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Prokaryotic microorganisms are at the base of marine food web and are sensitive to climate changes. In the Mediterranean Sea, an important link between surface water warming and microbial activities is hypothesized to exist on a seasonal and interannual scale, with high amounts of organic matter utilized by prokaryotes and re-cycled in warm periods. To test this hypothesis, from 1998 to 2013, the prokaryotic abundance and microbial activities (enzymatic activities, prokaryotic heterotrophic production and respiration), together with trophic parameters (chlorophyll, particulate organic carbon and nitrogen), were measured in the euphotic layer of the Ionian Sea (Central Mediterranean). A statistical simultaneous analysis of the environmental and prokaryotic data was performed with the aim of depicting the evolution of microbial activities and their implications in the biogeochemical cycles. Results showed that despite the large spatial and temporal variability, Temperature was the main factor affecting the variability of microbial abundance and metabolism with significant differences on both seasonal and interannual scales. Particularly interesting is the increase of production in summer and of the respiration in winter is very interesting. In spite of low levels of chlorophyll and organic carbon, an increase of prokaryotic heterotrophic production, coupled with that of alkaline phosphatase, was found. As a consequence, the increase of prokaryotic biomass was bottom-up sustained. Most of prokaryotic activities and respiration rates were significantly correlated to organic carbon pools; the process of organic matter decomposition was frequently enhanced at higher temperature, and both prokaryotic production and growth efficiency increased in summer. In view of climate changes, the rise in temperature was associated to the increase of heterotrophic activities and this trend can have a predictive value to describe the microbial processes in the Central Mediterranean Sea.

The Ross Sea is one of the most productive areas of the Southern Ocean and includes several functionally different marine ecosystems. With the aim of identifying signs and patterns of microbial response to current climate change, seawater microbial populations were sampled at different depths, from surface to the bottom, at two Ross Sea mooring areas southeast of Victoria Land in Antarctica. This oceanographic experiment, the XX Italian Antarctic Expedition, 2004-05, was carried out in the framework of the ABIOCLEAR project as part of LTER-Italy. Here, microbial biogeochemical rates of respiration, carbon dioxide production, total community heterotrophic energy production, prokaryotic heterotrophic activity, production (by3H-leucine uptake) and prokaryotic biomass (by image analysis) were determined throughout the water column. As ancillary parameters, chlorophylla, adenosine-triphosphate concentrations, temperature and salinity were measured and reported. Microbial metabolism was highly variable amongst stations and depths. In epi- and mesopelagic zones, respiratory rates varied between 52.4–437.0 and 6.3–271.5 nanol O2l-1h-1; prokaryotic heterotrophic production varied between 0.46–29.5 and 0.3–6.11 nanog C l-1h-1; and prokaryotic biomass varied between 0.8–24.5 and 1.1–9.0 µg C l-1, respectively. The average heterotrophic energy production ranged between 570 and 103 mJ l-1h-1in upper and deeper layers, respectively. In the epipelagic layer, the Prokaryotic Carbon Demand and Prokaryotic Growth Efficiency averaged 9 times higher and 2 times lower, respectively, than in the mesopelagic one. The distribution of plankton metabolism and organic matter degradation was mainly related to the different hydrological and trophic conditions. In comparison with previous research, the Ross Sea results, here, evidenced a relatively impoverished oligotrophic microbial community, throughout the water column.

The water quality of Sicilian transitional and coastal marine areas was investigated by evaluating the distribution of Enterococcus spp.; as a reference, two sewage samples were also analysed. The epidemiological implications of their presence were considered. Enterococcal strains isolated by culture methods were phenotypically and molecularly identified and their susceptibility to antibiotics tested. High phenotypic variability was detected, with low specific identification for 46% of the isolated strains. E. casseliflavus, isolated from transitional environments only, predominated; in coastal and sewage waters E. faecium prevailed, occurring also in transitional environments, where multiple antibiotic resistances were found. Ampicillin and vancomycin resistant enterococci were isolated exclusively from transitional environments. The dynamics of dissemination of antibiotic resistant enterococci in aquatic environments located across the South-North migration route of many wild birds could be very complex, emphasising the relevance of epidemiological studies on transitional aquatic environments as reservoirs of pathogenic enterococci, as well as on sedentary seagulls and migratory wild birds as carriers and spreaders of antibiotic resistant enterococci.

Microorganisms are considered as sentinels of environmental changes, but microbial response to the presence of contaminants, such as hydrocarbons, is still not fully understood. This study aimed at assessing the response of the microbial community to the presence of oil pollution, by comparison of two ecosystems, Milazzo Gulf (Sicily, Italy) and Caleta Cordova (Argentina), as representative of two different temperate Mediterranean and cold-temperate Patagonian ecoregions, respectively. Water and sediments were sampled at coastal stations characterized by different levels of hydrocarbon contamination and analyzed for their microbial enzymatic activity rates; the presence of hydrocarbon-degrading bacteria was also determined. The study approach allowed to determine that microbial metabolism was significantly reduced at the polluted stations, suggesting the detrimental effects of contaminants on organic matter degradation process. The isolation of strains of hydrocarbon-degrading bacteria confirmed that the oil contamination favoured the growth of strains specifically adapted to metabolize hydrocarbons and actively involved in the remediation processes.

