg and 100 mg/kg, respectively, their concentrations (except for the D sample) in the P3 plot halved when compared to their values at 0 d (Table 2). The highest decrease in heavy metal concentrations was found in topsoil at 25 cm from the poplar trunk (A) and in the rhizosphere soil. In the soil samples collected from the nonplanted area (control) at day 420 no significant decrease in heavy metal concentrations was observed (Table 2). 3.6. Heavy metal concentrations in roots and leaves Heavy metal concentrations in plant tissues (leaves and roots) were generally higher in roots than in leaves, except in the case of Pb and Sn in P1 and of Pb in P2, (Table 2). Consequently, the translocation factors (TF), calculated as the ratio of metal concentration in leaves to that in roots [56], were >1 only in the latter cases (Table 2).
3.7. Microbiological analysis The results of the microbiological analysis performed on soil sampled before the poplar planting (t = 0) and at day 420 in the rhizosphere and at different distances and depths from the poplar trunk soils (A–D) are shown in Table 3. The values of total microbial abundance and cell viability at day 0 were not directly related (ANOVA test not significant) either to OC content or to dehydrogenase activity (DHA). Although DHA is generally influenced positively by organic matter, because it stimulates microbial activity [49,52], the lowest values were found in the P1 (10.89 mg TPF/g) and control (19.05 mg TPF/g) plots, where the OC values were the highest (Table 3). Such low values of dehydrogenase activity indicate that the microbial community was not in a good condition [49,52,57] and are in line with the low quality state of the soil owing to the joint presence of PCBs and heavy metals, which presumably had a detrimental effect on microbial activity. At day 420 in the control, nonplanted soil microbiological parameters remained unvaried, demonstrating that the contamination persisted, while in the P1 rhizosphere a significant increase (p < 0.01) in DHA in line with a decrease in PCBs and heavy metals was observed. At the same time, the decrease in total microbial abundance and organic carbon content observed in the P1 rhizosphere may be ascribable to the stimulation of specific bacterial populations within the microbial community, that activated and transformed PCBs. Finally, the decrease in soil organic carbon is in line with its probable use as an electron donor for PCB reductive dehalogenation [58]. At day 420 a significant increase in dehydrogenase activity (DHA) was also found in the P3 and P4 plots, where a significant decrease in heavy metal pollution was observed. In these plots poplar planting also favoured an increase in soil organic carbon content. Finally, when plotting DHA values vs OC ones at day 420, a positive correlation was found (p < 0.01), suggesting a significant improvement in soil quality.
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V. Ancona et al. / New Biotechnology xxx (2016) xxx–xxx Table 2 Heavy metal concentrations (average values standard deviation) in soils and plant tissues sampled (leaves, roots; TF: translocation factors) at day 0 (t = 0) and 420 days after poplar-planting in different plots (P1, P2, P3 and P4). The metal concentrations above the National legal limit in D.Lgs 152/06 are in bold (V: 90 mg/kg; Cr: 150 mg/ kg; Sn: 1 mg/kg; Pb: 100 mg/kg). V
Cr
Sn
Pb
P1
t = 0 topsoil rhizosphere A B C D leaves roots TF
127.4 7.8 43.8 8.3 37.2 3.9 43.7 7.8 46.0 6.6 45.7 3.6 0.04 0.02 0.86 0.03 0.04
