BIODEGRADAZIONE DEGLI IDROCARBURI: DALLE COMUNITÀ MICROBICHE MARINE AI SISTEMI READY TO USE PER IL BIORISANAMENTO

Crude oil is one of the major hazardous pollutants. A promising technology for the treatment of contaminated sites is based on biodegradation of oil pollutants by microbial populations. Several microorganisms are known to use hydrocarbons as carbon sources. In the marine environment, obligate hydrocarbonoclastic bacteria (BIC) have been defined as being ecologically important for the aerobic removal of hydrocarbons. Recent advances in microbial ecology make it possible to combine molecular and culture-dependent approaches to identify bacterial community at environmental sites. In this thesis a microbiological survey of the Priolo Bay (eastern coast of Sicily, Ionian Sea), a chronically polluted marine coastal area, was carried out in order to discern its intrinsic bioremediation potential. Microbiological analysis, 16S rDNA-based Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting and analysis of hydrocarbons were performed on seawater, superficial sediment (0-5cm) and sub-superficial sediment (5-10 cm) samples from six stations on two transects. The analysis of organic contaminants revealed high concentrations of total hydrocarbons (HC) represented mainly by aliphatic hydrocarbons in all samples. High levels of Unresolved Complex Mixture of hydrocarbons (UCMs) were detected in sub-superficial sediments. The Denaturing Gradient Gel Electrophoresis analysis showed higher diversity and variability in superficial sediment than in water samples although seawater contains higher diversity of culturable hydrocarbon-degrading bacteria. Random sequencing of the DGGE bands revealed the presence of Alpha, Gammaproteobacteria, and uncultured Firmicutes HC degraders. A collection of 258 isolates, adapted to grow on mineral medium supplemented with crude oil as unique carbon source, was obtained from superficial sediments and seawater samples. The 16S rRNA analysis showed the presence of Gammaproteobacteria affiliated to genera of obligate hydrocarbonoclastic bacteria (Alcanivorax) and/or to generalist HC-degraders (Marinobacter, Thalassospira, Alteromonas) and Oleibacter (first isolation from the Mediterranean area). The HC-degrading microbial communities in sub-superficial sediment, generally operating under microaerophilic conditions are still not well understood. The microbial (archaea and bacteria) communities and metabolic potential in sub-superficial sediment of Priolo Bay was investigated. Bacterial communities show lower phylogenetic diversity than those of the superficial layer 0-5 cm but retain biodegradation capacities towards crude oil n-alkanes as shown by oil supplemented microcosms experiments. The DGGE analysis reveled that the bacterial communities responded to oil supplementation with a significant reduction in biodiversity and a shift in the composition that was different for each sampling station. Random sequencing of DGGE bands revealed the presence of obligate and generalist HC-degrading Gammaproteobacteria (Halomonas, Marinobacter, Pseudoalteromonas, Pseudomonas, Vibrio), Alphaproteobacteria (Thalassospira), genera frequently detected in oiled sediments (Idiomarina), anaerobic nitrate-reducers (Vallitalea) and sulphate-reducers (Peptoniphilus). Most of the detected bacteria were also isolated in pure culture. Interestingly Epsilonproteobacteria, exclusively represented by the genus Arcobacter, were identified in a unique sample. The composition of microbial communities inhabiting sub-superficial contaminated sediment shows features in communities with communities of subsurface petroleum reservoirs, produced water and hypersaline environments, where sinphrophic degradation between HC degrading bacteria and archaea has been proposed.The archaeal DGGE profiles exhibited a limited diversity and were less influenced by supplementing of oil. Random sequencing of DGGE bands revealed the presence of Crenarchaeota and Thaumarchaeota, identified in contaminated habitat. The subsuperficial sediment retain high biodegradation capacities and host hydrocarbon (HC) degraders that were isolated and identified. The presence of catabolic genes involved in the degradation of aliphatic and aromatic hydrocarbons was investigated by cloning and sequencing of amplified fragments. The retrieved alkane-monoxigenase (alkB) gene sequences clustered with those of uncultivated bacteria detected in many polluted marine sediments. Overall the results obtained on the characterization of microbial communities reveal that the Priolo bay hosts a rich community of HC degraders known for catabolic potentials which could contribute to natural attenuation of the area. Among the Priolo isolates, particularly interesting appears the isolation of Oleibacter marinus by enrichment culture on a n-alkane mixture (C16, C18, C24) from seawater, sampled at station N3. The strains belonging to the genus Oleibacter was described until now as a major degrader of petroleum aliphatic hydrocarbon spills and these isolates where further characterized as the first in tropical seas. The phylogenetic analyses of the strains, designated Oleibacter 3, 4 and 5, revelead that the isolates show 98-99% identity of their 16S rRNA gene with Oleibacter marinus type strain 2O1T. A fragment of the alkane monooxygenase encoding gene alkB, shows 76% identity with that of Pseudomonas nitroreducens and, among Oceanospirillaceae, with Thalassolituus oleivorans alkB gene (73% id.). The isolates grow on crude oil and n-alkanes and also on a limited number of C sources. Crude oil degradation, measured by Gas-Chromatography-FID analysis, is in the range of 82-99% of short and medium size chain n-alkanes. Low bio-surfactant production has been observed with three different methods (E24, drops collapse, oil spreading). The fatty acid methyl esters (FAMEs) of Oleibacter marinus 3, 4, 5 were detected by GC-FID analysis. Oleibacter and other Oceanobacter- related bacteria dominate in crude-oil impacted tropical seawater and their hydrocarbon-degradation activity was compared to that of the genus Alcanivorax with which they could compete. Oleibacter marinus might have a future key role in bioremediation of marine environments. In the last few years, the application of biotechnological processes that involvesing HC-degrading microorganisms, with the objective of solving environmental pollution problems, is rapidly growing. The use of immobilized cells has been investigated as an alternative technology for environmental applications. A novel biotechnological system based on immobilization of Oleibacter marinus and other hydrocarbon-degrading bacterial isolates (Alcanivorax SK2., Gordonia SoCg., Nocardia SoB) on biodegradable carrier (3D polycaprolactone scaffold, polylactic acid (PLA) and polycaprolactone (PCL) membranes), was developed to clean (sea)water contaminated by crude oil and its derivatives. A high capacity of adhesion and proliferation of bacterial cells within the whole three-dimensional structure of scaffold was observed after 48 h. The bacterial strains were able to attach to the PLA and PCL membranes using scanning electron microscopy (SEM) after 48h, reaching high proliferation and biofilm formation within the whole structure in 5 days. The biodegradation efficiency of HC-embedded scaffold and bacteria-membrane-systems was tested by GC-FID analysis and compared with planktonic cells. More than 50% of crude oil was removed by bacterial strains immobilized in biodegradable polycaprolactone (PCL) 3D scaffold, polylactic acid (PLA) and polycaprolactone (PCL) membranes; the degradative ability was between 7% and 20% higher that of planktonic cells. The bacterial immobilization is a promoting factor for biodegradation and a the novel ready to use systems be developed for promising toolsbioremediation of aquatic systems.