Several Italian and Chinese temperate lakes with soluble reactive phosphorus concentrations 20 ?g C L-1h-1) and could favor the release of inorganic phosphorus. High extracellular enzyme activities and phosphorus solubilizing bacteria abundance in sediments accelerated nutrient regeneration. In these conditions, the positive GLU-AP relationship suggested the coupling of carbon and phosphorus regeneration; an efficient phosphorus regeneration and high nitrogen levels (up to 0.067 and 0.059 mg L-1 NH4 and NO3 in Italy; 0.631 and 1.496 mg L-1 NH4 and NO3 in China) led to chlorophyll a peaks of 14.9 and 258.4 µg L-1 in Italy and China, respectively, and a typical algal composition. Conversely, in the oligo-mesotrophic lakes, very low nitrogen levels (in Italy, 0.001 and 0.005 mg L-1 NH4, and NO3, respectively, versus 0.053 and 0.371 mg L-1 in China) induced high LAP, while low phosphorus (33.6 and 46.3 µg L-1 total P in Italy and China, respectively) led to high AP. In these lakes nitrogen and phosphorus regeneration were coupled, as shown by positive LAP-AP relationship; however the nutrient demand could not be completely met without the supply from sediments, due to low enzymatic activity and phosphorus solubilizing bacteria found in this compartment.

Microbial processes involved in water biogeochemistry are linked to physical and chemical variability of water masses. While the occurrence of hydrological changes in the Eastern Mediterranean Sea has been documented, its effects on microbial dynamics and processes have been poorly investigated. An interdisciplinary survey was carried out in November 2011 to depict recent scenarios in the Ionian Mediterranean Sea. This study provides a snapshot of the hydrology and biogeochemistry of this basin, with particular emphasis on the abyssal waters, where two cores of the Eastern Mediterranean Deep Water, both of Adriatic origin, were found. The first core, located at about 3000 m, overlies the second one, which has a recent origin. Both water masses have Adriatic origin and the upper one is older than the lower one. A novel approach has been adopted to relate changes in microbial abundance and metabolism to new thermohaline and biogeochemical findings. A close relationship between surface processes driven by air-sea interaction, microbial variables, physical and chemical parameters (nutrients, dissolved organic carbon and oxygen) was observed. Being intimately coupled to DOC patterns, the microbial respiratory activity was likely the most responsive microbial parameter to hydrological changes, confirming this variable as a sentinel of environmental changes.

Background: The search of rapid methods for the detection of Escherichia coli in coastal marine waters is a topic of scientifi c interest to evaluate potential risks to human health related to their low bacteriological quality.

In coastal lakes the role of microorganisms in driving nutrients regeneration at different water depths and in sediments is not yet fully understood. The dynamics of microbial (algal and bacterial) abundance and bacterial activities involved in organic matter transformation were measured, together with nutrient concentrations, through a microcosm experiment set up using the oligotrophic Faro lake as a study model over a total period of 15 days and with a four-day frequency. Water column at different depths (surface, middle and bottom) and interstitial water obtained by sediment centrifugation were used in appropriate ratios (mixed 1:1 with surface waters) to fill 21-Litre plastic aquaria in order to simulate processes occurring in natural conditions. At early experimental period, the sharp decrease of dissolved organic nutrients and the abundant production of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) in correspondence with high phytoplankton abundance in bottom and interstitial water reflected the relevance of organic nutrients for inorganic nutrients regeneration and phytoplankton growth. Size fractionation of LAP and AP as well as the positive relationship between microbial compartments suggested that bacteria and phytoplankton worked in close reciprocal synergy, and coupling of nitrogen and phosphorus regeneration, especially in bottom and interstitial waters, was observed. At later experimental period, the change in bacterial community, especially the increase of filamentous shaped cells, together with a simultaneous increase of protozoan abundance indicated that nutrient replenishment made the microbial loop structure more competitive. In oligotrophic conditions, such as those in Faro lake, organic nutrient enrichment of bottom and interstitial waters was associated with changes in the bacterial community, with consequent stimulation of extracellular enzymes to support phytoplankton growth. Nutrient availability from microbial regeneration resulted in an increased complexity of the microbial loop structure, with bacteria and phytoplankton adopting specific strategies to respond to the changing environment.

Planktonic communities hold a relevant role within the EU Marine Strategy Framework Directive (MSFD). In view of reaching the Good Environmental Status of marine ecosystems, within this Directive the phyto- and zooplanktonic communities have received great attention, while Prokaryotes (Bacteria, Archaea, and Cyanobacteria) have been neglected. Indeed, the relevance of microbes (particularly of the faecal pollution indicators) as water quality indicators, the role that microorganisms play within the biogeochemical fluxes and in the whole ecosystem functioning, are all important features that deserve to be focused within the MSFD. The present study aims at reviewing the main issues where prokaryotic variables find useful application as descriptors of environmental status, trying to develop a panel of prokaryotic indicators suitable for the environmental quality assessment. From a survey of scientific literature and on field datasets, prokaryotic abundance and activities (enzymatic hydrolysis, prokaryotic production, and respiratory rates) are here chosen as early warning sentinels to detect changes in environmental quality, using an integrated approach with trophic (chlorophyll-a, particulate and dissolved organic matter) measurements.

