Phytoremediation of highly contaminated mining soils by Jatropha curcas L. and production of catalytic carbons from the generated biomass

Phytoremediation of highly contaminated mining soils by Jatropha 1 curcas L and production of catalytic carbons from the generated 2 biomass 3 4 Paloma Álvarez-Mateos, Francisco-Javier Alés-Álvarez, Juan Francisco García-Martín* 5 Department of Chemical Engineering, Faculty of Chemistry, University of Seville, C/ 6 Profesor García González, 1, 41012 Seville, Spain 7 *Corresponding author. E-mail address: [email protected] 8 9 10 Abstract 11 This paper deals with the removal of heavy metals from marginal soil mixtures from the 12 Cobre Las Cruces and Aznalcóllar mining areas containing high concentrations of metals 13 (Cr, Fe, Ni, Cu, Zn, Cd, Hg, Pb and As) by means of phytoremediation using Jatropha 14 curcas L., and the subsequent production of biocatalysts from the plant biomass. First, J. 15 curcas L. was sowed in eight mixtures of these mining soils to study its adaption to these 16 high-contaminated soils and its growth during 60 days in a greenhouse under conditions 17 simulating the South of Spain’s spring climate. Later, the most suitable soil mixtures for 18 plant growth were used for 120-day phytoremediation under the same conditions. Heavy 19 metal concentration in soils, roots, stems and leaves were measured by ICP-OES at the 20 beginning, at the middle and at the end of the phytoremediation period, thus calculating 21 the translocation and bioaccumulation factors. J. curcas L. was found to absorb great 22 amounts of Fe (> 3000 mg kg-1 plant) as well as notable amounts of Pb, Zn, Cu, Cr and 23 Ni, and traces of As. Other metals with lower initial concentrations such as Cd, Hg and 24 Sn were completely removed from soils. Finally, the plant biomass was subjected to 25 pyrolysis to obtain catalytic biocarbons, assessing the optimal temperature for the 26 pyrolytic process by means of thermogravimetric analysis and Raman spectroscopy. 27 28 Keywords: bioaccumulation; Jatropha curcas; mining soils; phytoremediation; 29 translocation factor. 30 31 Introduction 32 Soil contamination by heavy metals (As, Cd, Cr, Cu, Pb and Zn) is one of the major 33 environmental problems raising critical concerns for both human health and ecosystems 34 (Singh et al., 2011) due to their carcinogenic and mutagenic effects on animals and 35 humans (Sánchez-Chardi et al., 2009). Small quantities of these metals are required for 36 human health, however in higher concentrations they become toxic or dangerous, 37 affecting brain, kidney, lungs, liver and other important organs. Moreover, long-term 38 exposure to them can cause physical, muscular and neurological degenerative processes, 39 and even cancer (Järup, 2003). The clean-up of most of these soils is mandatory for the 40 area to be reclaimed and to minimize the entry of potentially toxic elements into the food 41 chain. 42 The main anthropogenic sources of heavy metals in soils are related to the mining 43 industry. The mining sector produces a whole range of gaseous pollutants both solid and 44 liquid. This can occur in different ways: by deposition from the atmosphere as sediment 45 particles or brought by rainwater; by direct discharge of the liquid products of mining and 46 metallurgical activity; or by infiltration of leachate from the mining environment. 47 Specifically, mining and mineral processing of sulphide ore deposits produce large 48 quantities of wastes, most of which are regarded as toxic or hazardous, due to the 49 formation of acid drainage, and to their heavy metal content (Jiménez-Moraza et al., 50 2006). 51 Phytoremediation basically refers to the use of plants and associated soil microbes to 52 reduce the concentrations or toxic effects of contaminants in the environment (Singh et 53 al., 2003; Suresh and Ravishankar, 2004). It can be used for the removal of not only heavy 54 metals but also organic pollutants such as polynuclear aromatic hydrocarbons, 55 polychlorinated biphenyls and pesticides. 