Intra- and inter-cores fungal diversity suggests interconnection of different habitats in an Antarctic frozen lake (Boulder Clay, Northern Victoria Land).

A perennially frozen lake at Boulder Clay site (Victoria Land, Antarctica), characterized by the presence of frost mounds, have been selected as an in situ model for ecological studies. Different samples of permafrost, glacier ice and brines have been studied as a unique habitat system. An additional sample of brines (collected in another frozen lake close to the previous one) was also considered. Alpha- and beta-diversity of fungal communities showed both intra- and inter-cores significant (p < 0.05) differences, which suggest the presence of interconnection among the habitats. Therefore, the layers of frost mound and the deep glacier could be interconnected while the brines could probably be considered as an open habitat system not interconnected with each other. Moreover, the absence of similarity between the lake ice and the underlying permafrost suggested that the lake is perennially frozen based. The predominance of positive significant (p < 0.05) co-occurrences among some fungal taxa allowed to postulate the existence of an ecological equilibrium in the habitats systems. The positive significant (p < 0.05) correlation between salt concentration, total organic carbon and pH, and some fungal taxa suggests that a few abiotic parameters could drive fungal diversity inside these ecological niches.

Cultivable Bacterial Communities in Brines from Perennially Ice-Covered and Pristine Antarctic Lakes: Ecological and Biotechnological Implications.

The diversity and biotechnological potentialities of bacterial isolates from brines of three Antarctic lakes of the Northern Victoria Land (namely Boulder Clay and Tarn Flat areas) were first explored. Cultivable bacterial communities were analysed mainly in terms of bacterial response to contaminants (i.e., antibiotics and heavy metals) and oxidation of contaminants (i.e., aliphatic and aromatic hydrocarbons and polychlorobiphenyls). Moreover, the biosynthesis of biomolecules (antibiotics, extracellular polymeric substances and enzymes) with applications for human health and environmental protection was assayed. A total of 74 and 141 isolates were retrieved from Boulder Clay and Tarn Flat brines, respectively. Based on 16S rRNA gene sequence similarities, bacterial isolates represented three phyla, namely Proteobacteria (i.e., Gamma- and Alphaproteobacteria), Bacteroidetes and Actinobacteria, with differences encountered among brines. At genus level, Rhodobacter, Pseudomonas, Psychrobacter and Leifsonia members were dominant. Results obtained from this study on the physiological and enzymatic features of cold-adapted isolates from Antarctic lake brines provide interesting prospects for possible applications in the biotechnological field through future targeted surveys. Finally, findings on contaminant occurrence and bacterial response suggest that bacteria might be used as bioindicators for tracking human footprints in these remote polar areas.

Dissolved organic matter in the deep TALDICE ice core: A nano-UPLC-nano-ESI-HRMS method.

Trace organic compounds in deep ice cores supply important paleoclimatic information. Untargeted analyses of dissolved organic matter provide an overview of molecular species in ice samples however, sample volumes usually required for these analyses are generally not available from deep ice cores. Here, we developed an analytical method using a nano-UPLC-nano-ESI-HRMS to detect major molecular species in ice cores. Samples (4 µL) from the TALos Dome Ice CorE (TALDICE), allowed investigating molecular species across a range of depths including during glacial and interglacial periods. We detected 317 chemical species that were tentatively assigned to fatty acids, hydroxy fatty acids and their degradation products (oxo-fatty acids and dicarboxylic acids), as well as oxidation byproducts of isoprene and monoterpenes. These compounds indicate that the main sources of the organic fraction are microbes as well as primary and secondary aerosols. Interglacial samples encompass a wide range of species including compounds from the oxidation of isoprene and monoterpenes as well as unsaturated fatty acids, while the glacial samples contained less diverse species. This difference may be due to decreased temperatures during the glacial period inhibiting terrestrial vegetation growth and increasing the sea ice extent, thereby weakening the emission sources.