As a part of a wider project on fisheries ecology, several biological and environmental parameters were monitored during two oceanographic cruises (BANSIC 2012 and NOVESAR 2013) in the Sicily Channel, which connects the Western and Eastern Mediterranean basins. The prokaryotic abundances and biomass as well as hydrolysis rates on organic matter were investigated in the euphotic layer of a retention area for fish larval stages including anchovy (Engraulis encrasicolus, Linnaeus, 1758) with the aim to investigate the different biogeochemical signatures in two seasonal conditions. The environmental parameters, particulate organic carbon and nitrogen together with heterotrophic production were also measured. Results showed significant increases for most of the studied parameters with increasing temperature during summer. This had effects on the Carbon cycle and recycling of nutrients; in fact total prokaryotic abundance and biomass, as well as carbon hydrolyzed by two enzymes (Leucine aminopeptidase and β-glucosidase), increased significantly during summer. Conversely Alkaline phosphatase activity, Chlorophyll concentration and Oxygen increased during winter. The same environmental parameters affected also the presence of fish eggs. Moreover high percentages of free enzymes (i.e., enzymes not associated with cells) were measured, accounting for percentages variable from 12 to 95 % of the total enzymatic activity, with values generally higher in summer than in winter. In this oligotrophic environment, the prokaryotic biomass was supported by the C hydrolyzed by enzymatic activities. The ratio between the hydrolyzed C and prokaryotic biomass was higher in winter than in summer, indicating that alkaline phosphatase activity contribute to an efficient incorporation of C into biomass in winter.

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.

The abundance, biomass and size-structure of planktonic populations, and the microbial metabolic processes were studied in the Sicily Channel, one of the most important spawning areas in the Mediterranean for anchovy (Engraulis encrasicolus), a pelagic species of commercial interest. Results showed that prokaryotes contribute for the 83% of total carbon biomass. Microphytoplankton abundances and biomasses were dominated by autotrophic nanoflagellates and dinoflagellates (36 identified species) and contribute 11% of total biomass. The microzooplanktonic biomass showed its maximum at the surface or subsurface and its contribution was low (4%). In agreement with the general oligotrophy of the investigated area, the study highlights the prevalence of pico-sized fractions within the whole phytoplankton biomass expressed as chlorophyll content, suggesting the importance of picophytoplankton in sustaining the microbial food web. At the same time, the levels of microbial hydrolytic activities are related to productive processes recycling the organic matter and releasing nutrients (P and N), indicating also an active functioning of ecosystem at low trophic levels. Autotrophic production exceeded oxidation by respiration; at the same time, the co-variation of prokaryotic activities and eggs distribution with temperature in summer was observed. The results obtained confirmed that the area acted as a nursery for small fish and both autotrophic and heterotrophic processes supported by microorganisms were in synergy.

This paper presents a winter carbon budget for the northern Adriatic Sea, obtained through direct measurements during two multidisciplinary cruises and literature data. A box model approach was adopted to integrate estimates of stocks and fluxes of carbon species over the total area. The oligotrophy at the basin scale and the start of primary productivity well before the onset of spring stratification were observed. In winter, the system underwent a complete reset, as the mixing of water masses erased any signal of previous hypoxia or anoxia episodes. The northern Adriatic Sea was phosphorus depleted with respect to C and N availability. This fact confirms the importance of mixing with deep-sea water for P supply to biological processes on the whole. Despite the abundant prokaryotic biomass, the microbial food web was less efficient in organic C production than phytoplankton. In the upper layer, the carbon produced by primary production exceeded the fraction respired by planktonic community smaller than 200 µm. On the contrary, respiration processes prevailed in the water column below the pycnocline. The carbon budget also proved that the northern Adriatic Sea can be an effective sink for atmospheric CO2 throughout the entire winter season.

The Western areas of the Adriatic Sea are subjected to inputs of inorganic nutrients and organic matter that can modify the trophic status of the waters and consequently, the microbiological processes involved in the carbon and phosphorus biogeochemical cycles, particularly in shallow coastal environments. To explore this topic, a survey was carried out during the spring of 2003 in a particular hydrodynamic area of the Gulf of Manfredonia, where the potential (P) and real (R) rates of four different microbial exoenzymatic activities (EEA) (alpha [alpha G] and glucosidases [G], leucine aminopeptidase [LAP], and alkaline phosphatase [AP]) as well as the P and R rates of prokaryotic heterotrophic production (PHP), AP as well as the P and R rates of PHP, primary production (PPnet), the prokaryotic and phototrophic stocks and basic hydrological parameters were examined. Three different water masses were found, with a thermohaline front (THF) being detected between the warmer and less saline coastal waters and colder and saltier offshore Adriatic waters. Under the general oligotrophic conditions of the entire Gulf, a decreasing gradient from the coastal toward the offshore areas was detected, with PHP, PPnet, stocks and EEA (alpha G, G, AP) being directly correlated with the temperature and inversely correlated with the salinity, whereas opposite relationships were observed for LAP activity. No enhancement of microbiological activities or stocks was observed at the THF. The use of P or R rates of microbiological activities, which decrease particularly for EEA, could result in discrepancies in interpreting the efficiency of several metabolic processes.

