Fecal bacteria contamination in the Adriatic Sea: Investigating environmental factors and modeling to manage recreational coastal waters.

This study is based on assessing fecal indicator bacteria contamination along meteorological, hydrological and physical-chemical variables after high rainy events during the summer period. The study focused on four different coastal sites in the western and eastern Adriatic coast characterized by various geomorphological and hydrological features, levels of urbanization and anthropogenic pressures, with the aim of finding appropriate and effective solutions to ensure the safety and sustainability of tourism and public health. Detailed in-situ survey revealed a wide range of fecal indicator bacterial (FIB) across the different river mouths with concentrations of E. coli ranging from 165 to 6700 CFU 100 mL-1. It was found that nitrogen compounds track microbial load and acted as tracers for fecal contaminants. Further, a modelling tool was also used to analyze the spatial and temporal distribution of fecal pollution at these coastal sites. The integrated monitoring through high frequent survey in river waters and modeling framework allowed for the estimation of fecal indicator bacterial load at the river mouth and examination of fecal pollutant dispersion in recreational waters, considering different scenarios of fecal dispersion along the coast. This study formed the basis of a robust decision support system aimed at improving the management of recreational areas and ensuring the protection of water bodies through efficient management of bathing areas.

Multi-benthic size approach to unveil different environmental conditions in a Mediterranean harbor area (Ancona, Adriatic Sea, Italy).

Harbors are hubs of human activity and are subject to the continuous discharge and release of industrial, agricultural, and municipal waste and contaminants. Benthic organisms are largely known to reflect environmental conditions they live in. Despite meio- and macrofauna interacting within the benthic system, they are ecologically distinct components of the benthos and as such may not necessarily respond to environmental conditions and/or disturbances in the same way. However, in a few field studies the spatial patterns of meio- and macrofauna have been simultaneously compared. In the present study, we assess the response and patterns in the abundance, diversity, and distribution of the two benthic size classes to the different environmental conditions they live in (i.e., sediment concentrations of selected trace metals and polycyclic aromatic hydrocarbons (PAHs); organic matter contents and grain size) characterizing the Ancona Harbor (Adriatic Sea). Meio- and macrofauna provided partially similar types of information depending on the indices used (univariate measures or community structure/species composition) and the different 'response-to-stress'. The community structure (i.e., taxa composition) of both benthic size components clearly showed differences among sampling stations located from inside to outside the harbor, reflecting the marked environmental heterogeneity and disturbance typically characterizing these systems. Notwithstanding, the univariate measures (i.e., meio- and macrofauna total abundance, diversity indices and equitability) didn't show similar spatial patterns. Meiofauna were likely to be more sensitive to the effects of environmental features and contaminants than macrofauna. Overall, trace metals and PAHs affected the community composition of the two benthic components, but only the meiofauna abundance and diversity were related to the environmental variables considered (i.e., quantity and quality of organic matter). Our results pinpoint the importance of studying both meio- and macrofauna communities, which could provide greater insight into the processes affecting the investigated area and reveal different aspects of the benthic ecosystems in response to harbor conditions.

Host-associated and Environmental Microbiomes in an Open-Sea Mediterranean Gilthead Sea Bream Fish Farm.

Gilthead seabream is among the most important farmed fish species in the Mediterranean Sea. Several approaches are currently applied to assure a lower impact of diseases and higher productivity, including the exploration of the fish microbiome and its manipulation as a sustainable alternative to improve aquaculture practices. Here, using 16S rRNA gene high-throughput sequencing, we explored the microbiome of farmed seabream to assess similarities and differences among microbial assemblages associated to different tissues and compare them with those in the surrounding environment. Seabream had distinct associated microbiomes according to the tissue and compared to the marine environment. The gut hosted the most diverse microbiome; different sets of dominant ASVs characterized the environmental and fish samples. The similarity between fish and environmental microbiomes was higher in seawater than sediment (up to 7.8 times), and the highest similarity (3.9%) was observed between gill and seawater, suggesting that gills are more closely interacting with the environment. We finally analyzed the potential connections occurring among microbiomes. These connections were relatively low among the host's tissues and, in particular, between the gut and the others fish-related microbiomes; other tissues, including skin and gills, were found to be the most connected microbiomes. Our results suggest that, in mariculture, seabream microbiomes reflect only partially those in their surrounding environment and that the host is the primary driver shaping the seabream microbiome. These data provide a step forward to understand the role of the microbiome in farmed fish and farming environments, useful to enhance disease control, fish health, and environmental sustainability.