179.5 10.6 82.0 8.0 67.6 7.7 79.3 9.2 57.5 3.6 79.4 7.4 11.3 6.58 31.5 2.44 0.35
5.9 2.1 2.4 1.3 2.8 2.0 2.1 0.4 2.9 0.4 2.5 0.9 0.99 1.20 0.41 0.19 2.41
74.5 7.7 14.1 1.0 15.0 0.4 16.0 3.5 23.4 9.5 14.8 1.9 12.7 2.9 6.3 7.1 2.00
P2
t = 0 topsoil rhizosphere A B C D leaves roots TF
66.6 4.1 43.8 2.7 40.0 0.4 41.6 5.7 41.8 2.8 40.6 6.3 0.05 0.01 0.40 0.05 0.12
60.3 15.4 48.9 6.1 47.4 3.0 46.0 3.6 41.9 6.3 45.1 8.3 9.7 5.0 20.4 7.5 0.48
60.8 7.6 3.5 0.2 3.0 1.0 4.1 2.3 3.1 0.6 5.9 1.6 <0.6 0.97 0.8 –
193.8 5.2 41.6 7.4 40.0 6.6 48.8 8.9 39.2 6.1 58.1 8.8 10.7 3.7 4.2 0.5 2.51
P3
t = 0 topsoil rhizosphere A B C D leaves roots TF
275.7 15.7 117.9 3.3 105.3 12.2 344.8 6.9 141.7 7.1 129.6 3.0 0.38 0.54 1.02 0.08 0.37
283.7 1.79 83.7 2.7 67.8 5.6 69.8 3.6 83.2 3.9 141.9 4.9 8.1 2.8 22.0 4.0 0.36
15.3 5.72 4.2 1.3 5.2 1.1 4.9 0.7 7.0 1.3 15.6 3.4 0.6 0.54 3.0 3.84 0.19
289.1 13.03 102.4 8.3 93.1 11.4 131.6 5.7 136.0 5.0 90.6 3.1 0.6 0.54 20.0 3.95 0.03
P4
t = 0 topsoil rhizosphere A B C D leaves roots TF
336.4 7.5 51.6 4.3 90.4 6.4 50.9 3.6 65.8 7.2 48.7 1.6 0.3 0.56 1.0 0.15 0.35
217.0 3.4 43.5 8.7 56.9 8.3 39.5 0.8 41.5 2.9 37.9 1.6 16.3 4.2 24.3 11.4 0.67
10.5 3.2 6.2 2.8 3.6 1.5 4.5 0.6 4.7 1.2 4.6 1.5 1.0 0.79 2.0 1.01 0.51
199.0 11.23 41.4 7.8 74.4 5.2 38.6 3.8 58.6 5.0 62.5 7.7 12.0 2.0 15.5 2.9 0.77
61.4 1.6
60.3 1.6
10.5 0.6
144.1 11.1
63.7 1.8
62.9 1.3
12.3 1.6
147.3 9.6
Control Topsoil t = 0 d Topsoil 420 d
Topsoil: 0–20 cm depth; Rhizosphere: soil attached and surrounding roots, 0– 30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0– 20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth
4. Discussion The significant decrease in all PCB congeners, to less than the Italian environmental law limit (60 ng/g), in the poplar-planted plot shows the effectiveness of the Monviso clone at promoting the degradation of these persistent compounds. Confirming this result is the fact that in the control plot soil, outside the poplar-planting area, PCBs had not decreased by day 420 (Table 1). As can be seen in Fig. 2, a decreasing trend in the percentage of PCB removal is observable from the soil points at higher distances (C and D) to those closer to the rhizosphere (A and B), showing a clear role in chemical removal. The overall low BAF values in leaves being just higher than 0.5 for the congener 28 pointed to there being a negligible transportation of PCBs into poplar tissue. Owing to their high hydrophobicity, PCBs are not expected to enter the transpiration stream. Nevertheless, in such cases transportation to within plants
7
may occur, owing to plant root exudates, which can act as surfactants forming a more hydrophilic complex with PCBs, making their transportation into plants possible [59,60]. Heavy metals were found in the poplar tissue, but their concentrations were generally higher in roots than in leaves (Table 2). The low values for translocation factors indicate that transport to upper parts of the plant was quite limited [56]. The translocation of metals can be toxic for photosynthetic activity, chlorophyll synthesis and antioxidant enzymes [61] and for this reason some authors report the presence of exclusion mechanisms in some plant species [62]. The Monviso clone was therefore not a hyperaccumulator plant, but merely able to bioconcentrate heavy metals, avoiding their translocation; consequently it was shown to be useful for heavy metal phyto-stabilisation [63]. This process exploits the ability of plant roots to change environmental conditions via root