Santina Santisi

Effective bioremediation strategy for rapid in situ cleanup of anoxic marine sediments in mesocosm oil spill simulation

The purpose of present study was the simulation of an oil spill accompanied by burial of significant amount of petroleum hydrocarbons (PHs) in coastal sediments. Approximately 1000 kg of sediments collected in Messina harbor were spiked with Bunker C furnace fuel oil (6500 ppm). The rapid consumption of oxygen by aerobic heterotrophs created highly reduced conditions in the sediments with subsequent recession of biodegradation rates. As follows, after 3 months of ageing, the anaerobic sediments did not exhibit any significant levels of biodegradation and more than 80% of added Bunker C fuel oil remained buried. Anaerobic microbial community exhibited a strong enrichment in sulfate-reducing PHs-degrading and PHs-associated Deltaproteobacteria. As an effective bioremediation strategy to clean up these contaminated sediments, we applied a Modular Slurry System (MSS) allowing the containment of sediments and their physical–chemical treatment, e.g., aeration. Aeration for 3 months has increased the removal of main PHs contaminants up to 98%. As revealed by CARD-FISH, qPCR, and 16S rRNA gene clone library analyses, addition of Bunker C fuel oil initially affected the activity of autochthonous aerobic obligate marine hydrocarbonoclastic bacteria (OMHCB), and after 1 month more than the third of microbial population was represented by Alcanivorax-, Cycloclasticus-, and Marinobacter-related organisms. In the end of the experiment, the microbial community composition has returned to a status typically observed in pristine marine ecosystems with no detectable OMHCB present. Eco-toxicological bioassay revealed that the toxicity of sediments after treatment was substantially decreased. Thus, our studies demonstrated that petroleum-contaminated anaerobic marine sediments could efficiently be cleaned through an in situ oxygenation which stimulates their self-cleaning potential due to reawakening of allochtonous aerobic OMHCB.

Effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics during assays of oil spill bioremediation: A microcosm study

Microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS₂₀₀₃) on microbial community dynamics. An experimental seawater microcosm, supplemented with crude oil and EPS₂₀₀₃ (SW+OIL+EPS₂₀₀₃), was monitored for 15 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out during all experimentation period. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil degradation was monitored by GC-MS analysis. Bioemulsificant addition stimulated an increase of the total bacterial abundance, change in the community structure and activity. The bioemulsificant also increased of 5 times the oil biodegradation rate. The data obtained from microcosm experiment indicated that EPS₂₀₀₃ could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Intrinsic bioremediation potential of a chronically polluted marine coastal area

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 DGGE fingerprinting and PLFAs analysis were performed on seawater and sediment samples from six stations on two transects. Higher diversity and variability among stations was detected by DGGE in sediment than in water samples although seawater revealed higher diversity of culturable hydrocarbon-degrading bacteria. The most polluted sediment hosted higher total bacterial diversity and higher abundance and diversity of culturable HC degraders. Alkane- and PAH-degrading bacteria were isolated from all stations and assigned to Alcanivorax, Marinobacter, Thalassospira, Alteromonas and Oleibacter (first isolation from the Mediterranean area). High total microbial diversity associated to a large selection of HC degraders is believed to contribute to natural attenuation of the area, provided that new contaminant contributions are avoided.

Presence of hydrocarbon-degrading bacteria in the gills of mussel Mytilus galloprovincialis in a contaminated environment: a mesoscale simulation study

A number of previous studies have shown that the relationships of symbiosis existing between mussels and microorganisms are directly dependent on the environmental conditions. However, little is known about existing relationships between mussels and bacteria in hydrocarbon-impacted marine environments. The aim of this preliminary study is to investigate the presence of oil-degrading bacteria in the mussel Mytilus galloprovincialis during growth in polluted ecosystems. All the experiments were carried out in a mesocosm system designed to simulate chronic pollution and to enable direct exposure of mussels to chemicals. Quantitative (4′,6-diamidino-2-phenylindole, colony-forming units, Most Probable Number) analyses and screening (presence/absence) of metabolic functional genes were performed to analyse bacterial populations inside the gills of mussels exposed and not exposed to hydrocarbons. The data obtained show that the presence of hydrocarbons affected the abundance of bacteria inside the gills of specimens and determines selection for specific (hydrocarbon-degrading) bacteria (i.e. Alcanivorax sp. and Marinobacter sp.). However, is not yet clear whether the presence of such genera of bacteria inside the mussel is due to symbiosis or as a result of filtration.

Quick stimulation of Alcanivorax sp. by bioemulsificant EPS2003 on microcosm oil spill simulation.

Oil spill microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on quick stimulation of hydrocarbonoclastic bacteria. Early hours of oil spill, were stimulated using an experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003); this system was monitored for 2 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out. Community composition of marine bacterioplankton was determined by 16S rRNA gene clone libraries. Data obtained indicated that bioemulsificant addition stimulated an increase of total bacterial abundance and, in particular, selection of bacteria related to Alcanivorax genus; confirming that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Microbial communities of polluted sub-surface marine sediments

Microbial communities of coastal marine sediment play a key role in degradation of petroleum contaminants. Here the bacterial and archaeal communities of sub-surface sediments (5-10 cm) of the chronically polluted Priolo Bay (eastern coast of Sicily, Italy), contaminated mainly by n-alkanes and biodegraded/weathered oils, were characterized by cultural and molecular approaches. 16S-PCR-DGGE analysis at six stations, revealed that bacterial communities are highly divergent and display lower phylogenetic diversity than the surface sediment; sub-surface communities respond to oil supplementation in microcosms with a significant reduction in biodiversity and a shift in composition; they retain high biodegradation capacities and host hydrocarbon (HC) degraders that were isolated and identified. HC-degrading Alfa, Gamma and Epsilon proteobacteria together with Clostridia and Archaea are a common feature of sub-surface communities. These assemblages show similarities with that of subsurface petroleum reservoirs also characterized by the presence of biodegraded and weathered oils where anaerobic or microaerophilic syntrophic HC metabolism has been proposed.

STRANgE, integrated physical-biological-mechanical system for recovery in of the "oil spill" in Antarctic environment.

Throughout the last century the increasing human activities in Antarctic region, particularly research expeditions, fishing, and tourism amplified the risk of oils spills at these high latitudes of the meridional hemisphere. A number of studies have been focused on chronic hydrocarbon contamination near Antarctic research stations revealing the presence and persistence of these human-derived contaminants. Marine ship-source oil spills in Antarctic region can have significant impacts on the marine environment. The key factors to effectively fight oil spills are a careful selection and proper use of the equipment and materials best suited to the critical local conditions. Despite the significant advances in the field of environmental recovery after an “oil spill” episode, research has recently shown that the usual techniques are often less effective than expected. This issue become much more relevant in the Antarctic case, not only for the incomparable environmental value of the Antarctic region but also for the extreme environmental conditions and the great distances from properly equipped centers, that make unfeasible sending naval vessels. Scope of the STRANgE Project is the preliminary design of a prototype floating platform, parachutable by plane, able to intervene as quickly as possible for the containment, removal and treatment/storage of the oil slick. New sorbent nanostructured materials and specialized Antarctic bacteria applications constitute the main innovations of this Project.