Renata Denaro

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature

Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.

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.

Bioremediation of benzene, toluene, ethylbenzene, xylenes-contaminated soil: a biopile pilot experiment

Aims:  In this study, we evaluated the removal efficiency of fuel hydrocarbons from a jet fuel contaminated area using bioaugmentation treatment in biopile.

New methodological strategies for detecting bacterial indicators

The development of monitoring strategies for the early warning of seawater pollution, with particular reference to faecal and hydrocarbon contamination, has been the specific goal of research carried out within the Cluster 10-SAM (Advanced Systems for Coastal Marine Monitoring) Project, funded by the Italian Ministry for University and Scientific Research. Advanced analytical approaches have been designed and applied to detect bacterial species that have been selected as potential indicators of pollution in seawater samples. We report the results obtained using the fluorescent antibody and enzymatic assays for the detection of Escherichia coli, and a real-time PCR protocol for monitoring marine areas for hydrocarbon pollution. Immunofluorescence and enzymatic methods revealed the occurrence of different faecal pollution levels, reaching 105 E. coli cells 100 ml−1 in the Straits of Messina. Real-time PCR results corresponded to the different degree of oil pollution of the analysed samples. The specificity and speed make these methods promising for the detection and evaluation of marine pollution.

Genome sequence of obligate marine polycyclic aromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME, isolated from petroleum deposits of the sunken tanker Amoco Milford Haven, Mediterranean Sea

Cycloclasticus sp. 78-ME isolated from petroleum deposits of the sunken tanker “Amoco Milford Haven” (Gulf of Genoa, Ligurian Sea, Italy) could effectively degrade polycyclic aromatic hydrocarbons of up to five condensed rings. The genome of 78-ME was sequenced and analysed to gain insights into its remarkable degrading capacities. It comprises two circular replicons, the 2,613,078 bp chromosome and the plasmid of 42,347 bp, with 41.84% and 53.28% of the G + C content respectively. A total of 2585 protein-coding genes were obtained, and three large operons with more than fifteen enzymes belonging to four different classes of ring-cleavage dioxygenases were found.

The culturable mycobiota of a Mediterranean marine site after an oil spill: isolation, identification and potential application in bioremediation

Bioremediation of marine environment could be the response to oil spills threats. In the present study the fungal community from a Mediterranean marine site chronically interested by oil spills was investigated. Sixty-seven taxa were isolated from water sample and 17 from sediments; for many of the identified species is the first report in seawater and sediments, respectively. The growth of 25% of the fungal isolates was stimulated by crude oil as sole carbon source. Four strains were selected to screen hydrocarbons degradation using the 2,6-dichlorophenol indophenol (DCPIP) colorimetric assay. A. terreus MUT 271, T. harzianum MUT 290 and P. citreonigrum MUT 267 displayed a high decolorization percentage (DP≥68%). A. terreus displayed also the highest decreases of hydrocarbons compounds (up to 40%) quantified by gas-chromatography analysis. These results suggest that the selected fungi could represent potential bioremediation agents with strong crude oil degradative capabilities.

Metaproteomics and metabolomics analyses of chronically petroleum-polluted sites reveal the importance of general anaerobic processes uncoupled with degradation

Crude oil is one of the most important natural assets for humankind, yet it is a major environmental pollutant, notably in marine environments. One of the largest crude oil polluted areas in the word is the semi‐enclosed Mediterranean Sea, in which the metabolic potential of indigenous microbial populations towards the large‐scale chronic pollution is yet to be defined, particularly in anaerobic and micro‐aerophilic sites. Here, we provide an insight into the microbial metabolism in sediments from three chronically polluted marine sites along the coastline of Italy: the Priolo oil terminal/refinery site (near Siracuse, Sicily), harbour of Messina (Sicily) and shipwreck of MT Haven (near Genoa). Using shotgun metaproteomics and community metabolomics approaches, the presence of 651 microbial proteins and 4776 metabolite mass features have been detected in these three environments, revealing a high metabolic heterogeneity between the investigated sites. The proteomes displayed the prevalence of anaerobic metabolisms that were not directly related with petroleum biodegradation, indicating that in the absence of oxygen, biodegradation is significantly suppressed. This suppression was also suggested by examining the metabolome patterns. The proteome analysis further highlighted the metabolic coupling between methylotrophs and sulphate reducers in oxygen‐depleted petroleum‐polluted sediments.