56 Phytoremediation employs on-site plants to absorb heavy metals and to prevent their 57 further transport. Plants generally handle the contaminants without affecting topsoil, thus 58 conserving its utility and fertility. They may improve soil fertility and increase organic 59 matter content (Abhilash et al., 2012; Cobbett, 2003). Therefore, phytoremediation is an 60 environmental ecotechnology that can be applied to remediate contaminated soils. It is a 61 novel, cost-effective, efficient, environmental and eco-friendly remediation strategy that 62 can be applied in situ and is solar-driven (LeDuc and Terry, 2005; Mukhopadhyay and 63 Maiti, 2010). What is more, it has already been demonstrated that phytoremediation is a 64 more ecological and economic technique than the conventional physical-chemical 65 alternatives (González-Chávez and Carrillo-González, 2013). 66 Recently, it has been reported that Jatropha curcas L. has a high capacity for 67 bioaccumulation, phytotranslocation and phytoremediation of heavy metals (Chang et al., 68 2014; González-Chávez et al., 2017; Marrugo-Negrete et al., 2015). J. curcas L. is a plant 69 belonging to the Euphorbiaceae family. It is cultivated in tropical and subtropical regions 70 around the world, becoming naturalized in some areas (Jamil et al., 2009; Pandey et al., 71 2012). This plant survives and grows on marginal, eroded and depleted lands. It requires 72 little water to grow, although it does not tolerate heavy rains. The implementation of these 73 fast growing plants in degraded or contaminated areas is important for improving soil 74 quality and preventing erosion. It also leads to organic matter enrichment and to greater 75 diversity of microorganisms in the soil (Abhilash et al., 2013). This plant can also 76 accumulate most metallic elements in quantities of up to 0.01% of its dry weight (1000 77 mg kg-1) (Ahmadpour et al., 2014; Ghavri and Singh, 2010). Other studies have shown 78 that Cr, Hg and Pb are not easily transferred to aerial plant biomass as they are mainly 79 stored in root cells (Bernabé-Antonio et al., 2015; Marrugo-Negrete et al., 2015), while 80 Zn is easily accumulated in green tissues (Yadav et al., 2009). 81 Although phytoremediation is a promising approach for the recovery of soils 82 contaminated with heavy metals, its main drawback is the later use of the contaminated 83 biomass generated over phytoremediation. As soon as the plants have absorbed the 84 pollutants from the soil, it is necessary to withdraw them from the contaminated zone. 85 The contaminated plants are regarded as residues and are kept in vegetable containers so 86 the problem is not solved. 87 The implementation of these fast growing plants in degraded or contaminated areas is 88 important for the soil quality, erosion prevention as well as organic matter enrichment 89 and diversity of microorganisms in soil. In addition to the beneficial effects on the soil, 90 the biomass obtained from J. curcas L. (stems, leaves and roots) could be used as a 91 potential source of energy and other resources (Chavan et al., 2015; Teo et al., 2015, 92 2014). A potential alternative is to perform thermochemical processes such as pyrolysis 93 to transform this biomass into high added-value products. Pyrolysis is a process for which 94 a material is thermally decomposed in the absence of oxygen or any other oxidizing agent. 95 The principal product obtained in the pyrolysis is pyrolytic carbon, which has numerous 96 potential applications, such as its use as acid heterogeneous catalysts (Yee et al., 2011). 97 Its properties depend mainly on the biomass composition, reactor type and the 98 experimental conditions in which pyrolysis is performed. 