Marine Fungal Diversity and Dynamics in the Gulf of Trieste (Northern Adriatic Sea).

Fungi contribute to different important ecological processes, including decomposition of organic matter and nutrient cycling, but in the marine environment the main factors influencing their diversity and dynamics at the spatial and temporal levels are still largely unclear. In this study, we performed DNA metabarcoding on seawater sampled monthly over a year and a half in the Gulf of Trieste (northern Adriatic Sea), targeting the internal transcribed spacer (ITS) and the 18S rRNA gene regions. The fungal communities were diverse, very dynamic, and belonged predominantly to marine taxa. Samples could be clustered in two groups, mainly based on the high (> 30%) or low relative proportion of the ascomycetes Parengyodontium album, which emerged as a key taxon in this area. Dissolved and particulate organic C:N ratio played important roles in shaping the mycoplankton assemblages, suggesting that differently bioavailable organic matter pools may be utilized by different consortia. The proportion of fungal over total reads was 31% for ITS and 0.7% for 18S. ITS had the highest taxonomic resolution but low power to detect early divergent fungal lineages. Our results on composition, distribution, and environmental drivers extended our knowledge of the structure and function of the mycobiome of coastal waters.

Genome-resolved metagenomics of Venice Lagoon surface sediment bacteria reveals high biosynthetic potential and metabolic plasticity as successful strategies in an impacted environment.

Bacteria living in sediments play essential roles in marine ecosystems and deeper insights into the ecology and biogeochemistry of these largely unexplored organisms can be obtained from 'omics' approaches. Here, we characterized metagenome-assembled-genomes (MAGs) from the surface sediment microbes of the Venice Lagoon (northern Adriatic Sea) in distinct sub-basins exposed to various natural and anthropogenic pressures. MAGs were explored for biodiversity, major marine metabolic processes, anthropogenic activity-related functions, adaptations at the microscale, and biosynthetic gene clusters. Starting from 126 MAGs, a non-redundant dataset of 58 was compiled, the majority of which (35) belonged to (Alpha- and Gamma-) Proteobacteria. Within the broad microbial metabolic repertoire (including C, N, and S metabolisms) the potential to live without oxygen emerged as one of the most important features. Mixotrophy was also found as a successful lifestyle. Cluster analysis showed that different MAGs encoded the same metabolic patterns (e.g., C fixation, sulfate oxidation) thus suggesting metabolic redundancy. Antibiotic and toxic compounds resistance genes were coupled, a condition that could promote the spreading of these genetic traits. MAGs showed a high biosynthetic potential related to antimicrobial and biotechnological classes and to organism defense and interactions as well as adaptive strategies for micronutrient uptake and cellular detoxification. Our results highlighted that bacteria living in an impacted environment, such as the surface sediments of the Venice Lagoon, may benefit from metabolic plasticity as well as from the synthesis of a wide array of secondary metabolites, promoting ecosystem resilience and stability toward environmental pressures. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00192-z.

Annual recurrence of prokaryotic climax communities in shallow waters of the North Mediterranean.

In temperate coastal environments, wide fluctuations of biotic and abiotic factors drive microbiome dynamics. To link recurrent ecological patterns with planktonic microbial communities, we analysed a monthly-sampled 3-year time series of 16S rRNA amplicon sequencing data, alongside environmental variables, collected at two stations in the northern Adriatic Sea. Time series multivariate analyses allowed us to identify three stable, mature communities (climaxes), whose recurrence was mainly driven by changes in photoperiod and temperature. Mixotrophs (e.g., Ca. Nitrosopumilus, SUP05 clade, and Marine Group II) thrived under oligotrophic, low-light conditions, whereas copiotrophs (e.g., NS4 and NS5 clades) bloomed at higher temperatures and substrate availability. The early spring climax was characterised by a more diverse set of amplicon sequence variants, including copiotrophs associated with phytoplankton-derived organic matter degradation, and photo-auto/heterotrophic organisms (e.g., Synechococcus sp., Roseobacter clade), whose rhythmicity was linked to photoperiod lengthening. Through the identification of recurrent climax assemblages, we begin to delineate a typology of ecosystem based on microbiome composition and functionality, allowing for the intercomparison of microbial assemblages among different biomes, a still underachieved goal in the omics era.