Biomass burning source identification through molecular markers in cryoconites over the Tibetan Plateau.

Cryoconite is a dark, dusty aggregate of mineral particles, organic matter, and microorganisms transported by wind and deposited on glacier surfaces. It can accelerate glacier melting and alter glacier mass balances by reducing the surface albedo of glaciers. Biomass burning in the Tibetan Plateau, especially in the glacier cryoconites, is poorly understood. Retene, levoglucosan, mannosan and galactosan can be generated by the local fires or transported from the biomass burning regions over long distances. In the present study, we analyzed these four molecular markers in cryoconites of seven glaciers from the northern to southern Tibetan Plateau. The highest levels of levoglucosan and retene were found in cryoconites of the Yulong Snow Mountain and Tienshan glaciers with 171.4 ±â€¯159.4 ng g-1 and 47.0 ±â€¯10.5 ng g-1 dry weight (d.w.), respectively. The Muztag glacier in the central Tibetan Plateau contained the lowest levels of levoglucosan and retene with mean values of 59.8 ng g-1 and 0.4 ±â€¯0.1 ng g-1 d.w., respectively. In addition, the vegetation changes and the ratios of levoglucosan to mannosan and retene indicate that combustion of conifers significantly contributes to biomass burning of the cryoconites in the Yulong Snow Mountain and Tienshan glacier. Conversely, biomass burning tracers in cryoconites of Dongkemadi, Yuzhufeng, Muztag, Qiyi and Laohugou glaciers are derived from the combustion of different types of biomass including softwood, hardwood and grass.

A thin ice layer segregates two distinct fungal communities in Antarctic brines from Tarn Flat (Northern Victoria Land).

Brines are hypersaline solutions which have been found within the Antarctic permafrost from the Tarn Flat area (Northern Victoria Land). Here, an investigation on the possible presence and diversity of fungal life within those peculiar ecosystems has been carried out for the first time. Brines samples were collected at 4- and 5-meter depths (TF1 and TF2, respectively), from two brines separated by a thin ice layer. The samples were analyzed via Illumina MiSeq targeting the ITS region specific for both yeasts and filamentous fungi. An unexpected high alpha diversity was found. Beta diversity analysis revealed that the two brines were inhabited by two phylogenetically diverse fungal communities (Unifrac value: 0.56, p value < 0.01; Martin's P-test p-value < 0.001) characterized by several specialist taxa. The most abundant fungal genera were Candida sp., Leucosporidium sp., Naganishia sp. and Sporobolomyces sp. in TF1, and Leucosporidium sp., Malassezia sp., Naganishia sp. and Sporobolomyces sp. in TF2. A few hypotheses on such differentiation have been done: i) the different chemical and physical composition of the brines; ii) the presence in situ of a thin layer of ice, acting as a physical barrier; and iii) the diverse geological origin of the brines.

An extracellular polymeric substance quickly chelates mercury(II) with N-heterocyclic groups.

A strain of Klebsiella oxytoca DSM 29614 is grown on sodium citrate in the presence of 50 mg l-1 of Hg as Hg(NO3)2. During growth, the strain produces an extracellular polymeric substance (EPS), constituted by a mixture of proteins and a specific exopolysaccharide. The protein components, derived from the outer membrane of cells, are co-extracted with the extracellular exopolysaccharide using ethanol. The extracted EPS contains 7.5% of Hg (total amount). This indicates that EPS is an excellent material for the biosorption of Hg2+, through chemical complexation with the EPS components. The binding capacity of these species towards Hg2+ is studied by cyclic voltammetry, and Hg L3-edge XANES and EXAFS spectroscopy. The results found indicate that Hg2+ is mainly bound to the nitrogen of the imidazole ring or other N-heterocycle compounds. The hydroxyl moities of sugars and/or the carboxyl groups of two glucuronic acids in the polysaccharide can also play an important role in sequestring Hg2+ ions. However, N-heterocyclic groups of proteins bind Hg2+ faster than hydroxyl and carboxyl groups of the polysaccharide.

