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Oceans emit ice-nucleating particles (INPs) which freeze supercooled cloud droplets, modifying clouds. We added dead biomass of three phytoplankton to seawater. Each time, this stimulated INP production in the water and INP emissions in sea spray.

Despite last decades’ interventions within local and communitarian programs, the Mediterranean Sea still receives poorly treated urban wastewater (sewage). Wastewater treatment plants (WWTPs) performing primary sewage treatments have poor efficiency in removing microbial pollutants, including fecal indicator bacteria, pathogens, and mobile genetic elements conferring resistance to antimicrobials. Using a combination of molecular tools, we investigated four urban WWTPs (i.e., two performing only mechanical treatments and two performing a subsequent conventional secondary treatment by activated sludge) as continuous sources of microbial pollution for marine coastal waters. Sewage that underwent only primary treatments was characterized by a higher content of traditional and alternative fecal indicator bacteria, as well as potentially pathogenic bacteria (especially Acinetobacter, Coxiella, Prevotella, Streptococcus, Pseudomonas, Vibrio, Empedobacter, Paracoccus, and Leptotrichia), than those subjected to secondary treatment. However, seawater samples collected next to the discharging points of all the WWTPs investigated here revealed a marked fecal signature, despite significantly lower values in the presence of secondary treatment of the sewage. WWTPs in this study represented continuous sources of antibiotic resistance genes (ARGs) ermB, qnrS, sul2, tetA, and blaTEM (the latter only for three WWTPs out of four). Still, no clear effects of the two depuration strategies investigated here were detected. Some marine samples were identified as positive to the colistin-resistance gene mcr-1, an ARG that threatens colistin antibiotics’ clinical utility in treating infections with multidrug-resistant bacteria. This study provides evidence that the use of sole primary treatments in urban wastewater management results in pronounced inputs of microbial pollution into marine coastal waters. At the same time, the use of conventional treatments does not fully eliminate ARGs in treated wastewater. The complementary use of molecular techniques could successfully improve the evaluation of the depuration efficiency and help develop novel solutions for the treatment of urban wastewater.

Benthic prokaryotes are the key-players in C-cycling at the sediment-seawater interface, one of the largest biologically active interfaces on Earth. Here, microbial-mediated processes, such as the degradation of organic matter and the incorporation of mobilized C into microbial biomass, depend on several factors such as environmental temperature and substrate availability, especially in shallow sediments at mid-high latitudes where seasonal fluctuations of these variables occur. In the present study, four degradative activities (β-glucosidase, lipase, chitinase and aminopeptidase), Heterotrophic C Production (HCP), Total Organic C (TOC), Total Nitrogen (TN) and Biopolymeric C (BPC) were investigated seasonally from April 2010 to April 2018 in the surface sediments of a shallow Long-Term Ecological Research (LTER) station of the northern Adriatic Sea. Significant temperature-dependences were described by Arrhenius-type equations for HCP and each of the degradative activities tested with the exception of aminopeptidase. The relatively low apparent Activation Energies suggested that these microbial-mediated processes were enhanced by the availability of palatable substrates over the study period. Nevertheless, a clear and tight dependence from such substrates was detected only for aminopeptidase, the most pronounced degradative activity observed. TN was identified by the stepwise multiple regression analysis as the environmental variable that mainly drove this exoenzymatic activity. Enhanced aminopeptidase rates mirrored peaks of TN that seemed, in turn, linked to the seasonal proliferation of benthic microalgae. By supplying prokaryotes with promptly available substrates, these autotrophs, represented mainly by diatoms, seemed to play an important role in the C-cycling regulation at the studied LTER station.

This study aims to validate the stable carbon isotopic composition (&delta, 13C) of phytoplankton as a tool for detecting submarine leakages of anthropogenic CO2(g), since it is characterised by &delta, 13C values significantly lower than the natural CO2 dissolved in oceans. Three culture experiments were carried out to investigate the changes in &delta, 13C of the diatom Thalassiosira rotula during growth in an artificially modified medium (ASW). Three different dissolved inorganic carbon (DIC) concentrations were tested to verify if carbon availability affects phytoplankton &delta, 13C. Simultaneously, at each experiment, T. rotula was cultured under natural DIC isotopic composition (&delta, 13CDIC) and carbonate system conditions. The available DIC pool for diatoms grown in ASW was characterised by &delta, 13CDIC values (&minus, 44.2 &plusmn, 0.9&permil, ) significantly lower than the typical marine range. Through photosynthetic DIC uptake, microalgae &delta, 13C rapidly changed, reaching significantly low values (until &minus, 43.4&permil, ). Moreover, the different DIC concentrations did not affect the diatom &delta, 13C, exhibiting the same trend in &delta, 13C values in the three ASW experiments. The experiments prove that phytoplankton isotopic composition quickly responds to changes in the &delta, 13C of the medium, making this approach a promising and low-impact tool for detecting CO2(g) submarine leakages from CO2(g) deposits.