Water quality integrated system: A strategic approach to improve bathing water management.

In the Adriatic Sea, massive rainfall events are causing flooding of rivers and streams, with severe consequences on the environment. The consequent bacterial contamination of bathing water poses public health risks besides damaging tourism and the economy. This study was conducted in the framework of WATERCARE, an EU Interreg Italy-Croatia Project, which aims at reducing the impact of microbial contamination on Adriatic bathing water due to heavy rainfall events drained in the local sewage network and; enhancing the quality of local waters; and providing support for the decision-making processes regarding the management of bathing water in line with EU regulations. The study involved the development of an innovative water quality integrated system that helps meet these objectives. It consists of four components: a real time hydro-meteorological monitoring system; an autosampler to collect freshwater samples during and after significant rainfall events; a forecast system to simulate the dispersion of pollutants in seawater; and a real-time alert system that can predict the potential ecological risk from the microbial contamination of seawater. The system was developed and tested at a pilot site (Fano, Italy). These preliminary results will be used to develop guidelines for urban wastewater and coastal system quality assessments to contribute to develop policy actions and final governance decisions.

Helium, inorganic and organic carbon isotopes of fluids and gases across the Costa Rica convergent margin.

In 2017, fluid and gas samples were collected across the Costa Rican Arc. He and Ne isotopes, C isotopes as well as total organic and inorganic carbon concentrations were measured. The samples (n = 24) from 2017 are accompanied by (n = 17) samples collected in 2008, 2010 and 2012. He-isotopes ranged from arc-like (6.8 RA) to crustal (0.5 RA). Measured dissolved inorganic carbon (DIC) δ13CVPDB values varied from 3.55 to -21.57‰, with dissolved organic carbon (DOC) following the trends of DIC. Gas phase CO2 only occurs within ~20 km of the arc; δ13CVPDB values varied from -0.84 to -5.23‰. Onsite, pH, conductivity, temperature and dissolved oxygen (DO) were measured; pH ranged from 0.9-10.0, conductivity from 200-91,900 µS/cm, temperatures from 23-89 °C and DO from 2-84%. Data were used to develop a model which suggests that ~91 ± 4.0% of carbon released from the slab/mantle beneath the Costa Rican forearc is sequestered within the crust by calcite deposition with an additional 3.3 ± 1.3% incorporated into autotrophic biomass.

Biostimulation of in situ microbial degradation processes in organically-enriched sediments mitigates the impact of aquaculture.

Fish farm deposition, resulting in organic matter accumulation on bottom sediments, has been identified as among the main phenomena causing negative environmental impacts in aquaculture. An in situ bioremediation treatment was carried out in order to reduce the organic matter accumulation in the fish farm sediments by promoting the natural microbial biodegradation processes. To assess the effect of the treatment, the concentration of organic matter in the sediment and its microbial degradation, as well as the response of the benthic prokaryotic community, were investigated. The results showed a significant effect of the treatment in stimulating microbial degradation rates, and the consequent decrease in the concentration of biochemical components beneath the cages during the treatment. During the bioremediation process, the prokaryotic community in the fish farm sediment responded to the overall improvement of the sediment conditions by showing the decrease of certain anaerobic taxa (e.g. Clostridiales, Acidaminobacteraceae and Caldilinaceae). This suggested that the bioactivator was effective in promoting a shift from an anaerobic to an aerobic metabolism in the prokaryotic community. However, the larger importance of Lachnospiraceae (members of the gut and faecal microbiota of the farmed fishes) in treated compared to non-treated sediments suggested that the bioactivator was not efficient in reducing the accumulation of faecal bacteria from the farmed fishes. Our results indicate that bioremediation is a promising tool to mitigate the aquaculture impact in fish farm sediments, and that further research needs to be oriented to identifying more successful interventions able to specifically target also fish-faeces related microbes.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

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.

Environmental Drivers Controlling Bacterial and Archaeal Abundance in the Sediments of a Mediterranean Lagoon Ecosystem.

