Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation: the case study at Ingurtosu mining site (Italy) Anna Rosa Sprocati, Chiara Alisi, Valentina Pinto, Maria Rita Montereali, Paola Marconi, Flavia Tasso, Katarzyna Turnau, et al. Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-013-2154-3
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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-013-2154-3
USING MICROBES FOR THE REGULATION OF HEAVY METAL MOBILITY AT ECOSYSTEM AND LANDSCAPE SCALE
Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation: the case study at Ingurtosu mining site (Italy) Anna Rosa Sprocati & Chiara Alisi & Valentina Pinto & Maria Rita Montereali & Paola Marconi & Flavia Tasso & Katarzyna Turnau & Giovanni De Giudici & Katarzyna Goralska & Marta Bevilacqua & Federico Marini & Carlo Cremisini
Received: 26 June 2013 / Accepted: 9 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The paper describes the fieldwork at the Italian test site of the abandoned mine of sphalerite and galena in Ingurtosu (Sardinia), with the aim to assess the applicability of a “toolbox” to establish the optimized techniques for remediation of soils contaminated by mining activities. A preliminary characterization—including (hydro)geochemistry, heavy metal concentration and their mobility in soil, bioprospecting for microbiology and botany—provided a data set for the development of a toolbox to deliver a microbially assisted phytoremediation process. Euphorbia pithyusa was selected as an endemic pioneer plant to be associated with a bacterial consortium, established with ten selected native strains, including metal-tolerant bacteria and producers of plant growth Responsible editor: Vera Slaveykova Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-2154-3) contains supplementary material, which is available to authorized users. A. R. Sprocati : C. Alisi : V. Pinto (*) : M. R. Montereali : P. Marconi : F. Tasso : C. Cremisini Environmental Characterization, Prevention and Recovery Unit, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Via Anguillarese, 301-00123 Rome, Italy e-mail:
[email protected] K. Turnau : K. Goralska Institute of Environmental Sciences and Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland G. De Giudici Department of Chemical and Geological Science, University of Cagliari, via Trentino 51, 09127 Cagliari, Italy M. Bevilacqua : F. Marini Department of Chemistry, University of Rome “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy
factors. The toolbox was firstly assessed in a greenhouse pot experiment. A positive effect of bacterial inoculum on E . pithyusa germination and total plant survival was observed. E . pithyusa showed to be a well-performing metallophyte species, and only inoculated soil retained a microbial activity with a high functional diversity, expanding metabolic affinity also towards root exudates. These results supported the decision to proceed with a field trial, investigating different treatments used singly or in combination: bioaugmentation with bacterial consortia, mycorrhizal fungi and a commercial mineral amendment. Microbial activity in soil, plant physiological parameters and heavy metal content in plants and in soil were monitored. Five months after the beginning, an early assessment of the toolbox under field conditions was carried out. Despite the cold season (October–March), results suggested the following: (1) the field setup as well as the experimental design proved to be effective; (2) plant survival was satisfactory; (3) soil quality was increased and bioaugmentation improved microbial activity, expanding the metabolic competences towards plant interaction (root exudates); and (4) multivariate analysis supported the data provided that the proposed toolbox can be established and the field trial can be carried forward. Keywords Microbially assisted phytoremediation . Field experiment . Heavy metals . Contaminated soils . Mycorrhizae . Mining sites . Mine wastes . Sardinia
Introduction Soil can be considered essentially as a non-renewable resource by the European Union, for a total of about 400 million hectares. Soil degradation is an acute problem, often resulting from human, industrial and mining activities (JRC 2012). The
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estimated cost of soil degradation could amount to 38 billion euros per year in the European Union where the area of soils affected by mining activities has been estimated at 0.6 %, compared to a world average of 0.2 %. The remediation of soils contaminated by mining activities is, therefore, a strategic objective for European policies. Indeed, high concentration of “heavy metals” (HM) in soils of mining areas constitutes a high risk of pollution, also for agricultural activities and villages lying in subsidiary areas (Garcia et al. 2008). In uncontaminated soils, HM concentrations vary in a very wide range (Kabata-Pendias 2001); these toxic elements are known to be adsorbed by clay minerals; phosphates; Fe, Al and Mn oxides and/or hydroxides; and by the organic matter (Kabata-Pendias 2001; Reimann et al. 2003). In mine ores, toxic elements can be incorporated in sulphide minerals such as, for example, As in arsenopyrite and pyrite (Sherman and Randall 2003). Due to the common extractive processes, soils of mining areas are characterized by severe alteration of the pH, absence or very low organic carbon content, loss of structure, extremely high concentrations of toxic elements (obviously related to the mine ores but quite often including the most toxic ones as As, Cd, and Pb in addition to Zn, Cu, Sb, etc.) and, last but not the least, drastic reduction of microbiological diversity (Giller et al. 1998). This means that any recovery activity has two priorities: first of all to reduce the risk of toxic element mobilization and transfer to underground and surface waters