99 In the present work, we have studied the growth and adaptation of J. curcas L. to highly 100 contaminated soils by heavy metals under the climatic conditions of the South of Spain 101 as well as the phytoremediation capacity (extraction of heavy metals from soil) of J. 102 curcas L. The distribution of extracted heavy metals in roots, stems and leaves was 103 analysed and quantified by ICP-OES. Bioaccumulation or bioconcentration factors (BAF) 104 and translocation factors (TF) of heavy metals were also compared. Finally, we have 105 assayed the formation of biocarbons, with catalytic properties, from J. curcas L. roots, 106 determining the structure of the biocarbons by Raman spectroscopy. 107 108 2. Materials and methods 109 2.1. Soil samples from the mining land 110 Soil samples were collected in Cobre Las Cruces and Aznalcóllar mining areas in 111 Andalusia, in the South of Spain. 112 In Cobre Las Cruces mine, 300 kg were taken from the storage zone for the original 113 topsoil in the mining region (Fig. S1.A). This soil (NCS) is currently used for the 114 subsequent restoration of the landscape and to minimize the visual impact of the mine. 115 In Aznalcóllar mine, samples were taken from the heap area with the highest 116 concentration of leachate metals. Two zones of well-differentiated soils were found in 117 this mine (Fig. S1.B and S1.C). One with yellow soil (YCS) and another one with grey 118 soil (GCS), so both samples were taken. A surface area of 50 m2 was selected placing an 119 imaginary mesh with 5 m sides (i.e. two adjacent 25-m2 grids for each soil). 5-kg samples 120 were taken at the centre of each grid at three different depths (0 – 30 cm, 30 – 60 cm and 121 60 – 90 cm, respectively, from the surface), which were subsequently mixed and 122 homogenized in a mixer for one hour. 123 Finally, the three soils were transported to a greenhouse. After their analysis, they were 124 used as substrate for the trials. 125 2.2. Germination of Jatropha curcas L. seeds 126 Seventy J. curcas L. seeds from Argentina were sown in vermiculite in 8-cm diameter 127 pots. They were germinated in a Fitoclima 18000EH climate chamber (Aralab, Portugal) 128 under the following conditions: 25 ºC, 80% humidity and continuous lighting (Fig. S1.D). 129 The plants were watered with Hoagland solution containing (in mmol L−1) 6.0 KNO3, 4.0 130 Ca(NO3)2·4H2O, 2.0 NH4H2PO4, 1.0 MgSO4·7H2O, 50.0 KCl, 25.0 H3BO3, 2.0 131 MnSO4·H2O, 2.0 ZnSO4·7H2O, 0.5 CuSO4·5H2O, 0.5 H2MoO4, and 20.0 Fe-EDTA, 132 providing K, Ca, N and P, Mg, Cl, B, Mn, Cl, B, Mn, Zn, Cu, Mo, and Fe, respectively. 133 Sixty seeds germinated, 85% of all those planted. The seedlings were kept in this climate 134 chamber for about 6 weeks until they were 25 cm tall (Fig. S1.E). Immediately afterwards 135 they were planted into 20-cm diameter plant pots containing peat moss (Universal Compo 136 Sana Substrate, Germany) and placed in a greenhouse. The climate in the greenhouse 137 simulated that of spring in the South of Spain; that is, a daytime temperature of 25 ± 3 ºC 138 and 18 ± 3 ºC at night, 60% humidity and between 8 and 14 h of light. The plants were 139 kept under these conditions for about six weeks to acclimate before the beginning of both 140 adaptation and absorption trials in mine soils, the resulting plant tall being 70 – 90 cm 141 (Fig. S1.F). 142 2.3. Preliminary assessment of Jatropha curcas L. adaptation to contaminated soils 143 First, the study of the adaptation of J. curcas L. to the three soils was made. Eight mixtures 144 of soils were assayed (Table 1), 5 replicas of each trial being performed. Plants were let 145 to grow in these soils for 60 days, being watered with tap water once a week. For further 146 comparison, 5 plants were sown in vermiculite (V100) and watered with Houlang 147 solution. Another 5 plants were analysed at day 0 as reference. Visual observations and 148 pH measures were carried out at days 0, 7, 14 and 60. Chemical analysis of the soil 149 mixtures as well as of the plant biomass (roots, stems and leaves) were performed at the 150 beginning and at the end of this adaptation period. 