A broad mercury resistant strain of Pseudomonas putida secretes pyoverdine under limited iron conditions and high mercury concentrations.

The Pseudomonas putida FB1, known as a broad-spectrum mercury resistant strain, becomes yellow-green due to the secretion of pyoverdine (PVDs) under limited iron conditions and high mercury concentrations. Different modified Nelson's media were obtained by adding mercury, iron, and the complexing agent nitrilotriacetic acid to demonstrate that the strain produces only the highest concentrations of PVDs due to the induction with 25 µM Hg2+. An amount of 250 mg PVDs was purified from the supernatant of 1 litre culture. The various forms of PVDs were characterized using different techniques such as fluorescence spectroscopy, high performance liquid chromatography coupled with high resolution mass spectrometry, and scanning electron microscope equipped with energy dispersive X-ray analyser. A set of "in vivo" experiments demonstrated that additions of Hg2+ to the cultures from 10 to 25 µM Hg2+ stimulate an over secretion of PVDs suggesting that the toxic cation strongly reduces the availability of apo-PVDs, because the complex mercuric-pyoverdine is very stable at neutral pH, and hinder the formation of PVDs-Fe(III).

Polysaccharide-based silver nanoparticles synthesized by Klebsiella oxytoca DSM 29614 cause DNA fragmentation in E. coli cells.

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO3 to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS(aer) and the AgNPs-EPS(anaer), were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS(aer), in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS(aer). In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS(aer) exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS(aer). The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag(+) release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.

Direct immersion solid-phase microextraction with gas chromatography and mass spectrometry for the determination of specific biomarkers of human sweat in melted snow.

To provide a reliable tool for investigating diffusion processes of the specific components of the human odor 3-hydroxy-3-methylhexanoic acid and 3-methyl-3-sulfanylhexan-1-ol through the snowpack, we developed and optimized an analytical method based on direct immersion solid-phase microextraction followed by gas chromatography with mass spectrometry. Direct immersion solid-phase microextraction was performed using polyacrylate fibers placed in aqueous solutions containing 3-hydroxy-3-methylhexanoic acid and 3-methyl-3-sulfanylhexan-1-ol. After optimization, absorption times of 120 min provided a good balance to shorten the analysis time and to obtain suitable amounts of extractable analytes. The extraction efficiency was improved by increasing the ionic strength of the solution. Although the absolute extraction efficiency ranged between 10 and 12% for 3-hydroxy-3-methylhexanoic acid and 2-3% for 3-methyl-3-sulfanylhexan-1-ol, this method was suitable for analyzing 3-hydroxy-3-methylhexanoic acid and 3-methyl-3-sulfanylhexan-1-ol concentrations of at least 0.04 and 0.20 ng/mL, respectively. The precision of the direct immersion solid-phase microextraction method ranged between 8 and 16%. The variability within a batch of six fibers was 10-18%. The accuracy of the method provided values of 88-95 and 86-101% for 3-hydroxy-3-methylhexanoic acid and 3-methyl-3-sulfanylhexan-1-ol, respectively. The limit of detection (and quantification) was 0.01 ng/mL (0.04 ng/mL) for 3-hydroxy-3-methylhexanoic acid and 0.06 ng/mL (0.20 ng/mL) for 3-methyl-3-sulfanylhexan-1-ol. The signal versus concentration was linear for both compounds (r(2) = 0.973-0.979). The stability of these two compounds showed that 3-hydroxy-3-methylhexanoic acid was more stable in water than 3-methyl-3-sulfanylhexan-1-ol. We applied the method to environmental samples in correspondence with an olfactory target buried previously.

GC-MS method for determining faecal sterols as biomarkers of human and pastoral animal presence in freshwater sediments.

