Occurrence of the black yeast Hortaea werneckii in the Mediterranean Sea.

The occurrence of cultivable fungi was investigated along the water column (25-2500 m depth) of four off-shore stations in the Mediterranean basin. An unexpected high abundance of fungi, accompanied by a scarce biodiversity, was observed up to 2500 m depth. The black yeast Hortaea werneckii, known to be one of the most salt tolerant eukaryotic organisms, was isolated for the first time from the Mediterranean Sea, and it was the dominant fungus present in seawater in almost all stations and depths, suggesting its ubiquitous distribution. Isolation of cultivable strains allowed their phylogenetic and taxonomic characterization, and demonstrated that almost all the retrieved fungal species should be considered of terrestrial origin, but well adapted to survive and reproduce at temperature and salinity conditions of the Mediterranean seawater.

Metal Resistance in Bacteria from Contaminated Arctic Sediment is Driven by Metal Local Inputs.

Anthropogenic impact over the Pasvik River (Arctic Norway) is mainly caused by emissions from runoff from smelter and mine wastes, as well as by domestic sewage from the Russian, Norwegian, and Finnish settlements situated on its catchment area. In this study, sediment samples from sites within the Pasvik River area with different histories of metal input were analyzed for metal contamination and occurrence of metal-resistant bacteria in late spring and summer of 2014. The major differences in microbial and chemical parameters were mostly dependent on local inputs than seasonality. Higher concentrations of metals were generally detected in July rather than May, with inner stations that became particularly enriched in Cr, Ni, Cu, and Zn, but without significant differences. Bacterial resistance to metals, which resulted from viable counts on amended agar plates, was in the order Ni2+>Pb2+>Co2+>Zn2+>Cu2+>Cd2+>Hg2+, with higher values that were generally determined at inner stations. Among a total of 286 bacterial isolates (mainly achieved from Ni- and Pb-amended plates), the 7.2% showed multiresistance at increasing metal concentration (up to 10,000 ppm). Selected multiresistant isolates belonged to the genera Stenotrophomonas, Arthrobacter, and Serratia. Results highlighted that bacteria, rapidly responding to changing conditions, could be considered as true indicators of the harmful effect caused by contaminants on human health and environment and suggested their potential application in bioremediation processes of metal-polluted cold sites.

The demosponge Halichondria (Halichondria) panicea (Pallas, 1766) as a novel source of biosurfactant-producing bacteria.

The Mediterranean sponge Halichondria (Halichondria) panicea was explored as a novel matrix for the isolation of biosurfactant-producing bacteria. A total of 38 (out of 56) isolates gave a good response to the employed screening tests (e.g., stable emulsion detection, surface tension measurement, hemolytic activity, and blue agar plate assay) and were selected for further analyses. The thin layer chromatography revealed a possible glucidic composition of biosurfactants. Most promising strains, i.e., those able to produce stable emulsion with percentage higher than 30% and yellow spots on TLC plates, were affiliated to the genera Pseudovibrio, Acinetobacter, and Bacillus. The biosurfactant production by two isolates (i.e., Acinetobacter sp. SpN134 and Pseudovibrio sp. SpE85) was evaluated under different culture conditions, in terms of temperature, NaCl concentration, and pH. Surface tension reduction ability was more stable than the emulsification, and resulted differently influenced by salinity, temperature, and pH. Acinetobacter sp. SpN134 resulted particularly efficient and competitive if compared with other well-known biosurfactant producers. Data suggest that sponges may represent a promising matrix for the isolation of biosurfactant-producing bacteria, reinforcing the growing interest towards filter-feeding organisms as underexplored sources of specialized bacteria.

Efficiency in hydrocarbon degradation and biosurfactant production by Joostella sp. A8 when grown in pure culture and consortia.

Joostella strains are emerging candidates for biosurfactant production. Here such ability was analyzed for Joostella strain A8 in comparison with Alcanivorax strain A53 and Pseudomonas strain A6, all previously isolated from hydrocarbon enrichment cultures made of polychaete homogenates. In pure cultures Joostella sp. A8 showed the highest stable emulsion percentage (78.33%), hydrophobicity rate (62.67%), and an optimal surface tension reduction during growth in mineral medium supplemented with diesel oil (reduction of about 12mN/m), thus proving to be highly competitive with Alcanivorax and Pseudomonas strains. During growth in pure culture different level of biodegradation were detected for Alcanivorax strain A53 (52.7%), Pseudomonas strain A6 (38.2%) and Joostella strain A8 (26.8%). When growing in consortia, isolates achieved similar abundance values, with the best efficiency that was observed for the Joostella-Pseudomonas co-culture. Gas-chromatographic analysis revealed an increase in the biodegradation efficiency in co-cultures (about 90%), suggesting that the contemporary action of different bacterial species could improve the process. Results were useful to compare the efficiencies of well-known biosurfactant producers (i.e. Pseudomonas and Alcanivorax representatives) with a still unknown biosurfactant producer, i.e. Joostella, and to confirm them as optimal biosurfactant-producing candidates.