The environmental factors controlling the abundance of Bacteria and Archaea in lagoon ecosystems are poorly understood. Here, an integrated physico-chemical, biogeochemical, and microbiological survey was applied in the Sacca di Goro lagoon (Po River Delta, Italy) to investigate the variation of bacterial and archaeal abundance, as assessed by Fluorescence In Situ Hybridization, along winter and summer environmental gradients. We hypothesised that bacterial and archaeal cells respond differentially to physico-chemical parameters of the sediment, which can be manifested in variations of total cells number. Our results suggest that Archaea are an important component of microbial communities (up to 20%) and they are also quite constant along the sediment depth investigated, while Bacteria tend to decrease in the subsurface sediments. The abiotic (i.e. temperature, ammonium, pH) and trophic parameters (i.e. chlorophyll a) explain differentially the variations of bacterial and archaeal distribution, and raise interesting questions about the ecological significance of the microbial composition in this area.

Diversity and Distribution of Prokaryotes within a Shallow-Water Pockmark Field.

Pockmarks are crater-like depression on the seafloor associated with hydrocarbon ascent through muddy sediments in continental shelves around the world. In this study, we examine the diversity and distribution of benthic microbial communities at shallow-water pockmarks adjacent to the Middle Adriatic Ridge. We integrate microbial diversity data with characterization of local hydrocarbons concentrations and sediment geochemistry. Our results suggest these pockmarks are enriched in sedimentary hydrocarbons, and host a microbial community dominated by Bacteria, even in deeper sediment layers. Pockmark sediments showed higher prokaryotic abundance and biomass than surrounding sediments, potentially due to the increased availability of organic matter and higher concentrations of hydrocarbons linked to pockmark activity. Prokaryotic diversity analyses showed that the microbial communities of these shallow-water pockmarks are unique, and comprised phylotypes associated with the cycling of sulfur and nitrate compounds, as well as numerous know hydrocarbon degraders. Altogether, this study suggests that shallow-water pockmark habitats enhance the diversity of the benthic prokaryotic biosphere by providing specialized environmental niches.

Large spatial scale variability in bathyal macrobenthos abundance, biomass, α- and ß-diversity along the Mediterranean continental margin.

The large-scale deep-sea biodiversity distribution of the benthic fauna was explored in the Mediterranean Sea, which can be seen as a miniature model of the oceans of the world. Within the framework of the BIOFUN project ("Biodiversity and Ecosystem Functioning in Contrasting Southern European Deep-sea Environments: from viruses to megafauna"), we investigated the large spatial scale variability (over >1,000 km) of the bathyal macrofauna communities that inhabit the Mediterranean basin, and their relationships with the environmental variables. The macrofauna abundance, biomass, community structure and functional diversity were analysed and the α-diversity and ß-diversity were estimated across six selected slope areas at different longitudes and along three main depths. The macrobenthic standing stock and α-diversity were lower in the deep-sea sediments of the eastern Mediterranean basin, compared to the western and central basins. The macrofaunal standing stock and diversity decreased significantly from the upper bathyal to the lower bathyal slope stations. The major changes in the community composition of the higher taxa and in the trophic (functional) structure occurred at different longitudes, rather than at increasing water depth. For the ß-diversity, very high dissimilarities emerged at all levels: (i) between basins; (ii) between slopes within the same basin; and (iii) between stations at different depths; this therefore demonstrates the high macrofaunal diversity of the Mediterranean basins at large spatial scales. Overall, the food sources (i.e., quantity and quality) that characterised the west, central and eastern Mediterranean basins, as well as sediment grain size, appear to influence the macrobenthic standing stock and the biodiversity along the different slope areas.

Large-scale distribution and activity of prokaryotes in deep-sea surface sediments of the Mediterranean Sea and the adjacent Atlantic Ocean.