and, second, to create the conditions to restore the area to a natural equilibrium. Reactions buffering pH in mine settings play a fundamental role (Jurjovec et al. 2004), but the other fundamental step is the restoration of the microbial diversity. Recovery could be achieved through natural attenuation which, however, is extremely slow. In addition, environments contaminated by metals generally suffer from low microbial activity; for this reason, any approach of bioremediation can be difficult to be implemented in the abandoned mining areas. On the other hand, studies on mining areas or other “extreme” environments are particularly useful to increase the scarce knowledge still existing about the extent of microbial diversity and cultivability, estimated in the order of 5–10 % and 0.1 %, respectively (Staley and Konopka 1985; Amann et al. 1995; Hugenholtz et al. 1998). The discovery of new microorganisms may increase the huge potential that microorganisms can contribute to the improvement of phytoextraction/stabilization technologies, inherent in their capacity to adapt, to communicate with plants and to accelerate chemical reactions up to 106 (Ortíz-Castro et al. 2009). The idea of the UMBRELLA project (7FP EU) to progress beyond the current state of knowledge and applications was to use an integrative approach, derived by “geobiological” processes, for soil remediation in mining and other HMcontaminated areas across Europe, coupling microorganisms with plants to reduce risks for the environment and human
health with a positive impact on downstream fluvial systems, including international waterways. This approach is based on the identification of appropriate strains of microorganisms native to the site and their use in combination with endemic plants for optimizing processes of phytoextraction or phytostabilization. For this purpose, microbial strains with suitable characteristics, HM tolerant and plant growth promoters (PGP), have been selected to establish microbial consortia to be employed as bioaugmentation agents in phytoremediation experiments. Mycorrhizae contribution was also investigated: mycorrhiza dependence is common among plants selected for phytoremediation of industrial tailings (Turnau et al. 2012); although, the practices commonly used often omit mycorrhiza propagation. Plants generally used in such practices include grasses that can develop without mycorrhiza (facultative mycorrhizal) but whose development would be still dependent on climatic conditions and human care, such as watering, which is an expensive alternative. In such cases, the phytoremediation starts to be sustainable and well progressing when arbuscular mycorrhizal fungi (AMF) appear (Turnau et al. 2012; Ryszka and Turnau 2007). Mycorrhizae were found to improve many aspects of plant life originating from scarce nutrition and low water availability, to reduce stresses from toxic metals, heat and UVand to enhance plant resistance to pathogens. Here we describe the experience developed at the Italian test site of the abandoned mine of sphalerite and galena in Ingurtosu (Sardinia) to assess the applicability of a “toolbox” developed to establish optimal techniques for coupling microorganisms and plants for the remediation of soils contaminated by mining activities. The experience is part of a set of fieldworks carried out in parallel in mining sites throughout Europe. The experimental activity on the Ingurtosu test site started with a preliminary characterization including (hydro)geochemistry, heavy metal concentration and their mobility in soil, bioprospecting for microbiology and botany. These data were the base for the development of a toolbox to deliver a microbially assisted phytoremediation process. Euphorbia pithyusa was selected as an endemic pioneer plant to be associated with the bacterial consortium, namely UIC, composed of ten native selected strains, metal-tolerant bacteria and effective producers of PGP compounds. The toolbox was tested in a greenhouse pot experiment to assess if the members associated were mutually compatible under the given specific soil conditions. In Wernitznig et al. (2013), results are shown with a special emphasis on plant physiological parameters and metal interaction, obtaining some positive and some ambiguous results. In this work, we analyse in more detail the positive results for soil microbial activity studied at the community level through Biolog ECOPlates, as a point of an absolute priority for assessing undergoing phytoremediation processes, due to the major role the microbial community plays as indicator of soil quality.
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The field trial was set up based on the outcome of the positive points of the pot experiment and began in October 2011. The experimental design of the field trial included different treatments used singly or in combination: bioaugmentation with bacterial consortia (BACT), mycorrhizae (MYC) and a commercial mineral amendment (VM) obtained from red mud, a by-product of the bauxite industry which is widely used, after adequate neutralization, for environmental remediation processes due to its metal-trapping capacity (Ma and Feng 2011; Liu et al. 2011). The trapping mechanism is controlled by various phenomena that are different for each metal as a function of its characteristics and of the form in which it is present (speciation in solution, formation of complexes, formation of oxyanions, solubility of its salt) (Brunori et al. 2005). Red mud is characterized by very high alkalinity, and its major constituents are crystalline hematite (Fe2O3), boehmite (γAlO(OH)), quartz (SiO2), sodalit e (Na4Al3Si3O12Cl) and gypsum (CaSO4·2H2O), with a minor presence of calcite (CaCO 3 ), whewellite (CaC 2O4·H 2O) and gibbsite (Al(OH) 3). Viromine™ is obtained from the reaction between red mud and sea water, thus reducing the resulting pH from >13 to pH