Improving environmental monitoring of Vibrionaceae in coastal ecosystems through 16S rRNA gene amplicon sequencing.

The Vibrionaceae family groups genetically and metabolically diverse bacteria thriving in all marine environments. Despite often representing a minor fraction of bacterial assemblages, members of this family can exploit a wide variety of nutritional sources, which makes them important players in biogeochemical dynamics. Furthermore, several Vibrionaceae species are well-known pathogens, posing a threat to human and animal health. Here, we applied the phylogenetic placement coupled with a consensus-based approach using 16S rRNA gene amplicon sequencing, aiming to reach a reliable and fine-level Vibrionaceae characterization and identify the dynamics of blooming, ecologically important, and potentially pathogenic species in different sites of the northern Adriatic Sea. Water samples were collected monthly at a Long-Term Ecological Research network site from 2018 to 2021, and in spring and summer of 2019 and 2020 at two sites affected by depurated sewage discharge. The 41 identified Vibrionaceae species represented generally below 1% of the sampled communities; blooms (up to ~ 11%) mainly formed by Vibrio chagasii and Vibrio owensii occurred in summer, linked to increasing temperature and particulate matter concentration. Pathogenic species such as Vibrio anguilllarum, Vibrio tapetis, and Photobacterium damselae were found in low abundance. Depuration plant samples were characterized by a lower abundance and diversity of Vibrionaceae species compared to seawater, highlighting that Vibrionaceae dynamics at sea are unlikely to be related to wastewater inputs. Our work represents a further step to improve the molecular approach based on short reads, toward a shared, updated, and curated phylogeny of the Vibrionaceae family.

Linking lifestyle and foraging strategies of marine bacteria: selfish behaviour of particle-attached bacteria in the northern Adriatic Sea.

Microbe-mediated enzymatic hydrolysis of organic matter entails the production of hydrolysate, the recovery of which may be more or less efficient. The selfish uptake mechanism, recently discovered, allows microbes to hydrolyze polysaccharides and take up large oligomers, which are then degraded in the periplasmic space. By minimizing the hydrolysate loss, selfish behaviour may be profitable for free-living cells dwelling in a patchy substrate landscape. However, selfish uptake seems to be tailored to algal-derived polysaccharides, abundant in organic particles, suggesting that particle-attached microbes may use this strategy. We tracked selfish polysaccharides uptake in surface microbial communities of the northeastern Mediterranean Sea, linking the occurrence of this processing mode with microbial lifestyle. Additionally, we set up fluorescently labelled polysaccharides incubations supplying phytodetritus to investigate a 'pioneer' scenario for particle-attached microbes. Under both conditions, selfish behaviour was almost exclusively carried out by particle-attached microbes, suggesting that this mechanism may represent an advantage in the race for particle exploitation. Our findings shed light on the selfish potential of particle-attached microbes, suggesting multifaceted foraging strategies exerted by particle colonizers.

Long-term patterns and drivers of microbial organic matter utilization in the northernmost basin of the Mediterranean Sea.

Marine heterotrophic prokaryotes degrade, transform, and utilize half of the organic matter (OM) produced by photosynthesis, either in dissolved or particulate form. Microbial metabolic rates are affected by a plethora of different factors, spanning from environmental variables to OM composition. To tease apart the environmental drivers underlying the observed organic matter utilization rates, we analysed a 21 year-long time series from the Gulf of Trieste (NE Adriatic Sea). Heterotrophic carbon production (HCP) time series analysis highlighted a long-term structure made up by three periods of coherent observations (1999-2007; 2008-2011; 2012-2019), shared also by OM concentration time series. Temporal patterns of HCP drivers, extracted with a random forest approach, demonstrated that a period of high salinity anomalies (2002-2008) was the main driver of this structure. The reduced river runoff and the consequent depletion of river-borne inorganic nutrients induced a long-term Chl a decline (2006-2009), followed by a steady increase until 2014. HCP driving features over the three periods substantially changed in their seasonal patterns, suggesting that the years following the draught period represented a transition between two long-term regimes. Overall, temperature and particulate organic carbon concentration were the main factors driving HCP rates. The emergence of these variables highlighted the strong control exerted by the temperature-substrate co-limitation on microbial growth. Further exploration revealed that HCP rates did not follow the Arrhenius' linear response to temperature between 2008 and 2011, demonstrating that microbial growth was substrate-limited following the draught event. By teasing apart the environmental drivers of microbial growth on a long-term perspective, we demonstrated that a substantial change happened in the biogeochemistry of one of the most productive areas of the Mediterranean Sea. As planktonic microbes are the foundation of marine ecosystems, understanding their past dynamics may help to explain present and future changes.