In order to determine sterols and stanols in freshwater sediments to reconstruct the past presence of humans and pastoral animals, we developed an analytical method based on pressurised liquid extraction (PLE), clean-up performed using solid phase extraction (SPE) and sterol determination using gas chromatography-mass spectrometry (GC-MS) analysis. PLE extraction conditions were optimised using dichloromethane (DCM) and DCM/methanol mixtures. Clean-up was performed with 2 g silica SPE cartridges, and the concentrated extracts were eluted with 70 mL DCM. Extraction yield was evaluated using an in-house reference material spiked with (13)C-labelled cholesterol and aged for 10 days. In comparison with pre-extraction, where the sediment is extracted and then spiked with a known analyte concentration, this approach preserves the original composition of the sediment. DCM and DCM/methanol mixtures resulted in high extraction yields ranging from 86 to 92 % with good reproducibility (relative standard deviation (RSD) 5-8 %). PLE extraction yields obtained with DCM as the extracting solvent were about 1.5 times higher than extractions using an ultrasonic bath. The solvent extraction mixture and matrix composition strongly affected the solvent extraction composition where higher overall recoveries (70-80 %) for each compound were obtained with DCM. The extraction mixture and matrix composition also affected the analyte concentrations, resulting in a method precision ranging from 1 to 18 %. Diatomaceous earth spiked with 10 to 100 ng of sterols, and environmental samples fortified with suitable amounts of sterols provided apparent recovery values ranging from 90 to 110 %. We applied the method to environmental samples both close to and upstream from sewage discharge zones, resulting in substantially higher faecal sterol (FeSt) concentrations near the sewage. In addition, we also applied the method to a 37-cm freshwater sediment core in order to evaluate its applicability for obtaining vertical sterol profiles.

Characterisation of biosynthesised silver nanoparticles by scanning electrochemical microscopy (SECM) and voltammetry.

Silver nanoparticles (AgNPs) were biosynthesised by a Klebsiella oxytoca strain BAS-10, which, during its growth, is known to produce a branched exopolysaccharide (EPS). Klebsiella oxytoca cultures, treated with AgNO3 and grown under either aerobic or anaerobic conditions, produced silver nanoparticles embedded in EPS (AgNPs-EPS) containing different amounts of Ag(0) and Ag(I) forms. The average size of the AgNPs-EPS was determined by transmission electron microscopy, while the relative abundance of Ag(0)- or Ag(I)-containing AgNPs-EPS was established by scanning electrochemical microscopy (SECM). Moreover, the release of silver(I) species from the various types of AgNPs-EPS was investigated by combining SECM with anodic stripping voltammetry. These measurements allowed obtaining information on the kinetic of silver ions release from AgNPs-EPS and their concentration profiles at the substrate/water interface.

Yttrium and lanthanide complexes of ß-dialdehydes: synthesis, characterization, luminescence and electrochemistry of coordination compounds with the conjugate base of bromomalonaldehyde.

Novel yttrium, europium and terbium coordination compounds having formulae [AsPh4][Ln(BrMA)4] (6LN), Ln(BrMA)3(bipyO2) (7Ln), Ln(NMA)3(phen) (8Ln) and Ln(NMA)3(terpy) (9Ln) (Ln = Y, Eu, Tb; BrMA = conjugate base of bromomalonaldehyde; bipyO2 = 2,2'-bipyridine-N,N'-dioxide; phen = 1,10-phenantroline; terpy = 2,2':6',2''-terpyridine) were synthesized and characterized by using spectroscopic and electrochemical techniques. Uncharged europium and, to a lesser extent, terbium complexes showed appreciable luminescence in the solid state upon excitation with UV light. Polymeric materials and ionic liquids containing BrMA and lanthanides were prepared and photoluminescence measurements were carried out. From an electrochemical point of view, europium(III) BrMA-complexes showed a quasi-reversible one-electron reduction process. The one electron transfer reaction Eu(III) to Eu(II) allowed the photoluminescence tuning of 8Eu deposited on the surface of a glassy carbon electrode.