exudates. Plants can immobilise heavy metals through absorption and accumulation by roots, adsorption onto roots or precipitation within the rhizosphere. This process reduces the mobility of the metals and leaching into ground water, as well as their bioavailability in the food chain. One advantage of this strategy is that the disposal of the metal-laden plant material is not required [63]. However, there needs to be research into the turnover of nutritive roots and the potential release of metals from decomposing roots [64]. The initially low values for dehydrogenase activity at all sampled points indicated that the microbial community was not in a good condition. DHA is a good indicator of soil quality state, because it is affected by different factors such as organic matter content and its quality [57], oxygen and contamination. Heavy metals can reduce microbial activity because they interact with enzyme-substrate complexes and can influence the synthesis of bacterial cellular enzymes [65]. Moreover, soil aeration may also influence this activity, being an oxidative process. In the soil investigated, many initial factors therefore co-existed, such as PCBs, heavy metals and the absence of both a pedologically evolved soil and a homogeneous soil texture. The overall increase in dehydrogenase activity 420 days after the poplar planting in the P1, P2, P3 and P4 plots is therefore ascribable to rhizosphere formation, which is known to favour microbial activity [66,51]. Moreover, the simultaneous occurrence of the increase in microbial activity and the PCB degradation suggests that the presence of poplar directly favoured some natural bacterial populations in their degradation and demonstrates a synergistic microbial-plant action in soil quality recovery. The use of poplar, for plant-assisted bioremediation of sites polluted by PCBs and heavy metals, has been tested in other experiments [67,42,13]. Although the Monviso clone selected has already been used in Central Italy for promoting hexachlorocyclohexane degradation [41,37], this is the first time that the same clone has been successfully applied in a multi-contaminated area in Southern Italy. Poplar-assisted bioremediation can be considered an economical, environmentally sustainable and landscapeenhancing technology, complying with green remediation, as it has a low impact on climate change and on the environment, in line with environmental sustainability as reported by the US EPA [68]. 5. Conclusions The Monviso clone was applied to a historically contaminated soil to promote PCB biodegradation through its root system. At about 1 year after planting, the overall results of the chemical analyses (PCBs and heavy metals) showed the effectiveness of this green technology at recovering degraded soil not only from organic, but also from inorganic contamination (phyto-containment, [69]). The soil remediation strategy is still being applied and the planting of the Monviso clone in other contaminated parts of
Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006
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V. Ancona et al. / New Biotechnology xxx (2016) xxx–xxx
Table 3 Average values (standard error, s.e.) of Total microbial abundance, Cell viability, Dehydrogenase activity and Organic carbon content in soils sampled at day 0 (t = 0) and 420 days after poplar-planting in different plots (P1, P2, P3 and P4). Total microbial abundance N. cells/g s.e.
Cell viability % s.e.
Dehydrogenase activity mg TPF/g s.e.
Organic carbon g/kg s.e.
