Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

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.

Bioremediation for the recovery of oil polluted marine environment, opportunities and challenges approaching the Blue Growth.

The Blue Growth strategy promises a sustainable use of marine resources for the benefit of the society. However, oil pollution in the marine environment is still a serious issue for human, animal, and environmental health; in addition, it deprives citizens of the potential economic and recreational advantages in the affected areas. Bioremediation, that is the use of bio-resources for the degradation of pollutants, is one of the focal themes on which the Blue Growth aims to. A repertoire of marine-derived bio-products, biomaterials, processes, and services useful for efficient, economic, low impact, treatments for the recovery of oil-polluted areas has been demonstrated in many years of research around the world. Nonetheless, although bioremediation technology is routinely applied in soil, this is not still standardized in the marine environment and the potential market is almost underexploited. This review provides a summary of opportunities for the exploiting and addition of value to research products already validated. Moreover, the review discusses challenges that limit bioremediation in marine environment and actions that can facilitate the conveying of valuable products/processes towards the market.

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.

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.

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.

Predominant growth of Alcanivorax during experiments on "oil spill bioremediation" in mesocosms.

Mesocosm experiments were performed to study the changes on bacterial community composition following oil spill in marine environment. The analysis of 16S crDNA revealed a shift in the structure of initial bacterial population that was drastically different from that one measured after 15 days. The results showed that, after 15 days, bacteria closely related to the genus Alcanivorax became the dominant group of bacterial community in petroleum-contaminated sea water nitrogen and phosphorus amended. This suggested that these bacteria played the most important role in the process of bioremediation of oil-contaminated marine environments.

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.

Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME.

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities.

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.

Bioremediation of oil polluted marine sediments: A bio-engineering treatment.

The fate of hydrocarbon pollutants and the development of oil-degrading indigenous marine bacteria in contaminated sediments are strongly influenced by abiotic factors such as temperature, low oxygen levels, and nutrient availability. In this work, the effects of different biodegradation processes (bioremediation) on oil-polluted anoxic sediments were analyzed. In particular, as a potential bioremediation strategy for polluted sediments, we applied a prototype of the "Modular Slurry System" (MSS), allowing containment of the sediments and their physical-chemical treatment (by air insufflations, temperature regulation, and the use of a slow-release fertilizer). Untreated polluted sediments served as the blank in a non-controlled experiment. During the experimental period (30 days), bacterial density and biochemical oxygen demand were measured and functional genes were identified by screening. Quantitative measurements of pollutants and an eco-toxicological analysis (mortality of Corophium orientale) were carried out at the beginning and end of the experiments. The results demonstrated the high biodegradative capability achieved with the proposed technology and its strong reduction of pollutant concentrations and thus toxicity.

Marine Microbial Secondary Metabolites: Pathways, Evolution and Physiological Roles.

Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology.

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.

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.

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 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.

Comparison of 16SrDNA and toxR genes as targets for detection of Vibrio anguillarum in Dicentrarchus labrax kidney and liver.

Vibrio anguillarum is a pathogen that causes high mortality in marine and freshwater fish. The aim of this study was to develop a real-time PCR assay for identification and quantification of V. anguillarum in fish tissue. The assay was carried out using two target genes, 16SrDNA and toxR, to evaluate the influence of differences in the operon copy number in quantitative assessment, both in pure cultures of V. anguillarum serovar O1 (strain 975/I), as a reference, and in the liver and kidney of a sea bass (Dicentrarchus labrax) specimen. Real-time PCR analysis showed high specificity for both target genes, with a detection limit of approximately 1-10 bacterial cells per reaction in pure culture and 10/100 V. anguillarum cells per reaction in fish tissue, which corresponds to 2 × 10(2)/2 × 10(3) cells g(-1) fish tissue. Moreover, both genes showed high specificity but differing sensitivity due to the different operon copy number; as a result, it is possible to target the high copy number gene to improve sensitivity. Our results suggest that the protocol we tested can be used as a sensitive and specific molecular method for the detection of the fish pathogen V. anguillarum in fish tissue.

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 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.