View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UniCA Eprints Università degli Studi di Cagliari DOTTORATO DI RICERCA Dottorato di ricerca in Sviluppo e Sperimentazione di Farmaci antinfettivi Ciclo XXVIII Characterization and comparison of microbial communities from different tourist ports in Mediterranean Sea and evaluation of applicability of bioremediation treatments Settore scientifico disciplinare di afferenza BIO19 Presentata da: Dott.ssa Enrica Bullita Coordinatore Dottorato Prof.ssa Alessandra Pani Tutor Dott.ssa Elena Tamburini Esame finale anno accademico 2014 – 2015 Index of contents ABSTRACT ................................................................................................................................................................................. 1 CHAPTER 1 ................................................................................................................................................................................ 4 1. Comparison of the pollution status and bacterial communities in seawater of different Mediterranean tourism ports .... 4 1.1. INTRODUCTION ......................................................................................................................................................................... 4 1.1.1. Petroleum hydrocarbons .......................................................................................................................................................... 4 1.1.2. Oil pollution in the Mediterranean by ship traffic and port activities ....................................................................................... 5 1.2. AIM OF THE WORK .................................................................................................................................................................... 5 1.3. MATERIALS AND METHODS ...................................................................................................................................................... 6 1.3.1. Study areas, samplings and physical parameters ...................................................................................................................... 6 1.3.2. Chemical parameters ................................................................................................................................................................ 7 1.3.3. Determination of bacterial viable titles .................................................................................................................................... 8 1.3.4. On-field processing, DNA extraction and purification, T-RFLP analysis ..................................................................................... 9 1.3.5. Statistical analysis ................................................................................................................................................................... 10 1.4. RESULTS AND DISCUSSION ...................................................................................................................................................... 10 1.4.1. Comparison of the pollution status in seawater ..................................................................................................................... 10 1.4.2. Comparison of the bacterial communities in seawater .......................................................................................................... 13 1.1. REFERENCES ............................................................................................................................................................................ 14 1.2. DISSEMINATION ...................................................................................................................................................................... 15 CHAPTER 2 .............................................................................................................................................................................. 16 2. Comparison of the effects of biostimulation treatment on bacterial communities in seawater from different Mediterranean tourist ports .................................................................................................................................................... 16 2.1. INTRODUCTION ....................................................................................................................................................................... 16 2.1.1. Behaviour of oil in the marine environments.......................................................................................................................... 16 2.1.2. Microbial network involved in oil degradation ....................................................................................................................... 16 2.1.3. Hydrocarbon bioremediation in marine environments .......................................................................................................... 18 2.1.4. Biostimulation in seawater ..................................................................................................................................................... 19 2.2. AIMS OF THE WORK ................................................................................................................................................................ 19 2.3. MATERIALS AND METHODS .................................................................................................................................................... 20 2.3.1. Design of microcosms ............................................................................................................................................................. 20 2.3.2. Residual hydrocarbon determination ..................................................................................................................................... 21 2.3.3. DNA extraction and purification for T-RFLP analysis ............................................................................................................... 21 2.3.4. Statistical analysis ................................................................................................................................................................... 22 2.4. RESULTS AND DISCUSSION ...................................................................................................................................................... 22 2.4.1. Preliminary tests of natural attenuation ................................................................................................................................. 22 2.4.2. Comparison effect of the biostimulation treatments ............................................................................................................. 22 2.4.3. Comparison of bacterial communities during biostimulation treatment................................................................................ 24 2.5. REFERENCES ............................................................................................................................................................................ 25 CHAPTER 3 .............................................................................................................................................................................. 26 3. Characterization and comparison of alkane-degrading bacteria from seawater of different Mediterranean tourism ports 26 3.1. INTRODUCTION ....................................................................................................................................................................... 26 3.1.1. Bacterial aerobic degradation of alkanes ................................................................................................................................ 26 3.1.2. Role of surface active compounds in the microbial access to hydrocarbons .......................................................................... 28 3.1.3. Obligate hydrocarbonoclastic bacteria ................................................................................................................................... 30 3.2. AIMS OF THE WORK ................................................................................................................................................................ 33 3.3. MATERIALS AND METHODS .................................................................................................................................................... 33 3.3.1. Medium carbon sources.......................................................................................................................................................... 33 3.3.2. Strain selection and isolation in pure culture .......................................................................................................................... 33 3.3.3. 16S rRNA genes sequencing and phylogenetic analysis .......................................................................................................... 34 3.3.4. Growth and SAC production on hydrocarbons ....................................................................................................................... 34 3.3.5. Hydrocarbon degradation test ................................................................................................................................................ 36 3.4. RESULTS AND DISCUSSION .....................................................................................................................................................