Hydrocarbon pollutants shape bacterial community assembly of harbor sediments.

Petroleum pollution results in co-contamination by different classes of molecules, entailing the occurrence of marine sediments difficult to remediate, as in the case of the Ancona harbor (Mediterranean Sea, Italy). Autochthonous bioaugmentation (ABA), by exploiting the indigenous microbes of the environment to be treated, could represent a successful bioremediation strategy. In this perspective we aimed to i) identify the main drivers of the bacterial communities' richness in the sediments, ii) establish enrichment cultures with different hydrocarbon pollutants evaluating their effects on the bacterial communities' composition, and iii) obtain a collection of hydrocarbon degrading bacteria potentially exploitable in ABA. The correlation between the selection of different specialized bacterial populations and the type of pollutants was demonstrated by culture-independent analyses, and by establishing a collection of bacteria with different hydrocarbon degradation traits. Our observations indicate that pollution dictates the diversity of sediment bacterial communities and shapes the ABA potential in harbor sediments.

Degradation Network Reconstruction in Uric Acid and Ammonium Amendments in Oil-Degrading Marine Microcosms Guided by Metagenomic Data.

Biostimulation with different nitrogen sources is often regarded as a strategy of choice in combating oil spills in marine environments. Such environments are typically depleted in nitrogen, therefore limiting the balanced microbial utilization of carbon-rich petroleum constituents. It is fundamental, yet only scarcely accounted for, to analyze the catabolic consequences of application of biostimulants. Here, we examined such alterations in enrichment microcosms using sediments from chronically crude oil-contaminated marine sediment at Ancona harbor (Italy) amended with natural fertilizer, uric acid (UA), or ammonium (AMM). We applied the web-based AromaDeg resource using as query Illumina HiSeq meta-sequences (UA: 27,893 open reading frames; AMM: 32,180) to identify potential catabolic differences. A total of 45 (for UA) and 65 (AMM) gene sequences encoding key catabolic enzymes matched AromaDeg, and their participation in aromatic degradation reactions could be unambiguously suggested. Genomic signatures for the degradation of aromatics such as 2-chlorobenzoate, indole-3-acetate, biphenyl, gentisate, quinoline and phenanthrene were common for both microcosms. However, those for the degradation of orcinol, ibuprofen, phenylpropionate, homoprotocatechuate and benzene (in UA) and 4-aminobenzene-sulfonate, p-cumate, dibenzofuran and phthalate (in AMM), were selectively enriched. Experimental validation was conducted and good agreement with predictions was observed. This suggests certain discrepancies in action of these biostimulants on the genomic content of the initial microbial community for the catabolism of petroleum constituents or aromatics pollutants. In both cases, the emerging microbial communities were phylogenetically highly similar and were composed by very same proteobacterial families. However, examination of taxonomic assignments further revealed different catabolic pathway organization at the organismal level, which should be considered for designing oil spill mitigation strategies in the sea.

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.

Conversion of Uric Acid into Ammonium in Oil-Degrading Marine Microbial Communities: a Possible Role of Halomonads.

Uric acid is a promising hydrophobic nitrogen source for biostimulation of microbial activities in oil-impacted marine environments. This study investigated metabolic processes and microbial community changes in a series of microcosms using sediment from the Mediterranean and the Red Sea amended with ammonium and uric acid. Respiration, emulsification, ammonium and protein concentration measurements suggested a rapid production of ammonium from uric acid accompanied by the development of microbial communities containing hydrocarbonoclastic bacteria after 3 weeks of incubation. About 80 % of uric acid was converted to ammonium within the first few days of the experiment. Microbial population dynamics were investigated by Ribosomal Intergenic Spacer Analysis and Illumina sequencing as well as by culture-based techniques. Resulting data indicated that strains related to Halomonas spp. converted uric acid into ammonium, which stimulated growth of microbial consortia dominated by Alcanivorax spp. and Pseudomonas spp. Several strains of Halomonas spp. were isolated on uric acid as the sole carbon source showed location specificity. These results point towards a possible role of halomonads in the conversion of uric acid to ammonium utilized by hydrocarbonoclastic bacteria.

Bioremediation of Southern Mediterranean oil polluted sites comes of age.

Mediterranean Sea is facing a very high risk of oil pollution due to the high number of oil extractive and refining sites along the basin coasts, and the intense maritime traffic of oil tankers. All the Mediterranean countries have adopted severe regulations for minimizing pollution events and bioremediation feasibility studies for the most urgent polluted sites are undergoing. However, the analysis of the scientific studies applying modern 'meta-omics' technologies that have been performed on marine oil pollution worldwide showed that the Southern Mediterranean side has been neglected by the international research. Most of the studies in the Mediterranean Sea have been done in polluted sites of the Northern side of the basin. Those of the Southern side are poorly studied, despite many of the Southern countries being major oil producers and exporters. The recently EU-funded research project ULIXES has as a major objective to increase the knowledge of the bioremediation potential of sites from the Southern Mediterranean countries. ULIXES is targeting four major polluted sites on the coastlines of Egypt, Jordan, Morocco and Tunisia, including seashore sands, lagoons, and oil refinery polluted sediments. The research is designed to unravel, categorize, catalogue, exploit and manage the diversity and ecology of microorganisms thriving in these polluted sites. Isolation of novel hydrocarbon degrading microbes and a series of state of the art 'meta-omics' technologies are the baseline tools for improving our knowledge on biodegradation capacities mediated by microbes under different environmental settings and for designing novel site-tailored bioremediation approaches. A network of twelve European and Southern Mediterranean partners is cooperating for plugging the existing gap of knowledge for the development of novel bioremediation processes targeting such poorly investigated polluted sites.