Prokaryotic Response to Phytodetritus-Derived Organic Material in Epi- and Mesopelagic Antarctic Waters.

Particulate organic matter (POM) export represents the underlying principle of the biological carbon pump, driving the carbon flux from the sunlit to the dark ocean. The efficiency of this process is tightly linked to the prokaryotic community, as >70% of POM respiration is carried out by particle-associated prokaryotes. In the Ross Sea, one of the most productive areas of the Southern Ocean, up to 50% of the surface primary production is exported to the mesopelagic ocean as POM. Recent evidence suggests that a significant fraction of the POM in this area is composed of intact phytoplankton cells. During austral summer 2017, we set up bottle enrichment experiments in which we amended free-living surface and deep prokaryotic communities with organic matter pools generated from native microplankton, mimicking the particle export that may derive from mild (1 µg of Chlorophyll a L-1) and intense (10 µg of Chlorophyll a L-1) phytoplankton bloom. Over a course of 4 days, we followed free-living and particle-attached prokaryotes' abundance, the degradation rates of polysaccharides, proteins and lipids, heterotrophic production as well as inorganic carbon utilization and prokaryotic community structure dynamics. Our results showed that several rare or undetected taxa in the initial community became dominant during the time course of the incubations and that different phytodetritus-derived organic matter sources induced specific changes in microbial communities, selecting for peculiar degradation and utilization processes spectra. Moreover, the features of the supplied detritus (in terms of microplankton taxa composition) determined different colonization dynamics and organic matter processing modes. Our study provides insights into the mechanisms underlying the prokaryotic utilization of phytodetritus, a significant pool of organic matter in the dark ocean.

Inorganic and Organic Carbon Uptake Processes and Their Connection to Microbial Diversity in Meso- and Bathypelagic Arctic Waters (Eastern Fram Strait).

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

Uptake-release dynamics of the inorganic and organic carbon pool mediated by planktonic prokaryotes in the deep Mediterranean Sea.

Understanding the ecosystem functioning in the dark portion of the ocean is a challenge that microbial ecologists are still facing. Due to the large volume, the global deep Ocean plays a central role in the regulation of climate, possibly buffering the rise of atmospheric carbon dioxide if processes of CO2 fixation compensate for respiration. We investigated the rates of several prokaryotic activities (dissolved and particulate primary production, heterotrophic carbon production and respiration) in meso- and bathypelagic waters of the Mediterranean Sea, covering all sub-basins. Chemosynthesis was the main process for C uptake. The rates of organic C (OC) excretion (or viral-induced cell lysis) inferred from the dissolved primary production measurements were noteworthy, being comparable to particulate primary production, and possibly contributing to the formation of non-sinking particulate organic matter. Inorganic C fixation rates were significantly higher than those reported for other deep-sea systems, probably as a consequence of the persistently higher temperature of dark Mediterranean waters or to phylogenetically diverse communities involved in the process. Primary production was negatively correlated with dissolved organic carbon concentration and showed an inverse pattern to heterotrophic carbon production, indicating a niche partitioning between heterotrophs and autotrophs. In sum, the deep Mediterranean Sea harbors active autotrophic communities able to fix inorganic carbon faster than the heterotrophic carbon production rates.

Aurelia aurita Ephyrae Reshape a Coastal Microbial Community.

