Accelerated nitrogen cycling on Mediterranean seagrass leaves at volcanic CO2 vents.

Seagrass meadows form highly productive and diverse ecosystems in coastal areas worldwide, where they are increasingly exposed to ocean acidification (OA). Efficient nitrogen (N) cycling and uptake are essential to maintain plant productivity, but the effects of OA on N transformations in these systems are poorly understood. Here we show that complete N cycling occurs on leaves of the Mediterranean seagrass Posidonia oceanica at a volcanic CO2 vent near Ischia Island (Italy), with OA affecting both N gain and loss while the epiphytic microbial community structure remains largely unaffected. Daily leaf-associated N2 fixation contributes to 35% of the plant's N demand under ambient pH, while it contributes to 45% under OA. Nitrification potential is only detected under OA, and N-loss via N2 production increases, although the balance remains decisively in favor of enhanced N gain. Our work highlights the role of the N-cycling microbiome in seagrass adaptation to OA, with key N transformations accelerating towards increased N gain.

The role of epiphytes in seagrass productivity under ocean acidification.

Ocean Acidification (OA), due to rising atmospheric CO2, can affect the seagrass holobiont by changing the plant's ecophysiology and the composition and functioning of its epiphytic community. However, our knowledge of the role of epiphytes in the productivity of the seagrass holobiont in response to environmental changes is still very limited. CO2 vents off Ischia Island (Italy) naturally reduce seawater pH, allowing to investigate the adaptation of the seagrass Posidonia oceanica L. (Delile) to OA. Here, we analyzed the percent cover of different epiphytic groups and the epiphytic biomass of P. oceanica leaves, collected inside (pH 6.9-7.9) and outside (pH 8.1-8.2) the CO2 vents. We estimated the contribution of epiphytes to net primary production (NPP) and respiration (R) of leaf sections collected from the vent and ambient pH sites in laboratory incubations. Additionally, we quantified net community production (NCP) and community respiration (CR) of seagrass communities in situ at vent and ambient pH sites using benthic chambers. Leaves at ambient pH sites had a 25% higher total epiphytic cover with encrusting red algae (32%) dominating the community, while leaves at vent pH sites were dominated by hydrozoans (21%). Leaf sections with and without epiphytes from the vent pH site produced and respired significantly more oxygen than leaf sections from the ambient pH site, showing an average increase of 47 ± 21% (mean ± SE) in NPP and 50 ± 4% in R, respectively. Epiphytes contributed little to the increase in R; however, their contribution to NPP was important (56 ± 6% of the total flux). The increase in productivity of seagrass leaves adapted to OA was only marginally reflected by the results from the in situ benthic chambers, underlining the complexity of the seagrass community response to naturally occurring OA conditions.

Zebra Mussel Holobionts Fix and Recycle Nitrogen in Lagoon Sediments.

Bivalves are ubiquitous filter-feeders able to alter ecosystems functions. Their impact on nitrogen (N) cycling is commonly related to their filter-feeding activity, biodeposition, and excretion. A so far understudied impact is linked to the metabolism of the associated microbiome that together with the host constitute the mussel's holobiont. Here we investigated how colonies of the invasive zebra mussel (Dreissena polymorpha) alter benthic N cycling in the shallow water sediment of the largest European lagoon (the Curonian Lagoon). A set of incubations was conducted to quantify the holobiont's impact and to quantitatively compare it with the indirect influence of the mussel on sedimentary N transformations. Zebra mussels primarily enhanced the recycling of N to the water column by releasing mineralized algal biomass in the form of ammonium and by stimulating dissimilatory nitrate reduction to ammonium (DNRA). Notably, however, not only denitrification and DNRA, but also dinitrogen (N2) fixation was measured in association with the holobiont. The diazotrophic community of the holobiont diverged substantially from that of the water column, suggesting a unique niche for N2 fixation associated with the mussels. At the densities reported in the lagoon, mussel-associated N2 fixation may account for a substantial (and so far, overlooked) source of bioavailable N. Our findings contribute to improve our understanding on the ecosystem-level impact of zebra mussel, and potentially, of its ability to adapt to and colonize oligotrophic environments.

Investigating microbial indicators of anthropogenic marine pollution by 16S and 18S High-Throughput Sequencing (HTS) library analysis.

High-Throughput Sequencing technologies are providing unprecedented inventories of microbial communities in aquatic samples, offering an invaluable tool to estimate the impact of anthropogenic pressure on marine communities. In this case study, the Mediterranean touristic site of Aci Castello (Italy) was investigated by High-Throughput Sequencing of 16S and 18S rRNA genes. The sampling area falls within a Marine Protected Area and, notwithstanding, features an untreated urban wastewater discharge. Seawater samples were collected close to the wastewater output (COL) and at a second station about 400 m further off (PAN), before and after a summer increase in population. Prokaryotic communities clustered according to stations, rather than to seasons. While PAN showed a typical, not impacted, marine microbial composition, COL was consistently enriched in Epsilonproteobacteria and Firmicutes. Protist communities showed a peculiar clustering, as COL at springtime stood alone and was dominated by Ciliophora, while the other samples were enriched in Dinophyta. Analysis of alternative, detectable by High-Throughput Sequencing, microbial indicators, including both faecal- and sewage-associated, allowed uncovering the different sources of pollution in coastal and anthropogenically impacted marine ecosystems, underpinning the relevance of High-Throughput Sequencing-based screening as rapid and precise method for water quality management.

Antibiotic disturbance affects aquatic microbial community composition and food web interactions but not community resilience.