Evaluating the effects of anthropogenic pressure on the marine environment is one of the focal objectives in identifying strategies for its use, conservation and restoration. In this paper, we assessed the effects of chemical pollutants, grain size and plastic litter on functional traits, biodiversity and biotic indices. The study was conducted on the benthic communities of three harbours in the central Mediterranean Sea: Malta, Augusta and Syracuse, subjected to different levels of anthropogenic stress (high, medium and low, respectively). Six traits were considered, subdivided into 22 categories: reproductive frequency, environmental position, mobility, life habit, feeding habit and bioturbation. Functional diversity indices analysed were: Functional Divergence, Quadratic Entropy, Functional Evenness and Functional Richness. To assess the trait responses to environmental gradients, we applied RLQ analysis, which considers simultaneously the relationship between three components: environmental data (R), species abundances (L) and species traits (Q). From our analyses, significant relationships (P-value = 0.0018 for permutation of samples, and P-value = 0.00027 for permutation of species) between functional traits and environmental data were highlighted. The trait categories significantly influenced by environmental variables were those representing feeding habits and mobility. In particular, the first category was influenced by chemical pollutants (organotin compounds and polycyclic aromatic hydrocarbons) and grain size (silt and sand), while the latter category was influenced only by chemical pollutants. Pearson correlations performed for functional vs biotic and diversity indices confirmed the validity of the chosen conceptual framework for harbour environments. Finally, linear models assessing the influence of stressors on functional parameters underlined the link between environmental data vs benthic and functional indices. Our results highlight the fact that functional trait analysis provides a useful and fast method for detecting in greater depth the effects of multiple stressors on functional diversity in marine ecosystems.

Marine benthic microalgae are a promising bioindicator of contamination. To date, however, investigations on the microbenthic communities subjected to multiple stressors in natural environments are still very rare. To assess whether the benthic processes of primary production and oxygen consumption, and the structure of active and resting microbenthos, were affected by sediment contamination, seven stations were sampled in different zones of the port of Trieste, subjected to multiple and diffuse contamination, and a reference site in the Marine Reserve of Miramare. No major differences in total abundance of active microbenthos were observed among sites, but the dominance of stress-resistant species and the reduction of more sensitive ones, were registered nearby the main productive activities. The densities of resting microbenthos were higher in polluted areas, and represented by key dinoflagellate species that were clearly linked to contamination. The analysis of similarity applied to both active and resting communities significantly separated the most contaminated stations from the other ones. The photosynthetic capability of active microbenthos did not seem to be affected by contamination. The maximum oxygen consumption rates observed in sediments nearby the productive activities were likely ascribable to high organic C contents and the presence of metals in reduced chemical form.

Microbial degradation of high molecular weight organic matter in the marine environment is dependent on the synthesis and activity of hydrolytic exoenzymes. These can be found both associated to the cell (i.e., attached to cell-wall or in the periplasmic space) and cell-free, dissolved in the surrounding waters. Recent evidences suggest that exoenzymes location is linked to different foraging strategies. ‘Selfish’ bacteria use cell-bound enzymes to obtain large oligomers which are then further degraded in the confined periplasmic space, whereas cell-free enzymes are thought to be advantageous for particle-attached prokaryotes, consequently ‘sharing’ the hydrolysis product with other members of the degrading consortium. We examined whether different bacterial isolates exhibit different exoenzymatic activity profiles when exposed to different growth condition. Seven bacterial strains, isolated from the Ross Sea, were screened for the production of β-glucosidase, alkaline-phosphatase, lipase, chitinase and leucine aminopeptidase, assaying the hydrolysis rates of both cell-bound and cell-free exoenzymatic fractions. Furthermore, to test whether bacteria growing on particles are more prone to produce cell-free exoenzymes, we set up a controlled experiment, amending one of the bacterial isolates with phytodetritus, in order to promote the colonization of aggregates. Cell specific hydrolytic rates were highly variable and different isolates expressed different dominant hydrolytic activities. These results demonstrate a specialization for different substrates in different bacteria, further suggesting that some of the tested isolates may have an intrinsic potential to copiously produce cell-free exoenzymes. Moreover, our results suggest that this specialization is not strictly genetically determined but varies according to growth conditions. The experiment performed with phytodetrital particles highlighted an increasing contribution of the dissolved exoenzymatic activity in samples bearing aggregates and associated bacteria. Also, the tested isolate further modulated its hydrolytic machinery, expressing a different enzymatic profile when exposed to phytodetrital particles. The continuation of these experimental activities, testing the response of different bacterial isolates to different substrates, will help to determine the conditions under which different hydrolysis patterns develop, deepening the current knowledge on the organic matter cycling in the ocean.