The response of both microbial activities and prokaryotic abundances to environmental variability was studied in a transitional Mediterranean system (Oliveri–Tindari, Italy) during two yearly surveys (1997–'98 and 2005–'06). The total enzymatic (leucine aminopeptidase, β-glucosidase, alkaline phosphatase) and respiratory activity rates as well as of the abundances of total prokaryotes, culturable heterotrophic bacteria, faecal coliforms and enterococci were measured in surface waters of four brackish ponds, together with temperature, salinity, dissolved oxygen, pH, inorganic nutrients, chlorophyll- a and particulate organic carbon and particulate nitrogen determinations. The seasonal and interannual patterns of microbial parameters were investigated in relation to environmental variations. Both the microbial activities and the abundances of culturable heterotrophic bacteria, faecal coliforms and enterococci varied significantly among the ponds, reflecting their haline and trophic variability. Significant seasonal and interannual variations were found in the patterns of microbial activity, but not in total prokaryotic abundance, in response to temperature, dissolved oxygen and trophic changes. Microbial parameters showed a different response to environmental variability in the two examined periods: during 1997–'98, increases in the microbial activity rates and culturable bacterial counts were positively related with the trophic changes, while during 2005–'06 the patterns of leucine aminopeptidase, alkaline phosphatase and respiratory activities and of total prokaryotic and culturable bacteria abundances were uncoupled with those of temperature, salinity, dissolved oxygen, particulate organic carbon and chlorophyll- a . Changes in the relative importance of environmental and trophic variables between the two considered periods explain this discrepancy.

This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by ß-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a two years period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and Temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6°C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effect of temperature and trophic conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.

This paper presents a winter carbon budget for the northern Adriatic Sea, obtained through direct measurements during two multidisciplinary cruises and literature data. A box model approach was adopted to integrate estimates of stocks and fluxes of carbon species over the total area. The oligotrophy at the basin scale and the start of primary productivity well before the onset of spring stratification were observed. In winter, the system underwent a complete reset, as the mixing of water masses erased any signal of previous hypoxia or anoxia episodes. The northern Adriatic Sea was phosphorus depleted with respect to C and N availability. This fact confirms the importance of mixing with deep-sea water for P supply to biological processes on the whole. Despite the abundant prokaryotic biomass, the microbial food web was less efficient in organic C production than phytoplankton. In the upper layer, the carbon produced by primary production exceeded the fraction respired by planktonic community smaller than 200?m. On the contrary, respiration processes prevailed in the water column below the pycnocline. The carbon budget also proved that the northern Adriatic Sea can be an effective sink for atmospheric CO2 throughout the entire winter season.

We report on investigations of prokaryotic abundance, biomass, extracellular enzymatic activity, heterotrophic production and respiration in the full water column (~ 1200 m) of a deep convection site (the Southern Adriatic Pit), carried out on six cruises in 2006-2008. Vertical distribution of prokaryotic abundance varied vertically and temporally and ranged from 1.2 to 20.4 x 105 cell ml-1. Cell volumes, generally increased with depth and in the deep layers the lowest and highest mean cell volumes were observed in a period of no convective process (Feb-07) and a period of stratification (Jun-08) following the convection process occurred in Feb-08. Prokaryotic biomass decreased with depth and was related with both the seasonal cycles of organic matter and the hydrological processes. The picophytoplankton ranged in the upper layer (UL) from 0.089 to 10.71 x 104 cell ml-1 and cells were also recorded till 500 m depth in Feb-08. The variations of enzymatic activities in UL as well as leucine- aminopeptidase/ß-glucosidase ratio showed a seasonal trend probably linked with the productive processes of the photic layer. An inverse relation between Alkaline phosphatase (AP) and phosphate concentrations was found (AP = 0.0003 PO4 -1.7714, R2 = 0.333, P < 0.05) indicating that the presence of inorganic phosphorus in deep waters induced a reduction of total AP; such evidence may be related to deep water formation.. Generally cell-specific enzymatic activities increased with depth as did cell-specific carbon dioxide production rates, while cell-specific prokaryotic heterotrophic production had an opposite trend. High values of prokaryotic growth efficiency registered in the deep layers in Nov-06 reflected a supply of preformed C transported within the deep water masses. Overall, in 2007 in which no convective phenomenon was observed, the variability of prokaryotes metabolism was governed by the seasonal cycle of the organic matter, while in Nov-06 and Jun-08 the dynamics of deep water ventilation influenced the trend along the water column of many microbial parameters. The yearly trophic balance of the study site appeared to move towards autotrophy only in UL, whilst in the whole water column, the prokaryotic carbon demand exceeded POC availability rained down from euphotic zone. This mismatch was balanced by the DOC entrapped in the "younger waters" of new formation that alters the normal flux of the biological pump and fuels the deep marine biota in this area of deep water convection.

The marine pelagic zone situated > 200 m below the sea level (bls) is the largest marine subsystem, comprising more than two-thirds of the oceanic volume. At the same time, it is one of the least explored ecosystems on Earth. Few large-scale environmental genomics studies have been undertaken to examine the phylogenetic diversity and functional gene repertoire of planktonic microbes present in mesopelagic and bathypelagic environments. Here, we present the description of the deep-sea microbial community thriving at > 4900 m depth in Matapan-Vavilov Deep (MVD). This canyon is the deepest site of Mediterranean Sea, with a deepest point located at approximately 5270 m, 56 km SW of city Pylos (Greece) in the Ionian Sea (36°34.00N, 21°07.44E). Comparative analysis of whole-metagenomic data revealed that unlike other deep-sea metagenomes, the prokaryotic diversity in MVD was extremely poor. The decline in the dark primary production rates, measured at 4908 m depth, was coincident with overwhelming dominance of copiotrophic Alteromonas macleodii'deep-ecotype' AltDE at the expense of other prokaryotes including those potentially involved in both autotrophic and anaplerotic CO(2) fixation. We also demonstrate the occurrence in deep-sea metagenomes of several clustered regularly interspaced short palindromic repeats systems.

