Microfungi in highly copper-contaminated soils from an abandoned Fe–Cu sulphide mine: Growth responses, tolerance and bioaccumulation

Chemosphere 117 (2014) 471–476

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Technical Note

Microfungi in highly copper-contaminated soils from an abandoned Fe–Cu sulphide mine: Growth responses, tolerance and bioaccumulation Mirca Zotti a, Simone Di Piazza a,⇑, Enrica Roccotiello a, Gabriella Lucchetti b, Mauro Giorgio Mariotti a, Pietro Marescotti b a b

Laboratory of Mycology, DISTAV Dipartimento di Scienze della Terra dell’Ambiente e della Vita, University of Genoa, Corso Dogali, 1 M, I 16136 Genova, Italy DISTAV Dipartimento di Scienze della Terra dell’Ambiente e della Vita, University of Genoa, Corso Europa, 26, I 16136 Genova, Italy

h i g h l i g h t s  Fungi can play a key role in metal-polluted ecosystems via colonization and decontamination.  Clonostachys rosea, Trichoderma harzianum, and Aspergillus alliaceus were tested at increasing Cu(II).  The strains showed a Cu(II)-tolerance capability ranging from 100 to 400 mg L

1

.

 The strains of T. harzianum and C. rosea presented a high Cu(II)-bioaccumulation capability.  These microfungi may be fruitfully exploited in mycoremediation protocols.

a r t i c l e

i n f o

Article history: Received 27 March 2014 Received in revised form 11 August 2014 Accepted 12 August 2014

Handling Editor: O. Hao Keywords: Fungi Metal tolerance Bare soil Derelict mine

a b s t r a c t Copper is one of the most dangerous soil contaminants. Soils affected by high copper concentrations show low biodiversity and, above all, inadequate environmental quality. Microorganisms such as fungi can play a key role in metal-polluted ecosystems via colonization and decontamination. The study is devoted to characterize the microfungal community in highly Cu-contaminated bare soil from derelict Fe–Cu sulphide mines and to isolate microfungal strains able to tolerate and accumulate Cu. 11 Different taxa to be isolated has been isolated during two sampling campaigns (in Autumn and in Spring). Among these, Clonostachys rosea, Trichoderma harzianum, and Aspergillus alliaceus were tested at increasing Cu(II) concentrations and showed a Cu(II)-tolerance capability ranging from 100 to 400 mg L 1. Moreover, the strains of T. harzianum and C. rosea presented a high Cu(II)-bioaccumulation capability, 19 628 and 22 222 mg kg 1, respectively. These microfungi may be fruitfully exploited in mycoremediation protocols. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction This work presents a multidisciplinary research carried out in Cu-contaminated soil of a derelict Libiola mine (Eastern Liguria, Italy). Typical background Cu concentrations in different soil types range from 2 and 100 mg kg 1 (Kabata-Pendias and Mukherjee, 2007; Alloway, 2013). In altered conditions as in Cu-sulphide mine, copper concentration in waste rock disposals and in surrounding soils strongly increases (e.g. in the Libiola mine area Cu 160– 13 347 mg kg 1; mean 1885 mg kg 1) (Marescotti et al., 2008, 2010, 2013). Such very high Cu-content is mainly related to the presence of Cu-bearing pyrite and chalchopyrite and Fe-oxides and Fe-oxyhydroxides formed as a consequence of sulphide oxida⇑ Corresponding author. Tel.: +39 0102099378. E-mail address: [email protected] (S. Di Piazza). http://dx.doi.org/10.1016/j.chemosphere.2014.08.057 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

tion processes (e.g. Acid Mine Drainage processes – AMD; Jambor and Blowes, 1994). This is extremely important for copper bioavailability, since Cu-adsorption/desorption on these minerals are the main processes governing Cu mobility in soils with little, if any, organic compounds (Hooda, 2010). Opposite to most soil conditions, Cu is highly mobile in the sulphidic waste-rock dumps which are characterized by low pH, low clay mineral and organic matter content, and high permeability; all these factors may significantly increase Cu concentration in soil solutions (Kabata-Pendias and Mukherjee, 2007), in the sulphidic waste-rock dumps suitable conditions (in particular low pH, low clay mineral and organic matter content, and sandy to gravelly soils) for significant Cu-leaching from the solid phases and for its concentration in the soil solutions can be present. As a matter of fact Cu-toxicity symptoms are reported for plants growing in acid soils of pH less than 6.5, such as those commonly found in soils formed from Cu sulphide-rich