The deep Arctic Ocean is increasingly vulnerable to climate change effects, yet our understanding of its microbial processes is limited. We collected samples from shelf waters, mesopelagic Atlantic Waters (AW) and bathypelagic Norwegian Sea Deep Waters (NSDW) in the eastern Fram Strait, along coast-to-offshore transects off Svalbard during boreal summer. We measured community respiration, heterotrophic carbon production (HCP), and dissolved inorganic carbon utilization (DICu) together with prokaryotic abundance, diversity, and metagenomic predictions. In deep samples, HCP was significantly faster in AW than in NSDW, while we observed no differences in DICu rates. Organic carbon uptake was higher than its inorganic counterpart, suggesting a major reliance of deep microbial Arctic communities on heterotrophic metabolism. Community structure and spatial distribution followed the hydrography of water masses. Distinct from other oceans, the most abundant OTU in our deep samples was represented by the archaeal MG-II. To address the potential biogeochemical role of each water mass-specific microbial community, as well as their link with the measured rates, PICRUSt-based predicted metagenomes were built. The results showed that pathways of auto- and heterotrophic carbon utilization differed between the deep water masses, although this was not reflected in measured DICu rates. Our findings provide new insights to understand microbial processes and diversity in the dark Arctic Ocean and to progress toward a better comprehension of the biogeochemical cycles and their trends in light of climate changes.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

A first synoptic and trans-domain overview of plankton dynamics was conducted across the aquatic sites belonging to the Italian Long-Term Ecological Research Network (LTER-Italy). Based on published studies, checked and complemented with unpublished information, we investigated phytoplankton and zooplankton annual dynamics and long-term changes across domains: from the large subalpine lakes to mountain lakes and artificial lakes, from lagoons to marine coastal ecosystems. This study permitted identifying common and unique environmental drivers and ecological functional processes controlling seasonal and long-term temporal course. The most relevant patterns of plankton seasonal succession were revealed, showing that the driving factors were nutrient availability, stratification regime, and freshwater inflow. Phytoplankton and mesozooplankton displayed a wide interannual variability at most sites. Unidirectional or linear long-term trends were rarely detected but all sites were impacted across the years by at least one, but in many case several major stressor(s): nutrient inputs, meteo-climatic variability at the local and regional scale, and direct human activities at specific sites. Different climatic and anthropic forcings frequently co-occurred, whereby the responses of plankton communities were the result of this environmental complexity. Overall, the LTER investigations are providing an unparalleled framework of knowledge to evaluate changes in the aquatic pelagic systems and management options.

The effects of different physical and biogeochemical drivers on the carbonate system were investigated in a semi-enclosed coastal area characterized by high alkalinity riverine discharge (Gulf of Trieste, Northern Adriatic Sea, Mediterranean Sea). Our 2-year time-series showed that a large part of the seasonal carbonate chemistry variation was controlled by the large seasonal change of seawater temperature, though air-sea CO 2 exchange, biological activity (primary production-respiration), and riverine inputs also exerted a significant influence. With the exception of summer, the Gulf of Trieste was a sink of atmospheric carbon dioxide, showing a very strong CO 2 fluxes from atmosphere into the sea (−16.10 mmol m −2 day −1 ) during high wind speed event of north easterly Bora wind. The CO 2 influx was particularly evident in winter, when the biological activity was at minimum and the low seawater temperature enhanced CO 2 solubility. During spring, the drawdown of CO 2 by primary production overwhelmed the CO 2 physical pump, driving a significant decrease of dissolved inorganic carbon (DIC), [CO 2 ], and increase of pH T25 °C . In summer the primary production in surface waters occurred with the same intensity as respiration in the bottom layer, so the net biological effect on the carbonate system was very low and the further reduction of seawater CO 2 concentration observed was mainly due to carbon dioxide degassing induced by high seawater temperature. Finally, during autumn the respiration was the predominant process, which determined an overall increase of DIC, [CO 2 ], and decrease of pH T25 °C . This was particularly evident when the breakdown of summer stratification occurred and a large amount of CO 2 , generated by respiration and segregated below the pycnocline, was released back to the whole water column. Local rivers also significantly affected the carbonate system by direct input of total alkalinity (A T ) coming from the chemical weathering of carbonate rocks, which dominate the river watershed. Our finding clearly demonstrates a high A T concentration in low salinity surface waters (A T max = 2742.8 μmol kg −1 ) and a negative A T -salinity correlation. As a result the Gulf of Trieste revealed a low Revelle factor (10.1) and one of the highest buffer capacities of the Mediterranean Sea (s DIC = 0.31 mmol kg −1 ), which allows the system to store a significant amount of atmospheric CO 2 with a small decrease of seawater pH.