The Ionian Sea represents a suitable basin for studying the biogeochemical processes mediated by microbial activities. Due to its characteristics of a crossing region between the Western and Eastern Mediterranean Sea, it is one of the most affected sites by changes in water mass composition and dynamics, caused by the Eastern Mediterranean Transient (EMT). To date, relatively few data exist on microbial activities in pelagic areas of the Ionian Sea. From 1998 to 2004 during different research cruises, prokaryotic parameters (abundance, extracellular enzyme activities -leucine aminopeptidase, ß-glucosidase, alkaline phosphatase-, bacterial production and respiration) were measured together with the culturable bacteria and the main physical, chemical and trophic parameters (temperature, salinity, particulated organic matter). The aim of the study was to describe the spatial and temporal variability of microbial activities involved in the carbon and phosphorus cycles, in different layers. Results showed that organic matter transformation mediated by the microbial community displayed a significant increase in Autumn, pointing out the occurrence of significant changes also at meso- and bathy- pelagic depths. Unlike the dark ocean, the bacterial growth efficiency in the Ionian Sea, which increased with depth, seemed to vary its role from a source to a sink of carbon in the epi- and in meso-and bathypelagic layers, respectively. The mechanism of phosphatase regulation showed a weak inverse correlation between specific phosphatase and inorganic P in all seasons except Autumn. It is worth mentioning that the reported results constitute, to the best of our knowledge, one of the available datasets, giving information about microbial activities in the Ionian Sea.

In the framework of the VECTOR DIVCOST Project, a 2-year investigation was started in 2006, with the aim of testing the sensibility of microbial parameters to environmental changes and of assessing whether they can provide information about functional changes in the carbon cycle. The investigation was performed in the surface waters of two small brackish ponds (Ganzirri and Faro), located in the Cape Peloro transitional area (Sicily, Italy). The seasonal changes in both the microbial compartment [bacterioplankton, vibrios, exoenzymatic hydrolysis of proteins and polysaccharides, bacterial secondary production (HBP) and community respiration] and the trophic state of suspended matter [total suspended matter (TSM), particulate organic carbon (POC), particulate organic nitrogen (PON), C ⁄N] were analysed in relation to the hydrological characteristics [temperature, salinity, oxygen, fluorescence, NH4 ,N O 2, NO3 ,P O4]. Despite marked differences in the nutritional input and the diversification in both carbon budget and trophic level, the two ponds show similar trends in many of the investigated factors, hardly influenced by seasonal variations. Temporally coupled trends were observed for some parameters (enzyme activities, vibrios abundances, respiratory activity), whereas others (POC, PON, heterotrophic bacterial production, bacterioplankton) showed a seasonal shift between the two lakes. The different behaviour found for the some biotic parameters suggests that their response to environmental conditions may be modulated differently between the two lakes, which, despite their spatial proximity and reciprocal connection, do not always show contemporaneous functional processes.

A direct, microscopic fluorescent antibody method was developed to detect the occurrence of Enterococcus faecium in coastal aquatic environments and was compared with the conventional membrane filtering method. The "in situ" application of the antibody-based protocol in the analysis of water samples collected from coastal polyhaline habitats demonstrated good sensitivity and ease of implementation. Data obtained with the microscopic technique were in agreement with those obtained from culture counts. The fluorescent antibody method proved to be a rapid and reliable technique for the detection of E. faecium. The advantages and limitations intrinsic to the method are discussed, highlighting the potential of this new technique for monitoring coastal aquatic environments.

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.

Deep-sea hypersaline anoxic lakes (DHALs) of the Eastern Mediterranean represent some of the most hostile environments on our planet. We investigated microbial life in the recently discovered Lake Medee, the largest DHAL found to-date. Medee has two unique features: a complex geobiochemical stratification and an absence of chemolithoautotrophic Epsilonproteobacteria, which usually play the primary role in dark bicarbonate assimilation in DHALs interfaces. Presumably because of these features, Medee is less productive and exhibits reduced diversity of autochthonous prokaryotes in its interior. Indeed, the brine community almost exclusively consists of the members of euryarchaeal MSBL1 and bacterial KB1 candidate divisions. Our experiments utilizing cultivation and [14C]-assimilation, showed that these organisms at least partially rely on reductive cleavage of osmoprotectant glycine betaine and are engaged in trophic cooperation. These findings provide novel insights into how prokaryotic communities can adapt to salt-saturated conditions and sustain active metabolism at the thermodynamic edge of life., This work was supported by the European Commission's program, under the MAMBA (Contract FP7-KBBE-2009-2B-226977) and MicroB3 (Contract FP7-OCEAN.2011-2-287589) Projects. M.F. gratefully acknowledges the financial support by the Spanish Ministry of Economy and Competitiveness (Project BIO2011-25012).