151 2.4. Heavy metal absorption trials 152 Three of the soil mixtures assayed in the adaptation trials, namely NCS100, YCS20 and 153 YCS50, were selected to assess the heavy metal absorption by J. curcas L. Plants were 154 sowed in 50-cm diameter pots and were let to grow for 120 days, being watered with tap 155 water once a week. Ten replicas of each trial were performed. Five replicas of soils and 156 plants were analysed after 60 days (first cut-off) and another 5 replicas after 120 days 157 (second cut-off). 158 2.5. Catalytic carbons production 159 The dried and sieved roots of the plants were subjected to pyrolysis in an inert nitrogen 160 atmosphere at 300, 350, 400, 450, 500, 550, 600, 650 and 700 °C, respectively. To do 161 this, 0.5 g sample were placed in a 25 x 300 quartz tube and introduced in a Carbolite 162 Tube Furnace MTF 12/38/250 equipped with a Eurotherm 2416CG temperature 163 controller. The outlet of the quartz tube was connected to two bubblers submerged in ice 164 to condense the flue gases (bio-oils), an extractor hood eliminating the uncondensed 165 gases. The flow of inert N2 gas was set to 6 L h-1, and the heating rate was set to 30 ºC 166 min-1. The working temperature was maintained for 2 h. The resulting catalytic carbons 167 were let to cool to room temperature. 168 2.6. Soil and plant analysis 169 Before being analysed, soils were weighted and dried in a stove at 60 ºC for 72 h. Once 170 dried, the samples were sieved using a 2 mm mesh, removing pebbles but not the clots of 171 soil. A mortar was used to reduce the size of the soil particles and to press them through 172 the sieve. After being sieved, samples were carefully homogenized. 173 As for plant analysis, they were first cut-up, separating roots, stems and leaves. Next, all 174 the parts of the plants were dried in a stove at 60 ºC to constant weight. Finally, they were 175 ground using a Culatti DFH48 mill with a 2 mm sieve. 176 2.6.1. pH 177 To measure the pH of the soils, 10 g soil were weighed in a beaker and distilled water 178 was added until a thick, homogeneous paste was obtained with no excess water. This was 179 let to stand for 30 min before introducing the pH meter electrode to measure the pH (Ali 180 et al., 2011). 181 2.6.2. Phosphates 182 The Olsen method was used to determine the amount of phosphates (PO43- or HPO42-) in 183 the soil samples. Using this method, it is possible to determine the phosphates that a plant 184 can assimilate. It is based on a colorimetric assay which measures the optical density of 185 the blue solution produced when reducing the phosphomolybdic complex 186 ((NH4)PO4(MoO3)12) formed by orthophosphoric acid (H3PO4) and ammonium 187 molybdate (NH4)MoO4 (Jackson, 1958). Firstly, seven standard solutions from 0.2 to 1 188 mg kg-1 KH2PO4 were prepared, to which 8 mL 0.03M (NH4)MoO4 solution were added. 189 The absorbance at 880 nm of the resulting solutions was measured in a Varian Cary 100 190 UV-visible spectrophotometer, and a calibration curve was obtained. To analyse 191 phosphates in soil samples, 2.5 g soil were weighed in a beaker, then 50 ml 0.5 N 192 NaHCO3 were added and the mixture was stirred for 30 min before filtering. Finally, 8 193 mL 0.03M (NH4)MoO4 solution were added to the filtered solution and the absorbance 194 at 880 nm was measured. Results were expressed as mg available phosphorus kg-1 soil. 195 2.6.3. Organic matter 196 The organic matter in soil samples was determined by oxidising it with potassium 197 dichromate (K2Cr2O7) and sulphuric acid (H2SO4), measuring the reduced chromium 198 (Cr3+) by UV-Vis absorption spectroscopy (Jackson, 1958). Before analysing the 199 samples, a calibration curve was made. Six standard solutions of glucose were prepared 200 from 5 to 50 mg mL-1, reacting 2 mL of each with 10 mL 0.17 M K2Cr2O7 and 10 mL 201 H2SO4. As the rate and intensity of oxidation depend on the amount and kind of organic 202 matter in the samples and the reaction temperature, all the reactions took place in a 100 203 ºC water bath for 30 min. The same procedure was followed for the reaction of 0.3 g of 204 each of the soil samples. After preparing the standard reactions and the samples, the 205 absorption spectra were recorded between 400 and 800 nm. Results were expressed in g 206 organic matter kg-1 soil. 207 2.6.4. Carbonates 208 The percentage of total carbonates (CaCO3) in soil samples was determined using a 209 gasometric method known as Bernard's calcimetry. This method involves the indirect 210 measurement of the carbonates in the soil by the gasometric determination of the CO2 211 released when the carbonates in soil samples react with HCl. To calibrate Bernard's 212 calcimetry, 0.25 g pure CaCO3 were weighed and were let to react with 5 ml 1:1 HCl 213 solution. The same procedure was followed to determine the carbonates in soil samples. 214 2.6.5. Nitrates 215 UV-visible absorption spectrophotometry was used to determine the content in nitrates 216 (NO3-) in soil samples, recording the absorption spectra of the samples at 220 nm in a 217 Varian Cary 100 UV-visible spectrophotometer (APHA et al., 2005). The calibration 218 curve was obtained from 10 standard solutions from 0.2 to 5.0 mg kg-1 KNO3 to which 1 219 mL 1 M HCl was added. To determine the content in nitrates in soil samples, 10 g soil 220 were weighed in a beaker and then 50 mL distilled water were added, the mixture being 221 stirred for 1 h before filtering. Finally, 1 mL 1 M HCl was added to the filtered solution. 222 Results were expressed as mg nitrates kg-1 soil. 223 et al., 2009). The concentration of Fe decreased by only 15-39% due to its high initial 370 concentration (> 30000 mg kg-1). 371 3.3.2. Analysis of the plants sowed in NCS100, YCS20 and YCS50 soils 372 While differences were hardly observed after 60 days, Fig. 5 clearly shows that the 373 biomass production after 120 days drastically decreased when increasing the percentage 374 of YCS in the soil. Thus, J. curcas L. barely grew in YCS50 from day 60 to day 120. The 375 elemental analysis of roots, stems and leaves after 120 days (Fig. 6) showed that the 376 absorption of these metals by the plants affects, above all, the content of N, which 377 increased its percentage in leaves and decreased in roots and stems when increasing the 378 percentage of YCS in the soil where the plant was sowed (Fig. 6.C). The higher 379 percentage of N in YCS20 and YCS50 leaves can be related to the chelating power of 380 metals such as Cu, Zn and Pb, which are able to fix nitrogen to form complexes (Wuana 381 et al., 2010). Percentages of C and H were similar in each biomass section and soil. 382 The ICP-OES analysis showed that plants mainly absorbed and accumulated Fe (Table 383 5) because this was the predominant element in soils, although they also absorbed to a 384 less extent Cr, Ni, Cu, Zn and Pb. The concentrations of these metals found can be 385 considered as promising, since they were quite high. The concentration of Fe in plants 386 was higher than the accumulation limit by J. curcas for most metallic elements (1000 mg 387 kg-1) reported by other authors (Ahmadpour et al., 2014; Ghavri and Singh, 2010). 