Over the last two decades, increasing attention has been paid to the impact of jellyfish blooms on marine communities. Aurelia aurita is one of the most studied of the Scyphozoans, and several studies have been carried out to describe its role as a top-down controller within the classical food web. However, little data are available to define the effects of these jellyfish on microbial communities. The aims of this study were to describe the predation impact of A. aurita ephyrae on a natural microplanktonic assemblage, and to determine any reshaping effects on the prokaryote community composition and functioning. Surface coastal water was used to set up a 24-h grazing experiment in microcosms. Samples were collected to determine the variations in prey biomass, heterotrophic carbon production (HCP), extracellular leucine aminopeptidase activity, and grazing pressure. A next-generation sequencing technique was used to investigate biodiversity shifts within the prokaryote and protist communities through the small subunit rRNA tag approach. This study shows that A. aurita ephyrae were responsible for large decreases in the abundances of the more motile microplankton groups, such as tintinnids, Dinophyceae, and aloricate ciliates. Bacillariophyceae and Mediophyceae showed smaller reductions. No evidence of selective predation emerged in the analysis of the community diversity down to the family level. The heterotrophic prokaryote biomass increased significantly (by up to 45%), in parallel with increases in HCP and leucine aminopeptidase activity (40%). Significant modifications were detected in prokaryotic community composition. Some classes of Gammaproteobacteria and Flavobacteriia showed higher relative abundances when exposed to A. aurita ephyrae, while there was a net decrease for Alphaproteobacteria. Overall, this study provides new insight into the effects of A. aurita on microbial communities, underlining their selective predation toward the more motile groups of microplankton and their impact on prokaryotic assemblages, by favoring blooms of copiotrophic taxa.

Foam production as a side effect of an offshore liquefied natural gas terminal: how do plankton deal with it?

The future growing demand of fossil fuels likely will lead to an increased deployment of liquefied natural gas terminals. However, some concerns exist about their possible effects on the marine environment and biota. Such plants showed to cause the production of foam, as occurred at the still operative terminal of Porto Viro (northern Adriatic Sea). Here, we present results from two microcosm experiments focused on the effects of such foam on microbially mediated degradation processes and its consequent incorporation within the pelagic food web. Such material could be considered as a heterogeneous matrix of both living and non-living organic matter, which constitutes an important substrate for exoenzymes as suggested by the faster hydrolytic rates measured in the treatment microcosms. In the second experiment, a quite immediate and efficient carbon transfer to planktonic biomass through prokaryotic incorporation and consequent predation by heterotrophic flagellates was highlighted. Although no negative effect was evidenced on the overall microbes' growth and foam-derived C seemed to be easily reworked and transferred to higher trophic levels, an important reduction in biodiversity was evidenced for microalgae. Among them, mixotrophic organisms seemed to be favoured suggesting that the addition of foam could cause a modification of the microbial community structure.

Assemblages' structure and activity of bacterioplankton in northern Adriatic Sea surface waters: a 3-year case study.

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

Organic-matter degradative potential of Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica).

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

Ecological advantages from light adaptation and heterotrophic-like behavior in Synechococcus harvested from the Gulf of Trieste (Northern Adriatic Sea).

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

Annual dynamics of bacterioplankton assemblages in the Gulf of Trieste (Northern Adriatic Sea).

Bacterioplankton community diversity was investigated monthly in coastal waters of the Gulf of Trieste (NE Adriatic Sea) throughout 2003. Superficial bacterial assemblages of two differently freshwater influenced stations were studied using PCR-DGGE fingerprinting techniques. Bacterial genetic diversity of the sampled area, as estimates of the number of DGGE bands was high (36-64) compared to that reported in other studies employing this fingerprint technique. The similarity index (Sorensen Index) between assemblages showed a defined operational taxonomic units (OTUs) succession pattern in the more typically marine station with stable winter communities and quickly changing summer ones. On the contrary in the station affected by riverine inputs no clear pattern was detected. In both sites, according to cluster analyses performed on the DGGE banding pattern, three seasonal assemblages were identified: winter-spring, summer and fall. Sequence analysis of fifty-six among the brightest gel bands led to the observation of bacteria affiliated to Gram positive, Cyanobacteria, Cytophaga-Flavobacteria-Bacteroides (CFB) lineages and the alpha-, gamma- and delta- subdivisions of the Proteobacteria. Gamma-Proteobacteria constituted the main fraction (60%) of sequences in the more typically marine station, whereas the river-influenced station was characterised by more heterogeneous assemblages (39% alpha-Proteobacteria, 32% Flavobacteria).