The southern- central Tyrrhenian Sea is a poorly studied area of the Mediterranean Sea, although its importance for the biological fluxes among the western and eastern basins is recognised. This note aimed at evaluating in the waters of the Southern Tyrrhenian Sea the efficiency of some steps of the organic matter processing (enzymatic hydrolysis, prokaryotic production and oxidation) and the role played by the prokaryotic community in this context. During the MEDBIO multidisciplinary cruise performed in July 2005, the distribution of prokaryotic abundance and activities (exo enzymatic activity, heterotrophic production and respiratory activity) was studied. The results showed the presence of high exo enzymatic activity rates (mostly leucine amino peptidase) in the deep sea, while heterotrophic production and respiratory activity rates were high at surface. Some speculations on the different behaviors of the Carbon fluxes occurring among the different epi, meso and bathypelagic layers are reported. At the epipelagic layer both the decomposition and biomass production processes were high. At the mesopelagic layer the organic matter was quickly mobilised but not efficiently incorporated into biomass, therefore the dissolved monomers remained available in the water column for export to other trophic levels or utilization in other chemical processes (lateral advection, oxidation). The bathypelagic layer was characterised by high levels of hydrolytic activity coupled with relatively high prokaryotic production; in this layer, exo enzymatic activity levels fall in an order of magnitude similar to that observed at the epipelagic one. This finding highlights the importance of microbially mediated processes in sustaining life in the marine depths.

A 1-year cycle of observations was performed in four Sicilian transitional water systems (Oliveri-Tindari, Cape Peloro, Vendicari and Marsala) to characterise their ecological status. A panel of variables among which trophic and microbial (enzyme activities, abundance of hetetrophic bacteria and of bacterial pollution indicators) parameters, were selected. Particulate organic carbon (POC) and nitrogen (PON) and chlorophyll-. a (Chl-. a) contents defined the trophic state, while microbial hydrolysis rates and abundance gave insights on microbial community efficiency in organic matter transformation and on allochthonous inputs. To classify the trophic state of examined waters, the synthetic trophic state index (TRIX) was calculated.Microbial hydrolysis rates correlated positively with POC and Chl-a, which increased along the eutrophication gradient. The significant relationships among TRIX, trophic and microbial parameters suggested the use of leucine aminopeptidase, alkaline phosphatase and POC as suitable parameters to implement the Water Framework Directive when assessing the ecological status of transitional water systems.

A synthesis of field data carried out in the Mediterranean Sea are presented, aimed at contributing to the knowledge of three prokaryotic-mediated processes and their implications on the Carbon cycle. The distribution of exoenzymatic activities, secondary production and respiration rates was studied together with the prokaryotic abundances. Particular attention was paid to the meso- and bathypelagic layers which play an important role in the Mediterranean carbon cycle. The study is noteworthy because of its large spatial scale spanning the entire Mediterranean Sea over 4 years. In addition, two Atlantic stations in front of the Gibraltar Strait were investigated. The longitudinal distribution of prokaryotic activities and abundance along the MED showed different trends along the depthlayers. In particular, higher exoenzymatic rates were detected in the Eastern basin compared to the Western one; carbon respiration rate showed patterns variable with the sampling periods in the epipelagic and bathypelagic layers, while a consistent Westwards decreasing trend at the mesopelagic layers occurred. Specific enzyme activities per cell showed high values in the deepest layers for leucine aminopeptidase. Comparison with Carbon respiration rate data collected before the 2000s showed changing patterns of microbial heterotrophic processes in the Mediterranean Sea.

Despite the fact that marine viruses have been increasingly investigated in the last decade, knowledge on virus abundance, biomass and distribution in mesopelagic and bathypelagic waters is limited. We report here the results of a large-spatial-scale study (covering more than 3000 km) on the virioplankton distribution in epi-, meso- and bathypelagic waters in 19 areas of the Mediterranean Sea, from the Alboran Sea and Western Mediterranean, to the Tyrrhenian Sea, Sicily Channel and Ionian Sea. Integrated viral abundance in epipelagic waters was significantly higher than in deep-sea waters (on average, 2.4 vs. 0.5 x 10(12) virusesm(-3)). However, abundance of viruses in the deep-Mediterranean waters was the highest reported so far for deep seas worldwide (7.0 and 3.1 x 10(11) viruses m(-3) in mesopelagic and bathypelagic waters, respectively) and their biomass accounted for 13-18% of total prokaryotic C biomass. The significant relationship between viral abundance and prokaryotic abundance and production in deep waters suggests that also deep-sea viruses are closely dependent on the abundance and metabolism of their hosts. Moreover, virus to prokaryote (and nucleoid -containing cell (NuCC)) abundance ratio increased with increasing depths suggesting that deep waters may represent optimal environments for viral survival or proliferation. Overall, our results indicate that deep waters may represent a significant reservoir of viruses and open new perspectives for future investigations of viral impact on the functioning of meso-bathypelagic ecosystems. (c) 2007 Elsevier Ltd. All rights reserved.

Aims: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. Methods and Results: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, β-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. Conclusions: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. Significance and Impact of the Study: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.

The northern Victoria Land coastal marine environment was investigated during the late summer 2004, within the framework of the Latitudinal Gradient Project (LGP), to describe the physical, chemical and biological patterns of the water column and their relationship with the pelagic and benthic compartments, and to determine to what extent they change with latitude. A latitudinal gradient from Cape Adare to the Terra Nova Bay–Cape Russell area was determined on the basis of abiotic and trophic factors. Cape Adare had lower values of organic matter (particulate organic carbon < 150 μg l−1) available for the benthic communities, but this organic matter had good trophic quality. In Terra Nova Bay the particulate organic matter was quantitatively higher (organic carbon > 400 μg l−1), presumably reaching the bottom via faecal pellets, but was more detrital, although its nutritive value was still high (carbon protein content nearly 40%), as confirmed by the great quantity of phytopigments in the sediments (> 4.0 μg g−1). The benthic communities changed with latitude as well, partially reflecting the environmental and trophic gradient, but also showing a large within-area variability (except for the Cape Adare area), due to a complex array of variables that did not change with latitude.