388 The concentrations of Cu and Zn after 120 days were in the same range than those found 389 in J. curcas established for 105 days on a mine residue amended or not amended with 390 biochar and inoculated or not inoculated with the mycorrhizal fungus Acaulospora sp 391 (González-Chávez et al., 2017). By contrast, these authors found much lower 392 concentrations of Pb (less than 25 mg kg-1) in their trials. Higher Cu concentrations (665 393 ± 1 mg kg-1), based on total plant dry biomass, were found in J. curcas planted for 5 394 months in soils spiked with 400 mg kg-1 Cu (Ahmadpour et al., 2014). However, our study 395 deals with much more contaminated soils with numerous heavy metals at high 396 concentrations at the same time, and the metal concentrations depend on plant growth. In 397 spite of achieving higher Cu concentrations in plant tissue, the maximum total Cu removal 398 from soils these authors achieved was 1.2% (Ahmadpour et al., 2014), while we achieved 399 Cu removal from soils of up to 72% (Table 4). Furthermore, J. curcas sowed in soils 400 artificially contaminated up to 10 mg Hg kg-1 soil (using mercury nitrate solution) was 401 able to solely absorb up to 7.3 mg Hg kg-1 biomass after 4-month trial (Marrugo-Negrete 402 et al., 2015). The low initial concentrations of trace elements (Cd, Hg and Sn) found in 403 the different parts of the plants (roots, stems and leaves) did not allow determining with 404 great precision their concentrations at the end of the heavy metal absorption trials. Since 405 these elements were not found in soils, it could be assumed that most of them were totally 406 absorbed by plants. Taken into account the different parts of the plant, 800.5, 633.3 and 407 872.1 mg metals kg-1 plant were found in roots, stems and leaves, respectively, of 408 NCS100 samples; 1262.4, 1192.9 and 607.6 mg metals kg-1 plant were found in roots, 409 stems and leaves, respectively, of YCS20 samples; and 2132.5, 446.4 and 421.2 mg 410 metals kg-1 plant were found in roots, stems and leaves, respectively, of YCS50 samples. 411 As it can be observed, plants sowed in the most initially contaminated soil (YCS50) were 412 the ones that more metals accumulated in roots, which agrees with other authors' findings 413 (Ahmadpour et al., 2014; Ghavri and Singh, 2010; González-Chávez et al., 2017; 414 Marrugo-Negrete et al., 2015; Yadav et al., 2009). The first authors (Ghavri and Singh, 415 2010) detected 49.3 – 64.9 mg Fe kg-1 J. curcas roots grown in garden soil for 100 days 416 while the Fe concentration in J. curcas roots grown in iron rich wasteland soil for 100 417 days was 6 folds higher (301.24 – 319.53 mg kg-1). In our case, Jatropha curcas L. roots 418 sowed in SCN100, YCS20 and YCS50 soils for 120 days absorbed 622.2, 1124.3 and 419 1929.1 mg kg-1 Fe. 420 Regarding the translocation of these metals in the plants, the metals are seen to remain 421 mainly in leaves and stems in most cases (Fig. 7). All the metals increased their 422 translocation to aerial parts of the plant over time for NSC100 and YCS20, except Cr in 423 plants sowed in NSC100 and Fe in plants sowed in YCS20. The metals translocation 424 factors for YCS50 showed and uneven behaviour, being the global translocation factor 425 for plants sowed in this soil lower than those for NCS100 and YSC20. The global 426 translocation factors (considering all the analysed metals) for NCS100, YCS20 and 427 YCS50 were 0.81, 1.95 and 1.37 at day 60, and 1.88, 1.43 and 0.41 at day 120, 428 respectively. Most of individual TF were higher than those found for the same plant for 429 Hg in mining soils artificially contaminated after the same phytoremediation time 430 (Marrugo-Negrete et al., 2015), for Cu in soils spiked with Cu in amounts of 0, 50, 100, 431 200, 300, and 400 mg kg-1 (Ahmadpour et al., 2014), and for Cu, Zn and Pb in mining 432 soils with biochar addition and under the effect of mycorrihizal inoculation (González-433 Chávez et al., 2017). Accumulation and distribution of heavy metals in plant tissues are 434 of major importance in phytoremediation of contaminated soils. If the objective is to 435 stabilize metals in roots for them not to enter in stems and leaves and therefore to prevent 436 metals from entering the ecosystem, low TF are desirable. Therefore, the low global TF 437 (0.41) obtained for the most contaminated soil (YCS50) at day 120 is a promising result. 