Knowledge on ecosystem functioning can largely contribute to promote ecosystem-based management and its application. The Mar Piccolo of Taranto is a densely populated area at a high risk of environmental crisis. Here, planktonic primary production (PP) and heterotrophic prokaryotic production (HPP) were measured as proxies of functioning in three sampling sites located in two inlets at different levels of industrial contamination, during three sampling surveys (June 2013, February and April 2014). To have a better overall view and provide some insights into the benthic-pelagic coupling, we integrated PP and HPP in the water column with those in the sediments and then discussed this with the origin of the organic matter pools based on analysis of stable isotopes. Heavy metals and polychlorobiphenyls (PCBs) were also analysed in the surface (1 cm) sediment layer and related to the overall ecosystem functioning. Multidimensional scaling (MDS) analysis, based on the main data, clearly separated the second inlet from the first one, more severely impacted by anthropogenic activities. The stable isotope mixing model suggested the prevalent terrestrial/riverine origin of the particulate organic matter pools (mean 45.5 %) in all sampling periods, whereas phytoplankton contributed up to 29 % in February. Planktonic PP and HPP rates followed the same pattern over the entire study period and seemed to respond to phytoplankton dynamics confirming this community as the main driver for the C cycling in the water column. On the contrary, benthic PP rates were almost negligible while HPP rates were lower or comparable to those in the water column indicating that although the Mar Piccolo is very shallow, the water column is much more productive than the surface sediments. The sediment resuspension is likely responsible for a pulsed input of contaminants into the water column. However, their interference with the proper functioning of the pelagic ecosystem seems to be limited to the bottom layers. © 2015, Springer-Verlag Berlin Heidelberg.

Notwithstanding the increasing amount of researches on the effect of ocean acidification (OA) on marine ecosystems, no consent has emerged on its consequences on many prokaryote-mediated processes. Two mesocosm experiments were performed in coastal Mediterranean areas with different trophic status: the summer oligotrophic Bay of Calvi (BC, Corsica, France) and the winter mesotrophic Bay of Villefranche (BV, France). During these experiments, nine enclosures (∼54 m 3 ) were deployed: 3 unamended controls and 6 elevated CO 2 , following a gradient up to 1250 μatm. We present results involving free-living viral and prokaryotic standing stocks, bacterial carbon production, abundance of highly active cells (CTC+), and degradation processes (beta-glucosidase, chitinase, leucine-aminopeptidase, lipase and alkaline phosphatase activities). The experiments revealed clear differences in the response of the two prokaryotic communities to CO 2 manipulation. Only abundances of heterotrophic prokaryotes, viruses and lipase activity were not affected by CO 2 manipulation at both locations. On the contrary, the percent of CTC+ was positively correlated to CO 2 only in BC, concomitantly to a bulk reduction of [ 3 H]-leucine uptake. The other tested parameters showed a different response at the two sites suggesting that the trophic regime of the systems plays a fundamental role on the effect of OA on prokaryotes through indirect modifications of the available substrate. Modified degradation rates may affect considerably the export of organic matter to the seafloor and thus ecosystem functioning within the water column. Our results highlight the need to further analyse the consequences of OA in oligotrophic ecosystems with particular focus on dissolved organic matter.

The subcellular effects of pollution were evaluated using two lysosomal biomarkers in mussels, Mytilus galloprovincialis, deployed periodically over a period of 5 years in a harbour area in the Bay of Muggia (Gulf of Trieste, North Adriatic Sea) that is strongly influenced by anthropogenic activities. Mussels were collected from a clean marine farm and analysed (sample T0). A sub-sample was transplanted to the harbour site (sample M) and analysed after about 12 weeks. An additional sub-sample was relocated within the farm as a control and was also tested at the end of the 12-week period (sample T1). The transplantation procedures were repeated twice yearly for 5 consecutive years, starting in 2009. Two well-established lysosomal biomarkers, i.e. lysosomal membrane stability and lipofuscin accumulation, were evaluated in hepatopancreas cells. The body condition index and mortality rate were also assessed. Moreover, various pollutants were determined in both mussel flesh, for a better comprehension of the biological response, and sediments, for a general characterization of the study area. As a whole, the applied biomarkers were found to be appropriate for determining the responses of mussels to environmental pollutant loads over time. Variations in lysosomal membrane stability and lipofuscin content were mostly related to total PAHs and metals respectively. Our results confirm the usefulness of active biomonitoring in evaluating pollution trends in marine coastal areas and in particular the value of lysosomal biomarkers as a rapid screening tool for highlighting pollutant effects at least at organism level.

Two oceanographic cruises encompassing the whole Adriatic Sea were carried out during February and October 2008. Selected stations were sampled at several depths to determine total prokaryotes and picocyanobacteria abundance using epifluorescence microscopy, and to estimate prokaryotic carbon production by 3 H-leucine incorporation. Biological data were related to physical parameters including temperature, salinity and fluorescence, and an attempt to associate bacterial dynamics to water mass characteristics was performed. In both seasons prokaryotic distribution and production showed a decreasing latitudinal gradient likely dependent on riverine inputs highlighted by a strong negative correlation with salinity ( P