Three microbial extracellular enzymes, leucine aminopeptidase (LAP), β-glucosidase (β-glu) and alkaline phosphatase (AP), were studied in six small Mediterranean littoral ponds, to evaluate the diversity of microbial activities relative to prevailing environmental conditions. The marked diversification of the trophic states, ranging from oligotrophy to eutrophy, in the ponds was reflected in a range of enzyme patterns at different spatial and temporal scales. There were higher levels and greater variability of microbial activity in the oldest and most ‘confined’ ponds (ranges: 0.55–4360.00 nm h−1, 0.15–76.44 nm h−1, 1.29–1600.00 nm h−1 for LAP, β-glu and AP respectively) compared with the youngest and most seaward ponds (ranges: 22.64–612.0 nm h−1, 0.06–48.89 nm h−1, 0.32–744.0 nm h−1 for LAP, β-glu and AP respectively). The close relationship of the degradative potential with chlorophyll-a and particulate organic carbon could be a consequence of the stimulating effect of phytoplankton-released polymeric compounds (organic matter) and/or a response of the microbial community to warm temperatures, which were recorded from July to September. Within an area less than 1 km2, different aquatic ecosystems coexist and maintain their distinctive properties in terms of microbial biogeochemical processes.

The uptakes of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by mussels (Mytilus galloprovincialis) and the times for depuration were investigated in order to determine the most useful indicator of vibrio contamination. The mussels were maintained in tanks of static seawater contaminated with bacteria at 5 log10 CFU/ml for bioaccumulation. Depuration was carried out by circulating fresh seawater through the tanks. Each organism was presented alone and with others to mussels, at temperatures of 14 and 21 degrees C. In water contaminated with either single or mixed organisms, the bacteria accumulated rapidly in the mussels reaching high concentrations after 1 h. With both single and mixed organisms, the maximum numbers of E. coli in mussels were 6.6 log10 CFU/g at 14 degrees C and 5.4 log10 CFU/g at 21 degrees C. Both V. cholerae non-O1 and E. durans alone or with other organisms reached a number ranging from 6.5 to 7 log10 CFU/g at both temperatures. During depuration the numbers of all the organisms slowly decreased, with E. coli alone, numbers ranged from 2.8 to 2 log10 CFU/g after 72 h at both 14 and 21 degrees C, and the organisms were undetectable after 144 h. With mixed organisms at 14 degrees C E. coli became undetectable after 168 h but at 21 degrees C no E. coli were recovered after 72 h. At 14 degrees C V. cholerae non-O1 alone also was undetectable after 168 h, but at 21 degrees C and with mixed organisms at both temperatures. V. cholerae was recovered after 168 h at numbers about 1 log10 CFU/g. After 168 h numbers of E. durans alone ranged from 2.6 log10 CFU/g at 14 degrees C to 1.5 log10 CFU/g at 21 degrees C, and with mixed organisms the numbers ranged from 2.3 to 2.0 log10 CFU/g at both temperatures. Of the three bacteria of faecal origin, E. durans is quickly acquired by mussels and released more slowly than the others, while E. coli quickly becomes undetectable. The results suggest that, for this kind of seafood, enterococci may be a more appropriate indicator than E. coli of risks to consumers from vibrios.

This paper concerns the current debate as to whether oceans are heterotrophic or autotrophic environments. Microbes are responsible for the assimilation and remineralization of CO2 in the sea and microbial processes are involved in the Earth climatic change. The variability of microbial biomass and activities were studied in the Mediterranean Sea which represents a suitable basin for mapping the trophic regimes by the study of biogeochemical processes. The surveys were carried out in the epi-, meso- and bathy-pelagic zones of the Central Mediterranean Sea from 1996 to 2002. Bacterial abundance, leucine aminopeptidase (LAP) and h-glucosidase (h-GLU) activities, bacterial carbon production (BCP), community respiration (R) and primary production (PP) were the main parameters investigated. Biomass and activities seasonally varied in epipelagic and, to some extent, in meso- and bathy-pelagic layers too and several trophic ratios were determined, showing different scenarios in the different areas and seasons. In the euphotic layer, bacterial growth efficiency (BGE) widely ranged between 4% and 50% showing a decreasing trend from estuaries towards pelagic areas in alignment with thetrophic gradient from theNorthernAdriatic to the IonianSea. However, BGE did not correlate to PP and temperature. The Central Mediterranean Sea showed different trophic regimes in the examined areas and seasons. In the Northern Adriatic Sea in winter, the trophic balance appeared to move towards autotrophy and a positive C budget resulted. An opposite scenario happened in summer when the trophic balance moved to heterotrophy and a negative C budget occurred. In the Ionian Sea, the overall balance turned always towards remineralization. However, on annual time scale, the Central Mediterranean Sea seems to show a tendency towards a heterotrophic system. D 2005 Elsevier B.V. All rights reserved.