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parent rocks, especially within or around Cu-mines (Hillel et al., 2004; Kabata-Pendias and Mukherjee, 2007). The physicochemical properties of the contaminated environment tend to inhibit soilforming processes and plant growth, affecting the area’s biodiversity and exerting a strong selective pressure on the local flora and mycoflora (Gadd, 1993; Pratas et al., 2005; Crane et al., 2010; Ceci et al., 2012). Microorganism communities and related biological activities play a key role in colonizing and decontaminating metal-polluted soil ecosystems and consequently have an active environmental and economic significance (Gadd, 1993, 2007; Fomina et al., 2005a,b). Metal uptake in filamentous fungi is a complicated biological process and several authors have hypothesized a process consisting in two phases (Ross, 1994; Yetis et al., 2000): (1) passive absorption, rapid and metabolic energy-independent, (2) active uptake, slow and metabolic energy-dependent (for example see lead accumulation in Clonostachys rosea (Link) Schroers, Samuels, Seifert and W. Gams, by Zucconi et al., 2003). Clearly, the amounts of actively absorbed metals are higher than those absorbed passively. Several studies showed how macro and microfungi could be successfully exploited in order to reforest barren and contaminated soils (Bojarczuk and Kieliszewska-Rokicka, 2010) or to decontaminate soils and waters from several ecotoxic metals, such as Cu (Yap et al., 2011; Wang and Wang, 2013), Pb (Zucconi et al., 2003), V (Ceci et al., 2012). Concerning copper-contaminated soils, recent studies have shown how macrofungi such as Trametes versicolor (L.) Lloyd (S ß ahan et al., 2010) and microfungi such as Aspergillus niger Tiegh. (Dursun et al., 2003), A. terreus Thom (Cerino-Córdova et al., 2012), A. versicolor (Vuill.) Tirab. (Tasßtan et al., 2010), Penicillium notatum Westling (Anjum et al., 2009), Rhizopus arrhizus A. Fisch. (Sag˘ et al., 2002), Trichoderma atroviride P. Karst. (Yap et al., 2011), T. viride Pers. (Wang and Wang, 2013) are able to absorb Cu from contaminated liquid and solid matrices. Nevertheless, few studies have been conducted on the relationship between saprotrophic fungi and copper-contaminated soil. The goals of this research were to test the growth responses and resistance threshold to copper in microfungal strains isolated from the Libiola Cu-sulphide mine. The novelty of the work consists of the isolation of two new fungal strains able to accumulate Cu, which may be possibly exploited in some future mycoremediation protocols. 2. Materials and methods 2.1. Study area The derelict Libiola mine (NW Italy) was one of Italy’s most important Fe–Cu sulphide mines. It was industrially exploited from 1864 until 1962 and, during this period, produced over 1 Mt of Fe–Cu sulphides with an average grade ranging from 7 to 14 Cu wt% (Marescotti et al., 2008, 2010). During exploitation, five major waste-rock dumps were built up through progressive accumulation of heterogeneous sterile rocks (derived from galleries and open-pit excavations) and non-valuable ore-fragments, with metal concentration below the economic cut-off, produced during beneficiation processes. The soils of the dumps are characterized by severe edaphic condition due to their peculiar physical and chemical properties (e.g. steep slopes, low moisture retainability, impermeabilization due to cementification and hardpan formation, high metal concentrations, low pH values, low availability of essential macronutrients). For these reasons most of the Libiola waste-rock dumps showed a complete absence of plants, while few zones show pioneer vegetation (Marsili et al., 2009; Roccotiello et al., 2010). Six sampling plots were randomly chosen in a flat part of the dump (about 60 m2) which is characterized by a complete absence

