Alma Mater Studiorum – Universitá di Bologna Dottorato di Ricerca in Scienze Biochimiche e Biotecnologiche Ciclo XXVII Settore Concorsuale 03/D1 Settore Scientifico Disciplinare CHIM/11 MICROBIAL ECOLOGY OF BIOTECHNOLOGICAL PROCESSES Presentata da: Dott.ssa Serena Fraraccio Coordinatore Dottorato Relatore Chiar.mo Prof. Chiar.mo Prof. Fabio Fava Santi Mario Spampinato Correlatori Giulio Zanaroli, Ph.D. Assoc. Prof. Ondřej Uhlík, Ph.D. Esame finale anno 2015 Abstract The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA- accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high- throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment. Keywords Microbial ecology; metagenomics; biotechnological process; bioremediation; aerobic cometabolism; chlorinated aliphatic hydrocarbons (CAHs); polycyclic aromatic hydrocarbons (PAHs); polyhydroxyalkanoates (PHA); denaturing gradient gel electrophoresis (DGGE); stable isotope probing (SIP). ii A mia madre… iii Acknowledgements The first thanks is for my parents and my sister. Thank you for your unconditional support, no matter how distant, no matter how tired…you have always been next to me, as emblem of honesty, simplicity and what really love is. Thank you zia Pina and zio Franco for all your teachings, patience and hilarity! I thank my other uncles and cousins and of course I thank my grandmother, she is almost a century of thoughts, joys, sorrows, wars…so many stories to hear! A very thankful hug to my closest friends Melania, Aurora, Martina, Luca and to all of the people that make my life special. I thank Lucie and her family for the warm welcome they gave to me everytime I needed and for the great time spent together in Prague. I am endlessly grateful, for the opportunities afforded to me, to Ondřej Uhlík, Professor Tomaš Macek and all of the great people around the Lab 209 at the Department of Biochemistry and Microbiology, University of Chemistry and Technology of Prague. A special thought is for Lucka, Jachym, Michal, Jitka, Terka, and Blanka. I also wish to thank all of the other people who allowed me to work at the research project in Prague and the Dr. Mary Beth Leigh of University of Alaska Fairbanks. The research activity conducted at the University of Bologna, Department of Civil, Chemical, Environmental and Material Engineering (DICAM), was supported by the European Commission under Grant Agreement No. 265946 (MINOTAURUS Project, 7th FP). I thank Professor Davide Pinelli and Dario Frascari, Ph.D, for their kindness and all of the things they taught me. Chapter Five of my dissertation was realized in the framework of a cooperation with the “Sapienza” University of Rome and the work was supported by the EU ROUTES project (Contract No 265156, FP7 2007–2013, THEME [ENV.2010.3.1.1-2] Innovative system solutions for municipal sludge treatment and management). iv Table of Contents Chapter 1. Introduction ..................................................................................................................... 1 1.1. General introduction............................................................................................................... 1 1.2. Research objectives and rationale ......................................................................................... 3 1.3. Dissertation organization ....................................................................................................... 4 Chapter 2. Literature review ............................................................................................................ 5 2.1. Bioremediation of aquifers contaminated by chlorinated aliphatic hydrocarbons (CAHs)............................................................................................................................................. 5 2.1.1. CAHs: distribution in the environment, chemical and physical properties, toxicity ......... 5 2.1.2. Biodegradation of CAHs .................................................................................................... 7 2.1.2.1. Anaerobic microbial dehalogenation .......................................................................... 8 2.1.2.2. Microbial aerobic oxidation of CAHs ....................................................................... 10 2.1.2.3. Aerobic cometabolic degradation of CAHs .............................................................. 11 2.1.2.4. Key enzymes in the aerobic cometabolism of CAHs ............................................... 14 2.1.2.4.1. Soluble di-iron monooxygenases (SDIMO) ...................................................... 14 2.1.2.4.2. Methane monooxygenase (MMO) ..................................................................... 14 2.1.2.4.3. Butane and propane monooxygenases ............................................................... 15 2.1.2.4.4. AlkB super-family .............................................................................................. 16 2.1.2.4.5. Bacterial cytochrome P450 ................................................................................ 17 2.1.3. In situ aerobic cometabolic bioremediation of CAH-contaminated aquifers .................. 18 2.1.3.1. Main technologies for the in situ bio-treatment of contaminated aquifers ............... 19 2.1.3.2. On-site treatment with immobilized-biomass systems ............................................. 20 2.2. Bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soils ........... 21 2.2.1. PAHs: distribution, chemical and physical properties, toxicity ....................................... 21 2.2.2. PAH biodegradation ......................................................................................................... 22 2.2.3. Bacterial aerobic biodegradation ..................................................................................... 23 2.2.4. Phytoremediation and rhizoremediation of PAH-contaminated soil ............................... 25 2.3. Production of polyhydroxyalkanoates (PHAs) ................................................................... 27 2.3.1. PHAs: main characteristics and applications ................................................................... 27 2.3.2. PHA production by pure microbial cultures and process implementation at industrial scale ............................................................................................................................................ 28 2.3.3. Other PHA production strategies: employment of mixed microbial cultures .................. 30 v 2.3.4. “Sludge minimization in municipal wastewater treatment by polyhydroxyalkanoates (PHA) production” (Valentino et al. 2014b) ............................................................................. 31 2.4. Molecular advances in biotechnology to characterize microbial communities ............... 33 2.4.1. Introduction to metagenomics and sequence analysis as powerful tools in microbial ecology ....................................................................................................................................... 33 2.4.2. Describing environmental microbial communities’ biodiversity ..................................... 34 2.4.3. Molecular fingerprinting of microbial communities ........................................................ 35 2.4.3.1. PCR-based techniques..............................................................................................