A late summer study of marine bacteria activities, and the interrelationships with the microbial loop and the microbial food chain was carried out from 22 January to 10 February 2000 in a coastal area of Terra Nova Bay (Ross Sea). The objective was to investigate the transition from the end of a phytoplanktonic bloom to the start of winter. Intense bacterial activities, comparable to those of temperate marine environments, were observed. The carbon potentially mobilized from proteinaceous matter was quantitatively the most important source of carbon for the bacterioplankton. The leucine aminopeptidase activity was higher in January samples and decreased towards 10 February whereas an opposite trend was observed for alkaline phosphatase and �-glucosidase activities. The bacterial production was supported by c. 0.2% of the amounts of dissolved organic carbon mobilised by hydrolytic activities and by 7% of inorganic phosphate mobilised by alkaline phosphatase activity. A sharp reduction in the bacterial biomass, possibly due to zooplankton grazing or viral lysis, was observed for the first time in Terra Nova Bay.

In late austral spring 1994, a study was carried out in the Ross Sea, with sampling in the ice-free, ice-covered and marginal ice zones. Samples were taken along two transverse transects, to determine spatial variations in some microbiological parameters. Some stations were repeatedly sampled to study the temporal development of microbial components. Microbial biomass was measured as fractionated ATP in the following size categories: micro- (250–10µm), nano- (10–2µm) and picoplankton (2–0.2µm); bacterial biomass was estimated on the basis of lipopolisac-charide concentration and microbial respiratory rate was estimated by the Electron Transport System activity of organisms

During the austral summer of 1991 a study was carried out on the presence and distribution of the genus Vibrio in the Straits of Magellan. Vibrios strains were isolated using membrane filters and Marine Agar 2216 in anaerobiosis. Variations of the populations of total heterotrophic bacteria and vibrios were observed both on the surface and along the column of water. All vibrios are psychrotrophic and were grouped in 4 cluster among which cluster 1, identified as presumed V. anguillarum, seems the most important including 73% of strains. A certain habitat segregation of clusters was noted. Cluster 4 was found only in a deep and permanently colder water mass. The relations between 20 environmental parameters and the bacterial population were also studied. Significant positive correlations were observed between the vibrios population and various fractions of suspended particulate matter.

A total of 42 vibrio strains isolated from sewage, together with 7 culture collection strains were studied by numerical taxonomy. A simplified clustering is shown in Fig. 1. Two important phena were determined. Phenon 1, identified as V. cholerae non-01, formed at 0.90% S, contains 32 sewage strains and 2 reference strains. Phenon 2, identified as V. metschnikovii, formed at 0.85% S, contains 5 sewage strains. No typical marine vibrio species were isolated. The phenotypic characteristics of 2 phena are shown in Table 2. All V. cholerae non-01 were haemolytic, proteolytic and lipolytic. They also showed some phenotypic characteristics (degradation of esculin, 81.2%; utilization of: D-glucuronate, 88.2%; cellobiose, 47.0% and lactose, 47.0%), and growth in NaCl 6%, 91.1%. According to literature references, the survival of V. cholerae non-01 in Mar del Plata marine environment is possible.

Thirty three samples from recreational waters from different areas of Río de la Plata were tested for Salmonella (Fig. 1). A 200 ml sample filtered through a diatom bed as well as 2 and 20 ml samples without filtering were studied using a simple and economical technique. The isolation probability was directly dependent on the water volume analyzed. Best results were obtained when tetrathionate broth with novobiocin and Salmonella shigella agar were used (Table 3). Salmonella was isolated from 36% of the samples studied and 12 serotypes were identified (Table 5). The relation between the NPM of each bacteriological indicator used and the NPM of Salmonella are shown in Table 4. Correlation values were calculated by relating mean point logarithms of the classes in Table 4 with the isolation percentages of Salmonella for each class. These values were low, except for fecal Streptococcus (R2 = 0.94). The high density of fecal coliforms found in Bagliardi and Pescadores Beaches (rarely less than 10.000 bacteria/100 ml) and the incidence of Salmonella, lead us to conclude that these waters are unsafe for bathing and swimming, specially for children, according with the international recommendation on the quality of recreational waters. Waters with the characteristics described here should be periodically analyzed for Salmonella concentration. The lack of correlation values between the MPN of total and fecal coliforms and the MPN of Salmonella, also supports this advise.

[1] Dissolved and particulate organic carbon, bacterial biomass, microbial enzymatic activities (EEA: leucine aminopeptidase, β-glucosidase, and alkaline phosphatase), bacterial production, respiration rates, and bacterial growth efficiency were determined in 10 stations of the Ionian Sea (winter 1998–1999) with the aim of characterizing the recycling of biogenic carbon and phosphorus in the different water masses, previously identified on the basis of their hydrographical properties. All microbial activities decreased markedly with depth, with a sharp increase in the benthic boundary layer, where potential remineralization rates of phosphorus up to 1.03 μg P·dm−3d−1 and bacterial carbon production of 0.078 μg C·dm−3 d−1 were recorded. Those rates were close to the surface ones; the bacterial growth efficiency was also around 20%, similar to the surface value, sustaining the microbial food chain at the bottom. The daily hydrolysis of the organic carbon pool estimated by EEA varied from 0.67% (Ionian Surface Water) to 0.02% (Deep Water). Alkaline phosphatase activity was generally low in the intermediate and deep layers, in relation to the higher inorganic P content. The last facts support the hypothesis that deep waters of Ionian Sea, and in general of the entire Mediterranean basin, because of their young age, carry a larger amount of labile dissolved organic carbon, which reduces the need for a high recycling activity by bacterial community. As a matter of fact, a relatively higher activity per cell in carbon production rates was found in the deep layer where a large volume of the very recently formed Cretan Sea Outflow Water was present.