of vegetation, namely barren substrate. A soil sample (about 2 kg) for each plot was collected in Spring and in Autumn between 5 and 20 cm depth by preliminarily removing the fraction >2 cm. The samples were then split into 2 identical parts for the mycological and mineralogical/geochemical analyses. 2.2. Analytical methods The particle size distribution was determined by dry sieving (sieve diameter from 10 mm to 0.063 mm) following the standard ASTM size classes. The mineralogical and lithological characterization of the main constituents was performed by means of optical (transmitted- and reflected-light) microscopy. The modal abundance was estimated by image analysis with the software Olympus C-View II-Bund-Cell^B. The pH values were determined in laboratory by WTW Multiline P3 Set after equilibrating the soil fraction <2 mm in deionized water for 12–16 h (or overnight), at a solidto-water ratio of 1:2.5 (w/w). For the geochemical analyses, the soil fraction <2 mm was ground in an agate jar; selected potentially toxic elements (PTEs) (Cu, Zn, Ag, Ni, Co, Mn, and Cr) were determined by ICP-MS at ACME Analytical Lab (Canada) on solutions obtained by acid digestion. The modified EPA Method 1312 (U.S. EPA, 1994) was employed as qualitative leaching test. With this method, slightly acidified deionized water, used to simulate natural precipitations, interacted in a rotary agitator for 18 hours with the <2 mm fraction of the sample, at a solid-to-water ratio 1:20 (w/ w). The total trace-element contents of the filtered solution were then analyzed by ICP-AES at ACME Analytical Lab (Canada). The element concentrations were measured in triplicate for quality measurement assurance and the percentage coefficients of relative standard deviation were below 10%, reaching maximum values of about 25% only for those concentrations close to the detection limit of the element. 2.3. Isolation and identification of the microfungi Microfungi were counted as MTUs (Morphological Taxonomic Units) to calculate MTU g 1 dry soil and isolated with the dilution plate technique (Gams et al., 1987) using two media MEA + C (Malt Extract Agar added with Chloramphenicol; and Rose Bengal agar, RB). The initial dilution was obtained by mixing 1 g of soil with 100 mL of sterile water. Each soil sample was plated in duplicate, once for each dilution (1:50 000 and 1:100 000). The fungi were identified according to conventional mycology methods by observing their micro- and macromorphological characteristics and considering the different trophic and physiological requirements. Moreover, in order to identify critical microfungal species, molecular analyses (ß-tubulin locus DNA sequence) were performed. The isolated strains were conserved in the culture collection of Mycological Laboratory of DISTAV (University of Genoa, Italy). 2.4. Cu resistance and accumulation Growth response screening in Cu-enriched media of all filamentous fungi isolated were carried out to select the most tolerant strains. The inoculum solution was prepared by diluting fungal conidia (obtained by scraping the surface of 12-d-old cultures with a loop) in a semisolid suspension of Tween 80 (polysorbitan 80). The conidia were counted using a Burker chamber to quantify the inoculums (8  105 conidia). 2.5. Cu screening test Metal solutions were prepared by dissolving CuSO4 5 H2O in deionized water. The fungal screening was done at Cu(II) 0 and

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100 mg L 1. The screening medium was prepared by mixing the metal solution separately autoclaved (at 121 °C, 20 min). The pH of the final solution was adjusted to the desired value by using sterile HCl (1N) and NaOH (1N) solutions and measured with a pH meter. The isolated fungi (5 replicates each isolate) were inoculated on solid MEA (Petri dishes 12 cm Ø) and kept at 24 °C for 7 d, to verify the growing capability of each strain under Cu stress.

Table 1 Statistical summary of bulk elemental composition (selected metals; mg kg bioavailable (mg L 1) and pH in the studied samples.

Mean SD Min value Max value

1

), Cu-

Cu bioavailable

Cu

Zn

Ag

Ni

Co

Mn

Cr

pH

4 0.6 3 4

25 784 124 2404 2729

123 11 109 139

2.9 0.2 2.6 3.1

67 1 65 69

45 2 42 47

320 15 302 341

188 5 178 191

4 0.2 3.5 4.2

2.6. Cu accumulation test Three microfungal strains were selected to test the effects of Cu(II) (0, 200 and 400 mg L 1 of CuSO4 5
H2O dissolved in deionized water) on the growth and metal ion uptake by fungi. Experiments were carried out in 100 mL conical flask with 80 mL working volume. The medium was inoculated following the protocol above mentioned and were daily observed for 7 d. Subsequently, colonies were washed with tap water, then with distilled water and filtered using filter paper 0.45 lm (Sartorius). To determine dry weight (DW), mycelia were oven-dried at 60 °C 48 h, samples were weighted and then powdered using a glass mortar and pestle. The Cu concentration of each sample was assessed by acid digestion (0.5 g powder leached with 3 mL 2–2–2 HCl–HNO3–H2O, 95 °C, 1 h) followed by ICP-MS analysis at ACME Analytical Lab (Canada). The Cu-tolerance in the selected strains was assessed by means of the tolerance index (TI) (based on the dry weights of fungal biomass after 7 d) calculated as reported by Fomina et al. (2003) and Crane et al. (2010). The TI evaluates the inhibition of biomass production on the Cu-enriched media compared with the Cu(II) 0 mg L 1. The lower the TI, the greater the Cu toxicity. 3. Results