From July 2010 to July 2012, free-living nematodes were investigated in terms of abundance, genera and trophic composition at the long term St. C1 (depth 18 m), located in the Gulf of Trieste (northern Adriatic Sea). The integration of these results with environmental (e.g. sediment grain-size, Biopolymeric C and Chl a) and biological variables (benthic diatom biomass and composition) collected synoptically with nematodes, allowed to clarify the linkage between these organisms and another ecosystem component, i.e. benthic diatoms. The observed peaks of nematode abundance in summer samplings were ascribable almost exclusively to piercing nematodes that feed on microalgae (Chromadoridae and, among them, Ptycholaimellus) and were concomitant with the highest numbers of benthic diatoms. DISTLM outputs further corroborated this interaction by indicating Chl a, i.e. a proxy of benthic diatoms, as the only environmental variable that significantly shaped nematode assemblage over the 2-year period. This linkage was not explained only quantitatively (i.e. more diatoms supported more nematodes) but also qualitatively. During winter, in fact, the presence of heavily silicified diatoms co-occurred with nematode minima and the lowest percentage of piercing organisms, suggesting an overall minor ability of the assemblage in using this feeding strategy. In a benthic ecosystem functioning point of view, the observed seasonal pattern of variation in both nematodes and benthic diatoms suggests that more of the energy flowing to nematodes during the summer derives directly from benthic diatom primary production while in the winter this linkage is less important.

The bacterial community, both in terms of community structure (denaturing gradient gel electrophoresis fingerprinting) and activity (exoenzymatic hydrolysis of proteins, polysaccharides and phosphorylated molecules and leucine uptake), was investigated seasonally for 3 years (2004–2006) in a large-scale grid in the northern Adriatic Sea. A high variability characterized the spatial structure of bacterial assemblages and a scarce seasonality was found in all the nine studied stations. Bacterial communities were substantially diverse in the same season of the 3 years, in contrast to what was reported previously for oceanic sites. Assemblages were in general strongly affected by river inputs, especially in spring, when freshwater loads were higher. Finally, a close relationship was found between given assemblages and their patterns of degradation/production activities by applying a multivariate analysis (linear discriminant analysis) to the dataset. The high variability of bacterial community structures and patterns of activity may indicate an ecological response to the high dynamism that characterizes the basin both on a physical and on a biological basis.

The growth, activity and structure of a marine plankton assemblage from the Gulf of Trieste (NE Adriatic Sea) were monitored in a short-term mesocosm experiment (25 L) in order to investigate the influence of phosphorous availability on the production and utilization of organic matter. The coastal community, dimensionally selected (b200 μm), was grown under different P-concentrations. The results obtained from the nutrient enriched enclosures highlighted the microbial food web potential in high productivity systems: the phytoplankton community grew to a bloom-like state and resulted particularly productive enhancing the development of the prokaryotic population (especially of its active fraction). Glucidic exoenzymes operated at high hydrolysis rates in order to recycle extracellular carbohydrate/exudates produced by the abundant phytoplankton community. Intense prokaryotic production, directed prevalently to replication rather than to biomass production, increased the density of prokaryotic cells on levels that particularly favoured virus proliferation, acting thus as feedback control on the bacterial population. Different P concentrations also strongly influenced the structure of the bacterial community (either directly or indirectly) analyzed through denaturing gradient gel electrophoresis (DGGE), by selecting two diverse assemblages at the end of the experiment. Conversely to high N:P ratios observed throughout the year in the Northern Adriatic Sea, our study simulated a phosphorous-balanced condition and pointed out how nutrient limitation affects the entire microbial plankton community, reducing the trophic status of the environment through the control of phytoplankton production.

The strength of the bacteria-phytoplankton coupling and the importance of the microbially mediated carbon fluxes have been investigated in a microtidal lagoon (Lagoon of Venice), with emphasis on the trophic variations, in a within-system perspective. The large trophic heterogeneity of the three stations considered corresponded to an elevated variability of phytoplankton biomass and production (from 0.1 up to 300 microg C L(-1) h(-1)), while bacteria standing stock and production (from 2 to 8 microg C L(-1) h(-1)) appeared, in comparison, to be much more constant. The relationships between bacteria and the phytoplankton community could not be related to the trophic state in a straightforward way; rather, some patterns common to the three stations could be evidenced. In particular, the two communities appeared to be clearly uncoupled, bacterial carbon demand (BCD) always exceeding dissolved primary production (DPP) and, mostly, also total primary production, independent of the station and the season considered. The occurrence of situations in which bacterial production was larger than primary production and the continuous prevalence of BCD over DPP implied that, quite independent of the trophic variability, sources of organic carbon other than phytoplankton production were necessary and available to sustain the bacterial metabolism in the Lagoon of Venice.

Culturable vibrios were isolated from seawater collected during an annual sampling study performed along the Gulf of Trieste coast (Northern Adriatic Sea), and conventional culturing and identification methods were used to investigate the presence of Vibrio parahaemolyticus. Biochemically selected Vibrio strains were subjected to phenotypical identification performed using Alsina's scheme, API 20E and API 20NE. PCR and sequence analysis of the 16S rRNA gene and detection of the species-specific toxR and tlh genes were carried out on strains presumptively identified as V. parahaemolyticus and on a set of unidentified strains to confirm biochemical characterizations. In addition, PCR assays targeting the virulence genes, tdh and trh, were carried out to detect pathogenic strains. PCR results were compared with phenotypic characterizations to evaluate the accuracy of the biochemical methods applied. False-negative identifications were obtained by all phenotypic-based procedures, while API 20E yielded only one false positive. Because the amplification of the 16S rRNA gene produced uncertain results, toxR and tlh gene detections were necessary to confirm the biochemical identifications. Finally, molecular characterization demonstrated the presence of V. parahaemolyticus trh-positive strains and underlined the difficulty in the recognition of the pathogenic environmental organism using conventional methods.

