Francesca Crisafi

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.

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.

Heterotrophic bicarbonate assimilation is the main process of de novo organic carbon synthesis in hadal zone of the Hellenic Trench, the deepest part of Mediterranean Sea

Ammonium-oxidizing chemoautotrophic members of Thaumarchaea are proposed to be the key players in the assimilation of bicarbonate in the dark (ABD). However, this process may also involve heterotrophic metabolic pathways, such as fixation of carbon dioxide (CO2) via various anaplerotic reactions. We collected samples from the depth of 4900 m at the Matapan-Vavilov Deep (MVD) station (Hellenic Trench, Eastern Mediterranean) and used the multiphasic approach to study the ABD mediators in this deep-sea ecosystem. At this depth, our analysis indicated the occurrence of actively CO2-fixing heterotrophic microbial assemblages dominated by Gammaproteobacteria with virtually no Thaumarchaea present. [14C]-bicarbonate incorporation experiments combined with shotgun [14C]-proteomic analysis identified a series of proteins of gammaproteobacterial origin. More than quarter of them were closely related with Alteromonas macleodii ‘deep ecotype’ AltDE, the predominant organism in the microbial community of MVD. The present study demonstrated that in the aphotic/hadal zone of the Mediterranean Sea, the assimilation of bicarbonate is associated with both chemolithoauto- and heterotrophic ABD. In some deep-sea areas, the latter may predominantly contribute to the de novo synthesis of organic carbon which points at the important and yet underestimated role heterotrophic bacterial populations can play the in global carbon cycle/sink in the ocean interior.

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.

Bioremediation technologies for polluted seawater sampled after an oil-spill in Taranto Gulf (Italy): A comparison of biostimulation, bioaugmentation and use of a washing agent in microcosm studies

One of the main challenges of bioremediation is to define efficient protocols having a low environmental impact. We have investigated the effect of three treatments in oily-seawater after a real oil-spill occurred in the Gulf of Taranto (Italy). Biostimulation with inorganic nutrients allowed the biodegradation of the 73±2.4% of hydrocarbons, bioaugmentation with a selected hydrocarbonoclastic consortium consisting of Alcanivorax borkumensis, Alcanivorax dieselolei, Marinobacter hydrocarbonoclasticus, Cycloclasticus sp. 78-ME and Thalassolituus oleivorans degraded 79±3.2%, while the addition of nutrients and a washing agent has allowed the degradation of the 69±2.6%. On the other hand, microbial community was severely affected by the addition of the washing agent and the same product seemed to inhibit the growth of the majority of strains composing the selected consortium at the tested concentration. The use of dispersant should be accurately evaluated also considering its effect on the principal actors of biodegradation.

Alcanivorax borkumensis produces an extracellular siderophore in iron-limitation condition maintaining the hydrocarbon-degradation efficiency

Obligate marine hydrocarbonoclastic bacteria possess genetic and physiological features to use hydrocarbons as sole source of carbon and to compete for the uptake of nutrients in usually nutrient-depleted marine habitats. In the present work we have studied the siderophore-based iron uptake systems in Alcanivorax borkumensis SK2 and their functioning during biodegradation of an aliphatic hydrocarbon, tetradecane, under iron limitation conditions. The antiSMASH analysis of SK2 genome revealed the presence of two different putative operons of siderophore synthetases. Search for the predicted core structures indicated that one siderophore is clearly affiliated to the family of complex oligopeptidic siderophores possessing an Orn-Ser-Orn carboxyl motif whereas the second one is likely to belong to the family of SA (salicylic acid)-based siderophores. Analyzing the supernatant of SK2 culture, an extracellular siderophore was identified and its structure was resolved. Thus, along with the recently described membrane-associated amphiphilic tetrapeptidic siderophore amphibactin, strain SK2 additionally produces an extracellular type of iron-chelating molecule with structural similarity to pseudomonins. Comparative Q-PCR analysis of siderophore synthetases demonstrated their significant up-regulation in iron-depleted medium. Different expression patterns were recorded for two operons during the early and late exponential phases of growth, suggesting a different function of these two siderophores under iron-depleted conditions.

Fast pesticide pre-screening in marine environment using a green microalgae-based optical bioassay

The present study evaluates an optical bioassay based on green photosynthetic microalgae as a promising alternative for monitoring of relevant seawater pollutants. Photosystem II fluorescence parameters from several microalgae species were examined in the presence of three common marine pesticides that act as photosynthesis inhibitors. The three pollutants were detected within 10 min in concentrations between ng/L-μg/L. The different algae species showed slightly diverse pesticide sensitivities, being Chlorella mirabilis the most sensitive one. Potential interferences due to oil-spill pollutants were discarded. The lipid content was characterized to identify microorganisms with suitable mechanisms that could facilitate stress acclimatization. C. mirabilis presented elevated content of unsaturated lipids, showing a promising potential for biosensing in saline stress conditions. The optimized microalgae-based bioassay was preliminarily incorporated into a marine buoy for autonomous pre-screening of pesticides in coastal areas, demonstrating its suitability for real-time monitoring of marine water and quantitative evaluation of total biotoxicity.

Contribution of Bicarbonate Assimilation to Carbon Pool Dynamics in the Deep Mediterranean Sea and Cultivation of Actively Nitrifying and CO2-Fixing Bathypelagic Prokaryotic Consortia

Covering two-thirds of our planet, the global deep ocean plays a central role in supporting life on Earth. Among other processes, this biggest ecosystem buffers the rise of atmospheric CO2. Despite carbon sequestration in the deep ocean has been known for a long time, microbial activity in the meso- and bathypelagic realm via the “assimilation of bicarbonate in the dark” (ABD) has only recently been described in more details. Based on recent findings, this process seems primarily the result of chemosynthetic and anaplerotic reactions driven by different groups of deep-sea prokaryoplankton. We quantified bicarbonate assimilation in relation to total prokaryotic abundance, prokaryotic heterotrophic production and respiration in the meso- and bathypelagic Mediterranean Sea. The measured ABD values, ranging from 133 to 370 μg C m−3 d−1, were among the highest ones reported worldwide for similar depths, likely due to the elevated temperature of the deep Mediterranean Sea (13–14°C also at abyssal depths). Integrated over the dark water column (≥200 m depth), bicarbonate assimilation in the deep-sea ranged from 396 to 873 mg C m−2 d−1. This quantity of produced de novo organic carbon amounts to about 85–424% of the phytoplankton primary production and covers up to 62% of deep-sea prokaryotic total carbon demand. Hence, the ABD process in the meso- and bathypelagic Mediterranean Sea might substantially contribute to the inorganic and organic pool and significantly sustain the deep-sea microbial food web. To elucidate the ABD key-players, we established three actively nitrifying and CO2-fixing prokaryotic enrichments. Consortia were characterized by the co-occurrence of chemolithoautotrophic Thaumarchaeota and chemoheterotrophic proteobacteria. One of the enrichments, originated from Ionian bathypelagic waters (3,000 m depth) and supplemented with low concentrations of ammonia, was dominated by the Thaumarchaeota “low-ammonia-concentration” deep-sea ecotype, an enigmatic and ecologically important group of organisms, uncultured until this study.