sites (http://www.camera.it/parlam/leggi/deleghe/06152dl.htm); and the reported ranges for different types of uncontaminated soils (Kabata-Pendias and Mukherjee, 2007; Alloway, 2013). 3.2. Microfungi 96 Petri dishes were inoculated; 301 MTUs belonging to 11 different taxa have been isolated (99 in Autumn and 202 in Spring). The MTUs per gram of soil distributed in two different sample seasons shows a substantial disparity: 2.06  105 MTU g 1 in Autumn and 4.20  105 MTU g 1 in Spring. The most recurring species of filamentous fungi are Trichoderma harzianum Rifai (22%), C. rosea (Link) Schroers, Samuels, Seifert and W. Gams (14%) and Aspergillus alliaceus Thom & Church (11.5%). These three species occur in each sample point and account for about 50% of total MTUs isolated. The comparison between total MTUs occurrence and bioavailable Cu (r = 0.57) in each sample point shows that the total MTUs amount decreases if the Cu bioavailable increases (Fig. 3a). Conversely, Pearson’s correlation between three species of most present filamentous fungi shows that the MTUs increases slightly despite the rise of bioavailable Cu (Fig. 3b–d) (T. harzianum r = 0.52, C. rosea r = 0.94, and A. alliaceus r = 0.70).

3.1. Mineralogy and chemistry 3.3. Cu tolerance and accumulation The soil samples (Fig. 1a, 1b) are gravelly-sandy sediments and the sandy-, silty- and clayey-fractions represent as much as 25% of the total. The pH of soils is acid and generally homogeneous across the samples (3–4; Table 1). The mineralogical and lithological composition of the studied waste-rock soils is summarized in Fig. 2a and b. From the chemical point of view, the soils evidence significant concentrations of several metals (Table 1); as expected, copper concentrations are very high in all samples, up to one order of magnitude higher than the Italian limits for residential and industrial

Table 2 summarizes the growth response (expressed in terms of TI values) shown by the analyzed strains at different levels of Cu concentrations. It is worth noting that increasing Cu(II) concentration determines a progressive selection of resistant fungal strains. Moreover, a Cu(II)-enriched media with concentration at 100 mg L 1 allows growing only the three strains (T. harzianum, C. rosea and A. alliaceus). Table 2 also highlights that at Cu(II) 200 mg L 1 only two (T. harzianum, C. rosea) strains are vital and, eventually, sole T. harzianum is able to resist at Cu(II) 400 mg L 1.

Fig. 1. (a) Aerial view of the Libiola mining area (dotted circle) with the position of the study area (white arrow) with the geographical position (box) and (b) barren soil in the sampling area.

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Fig. 2. (a) Modal abundance (%) of the main mineral species and (b) of the main mineral lithotypes.

Fig. 3. Relationship between bioavailable copper and: total MTU 105 of microfungi in each soil sample; T. harzianum in each soil sample; C. rosea in each soil sample; A. alliaceus in each soil sample.

Table 2 Growth response (G) and relative Tolerance Index (TI) shown by the analyzed strains at different levels of Cu(II) concentrations. Cu(II) concentration (mg kg 0

100

1

)

200

400

Species

G

TI

G

TI

G

TI

Aspergillus alliaceus Clonostachys rosea Penicillium chrysogenum P. rubrum Trichoderma harzianum Sterile mycelia type 1 Sterile mycelia type 2 Sterile mycelia type 3 Yeast type 1 Yeast type 2 Yeast type 3

+ + + + + + + + ND ND ND

100 100 100 100 100 100 100 100 ND ND ND

+ +

31.3 40.2

+ +

6.5 29.9

+

92.2

+

89.1

G

TI

+

24.2

The TI for each concentration decreases in the microfungal strains when the Cu(II) concentration increases. Data on accumulation capability (Table 3) show that T. harzianum and C. rosea have high bioaccumulation capability at Cu(II) 200 mg mg L 1 while at Cu(II) 400 mg mg L 1 their biomass grown in the flask was insufficient for ICP-MS analysis. On the base of the occurrence and the growth response, we have decided to focus our investigation only on the following strains: T. harzianum, C. rosea and, A. alliaceus, which present the highest occurrence and are able to grow at Cu(II) 100 mg L 1. 4. Discussion