During the summer 2005/06, an oceanographic cruise was carried out in the Ross Sea, from Cape Adare, through the Terra Nova Bay polynya to the eastern edge of the Ross Ice Shelf. We analysed microbial activities (prokaryotic carbon production, protease, phosphatase, beta-glucosidase and lipase activity) and bacterial community structure (using Denaturing Gradient Gel Electrophoresis - DGGE) in order to establish if differences in bacterioplankton assemblages and their metabolic requirements occur within the five Ross Sea water masses: Antarctic Surface Waters (AASW), High Salinity Shelf Water (HSSW), Ice Shelf Water (ISW), Antarctic Bottom Water (AABW), Circumpolar Deep Water (CDW). Differences in activities were found between the highly active AASW and all the other water bodies. A Principal Component Analysis highlighted two main gradients: in the Cape Adare area (AASWn, CDW and AABW) higher phosphatase, lipase and glycolytic activities, increasing towards the surface, were identified, whereas in the southern sector of the basin [AASWs and (m)HSSW] higher leucine uptake and polypeptide degradation characterized the second gradient. DGGE fingerprinting showed for the first time that different water masses harboured diverse bacterial communities, highlighting the high specificity of deep water assemblages.Alpha- andGammaproteobacteriarepresented the main phylogenetic groupings in all samples and no substantial difference in the phylogenetic composition of assemblages was found between different water masses.

Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica) during austral summer 2003 was phenotypically characterized and its capability of degrading organic matter was tested. We evaluated specific bacterial growth rates (mu) to understand at which temperatures bacterial growth shows a linear and direct relationship with the available substrate (4-22 degrees C) and afterwards we tested H. glaciei growth curves and degradative potential at 0, 10 and 37 degrees C using two different media (one enriched and one depleted in PO(4)). The strain grew exponentially only at 10 degrees C. The fastest hydrolysis rates were expressed by enzymes aimed at polysaccharide degradation (alpha-D-glucosidase, beta-D-glucosidase and beta-D-galactosidase) while alkaline phosphatase and aminopeptidase activities were rather low. Our data suggest a preferential demand for carbon derived from carbohydrates rather than from proteins: ectoenzyme activities transformed into carbon mobilization from organic polymers, showed that the total carbon potentially released from polysaccharides can be almost one order of magnitude higher than the protein carbon mobilization. Principal component analysis of the enzyme affinity separated the six experimental conditions, highlighting how different physical (temperature) and chemical (PO(4) enrichment or depletion) features actively lead to a differentiation in the efficiency of the ectoenzymes produced, resulting in preferential degradation of diverse kinds of organic substrates.

A preliminary study was carried out on a picocyanobacterial mixed culture harvested from the Gulf of Trieste (Northern Adriatic) and identified as Synechococcus spp. both by transmission electron microscopy observations, biliprotein composition and molecular analyses. Absorption and fluorescence spectra revealed phycourobilin and phycoerythrobilin chromophores, suggesting the presence of both CU- and C-phycoerythrin, besides phycocyanobilin chromophores typical for phycocyanins and allophycocyanins. Both biliprotein analyses and molecular identification indicated the presence of at least two Synechococcus subgroups presumably differing either in phycoerythrin type or in physiological traits. Among the exoenzymatic activities acting on different substrates, only aminopeptidase showed high hydrolysis rates and the uptake of organic molecules was positive for leucine but not for thymidine. The protein carbon mobilized was high compared with the leucine incorporation rates, resulting in low percentages of newly mobilized carbon utilized by cultures. The organic carbon incorporated as leucine was compared with the photosynthetically produced one, and the balance between the phototrophic- and heterotrophic-like processes was c. 3 : 1. Our findings suggest that the Synechococcus heterotrophy plays an important role in cell's metabolism, and that the photoheterotrophic behavior, together with their chromatic adaptation capability, might represent the key for the absolute dominance of this genus in the Adriatic Sea.

In view of the general paucity of integrated information on offshore benthic communities in the Adriatic Sea and given the vulnerability of this particular coastal system, microphytobenthos, meiofauna and macrofauna were synoptically investigated in front of the Emilia-Romagna coast (northern Adriatic Sea) in September 2010 and March 2011. As required by the Marine Strategy Framework Directive, which extends its action beyond the territorial waters (within 12 nmi) of the Member States, our findings could help to fill the gap of knowledge on the environmental status in offshore areas since the study was carried out also at >12 nmi from the coastline. In fact, sediment samples for the analysis of the benthic communities were collected from a 10-point-station grid that covered an area of about 400 km2 with water depths ranging from 13 to 50 m. The variability of the sediment grain size and other chemical variables in the sediment suggests the presence of two distinct environmental contexts that enhance the proliferation of different benthic communities. At the inshore stations (depth Corbula gibba. Offshore stations (depth >20 m) had muddier sediments, which likely exerted a greater retention of sediment-bound organic matter. These conditions seemed to favour benthic deposit feeders like meio- and macrofaunal annelids. Surprisingly, a conspicuous microphytobenthic community, mainly represented by the diatom Paralia sulcata, has been observed even at remarkable depths (~50 m) opening new questions regarding the role of these organisms in dim-light conditions. Although the investigated benthic communities were, to some extent, moderately influenced by multiple stressors, the co-existence of sensitive taxa (such as meiofaunal copepods and the amphipod Ampelisca spp.) with opportunistic ones (i.e., C. gibba) suggests that the benthic domain in the investigated area is not severely impacted.