Viral decay and viral production rates in continental-shelf and deep-sea sediments of the Mediterranean Sea.

Here, for the first time, we have carried out synoptic measurements of viral production and decay rates in continental-shelf and deep-sea sediments of the Mediterranean Sea to explore the viral balance. The net viral production and decay rates (1.1-61.2 and 0.6-13.5 x 10(7) viruses g(-1) h(-1), respectively) were significantly correlated, and were also related to prokaryotic heterotrophic production. The addition of enzymes increased the decay rates in the surface sediments, but not in the subsurface sediments. Both the viral production and the decay rates decreased significantly in the deeper sediment layers, while the virus-to-prokaryote abundance ratio increased, suggesting a high preservation of viruses in the subsurface sediments. Viral decay did not balance viral production at any of the sites investigated, accounting on average for c. 32% of the gross viral production in the marine sediments. We estimate that the carbon (C) released by viral decay contributed 6-23% to the total C released by the viral shunt. Because only c. 2% of the viruses produced can infect other prokaryotes, the majority is not subjected to direct lysis and potentially remains as a food source for benthic consumers. The results reported here suggest that viral decay can play an important role in biogeochemical cycles and benthic trophodynamics.

Determination of viral production in aquatic sediments using the dilution-based approach.

Viruses are the most abundant and dynamic biological entities in the world's ecosystems. Marine sediments, the largest biome in the world, have the potential to represent an optimal environment for viral development. To assess the viral effect on their hosts, and to understand the ecological role of the viruses in the benthic food webs and biogeochemical cycles, measurements of benthic viral production are needed. Different direct and indirect approaches have been proposed to estimate viral production in aquatic sediments, but a standardized protocol is not available yet. The method presented in this protocol relies on the short-time incubations of sediment samples with virus-free seawater, and the subsequent determination of the increase in viral abundance over time by epifluorescence microscopy. The protocol described here is highly reliable, inexpensive and easy to use. The entire procedure takes approximately 3 days to be completed, but the method allows the parallel processing of several sediment samples, which is recommended in ecological studies.

Major viral impact on the functioning of benthic deep-sea ecosystems.

Viruses are the most abundant biological organisms of the world's oceans. Viral infections are a substantial source of mortality in a range of organisms-including autotrophic and heterotrophic plankton-but their impact on the deep ocean and benthic biosphere is completely unknown. Here we report that viral production in deep-sea benthic ecosystems worldwide is extremely high, and that viral infections are responsible for the abatement of 80% of prokaryotic heterotrophic production. Virus-induced prokaryotic mortality increases with increasing water depth, and beneath a depth of 1,000 m nearly all of the prokaryotic heterotrophic production is transformed into organic detritus. The viral shunt, releasing on a global scale approximately 0.37-0.63 gigatonnes of carbon per year, is an essential source of labile organic detritus in the deep-sea ecosystems. This process sustains a high prokaryotic biomass and provides an important contribution to prokaryotic metabolism, allowing the system to cope with the severe organic resource limitation of deep-sea ecosystems. Our results indicate that viruses have an important role in global biogeochemical cycles, in deep-sea metabolism and the overall functioning of the largest ecosystem of our biosphere.

Viral production, decay rates, and life strategies along a trophic gradient in the North Adriatic Sea.

Although the relationships between trophic conditions and viral dynamics have been widely explored in different pelagic environments, there have been few attempts at independent estimates of both viral production and decay. In this study, we investigated factors controlling the balance between viral production and decay along a trophic gradient in the north Adriatic basin, providing independent estimates of these variables and determining the relative importance of nanoflagellate grazing and viral life strategies. Increasing trophic conditions induced an increase of bacterioplankton growth rates and of the burst sizes. As a result, eutrophic waters displayed highest rates of viral production, which considerably exceeded observed rates of viral decay (up to 2.9 x 10(9) VLP liter(-1) h(-1)). Viral decay was also higher in eutrophic waters, where it accounted for ca. 40% of viral production, and dropped significantly to 1.3 to 10.7% in oligotrophic waters. These results suggest that viral production and decay rates may not necessarily be balanced in the short term, resulting in a net increase of viruses in the system. In eutrophic waters nanoflagellate grazing, dissolved-colloidal substances, and lysogenic infection were responsible together for the removal of ca. 66% of viral production versus 17% in oligotrophic waters. Our results suggest that different causative agents are primarily responsible for the removal of viruses from the water column in different trophic conditions. Factors other than those considered in the past might shed light on processes responsible for the removal and/or decay of viral particles from the water column.