Biosurfactant production by hydrocarbon-degrading Brevibacterium and Vibrio isolates from the sea pen Pteroeides spinosum (Ellis, 1764).

Among filter-feeders, pennatulids are the most complex and polymorphic members of the cnidarian class Anthozoa. They display a wide distribution throughout all the oceans, constituting a significant component of the sessile megafauna from intertidal to abyssal depths. In this study, a total of 118 bacterial isolates from enrichment cultures, carried out with homogenates of the sea pen Pteroeides spinosum (Ellis, 1764), were screened for hydrocarbon utilization by using the 2,6-dichlorophenol indophenol assay. Among them, 83 hydrocarbon-oxidizing isolates were analyzed for biosurfactant production by standard screening tests (i.e., emulsifying activity, E24 detection, surface tension measurement, microplate assay). The 16S rRNA gene sequencing revealed the affiliation of the most promising isolates to the genera Brevibacterium and Vibrio. Biosurfactant production resulted strongly affected by salinity and temperature conditions, and occurred in the presence of diesel oil and/or crude oil, whereas no production was observed when isolates were grown on tetradecane. The strains resulted able to create stable emulsions, thus suggesting the production of biosurfactants. Further analyses revealed a glycolipidic nature of the biosurfactant extracted from Vibrio sp. PBN295, a genus that has been only recently reported as biosurfactant producer. Results suggest that pennatulids could represent a novel source for the isolation of hydrocarbon-oxidizing bacteria with potential in biosurfactant production.

Biosurfactant activity, heavy metal tolerance and characterization of Joostella strain A8 from the Mediterranean polychaete Megalomma claparedei (Gravier, 1906).

The effect of heavy metals on the activity of biosurfactants produced by Joostella strain A8 from the polychaete Megalomma claparedei was investigated. Biosurfactant activity was first improved by evaluating the influence of abiotic parameters. Higher E(24) indices were achieved at 25 °C in mineral salt medium supplemented with 2 % glucose, 3 % sodium chloride (w/v) and 0.1 % ammonium chloride (w/v). Considerable surface tension reduction was never recorded. Heavy metal tolerance was preliminarily assayed by plate diffusion method resulting in the order of toxicity Cd > Cu > Zn. The activity of biosurfactants was then evaluated in the presence of heavy metals at different concentrations in liquid cultures that were incubated under optimal conditions for biosurfactant activity. The production of stable emulsions resulted generally higher in the presence of metals. These findings suggest that biosurfactant production could represent a bacterial adaptive strategy to defend cells from a stress condition derived from heavy metals in the bulk environment.

Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy).

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

Heavy metal tolerance and polychlorinated biphenyl oxidation in bacterial communities inhabiting the Pasvik River and the Varanger Fjord area (Arctic Norway).

Heavy metals (HMs) and polychlorobiphenyls (PCBs) enter the Arctic environment through a variety of anthropogenic sources with deleterious effects towards biota and public health. Bacteria first transfer toxic compounds to higher trophic levels and, due to the tight link existing between prokaryotic community functions and the type and concentration of contaminants, they may be useful indicator of pollution events and potential toxicity to other forms of life. The occurrence and abundance of HM-tolerant and PCB-oxidizing bacteria in the sub-Arctic Pasvik river area, heavily impacted by anthropogenic modifications, was related to HM and PCB contamination. This latter more likely derived from local inputs rather than a global contamination with higher PCB and HM amounts (and higher bacterial viable counts) that were determined in inner and middle sections of the River. Finally, a panel of bacteria with potential applications in the bioremediation of cold environments were selected and phylogenetically identified.

Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands).