The northern Adriatic Sea is among the most productive areas within the Mediterranean Sea, mainly because of the important river discharges that characterise the area. During an oceanographic cruise in April 2011, 12 stations located in the western sector of the basin were sampled in order to deepen the current knowledge on polysaccharide utilisation within phytoplankton blooms. The bloom detected in the proximity of the Po river delta was mainly supported by Skeletonema marinoi and several species of Chaetoceros and determined the spatial distribution of total carbohydrates in surface and subsurface waters. The kinetics of four glycolytic exoenzymes were tested (alpha- and beta-glucosidase together with alpha- and beta-galactosidase), and cellulose-degrading and cellobiose-utilising microbes were enumerated. The fastest enzymatic cleavage was performed by alpha-galactosidase in low-chlorophyll areas. The highest abundance of cellulose-degrading microorganisms corresponded to the lowest degradation rates. Relatively slow hydrolysis was compensated by a pronounced affinity of the enzymes for their substrates. This metabolic strategy would minimise energy consumption, by producing few, high-performance enzymes. No evident relationship between phytoplankton assemblages and polysaccharide degradation dynamics was found.

In the summers of 2000, 2001 and 2002, large amounts of sticky mucilaginous material aggregated to form masses of impressive dimensions over large areas of the Adriatic Sea, particularly in its northern part. Aggregates differing in size were sampled by SCUBA divers and submitted to chemical (nutrient and organic matter concentrations) and biological analysis (virus, bacteria and phytoplankton abundances and bacterial metabolism). Suspended and sinking mucilaginous aggregates were biota-rich environments where the abundance of planktonic organisms and the concentration of nutrients were orders of magnitude higher than in the surrounding seawater. The embedded phytoplankton was mostly composed of diatoms, but the dinoflagellate Gonyaulax fragilis, previously reported in association with the aggregates, was also present. A variety of processes occurred within the mucilaginous aggregates which resulted in the transformation of the organic matter composition and lability and contributed to a partial degradation of mucilage. For an efficient degradation of mucilage, several conditions are necessary: high bacterial abundance and activity and an efficient recycling of nutrients within the aggregates. Most of these conditions, appear to change depending on the type and age of the aggregate. During the first phase of aggregation (cobwebs and ribbons), bacterial activities addressed the degradation of organic matter, particularly that of the nitrogen fraction. The degradation products were rapidly taken up by bacteria, supporting an increase in their abundance and production. In aged mucilage (clouds), the degradation processes decreased and the bacterial metabolism suggested the presence of new organic labile compounds probably due to phytoplankton production. On the basis of our results, stringers, generally considered the first step of the aggregation process, seemed to be the result of a mechanical disruption of other types of aggregates.

Viruses and prokaryotes represent the smallest and the most abundant biological entities in marine environments. The interest for viruses and their interactions with marine organisms is continuously rising but the studies are generally limited to short-time investigations. This study conducted in the Gulf of Trieste on monthly resolution investigates for the very first time relationships between viruses and prokaryotes (both heterotrophs-HP and autotrophs-AP) over ten years (2000–2010). From our results emerged that no clear relationship between the abundances of viruses and prokaryotes is observed unless for rather restricted time intervals. Some of the sporadic peaks of viral abundances can be attributable to infections occurred during the autumn phytoplankton blooms, thus probably contributing to the end of the bloom. We infer that the general uncoupling between viruses and prokaryotes in the Gulf of Trieste is due to the variety of factors that regulate viral infection, proliferation and persistence such as the diversity of viral life cycles that are determined by environmental factors, the abundance and the physiological status of their hosts.

The EU Marine Strategy Framework Directive 2008/56/EC (MSFD) defines a framework for Community actions in the field of marine environmental policy in order to achieve and/or maintain the Good Environmental Status (GES) of the European seas by 2020. Microbial assemblages (from viruses to microbial-sized metazoa) provide a major contribution to global biodiversity and play a crucial role in the functioning of marine ecosystems, but are largely ignored by the MSFD. Prokaryotes are only seen as "microbial pathogens", without defining their role in GES indicators. However, structural or functional prokaryotic variables (abundance, biodiversity and metabolism) can be easily incorporated into several MSFD Descriptors (i.e. D1. Biodiversity, D4. Food webs, D5. Eutrophication, D8. Contaminants and D9. Contaminants in seafood) with beneficial effects. The present review provides a critical analysis of the current MSFD descriptors and illustrates the reliability and advantages of the potential incorporation of some prokaryotic variables within the set of indicators of marine environmental quality. Following a cost/benefit analysis against scientific and economic criteria, we conclude that marine microbial components, and particularly prokaryotes, are highly effective for detecting the effects of anthropogenic pressures on marine environments and for assessing changes in the environmental health status. We thus recommend the inclusion of these components in future implementations of the MSFD.

