Organic and inorganic Pb affects autochthonous bacterial communities and phytoextraction potentials of plant species grown in a formerly industrial soil contaminated by tetraethyl-lead

Lead is worldwide one of the pollutants of major concern due to its widespread diffusion, persistence and toxicity. In particular Tetra-ethyl lead (TEL) has been used for almost a century as anti-knocking additive for petrol, leaving an ubiquitous pollution of both inorganic and more toxic organic Pb in low concentrations and more severe contaminations at manufacturing level, implying the need for remediation. An interesting alternative or complement to the expensive and invasive physical-chemical remediation conventional technologies is the Bioremediation – the use of microorganisms and/or plant to degrade, remove or detoxify the contaminants. In particular Phytoremediation enhanced by microorganisms - based on the use of plant in synergy with microorganisms – offers a low cost in-situ applicable method to remediate and restore contaminated areas. In this context the present PhD study - which can be divided in two main sections - dealt with a real case of a soil highly contaminated by both organic and inorganic lead in a former industrial area in Trento, where the company SLOI produced TEL for almost 40 years till the end of the ’70. The first section of the study focused on the soil autochthonous bacterial community selected in the Ex-SLOI area with the aim of evaluating its biodiversity and composition, resistance, bioremediation potential and the impact of the long-term Pb contamination; it also aimed at isolating and characterizing community members in relation to their resistance and plant growth promoting (PGP) potential. On the other side the second section of the PhD study focused on the interaction of the examined autochthonous soil micro flora and plants in relation to Pb contamination in a Phytoremediation approach, examining 2 plants:Brassica juncea - crop plant with high biomass production known to accumulate various metals - and Apocynum cannabinum - suggested for both organic and inorganic Pb phytoextraction although only few literature data are available. The term Phytoextraction specifically refers to the use of pollutant-accumulating plants able to extract and translocate contaminants to the harvestable aerial portion, which can then be removed from the site; the efficiency of a phytoremediation/phytoextraction process depends directly on both biomass production and accumulation of the contaminant by the plant. Therefore both a lab-scale and a field-scale trial were performed to evaluate the interaction and possible synergistic role of the examined soil autochthonous micro flora in a Phytoremediation approach, and to study the tolerance and basic phytoremediation potential of the 2 plants in relation to the examined contamination. In the context of the characterization of the soil autochthonous bacterial community the results obtained indicated that the high Pb contamination in the Ex-SLOI area exerted a selection for a tolerant and well adapted community, characterized by a heterogenic structure and - for a perturbed soil - rich biodiversity, resistance and degrading potential. In particular both the molecular and the culture-dependent study performed indicated the predominance of the gram-negative Proteobacteria phylum, which included the majority of OTUs directly isolated from the most contaminated examined sampling points and accounted for the totality of strains isolated in the strictest condition of Enrichment culture - performed with TEL as sole carbon source, to select most resistant and metabolically potential strains. This phylum also included all 8 strains which displayed - in a molecular analysis - heavy metal (HM) resistance determinants, including a strain of Cupriavidus campinensis with 4 determinants (for resistance to Pb, Hg, Cr and Cd-Zn-Co). Considering also the PGP traits examined, 3 OTUs - of the Gamma and Beta Proteobacteria classes - reported both HM resistance determinants and PGP traits, namely Delftia sp., Pseudomonas putida and Stenotrophomonas maltophilia, consequently particularly interesting even in a Phytoremediation perspective with a biaugmentation protocol. The study performed indicated therefore the selection imposed by the contamination of a resistant microbial cenosis, with members of particular high resistance and PGP potential belonging to the most represented Proteobacteria phylum. Examining the interaction of indigenous micro flora with the plants B.juncea and A.cannabinum in the lab-scale study - performed in parallel with the soil from the Ex-SLOI area untreated and sterilized - the results pointed out the micro flora positive influence on both plant species, improving plant growth and the Phytoremediation efficiency; in B.juncea it also positively affected Pb uptake. Moreover, the analyses performed on the speciation of the organic Pb compounds in soil along the lab-scale trial with B.juncea pointed out a progressive degradation of organic lead during the phytoremediation process, suggesting the promotion by the plant-micro flora system of the mineralization of organic Pb to the less toxic and mobile inorganic lead. Obtained data also indicated tolerance and resistance of both plant species towards inorganic and organic Pb, reaching good Pb concentrations in their tissues, despite Pb is a challenging metal which tightly bind to soils particles and plant materials. However A. cannabinum showed a much lower and slower shoot biomass production and, although it reached the highest detected Pb concentration within its tissues, it displayed a sharp preferential Pb accumulation within roots, while accumulation in the harvestable shoots is the main objective in a phytoextraction process. Besides almost one order of magnitude decrease was detected in its shoot Pb concentrations in field conditions. However in field-trial performed at the Ex-SLOI area both species showed higher values of biomass production and consequently of Pb phytoextracted - defined as µg Pb/plant and calculated as the product of Pb concentrations reached in plant tissues and biomass produced - which evaluates the actual Phytoremediation efficiency. Comparing the 2 plant species, B.juncea showed a higher Phytoextraction efficiency accumulating a comparable amount of Pb in the harvestable shoot both in lab-scale mesocosms and in open field conditions, but in a much shorter time. Values obtained indicated that, despite not applicable at the high contaminations as those detected at the examined points within the Ex-SLOI, a Phytoremediation process with B.juncea could be interesting at lower/shallow inorganic/organic Pb contamination and in a combined approach with physical-chemical techniques. Accordingly to literature the main drawbacks of Phytoremediation are the need of time and low efficiency - associated to pollutant fixation by soil particles and low absorption and/or transportation by plants - and growing interest has been drawn by microorganisms with PGP activity, able to increase plant biomass and/or metal uptake, and by the use of higher plants such as Poplar (Populus sp.). Interesting prospective - and trial in progress - is in fact the application of this arboreal plant – with extensive root system and great biomass production - in a biaugmentation protocol with strains isolated from the examined indigenous micro flora selected within the Ex-SLOI area.