438 On the contrary, the high TF obtained for some metals such as Cr, Ni, Zn and Pb cannot 439 be regarded as good results if the purpose is the phytoremediation of soils contaminated 440 with these metals. 441 With regard to bioaccumulation factors, is spite of the great Fe concentration absorbed by 442 plants (Table 5), BAF of Fe was very low for the 3 soils in the 2 days of sampling because 443 of the huge initial concentration of Fe (between 32213 and 265357 mg kg-1), which could 444 not be removed in 120 days, so a great amount of Fe remained in the soils at the end of 445 this period of time (Table 4). This also led to low global bioaccumulation factors. The 446 global BAF for NCS100, YCS20 and YCS50 were 0.142, 0.097 and 0.041 at day 60, and 447 0.117, 0.083 and 0.049 at day 120, respectively. As can be noted, BAF decreased over 448 time. This was due to the huge plant growth from day 60 to day 120 (Fig. 5). BAF (except 449 for Fe) were in the range of those found for Hg, Cu, Zn and Pb in mining soils with J. 450 curcas for a similar cultivation period (González-Chávez et al., 2017; Marrugo-Negrete 451 et al., 2015), these soils having initial metal concentrations much lower than those used 452 in this study, which highlights the good results achieved. 453 454 3.4. Characterization of the catalytic carbons. 455 Two main phases can be distinguished in the thermogravimetric curves obtained in the 456 pyrolysis of the contaminated root samples at different temperatures. Fig. 9 depicts the 457 weight losses obtained for the roots of plants sowed in YCS50 soil, which had the highest 458 concentration of heavy metals (2132.5 mg kg-1). The first, between room temperature and 459 350 ºC, is the active zone and is related to the degradation of polymers of hemicellulose 460 and cellulose. The second phase, the passive zone, showed a decrease in the slope until 461 600 ºC as a result of the decomposition of lignin. Beyond this phase, the slope steepened 462 again due to the loss of mass caused by the total degradation of the biomass. 463 Fig. S3 shows the Raman spectra of the pyrolytic carbons for this sample obtained from 464 300 ºC to 650 ºC and the spectrum of the standard graphite. For C-based materials 465 containing C-sp2 sites, the most intense features can be observed at about 1582, 1350, 466 1620 and 2700 cm-1, which are called G, D, D’ and 2D (also G’) (Reich and Thomsen, 467 2004; Tai et al., 2009; Veres et al., 2008; Viana and Marques, 2015). The G band appears 468 from a double degenerated phonon mode with E2g symmetry, while the D and D’ bands 469 emerge from the phonon modes with A1g symmetry (Ferrari and Robertson, 2000; 470 Pimenta et al., 2007; Reich and Thomsen, 2004). The G band appears in all graphite 471 samples, while the D and D’ bands only appear in the spectra of disordered samples, and 472 highly crystalline graphite does not show these bands. So, the D band is attributed to 473 disorders and defects in the graphite structure. Comparing the G-band (~ 1575 cm-1) and 474 the D-band (~ 1355 cm-1) obtained for pyrolytic carbons with that of commercial graphite 475 (Fig. S5), it can be observed that the sample pyrolysed at 550 ºC is the one that presents 476 the intensity ratio of G and D bands, as well as their positions and widths, more similar 477 to those of graphite. Samples pyrolysed at temperature lower than 550 ºC showed wider 478 bands that overlap each other while samples pyrolysed at temperatures higher than 550 479 ºC showed lower intensity bands, leading to the formation of amorphous carbonaceous 480 materials. 