Open fjords are subject to contrasting environmental conditions, owing to meltwater glacial inputs, terrestrial runoff, and marine water mass exchanges, which are exacerbated by anthropogenic and climate perturbations. Following a slope-dependent water circulation, the subtidal sandy sediment belt regulates the convergent transport of nutrients downward the fjord depths, and the effective entrapment of suspended particles and microorganisms. In this study, we aimed at testing how glacial and seawater inputs may influence the bacterial community structure of subtidal sand deposits in the Kongsfjorden. Through total and viable cell counting and an amplicon sequencing approach, we found relevant differences in bacterial community structure along the glacio-marine sampling transect. Viable and high nucleic acid content (HNA) cells represented an important fraction of the total community, generally decreasing toward the glacier front. Besides the predominance of Alpha- and Gammaproteobacteria, Bacteroidetes, Firmicutes and Parcubacteria, the bacterial community structure was likely affected by the glacial activity in the inner fjord, with the occurrence of distinctive phylotypes belonging to Gemmatimonadates, Nitrospirae, Acidobacteria, and Chloroflexi. Overall, our outcomes highlighted that exploring the bacterial community distribution and structure can provide new insights into the active role of sand deposits in coastal cold environments.

Enrichment, isolation and biodegradation potential of psychrotolerant polychlorinated-biphenyl degrading bacteria from the Kongsfjorden (Svalbard Islands, High Arctic Norway).

Persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs), have been detected in abiotic Arctic matrices: surface sediments and seawater from coastal areas in the Kongsfjorden were collected and analyzed. Levels of PCBs varied depending on the sampling site. Total PCB concentrations were between 11.63 (site C2W) and 27.69pgl-1 (site AW). These levels were comparable to those reported previously in lake sediments from the northern Svalbard. The occurrence and biodegradation potential of cold-adapted PCB-oxidizing bacteria in seawater and sediment along the fjord was also evaluated. After enrichment with biphenyl, 246 isolates were obtained with 45 of them that were able to grow in the presence of the PCB mixture Aroclor 1242, as the sole carbon source. The catabolic gene bphA was harbored by 17 isolates with affiliates to the genera Algoriphagus, Devosia and Salinibacterium that have been never reported as able to utilize PCBs, thus deserving further investigation. The total removal of Aroclor 1242 and selected PCB congeners was evaluated at 4 and 15°C for eight bphA-harboring isolates and Gelidibacter sp. DS-10. With few exceptions, tested strains showed greater efficiency at 15 than at 4°C. Isolates were able to reduce most chromatographic peaks by >50%, with some di- and trichlorobiphenyls that were quite totally removed (>90%).

Long-Term Monitoring of Dolphin Biosonar Activity in Deep Pelagic Waters of the Mediterranean Sea.

Dolphins emit short ultrasonic pulses (clicks) to acquire information about the surrounding environment, prey and habitat features. We investigated Delphinidae activity over multiple temporal scales through the detection of their echolocation clicks, using long-term Passive Acoustic Monitoring (PAM). The Istituto Nazionale di Fisica Nucleare operates multidisciplinary seafloor observatories in a deep area of the Central Mediterranean Sea. The Ocean noise Detection Experiment collected data offshore the Gulf of Catania from January 2005 to November 2006, allowing the study of temporal patterns of dolphin activity in this deep pelagic zone for the first time. Nearly 5,500 five-minute recordings acquired over two years were examined using spectrogram analysis and through development and testing of an automatic detection algorithm. Echolocation activity of dolphins was mostly confined to nighttime and crepuscular hours, in contrast with communicative signals (whistles). Seasonal variation, with a peak number of clicks in August, was also evident, but no effect of lunar cycle was observed. Temporal trends in echolocation corresponded to environmental and trophic variability known in the deep pelagic waters of the Ionian Sea. Long-term PAM and the continued development of automatic analysis techniques are essential to advancing the study of pelagic marine mammal distribution and behaviour patterns.

Size Distribution of Sperm Whales Acoustically Identified during Long Term Deep-Sea Monitoring in the Ionian Sea.

The sperm whale (Physeter macrocephalus) emits a typical short acoustic signal, defined as a "click", almost continuously while diving. It is produced in different time patterns to acoustically explore the environment and communicate with conspecifics. Each emitted click has a multi-pulse structure, resulting from the production of the sound within the sperm whale's head. A Stable Inter Pulse Interval (Stable IPI) can be identified among the pulses that compose a single click. Applying specific algorithms, the measurement of this interval provides useful information to assess the total length of the animal recorded. In January 2005, a cabled hydrophone array was deployed at a depth of 2,100 m in the Central Mediterranean Sea, 25 km offshore Catania (Ionian Sea). The acoustic antenna, named OνDE (Ocean noise Detection Experiment), was in operation until November 2006. OνDE provided real time acoustic data used to perform Passive Acoustic Monitoring (PAM) of cetacean sound emissions. In this work, an innovative approach was applied to automatically measure the Stable IPI of the clicks, performing a cepstrum analysis to the energy (square amplitude) of the signals. About 2,100 five-minute recordings were processed to study the size distribution of the sperm whales detected during the OνDE long term deep-sea acoustic monitoring. Stable IPIs were measured in the range between 2.1 ms and 6.4 ms. The equations of Gordon (1991) and of Growcott (2011) were used to convert the IPIs into measures of size. The results revealed that the sperm whales recorded were distributed in length from about 7.5 m to 14 m. The size category most represented was from 9 m to 12 m (adult females or juvenile males) and specimens longer than 14 m (old males) seemed to be absent.