The deep-sea represents a substantial portion of the biosphere and has a major influence on carbon cycling and global biogeochemistry. Benthic deep-sea prokaryotes have crucial roles in this ecosystem, with their recycling of organic matter from the photic zone. Despite this, little is known about the large-scale distribution of prokaryotes in the surface deep-sea sediments. To assess the influence of environmental and trophic variables on the large-scale distribution of prokaryotes, we investigated the prokaryotic assemblage composition (Bacteria to Archaea and Euryarchaeota to Crenarchaeota ratio) and activity in the surface deep-sea sediments of the Mediterranean Sea and the adjacent North Atlantic Ocean. Prokaryotic abundance and biomass did not vary significantly across the Mediterranean Sea; however, there were depth-related trends in all areas. The abundance of prokaryotes was positively correlated with the sedimentary concentration of protein, an indicator of the quality and bioavailability of organic matter. Moving eastwards, the Bacteria contribution to the total prokaryotes decreased, which appears to be linked to the more oligotrophic conditions of the Eastern Mediterranean basins. Despite the increased importance of Archaea, the contributions of Crenarchaeota Marine Group I to the total pool was relatively constant across the investigated stations, with the exception of Matapan-Vavilov Deep, in which Euryarchaeota Marine Group II dominated. Overall, our data suggest that deeper areas of the Mediterranean Sea share more similar communities with each other than with shallower sites. Freshness and quality of sedimentary organic matter were identified through Generalized Additive Model analysis as the major factors for describing the variation in the prokaryotic community structure and activity in the surface deep-sea sediments. Longitude was also important in explaining the observed variability, which suggests that the overlying water masses might have a critical role in shaping the benthic communities.

Diversity and phylogenetic analyses of bacteria from a shallow-water hydrothermal vent in Milos island (Greece).

Studies of shallow-water hydrothermal vents have been lagging behind their deep-sea counterparts. Hence, the importance of these systems and their contribution to the local and regional diversity and biogeochemistry is unclear. This study analyzes the bacterial community along a transect at the shallow-water hydrothermal vent system of Milos island, Greece. The abundance and biomass of the prokaryotic community is comparable to areas not affected by hydrothermal activity and was, on average, 1.34 × 10(8) cells g(-1). The abundance, biomass and diversity of the prokaryotic community increased with the distance from the center of the vent and appeared to be controlled by the temperature gradient rather than the trophic conditions. The retrieved 16S rRNA gene fragments matched sequences from a variety of geothermal environments, although the average similarity was low (94%), revealing previously undiscovered taxa. Epsilonproteobacteria constituted the majority of the population along the transect, with an average contribution to the total diversity of 60%. The larger cluster of 16S rRNA gene sequences was related to chemolithoautotrophic Sulfurovum spp., an Epsilonproteobacterium so far detected only at deep-sea hydrothermal vents. The presence of previously unknown lineages of Epsilonproteobacteria could be related to the abundance of organic matter in these systems, which may support alternative metabolic strategies to chemolithoautotrophy. The relative contribution of Gammaproteobacteria to the Milos microbial community increased along the transect as the distance from the center of the vent increased. Further attempts to isolate key species from these ecosystems will be critical to shed light on their evolution and ecology.

Benthic-pelagic coupling: effects on nematode communities along southern European continental margins.

Along a west-to-east axis spanning the Galicia Bank region (Iberian margin) and the Mediterranean basin, a reduction in surface primary productivity and in seafloor flux of particulate organic carbon was mirrored in the in situ organic matter quantity and quality within the underlying deep-sea sediments at different water depths (1200, 1900 and 3000 m). Nematode standing stock (abundance and biomass) and genus and trophic composition were investigated to evaluate downward benthic-pelagic coupling. The longitudinal decline in seafloor particulate organic carbon flux was reflected by a reduction in benthic phytopigment concentrations and nematode standing stock. An exception was the station sampled at the Galicia Bank seamount, where despite the maximal particulate organic carbon flux estimate, we observed reduced pigment levels and nematode standing stock. The strong hydrodynamic forcing at this station was believed to be the main cause of the local decoupling between pelagic and benthic processes. Besides a longitudinal cline in nematode standing stock, we noticed a west-to-east gradient in nematode genus and feeding type composition (owing to an increasing importance of predatory/scavenging nematodes with longitude) governed by potential proxies for food availability (percentage of nitrogen, organic carbon, and total organic matter). Within-station variability in generic composition was elevated in sediments with lower phytopigment concentrations. Standing stock appeared to be regulated by sedimentation rates and benthic environmental variables, whereas genus composition covaried only with benthic environmental variables. The coupling between deep-sea nematode assemblages and surface water processes evidenced in the present study suggests that it is likely that climate change will affect the composition and function of deep-sea nematodes.