481 Conclusions 482 The high percentage of germination obtained from the seeds of Jatropha curcas L. (85%) 483 and the rapid adaptation to the spring climate simulated in the greenhouse make this 484 species a plant with wide possibilities for phytoremediation of marginal soils. 485 Nevertheless, J. curcas L. did solely survive in soil mixtures containing up to 50% of the 486 highly contaminated mining soils assayed in this work. 487 With regard to metal removal from soils, metals with low initial concentrations (<10 mg 488 kg-1) such as Hg, Sn and Cd were completely removed, while the concentrations of metals 489 within the range 10 – 1000 mg kg-1 (Cr, Ni, Cu, Zn and Pb) were reduced between 30 – 490 70%, except As. The concentration of Fe in soils was solely reduced 15% as consequence 491 of its high initial concentration (> 30000 mg kg-1). On the other hand, the translocation 492 factors of Cr, Ni, Zn and Pb were high, which can be a hindrance for the phytoremediation 493 of soils containing high concentrations of these metals. 494 Finally, the structural characterization by Raman spectroscopy of pyrolytic coal obtained 495 from the contaminated roots of the J. curcas L. showed that the most suitable pyrolysis 496 temperature to obtain a graphite structure was 550 ºC. 497 498 Acknowledgments 499 Authors gratefully thank Camposur Investiga S.L. for its financial support to this research 500 work 501 502 References 503 Abhilash, P.C., Powell, J.R., Singh, H.B., Singh, B.K., 2012. Plant-microbe 504 interactions: Novel applications for exploitation in multipurpose remediation 505 technologies. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2012.04.004 506 Abhilash, P.C., Singh, B., Srivastava, P., Schaeffer, A., Singh, N., 2013. 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Percentage of growth of leaves (black bars), stems (open bars) and roots (grey bars) of Jatropha curcas L. in each soil at the end of the 60-day adaptation period. 0 10 20 30 40 50 60 Biomass growth (%) Figure 3: Changes in pH in soils at days 0 (black bars), 60 (open bars) and 120 (grey bars) during the heavy metal absorption trials. Figure 4: Evolution of the contents of (A) carbonates, (B) phosphates, (C) nitrates and (D) organic matter in NCS100, YCS20 and YCS50 soils at the beginning (black bars), after 60 days (open bars) and after 120 days (grey bars). 0 2 4 6 8 10 12 14 NCS100 YCS20 YCS50 Carbonates (%) (A) 0 5 10 15 20 25 30 NCS100 YCS20 YCS50 Phosphates (mg kg -1) (C) 0 5 10 15 20 25 NCS100 YCS20 YCS50 Nitrates (mg kg -1) (B) 0 5 10 15 20 NCS100 YCS20 YCS50 Organic matter (g kg-1) (D) Figure 5: Percentage of biomass increased of plants sowed in NCS100, YCS20 and YCS50 soils after 60 (black bars) and 120 (open bars) days. 0 5 10 15 20 25 30 35 40 45 NCS100 YCS20 YCS50 Biomass growth (%) Figure 6. Elemental analysis of carbon (A), hydrogen (B) and nitrogen (C) in leaves (black bars), stems (open bars) and roots (grey bars) of plants sowed for 120 days in 34 36 38 40 42 44 46 48 NCS100 YCS20 YCS50 Carbon (%) (A) 5,0 5,2 5,4 5,6 5,8 6,0 NCS100 YCS20 YCS50 Hydrogen (%) (B) 0 1 2 3 4 5 NCS100 YCS20 YCS50 Nitrogen (%) (C) NCS100, YCS20 and YCS50 soils. Fig. 7: Translocation factors of Cr, Fe, Ni, Cu, Zn and Pb of Jatropha curcas L. plants sowed in NCS100 (black bars), YSC20 (open bars) and YCS50 (grey bars) soils after 60 and 120 days. Fig. 8: Bioaccumulation factors of Cr, Fe, Ni, Cu, Zn and Pb of Jatropha curcas L. plants sowed in NCS100 (black bars), YSC20 (open bars) and YCS50 (grey bars) soils after 60 and 120 days. Figure 9. Thermogravimetric curve obtained in the pyrolysis of the roots of Jatropha curcas L. planted in the YCS50 soil. 0,20 0,25 0,30 0,35 0,40 0,45 0,50 300 400 500 600 700 Mass loss (g) T (°C)