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

The soil of the barren part of the studied waste-rock dump is characterized by homogeneous, very high concentrations of copper, up to one order of magnitude higher than the natural

M. Zotti et al. / Chemosphere 117 (2014) 471–476 Table 3 Cu(II)-accumulation capability (mg kg 1) of T. harzianum and C. rosea submitted to different Cu(II) concentrations (mg L 1).

T. harzianum Medium T. harzianum C. rosea Medium C. rosea

Cu0

Cu200

Cu400

BF 200

BF 400

0 ± 0.0 0 ± 0.0 0 ± 0.0 0 ± 0.0

19 628 ± 1353 181 ± 1 22 222 ± 655 205 ± 3

– 453 ± 33 – 407 ± 2

108 ± 3

–

108 ± 3

–

background of the vegetated soils of the Libiola Area (160– 329 mg kg 1; Marescotti et al., 2010, 2013). Copper, as well as other metals (in particular Zn), can also be taken up by Fe-oxide and oxyhydroxides which are widespread over the Libiola mine and extensively form during oxidation primary sulphides (Carbone et al., 2005; Marescotti et al., 2008, 2010). These results suggest that Cu-bioavailability is mainly governed by the oxidation processes triggering sulphide weathering and by Cu-adsorption/ desorption processes on goethite. Since both processes are mainly controlled by the availability of circulating waters and by highly variable chemical parameters (e.g. pH, Eh) it can be inferred that Cu-bioavailability can significantly change according to season, i.e. during dry and wet periods, exerting a strong selective pressure on native biota, especially mycobiota. Data on the fungal community show, on the one hand, a low number of taxa (11) and, on the other hand, a rather high number of MTUs (3.1  105). Therefore, it may be assumed that fungal germination is inhibited by very high levels of bioavailable Cu(II), as a consequence only few taxa are able to germinate in these extreme conditions. Moreover, the lack of organic matter could facilitate the growth of more competitive species. Indeed, data on taxa finding frequency show that only T. harzianum, C. rosea and A. sclerotiorum has been collected more than 10% of total finding. Furthermore, the correlations between the previously mentioned species and bioavailable Cu(II) concentration is positive. Although previous works about mycobiota on mine waste dumps (Marescotti et al., 2010; Roccotiello et al., 2010) hypothesized the possible tolerance/resistance to Cu by T. harzianum and C. rosea, none of these microfungal species has been tested for these properties. On the base of growth response, the achieved results highlight that T. harzianum, C. rosea and A. sclerotiorum are able to germinate and grow on high Cu-enriched media. In addition, our study demonstrates the species T. harzianum and C. rosea as able to accumulate Cu. The capability of these species, never observed before, represents the most novel aspect of our work. The previous microfungal strains belong to three genus which include other species that are known to be resistant and/ or tolerant to metals (Gadd, 2001; Iskandar et al., 2011; Ceci et al., 2012), with a good growth capability in metal-polluted soil (Al-Rajhi, 2013; Fomina et al., 2003). Tolerance can help assess the capability of fungi to be exploited as a bioremediation tool (Carrillo-González and González-Chávez, 2012; Ceci et al., 2012). The TI value decreased with Cu treatments, but is was higher in T. harzianum, indicating that (i) this species was able to tolerate Cu at the chosen treatment levels, (ii) it was very effective in extracting Cu and (iii) it protects itself from adverse effects of the metal. However, beyond Cu 200 mg L 1 treatments, the TI declined, indicating that Cu had become deleterious for the microfungal strain. The high TI of T. harzianum was also substantiated by a high Cu accumulation displaying an efficient Cu uptake. This might be due to the existence of some tolerance mechanisms, which help the species in coping with high metal concentrations. This has already been demonstrated by Al-Rajhi (2013) where T. harzianum was employed in pot soil to minimize the uptake and concentration of metals like copper and zinc in a crop plant, confirming its ability to live in metal-contaminated soils.