Galenea microaerophila gen. nov., sp. nov., a mesophilic, microaerophilic, chemosynthetic, thiosulfate-oxidizing bacterium isolated from a shallow-water hydrothermal vent.

A mesophilic, strictly microaerophilic, chemosynthetic bacterium, designated strain P2D(T), was isolated from the sediment of an active shallow-water hydrothermal vent in Paleochori Bay, on the Greek island of Milos. The cells were Gram-staining-negative rods that measured approximately 0.8-1.3 µm in length and 0.4-0.5 µm in width. Strain P2D(T) grew at 20-50 °C (optimum 35 °C), with 1.0-5.0% (w/v) NaCl (optimum 3.0%), and at pH 4.5-8.0 (optimum pH 5.5). The generation time under optimal conditions was 1.1 h. Growth occurred under chemolithoautotrophic conditions with S2O3²â» and CO(2) as the energy and carbon sources, respectively. Oxygen (5%) was used as sole terminal electron acceptor. No growth was observed in the presence of acetate, formate, lactate, tryptone or peptone. Chemolithoheterotrophic growth occurred when d-glucose or sucrose were present as carbon sources. None of the organic compounds tested was used as an electron donor. The genomic DNA G+C content of the novel strain was 44.9 mol%. In a phylogenetic analysis based on 16S rRNA gene sequences, strain P2D(T) was found to be most closely related to Thiomicrospira psychrophila DSM 13453(T) (92.8% sequence similarity). Based on the phylogenetic, physiological and chemotaxonomic evidence, strain P2D(T) represents a novel species of a new genus within the class Gammaproteobacteria of the family Piscirickettsiaceae, for which the name Galenea microaerophila gen. nov., sp. nov. is proposed. The type strain of the type species is P2D(T) ( = DSM 24963(T) = JCM 17795(T)).

Reliability of CARD-FISH procedure for enumeration of Archaea in deep-sea surficial sediments.

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1 M) and proteinase K (0.004 U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat-both abiotic environmental setting and sedimentary organic matter quality-explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.

Benthic bacterial response to variable estuarine water inputs.

Estuarine waters are known to enhance productivity in coastal environments, but little is known about the impact that nutrient-rich waters can have on benthic microbial processes. After intensive surface sediment sampling in a wide coastal area impacted by river plume waters, we performed on-site microcosm experiments in which we added estuarine waters, rich in both organic and inorganic N and P, to pristine offshore sediments. This experimental approach has been conducted in different coastal areas for two consecutive years, in which the river-plume waters differed in both inorganic and organic N and P content. Benthic bacterial response (total bacterial abundance and biomass, aminopeptidase, beta-d-glucosidase and bacterial C production) was investigated in treated and non-treated sediments. All structural and functional microbial variables increased significantly after plume-water supply (within 12-24 h bacterial abundance in the sediment doubled and enzymatic activities increased up to >50%). Results indicate that inorganic N supply from plume waters was sufficient to induce a significant response in benthic bacterial abundance, independently from the presence of high and/or variable N:P ratios. However, bacterial carbon production and exo-enzymatic activities increased significantly when the supply of organic P from plume waters was associated with a decrease of organic N:P ratios. We conclude that plume waters have important effects on benthic bacterial dynamics, but the extent of their biogeochemical implications is largely dependent upon their organic P availability and on stoichiometric ratios of organic nutrients supplied by plume waters.

Higher abundance of bacteria than of viruses in deep Mediterranean sediments.

The interactions between viral abundance and bacterial density, biomass, and production were investigated along a longitudinal transect consisting of nine deep-sea stations encompassing the entire Mediterranean basin. The numbers of viruses were very low (range, 3.6 x 10(7) to 12.0 x 10(7) viruses g(-1)) and decreased eastward. The virus-to-bacterium ratio was always < 1.0, indicating that the deep-sea sediments of the Mediterranean Sea are the first example of a marine ecosystem not numerically dominated by viruses. The lowest virus numbers were found where the lowest bacterial metabolism and turnover rates and the largest cell size were observed, suggesting that bacterial doubling time might play an important role in benthic virus development.