Notwithstanding the fundamental role that environmental microbes play for ecosystem functioning, data on how microbes react to disturbances are still scarce, and most factors that confer stability to microbial communities are unknown. In this context, antibiotic discharge into the environment is considered a worldwide threat for ecosystems with potential risks to human health. We therefore tested resilience of microbial communities challenged by the presence of an antibiotic. In a continuous culture experiment, we compared the abundance, composition and diversity of microbial communities undisturbed or disturbed by the constant addiction of tetracycline in low (10 µg/L) or intermediate (100 µg/L) concentration (press disturbance). Further, the bacterial communities in the three treatments had to face the sudden pulse disturbance of adding an allochthonous bacterium (Escherichia coli). Tetracycline, even at low concentrations, affected microbial communities by changing their phylogenetic composition and causing cell aggregation. This, however, did not coincide with a reduced microbial diversity, but was mainly caused by a shift in dominance of specific bacterial families. Moreover, the less disturbed community (10 µg/L tetracycline) was sometimes more similar to the control and sometimes more similar to heavily disturbed community (100 µg/L tetracycline). All in all, we could not see a pattern where the communities disturbed with antibiotics were less resilient to a second disturbance introducing E. coli, but they seemed to be able to buffer the input of the allochthonous strain in a similar manner as the control.

Understanding the association of Escherichia coli with diverse macroalgae in the lagoon of Venice.

Recent studies provided evidence that the macroalga Cladopohora in lakes hosts associated Escherichia coli, with consequences on the environmental and human health. We expanded these investigations to other macroalgae (Ulva spp., Sargassum muticum and Undaria pinnatifida) widespread in the lagoon of Venice (Italy). Attached E. coli were abundant, accounting up to 3,250 CFU gram(-1) of alga. Macroalgal-associated isolates belonged to all E. coli phylogroups, including pathogenic ones, and to Escherichia cryptic clades. Attached E. coli showed potential to grow even at in situ temperature on macroalgal extracts as only source of carbon and nutrients, and ability to produce biofilm in vitro. The genotypic diversity of the attached isolates was high, with significant differences between algae and the overlying water. Our evidences suggest that attached populations consist of both resident and transient strains, likely resulting from the heterogeneous input of fecal bacteria from the city. We report that cosmopolitan and invasive macroalgae may serve as source of E. coli, including pathogenic genotypes, and that this habitat can potentially support their growth. Considering the global diffusion of the macroalgae here studied, this phenomenon is likely occurring in other coastal cities worldwide and deserves further investigations from either the sanitary and ecological perspectives.

The genus Weissella: taxonomy, ecology and biotechnological potential.

Bacteria assigned to the genus Weissella are Gram-positive, catalase-negative, non-endospore forming cells with coccoid or rod-shaped morphology (Collins et al., 1993; Björkroth et al., 2009, 2014) and belong to the group of bacteria generally known as lactic acid bacteria. Phylogenetically, the Weissella belong to the Firmicutes, class Bacilli, order Lactobacillales and family Leuconostocaceae (Collins et al., 1993). They are obligately heterofermentative, producing CO2 from carbohydrate metabolism with either d(-)-, or a mixture of d(-)- and l(+)- lactic acid and acetic acid as major end products from sugar metabolism. To date, there are 19 validly described Weissella species known. Weissella spp. have been isolated from and occur in a wide range of habitats, e.g., on the skin and in the milk and feces of animals, from saliva, breast milk, feces and vagina of humans, from plants and vegetables, as well as from a variety of fermented foods such as European sourdoughs and Asian and African traditional fermented foods. Thus, apart from a perceived technical role of certain Weissella species involved in such traditional fermentations, specific Weissella strains are also receiving attention as potential probiotics, and strain development of particularly W. cibaria strains is receiving attention because of their high probiotic potential for controlling periodontal disease. Moreover, W. confusa and W. cibaria strains are known to produce copius amounts of novel, non-digestible oligosaccharides and extracellular polysaccharides, mainly dextran. These polymers are receiving increased attention for their potential application as prebiotics and for a wide range of industrial applications, predominantly for bakeries and for the production of cereal-based fermented functional beverages. On the detrimental side, strains of certain Weissella species, e.g., of W. viridescens, W. cibaria and W. confusa, are known as opportunistic pathogens involved in human infections while strains of W. ceti have been recently recongnized as etiological agent of "weissellosis," which is a disease affecting farmed rainbow trouts. Bacteria belonging to this species thus are important both from a technological, as well as from a medical point of view, and both aspects should be taken into account in any envisaged biotechnological applications.

Novel PCR-based identification of Weissella confusa using an AFLP-derived marker.

An extensive use of Weissella (W.) confusa is currently being made for the production of a variety of fermented foods and beverages although some strains of this species have emerged as opportunistic pathogens for humans and animals. Nevertheless, no rapid methods are available for the reliable identification of W. confusa. We developed a novel PCR using AFLP (Amplified Fragment Length Polymorphism)-derived primers for the rapid and unequivocal identification of W. confusa. Fluorescent AFLP of 30 strains of W. confusa, Leuconostoc citreum, Lactobacillus (Lb.) brevis, Lb. rossiae, Lb. plantarum and Lb. buchneri allowed us to detect, purify and sequence several W. confusa specific AFLP fragments. The homology search in BLAST of a 303 bp nucleotide sequence revealed a ≤ 77% identity of the purified fragment with the lepA gene of several lactic acid bacteria. A PCR assay targeting 225 bp of this fragment was developed and tested against the DNA of 109 strains, including 34 foodborne and clinical W. confusa and 75 strains of 47 phylogenetically closely and distantly related species, resulting in 100% specificity with a detection limit of 16 pg. Being the first species-specific PCR to date developed for the rapid and unambiguous identification of W. confusa, this novel assay could be a reliable and efficient tool for detecting W. confusa not only in food and beverages, but also in clinical specimens, thus contributing to clarify its real significance in human and animal infections.