UNIVERSITÀ DEGLI STUDI DI VERONA DIPARTIMENTO DI BIOTECNOLOGIE DOTTORATO DI RICERCA IN: BIOTECNOLOGIE MOLECOLARI INDUSTRIALI ED AMBIENTALI XXIII CICLO Organic and inorganic Pb affects autochthonous bacterial communities and phytoextraction potentials of plant species grown in a formerly industrial soil contaminated by tetraethyl-lead S.S.D. AGR16 Coordinatore: Prof. Roberto Bassi Tutor: Prof. Giovanni Vallini Co-Tutor: Dott.ssa Silvia Lampis Dottoranda: Dott.ssa Anita Ferrari Table of contents i Table of Contents Table of contents Table of contents ................................................................................................................................. i Table of contents .............................................................................................................................ii Abbreviations .................................................................................................................................. vi Abstract/Riassunto .............................................................................................................................. 7 Abstract .......................................................................................................................................... 8 Riassunto ...................................................................................................................................... 16 1. Introduction .................................................................................................................................. 25 1.1 Lead in the environment .......................................................................................................... 26 1.1.1 Pb general characteristics .................................................................................................. 26 1.1.2 Pb natural occurrence and ore extraction ............................................................................ 27 1.1.3 Use of Inorganic lead ........................................................................................................ 27 1.1.4 Organic lead ..................................................................................................................... 27 1.1.4.1 Use of Organic lead ........................................................................................................ 28 1.1.4.2 Tetraethyl-lead (TEL) ...................................................................................................... 29 1.1.5 Pb releases to the environment .......................................................................................... 32 1.1.5.1 Pb in soil ........................................................................................................................ 33 1.1.6 Human toxicity of lead ....................................................................................................... 34 1.1.6.1 Toxicity of Inorganic lead ................................................................................................ 35 1.1.6.2 Toxicity of Organic lead .................................................................................................. 35 1.2 Pb interaction with biological systems ....................................................................................... 37 1.2.1 Heavy metals and microorganisms ..................................................................................... 37 1.2.1.1 Lead resistance strategies by microorganisms .................................................................. 38 1.2.1.2 Microorganisms and Organic lead .................................................................................... 43 1.2.2 Pb in higher plants ............................................................................................................ 44 1.2.2.1 Pb toxicity in higher plants .............................................................................................. 44 1.2.2.2 Pb uptake and transport in plants .................................................................................... 45 1.2.2.3 Pb tolerance in plants ..................................................................................................... 45 1.2.2.4 Interactions plant-microorganisms ................................................................................... 49 1.3 Bioremediation ........................................................................................................................ 51 1.3.1 Remediation Background and Bioremediation ...................................................................... 51 1.3.2 Microbe-induced bioremediation ......................................................................................... 52 1.3.2.1 Organic lead Microbe-induced Biodegradation cases ......................................................... 53 1.3.3 Phytoremediation .............................................................................................................. 53 1.3.3.1 Plants and organic compounds ........................................................................................ 54 1.3.3.2 Pb Phytoextraction ......................................................................................................... 55 1.3.4 Phytoremediation assisted by soil rhizobacteria ................................................................... 56 1.3.5 Combined approach of conventional and biological remediation ........................................... 57 1.4 The SLOI Srl (Trento-Nord site) case of study............................................................................ 59 ii Table of Contents 1.4.1 The history ....................................................................................................................... 59 1.4.2 Remediation priority .......................................................................................................... 61 1.5 Aims of the study ..................................................................................................................... 62 2. Material and Methods .................................................................................................................... 65 2.1 Chemicals, culture media and solutions ..................................................................................... 66 2.1.1 Growth media ................................................................................................................... 66 2.1.2 Solutions ........................................................................................................................... 66 2.2 The Ex- SLOI sampling area ..................................................................................................... 67 2.2.1 Trento-Nord area‟s geological characteristics ....................................................................... 67 2.2.2 Sampling locations within Ex-SLOI area .............................................................................. 68 2.3 Culture-dependent study .......................................................................................................... 71 2.3.1 Enumeration of cultivable microflora ................................................................................... 71 2.3.2 Isolation of soil bacterial strains ......................................................................................... 71 2.3.2.1 Direct isolation ............................................................................................................... 71 2.3.2.1 Enrichment Cultures ....................................................................................................... 71 2.3.3 MIC (Minimum inhibitory concentration) determination for organic and inorganic Pb ............. 72 2.3.4 Growth Test with Tel as sole carbon source ........................................................................ 72 2.4 Plant growth promoting (PGP) traits analysis ............................................................................. 73 2.4.1 Assay for 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase activity ......................... 73 2.4.2 Assay for indoleacetic acid (IAA) production ........................................................................ 73 2.5 Phylogenetic analysis through molecular taxonomic techniques .................................................. 74 2.5.1 ARDRA (Amplified Ribosomal DNA Restriction Analysis) ....................................................... 74 2.5.1.1 Genomic DNA extraction with the bead-beater method ..................................................... 74 2.5.1.2 PCR amplification of 16S ribosomal RNA gene .................................................................. 74 2.5.1.3 Restriction digestion ....................................................................................................... 74 2.5.1.4 OTUs determination ........................................................................................................ 75 2.5.2 Sequence analysis and phylogenetic tree construction ......................................................... 75 2.6 Primer-specific PCR for heavy metal resistances and degrading capabilities towards hydrocarbons 76 2.6.1 DNA extraction .................................................................................................................. 76 2.6.2 PCR conditions ................................................................................................................. 76 2.6.2.1 Purification