P1
topsoil t = 0 Rhizosphere A B C D
2.11 * 107 2.4 * 105 4.66 * 106 5.1 * 105 1.38 * 107 1.1 * 106 2.17 * 107 1.1 * 106 1.17 * 107 1.1 * 106 1.74 * 107 1.2 * 106
59.7 6.2 36.6 5.1 20.2 3.2 17.9 4.5 20.2 1.5 46.1 5.5
8.96 3.5 31.9 1.2 37.3 4.5 28.9 11.8 32.9 0.9 27.9 2.0
10.9 1.2 4.8 0.6 7.8 0.4 6.0 0.5 6.4 1.0 5.9 0.2
P2
topsoil t = 0 Rhizosphere A B C D
9.84 * 106 1.8 * 105 1.33 * 107 1.4 * 106 1.57 * 107 9.7 * 105 1.52 * 107 4.1 * 105 9.84 * 106 4.0 * 105 1.03 * 107 1.9 * 106
57.5 7.0 19.7 2.0 20.0 1.2 32.3 6.2 15.7 1.8 38.6 5.2
84.5 7.2 82.4 5.6 47.27 1.1 44.8 5.7 37.5 1.3 35.06 0.7
9.5 0.9 12.5 0.5 8.5 0.6 8.4 0.5 9.5 0.4 8.6 0.4
P3
topsoil t = 0 Rhizosphere A B C D
1.40 * 107 1.5 * 106 3.89 * 106 2.6 * 105 7.47 * 106 1.0 * 106 2.31 * 107 2.1 * 106 1.19 * 107 9.2 * 105 7.85 * 106 9.6 * 105
43.2 5.3 19.5 2.2 27.3 3.5 20.2 3.2 52.1 4.2 20.2 1.6
125.2 10.0 213.0 12.1 143.4 19.0 140.5 49.3 197.9 11.9 143.0 19.8
10.7 0.4 12.7 0.2 12.4 0.1 11.4 0.2 12.4 0.1 11.5 0.2
P4
topsoil t = 0 Rhizosphere A B C D t=0 d t = 420 d
5.37 * 106 6.4 * 105 9.12 * 106 4.6 * 105 1.63 * 107 8.7 * 105 1.17 * 107 9.8 * 105 7.71 * 106 3.1 * 105 4.74 * 106 3.8 * 105 4.88 * 106 3.9 * 105 4.00 * 106 4.2 * 105
46.5 3.7 53.5 6.1 20.2 2.6 20.2 1.8 20.2 1.8 29.8 1.9 53.2 7.5 51.5 6.1
74.1 9.4 192.9 13.0 131.2 9.5 132.6 8.7 168.6 4.2 113.1 4.4 27.8 3.5 28.2 3.6
11.3 0.5 14.7 0.9 15.0 1.2 15.6 1.2 16.3 0.8 14.0 1.1 19.0 2.1 18.7 1.8
Control soil
Rhizosphere: soil attached and surrounding roots, 0–30 cm depth; A: 0.25 m distance from the trunk and 0–20 cm depth; B: 0.25 m distance from the trunk and 20–40 cm depth; C: 1 m distance from the trunk and 0–20 cm depth; D: 1 m distance from the trunk and 20–40 cm depth.
the same area has been performed. An annual sampling assessment of contaminants, microbial activity and plant biomass production will be carried out for at least the next 3–4 years. Currently, further analysis of the microbial structure and the composition of the microbial populations directly involved in the process of bioremediation is in progress. Acknowledgments The authors thank Giuseppe Bagnuolo (CNR-IRSA) for his technical support in the chemical analyses of the pollutants, Guido Del Moro, Andrea Decembrino and Vito Nicola Palmisano (CNRIRSA) for their useful assistance in the poplar planting, and Francesca Falconi (CNR-IRSA) for the microbiological analysis. The authors also thank Daniele Bianconi (CNR-IBAF) for his scientific support in the poplar plantation design and the Reverend Nicola Preziuso and Michele Arcangelo D’Alessandro (CEM of Taranto) and Pasquale Carmigiano (CODITA, Taranto) for their technical assistance during the field activities in the study area. Moreover, the authors thank CISA S.p.A. (Massafra, Italy) which partially funded this research within the Research Project “Applicazione di tecniche di fitorimedio a basso costo in località excampo Cimino-Manganecchia a Taranto” Prot. IRSA-CNR N. 0005159, 04/12/2012 . References [1] Furukawa K, Fujihara H. Microbial degradation of polychlorinated biphenyls: biochemical and molecular features. J Biosci Bioeng 2008;105:433–49. [2] Robertson LW, Hansen LG. PCBs recent advances in environmental toxicology and health effects. Lexington: University Press of Kentucky; 2015.
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Please cite this article in press as: V. Ancona, et al., Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.09.006