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Our study confirms the bioaccumulation ability of selected fungal strains and increases information concerning mycoflora in highly Cu-contaminated bare soils. A possible follow-up could be represented by a future development of efficient fungi-based protocols to be exploited in decontaminating mine sites. References Alloway, B.J., 2013. Heavy metals in soils. In: Trace Metals and Metalloids in Soils and their Bioavailability, third ed. Springer Science Business Media, Dordrecht, The Netherlands. pp. 613. Al-Rajhi, A.M.H., 2013. Impact of biofertilizer Trichoderma harzianum Rifai and the biomarker changes in Eruca sativa L. plant grown in metal-polluted soils. World Appl. Sci. J. 22, 171–180. Anjum, F., Bhatti, H.N., Ghauri, M.A., Bhatti, I.A., Asgher, M., Asi, M.R., 2009. Bioleaching of copper, cobalt and zinc from black shale by Penicillium notatum. Afr. J. Biotechnol. 8, 5038–5045. Bojarczuk, K., Kieliszewska-Rokicka, B., 2010. Effect of ectomycorrhiza on Cu and Pb accumulation in leaves and roots of silver birch (Betula pendula Roth.) seedlings grown in metal-contaminated soil. Water Air Soil Pollut. 207, 227–240. Carbone, C., Di Benedetto, F., Marescotti, P., Sangregorio, C., Sorace, L., Lima, N., Romanelli, M., Lucchetti, G., Cipriani, C., 2005. Natural Fe-oxides and oxyhydroxides nanoparticles: an EPR and SQUID investigation. Miner. Petrol. 85, 19–32. Carrillo-González, R., González-Chávez, A., 2012. Tolerance to and accumulation of cadmium by the mycelium of the fungi Scleroderma citrinum and Pisolithus tinctorius. Biol. Trace Elem. Res. 146, 388–395. Ceci, A., Maggi, O., Pinzari, F., Persiani, A.M., 2012. Growth responses to and accumulation of vanadium in agricultural soil fungi. Appl. Soil Ecol. 58, 1–11. Cerino-Córdova, F.J., García-León, A.M., Soto-Regalado, E., Sánchez-González, M.N., Lozano-Ramírez, T., García-Avalos, B.C., Loredo-Medrano, J.A., 2012. Experimental design for the optimization of copper biosorption from aqueous solution by Aspergillus terreus. J. Environ. Manage. 95, S77–S82. Crane, S., Dighton, J., Barkay, T., 2010. Growth responses to and accumulation of mercury by ectomycorrhizal fungi. Fungal Biol. 114, 873–880. Dursun, A.Y., Uslua, G., Cucia, Y., Aksub, Z., 2003. Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger. Process Biochem. 38, 1647–1651. Fomina, M., Ritz, K., Gadd, G.M., 2003. Nutritional influence on the ability of fungal mycelia to penetrate toxic metal-containing domains. Mycol. Res. 107, 861– 871. Fomina, M.A., Alexander, I.J., Colpaert, J.V., Gadd, G.M., 2005a. Solubilization of toxic metal minerals and metal tolerance of mycorrhizal fungi. Soil Biol. Biochem. 37, 851–866. Fomina, M.A., Gadd, G.M., Burford, E.P., 2005b. Toxic metals and fungal communities. In: Dighton, J., White, J.F., Oudemans, P. (Eds.), The Fungal Community: Its Organization and Role in the Ecosystem, third ed. CRC Press, pp. 733–758. Gadd, G.M., 1993. Interactions of fungi with toxic metals. New Phytol. 124, 25–60. Gadd, G.M., 2001. Metal transformations. In: Gadd, G.M. (Ed.), Fungi in Bioremediation. Cambridge University Press, UK, pp. 359–382. Gadd, G.M., 2007. Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycol. Res. 111, 3–49. Gams, W., Aa, H.A., van der Plaats-Niterink, A.J., van der Samson, R.A., Stalpers, J.A., 1987. CBS Course of Mycology, third ed. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Hillel, D., Rosenzweig, C., Powlson, D., Scow, K., Singer, M., Sparks, D., 2004. Encyclopedia of Soils in the Environment, vol. 1. Academic Press, Oxford, U.K.. 548 pp. Hooda, P.S., 2010. Trace Elements in Soils. John Wiley and Sons, Ltd., Chichester, West Sussex, U.K.. Iskandar, N.L., Mohd Zainudin, N.A.I., Tan, S.G., 2011. Tolerance and biosorption of copper (Cu) and lead (Pb) by filamentous fungi isolated from a freshwater ecosystem. J. Environ. Sci. 23, 824–830. Jambor, J.L., Blowes, D.W., 1994. The environmental geochemistry of sulfide minewastes. Short-Course Handbook, vol. 22. Mineralogical Association of Canada, Waterloo, Ontario, Canada. Kabata-Pendias, A., Mukherjee, A.B., 2007. Trace Elements From Soil To Human. Springer-Verlag, Berlin, Heidelberg, Germany. Marescotti, P., Carbone, C., De Capitani, L., Grieco, G., Lucchetti, G., Servida, D., 2008. Mineralogical and geochemical characterisation of open-air tailing and wasterock dumps from the Libiola Fe–Cu sulphide mine (Eastern Liguria, Italy). Environ. Geol. 53, 1613–1626. Marescotti, P., Azzali, E., Servida, D., Carbone, C., Grieco, G., De Capitani, L., Lucchetti, G., 2010. Mineralogical and geochemical spatial analyses of a waste-rock dump at the Libiola Fe–Cu sulphide mine (Eastern Liguria, Italy). Environ. Earth Sci. 61, 187–199. Marescotti, P., Roccotiello, E., Zotti, M., De Capitani, L., Carbone, C., Azzali, E., Mariotti, M.G., Lucchetti, G., 2013. Influence of soil mineralogy and chemistry on fungi and plants in a waste-rock dump from the Libiola mine (eastern Liguria, Italy). Per. Mineral. 82, 141–162.