The bacterial community, both in terms of community structure (denaturing gradient gel electrophoresis fingerprinting) and activity (exoenzymatic hydrolysis of proteins, polysaccharides and phosphorylated molecules and leucine uptake), was investigated seasonally for 3 years (2004-2006) in a large-scale grid in the northern Adriatic Sea. A high variability characterized the spatial structure of bacterial assemblages and a scarce seasonality was found in all the nine studied stations. Bacterial communities were substantially diverse in the same season of the 3 years, in contrast to what was reported previously for oceanic sites. Assemblages were in general strongly affected by river inputs, especially in spring, when freshwater loads were higher. Finally, a close relationship was found between given assemblages and their patterns of degradation/production activities by applying a multivariate analysis (linear discriminant analysis) to the dataset. The high variability of bacterial community structures and patterns of activity may indicate an ecological response to the high dynamism that characterizes the basin both on a physical and on a biological basis.

During the 2006 Italian Antarctic expedition a diel sampling was performed close to Cape Hallett (Ross Sea) during the Austral summer. Under-ice seawater samples (∼4 m) were collected every 2 h for 28 h in order to estimate prokaryotic processes' variability and community structure dynamics. Prokaryotic and viral abundances, exoenzymatic activities (β-glucosidase, chitinase, lipase, alkaline phosphatase and leucine aminopeptidase), prokaryotic carbon production ( 3 H -leucine incorporation) and community structure (Denaturing Gradient Gel Electrophoresis – DGGE fingerprints) were analysed. Results showed that the diel variability of the prokaryotic activity followed a variation in salinity, probably as a consequence of the periodical thawing of sea ice (driven by solar radiation and air temperature cycles), while negligible variation in viral and prokaryotic abundances occurred. The Bacterial and Archaeal community structures underwent an Operational Taxonomic Units (OTUs) temporal shift from the beginning to the end of the sampling, while Flavobacteria -specific primers highlighted high variations in this group possibly related to sea ice melting and substrate release.

A preliminary study was carried out on a picocyanobacterial mixed culture harvested from the Gulf of Trieste (Northern Adriatic) and identified as Synechococcus spp. both by transmission electron microscopy observations, biliprotein composition and molecular analyses. Absorption and fluorescence spectra revealed phycourobilin and phycoerythrobilin chromophores, suggesting the presence of both CU- and C-phycoerythrin, besides phycocyanobilin chromophores typical for phycocyanins and allophycocyanins. Both biliprotein analyses and molecular identification indicated the presence of at least two Synechococcus subgroups presumably differing either in phycoerythrin type or in physiological traits. Among the exoenzymatic activities acting on different substrates, only aminopeptidase showed high hydrolysis rates and the uptake of organic molecules was positive for leucine but not for thymidine. The protein carbon mobilized was high compared with the leucine incorporation rates, resulting in low percentages of newly mobilized carbon utilized by cultures. The organic carbon incorporated as leucine was compared with the photosynthetically produced one, and the balance between the phototrophic- and heterotrophic-like processes was c. 3 : 1. Our findings suggest that the Synechococcus heterotrophy plays an important role in cell's metabolism, and that the photoheterotrophic behavior, together with their chromatic adaptation capability, might represent the key for the absolute dominance of this genus in the Adriatic Sea.

Comprehensive, multi-year studies of microbial processes in an ecosystem context are important in understanding microbial regulation of ecosystem structure, function and variability. A 3 yr study on microbial community was carried out in a coastal area in the northern Adriatic Sea (Gulf of Trieste). It is a semi-enclosed shallow system, mostly controlled by pulsed external inputs, which determine a high variability of plankton communities. Samples were collected monthly at 2 stations from January 1999 to December 2001. Data illustrate remarkable inter- and intra-annual variability in parameters relevant to carbon biogeochemistry and ecosystem energy flow patterns. Integrated primary production (PP; 135 g C m-2 yr -1 ) in 1999 tripled (414 g C m -2 yr -1 ) in 2000, return- ing to a low level (150 g C m -2 yr -1 ) in 2001. Bacterial production (by 3 H-leucine incorporation) accounted, on average, for 35.3% of net PP in 1999, 9.5% in 2000 and 29.4% in 2001. Flux into the microbial loop via bacterial C demand, assuming 30% growth efficiency, was the dominant C flow pathway in 1999 and 2001, accounting for almost 100% of the net PP but a lesser fraction (40%) of net PP in 2000. Total microbial respiration (