Annual Acoustic Presence of Fin Whale (Balaenoptera physalus) Offshore Eastern Sicily, Central Mediterranean Sea.

In recent years, an increasing number of surveys have definitively confirmed the seasonal presence of fin whales (Balaenoptera physalus) in highly productive regions of the Mediterranean Sea. Despite this, very little is yet known about the routes that the species seasonally follows within the Mediterranean basin and, particularly, in the Ionian area. The present study assesses for the first time fin whale acoustic presence offshore Eastern Sicily (Ionian Sea), throughout the processing of about 10 months of continuous acoustic monitoring. The recording of fin whale vocalizations was made possible by the cabled deep-sea multidisciplinary observatory, "NEMO-SN1", deployed 25 km off the Catania harbor at a depth of about 2,100 meters. NEMO-SN1 is an operational node of the European Multidisciplinary Seafloor and water-column Observatory (EMSO) Research Infrastructure. The observatory was equipped with a low-frequency hydrophone (bandwidth: 0.05 Hz-1 kHz, sampling rate: 2 kHz) which continuously acquired data from July 2012 to May 2013. About 7,200 hours of acoustic data were analyzed by means of spectrogram display. Calls with the typical structure and patterns associated to the Mediterranean fin whale population were identified and monitored in the area for the first time. Furthermore, a background noise analysis within the fin whale communication frequency band (17.9-22.5 Hz) was conducted to investigate possible detection-masking effects. The study confirms the hypothesis that fin whales are present in the Ionian Sea throughout all seasons, with peaks in call detection rate during spring and summer months. The analysis also demonstrates that calls were more frequently detected in low background noise conditions. Further analysis will be performed to understand whether observed levels of noise limit the acoustic detection of the fin whales vocalizations, or whether the animals vocalize less in the presence of high background noise.

Influence of salinity and temperature on the activity of biosurfactants by polychaete-associated isolates.

Influence of different parameters on biosurfactant (BS) activity was carried out on strains that were isolated from the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum and additional 30 strains that were previously identified as potential BS producers from crude oil enrichments of the same polychaete specimens. The selection of BS-producing strains from polychaete natural samples was carried out by using standard screening tests. The BS activity by each isolate was evaluated for the effect of salinity and temperature on emulsion production and surface tension reduction, during incubation in mineral medium supplemented with tetradecane or diesel oil. All isolates showed a similar time course of BS activity, and the latter was more influenced by salinity rather than temperature. Some of the BS producers belonged to genera that have not (i.e. Citricoccus, Cellulophaga, Tenacibaculum and Maribacter) or have poorly been (Psychrobacter, Vibrio, and Pseudoalteromonas) reported as able to produce BSs. This is remarkable as some of them have previously been detected in hydrocarbon-enriched samples. Results confirm that filter-feeding polychaetes are an efficient source for the isolation of BS producers.

Bacteria associated with sabellids (Polychaeta: Annelida) as a novel source of surface active compounds.

A total of 69 bacteria were isolated from crude oil enrichments of the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum, and screened for biosurfactant (BS) production by conventional methods. Potential BS-producers (30 isolates) were primarily selected due to the production of both interesting spots on thin layer chromatography (TLC) plates and highly stable emulsions (E24 ≥ 50%). Only few strains grew on cetyltrimethylammonium bromide and blood agar plates, indicating the probable production of anionic surfactants. The 16S rRNA gene sequencing revealed that selected isolates mainly belonged to the CFB group of Bacteroidetes, followed by Gammaproteobacteria and Alphaproteobacteria. A number of BS-producers belonged to genera (i.e., Cellulophaga, Cobetia, Cohaesibacter, Idiomarina, Pseudovibrio and Thalassospira) that have been never reported as able to produce BSs, even if they have been previously detected in hydrocarbon-enriched samples. Our results suggest that filter-feeding Polychaetes could represent a novel and yet unexplored source of biosurfactant-producing bacteria.