476

M. Zotti et al. / Chemosphere 117 (2014) 471–476

Marsili, S., Roccotiello, E., Carbone, C., Marescotti, P., Cornara, L., Mariotti, M.G., 2009. Plant colonization on a contaminated serpentine site. Northeast Nat. 16, 297–308. Pratas, J., Prasad, M.N.V., Freitas, H., Conde, L., 2005. Plants growing in abandoned mines of Portugal are useful for biogeochemical exploration of arsenic, antimony, tungsten and mine reclamation. J. Geochem. Explor. 85, 99–107. Roccotiello, E., Zotti, M., Mesiti, S., Marescotti, P., Carbone, C., Cornara, L., Mariotti, M.G., 2010. Biodiversity in metal-polluted soils. Fresenius Environ. Bull. 19 (10b), 2420–2425. Ross, I.S., 1994. Uptake of zinc by fungi. In: Winkelmann, G., Winge, D.R. (Eds.), Metal Ions in Fungi. Mycology Series, vol. 11. Dekker, New York, pp. 237–257. Sag˘, Y., Akçael, B., Kutsal, T., 2002. Ternary biosorption equilibria of chromium (VI), copper (II), and cadmium (II) on Rhizopus arrhizus. Sep. Sci. Technol. 37, 279–309. Sßahan, T., Ceylan, H., Sßahiner, N., Aktasß, N., 2010. Optimization of removal conditions of copper ions from aqueous solutions by Trametes versicolor. Bioresour. Technol. 101, 4520–4526.

Tasßtan, B.E., Ertug˘rul, S., Dönmez, G., 2010. Effective bioremoval of reactive dye and heavy metals by Aspergillus versicolor. Bioresour. Technol. 101, 870–876. U.S. Environmental Protection Agency, 1994. Test methods for evaluating solid waste, physical/chemical methods (SW-846) 3rd ed, update B. Environmental Protection Agency, National Center for Environmental Publications, Cincinnati. Wang, B., Wang, K., 2013. Removal of copper from acid wastewater of bioleaching by adsorption onto ramie residue and uptake by Trichoderma viride. Bioresour. Technol. 136, 244–250. Yap, C.K., Yazdani, M., Abdullah, F., Tan, S.G., 2011. Is the high Cu tolerance of Trichoderma atroviride isolated from the Cu-polluted sediment due to adaptation? an in vitro toxicological study. Sains Malaysiana 40 (2), 119–124. Yetis, Ü., Dolek, A., Dilek, F.B., Özcengiz, G., 2000. The removal of Pb(II) by Phanerochaete chrysosporium. Water Res. 34, 4090–4100. Zucconi, L., Ripa, C., Alianiello, F., Benedetti, A., Onofri, S., 2003. Lead resistance, sorption and accumulation in a Paecilomyces lilacinus strain. Biol. Fertil. Soils 37, 17–22.