A rapid electrochemical procedure for the detection of Hg(0) produced by mercuric-reductase: application for monitoring Hg-resistant bacteria activity.

In this work, gold microelectrodes are employed as traps for the detection of volatilized metallic mercury produced by mercuric reductase (MerA) extracted from an Hg-resistant Pseudomonas putida strain FB1. The enzymatic reduction of Hg (II) to Hg (0) was induced by NADPH cofactor added to the samples. The amount of Hg(0) accumulated on the gold microelectrode surface was determined by anodic stripping voltammetry (ASV) after transferring the gold microelectrode in an aqueous solution containing 0.1 M HNO(3) + 1 M KNO(3). Electrochemical measurements were combined with spectrofluorometric assays of NADPH consumption to derive an analytical expression for the detection of a relative MerA activity of different samples with respect to that of P. putida. The method developed here was employed for the rapid determination of MerA produced by bacteria harbored in soft tissues of clams (Ruditapes philippinarum), collected in high Hg polluted sediments of Northern Adriatic Sea in Italy.

Electrochemical mapping reveals direct correlation between heterogeneous electron-transfer kinetics and local density of states in diamond electrodes.

Conducting carbon materials: a multi-microscopy approach shows that local heterogeneous electron-transfer rates at conducting diamond electrodes correlate with the local density of electronic states. This model of electroactivity is of considerable value for the rational design of conducting diamond electrochemical technologies, and also provides key general insights on electrode structure controls in electrochemical kinetics.

In situ scanning electrochemical microscopy (SECM) detection of metal dissolution during zinc corrosion by means of mercury sphere-cap microelectrode tips.

This work presents a scanning electrochemical microscopy (SECM)-based in situ corrosion probing methodology that is capable of monitoring the release of zinc species in corrosion processes. It is based on the use of Hg-coated Pt microelectrodes as SECM tips, which offer a wider negative potential range than bare platinum or other noble-metal tips. This allows for the reduction of zinc ions at the tip to be investigated with low interference from hydrogen evolution and oxygen reduction from aqueous solutions. The processes involved in the corrosion of zinc during its immersion in chloride-containing solutions were successfully monitored by scanning the SECM tip, set at an adequate potential, across the sample either in one direction or in the X-Y plane parallel to its surface. In this way, it was possible to detect the anodic and cathodic sites at which the dissolution of zinc and the reduction of oxygen occurred, respectively. Additionally, cyclic voltammetry (CV) or constant potential measurements were used to monitor the release of zinc species collected at the tip during an SECM scan.

Simple and clear evidence for positive feedback limitation by bipolar behavior during scanning electrochemical microscopy of unbiased conductors.

On the basis of an experimentally validated simple theoretical model, it is demonstrated unambiguously that when an unbiased conductor is probed by a scanning electrochemical tip (scanning electrochemical microscopy, SECM), it performs as a bipolar electrode. Though already envisioned in most recent SECM theories, this phenomenon is generally overlooked in SECM experimental investigations. However, as is shown here, this may alter significantly positive feedback measurements when the probed conductor is not much larger than the tip.

Effect of the insulating shield thickness on the steady-state diffusion-limiting current of sphere cap microelectrodes.

The effect of the insulating shield thickness on the steady-state diffusion-limiting current of sphere cap microelectrodes is investigated. Theoretical steady-state limiting currents are obtained by using a simulation procedure, which relies on the explicit finite difference method with a fixed time grid and an exponentially spatial grid. The results obtained indicate that the current increases by decreasing the thickness of the insulating sheath or by increasing the aspect ratio of the sphere cap (h/a, where h is the height of the sphere cap and a is the electrode basal radius), similarly to other types of microelectrodes with different electrode geometry, such as disks and finite cones. The simulated data are fitted to approximate analytical expressions to describe the dependence of the limiting current on both h/a and RG (RG=b/a, where b is the overall tip radius) parameter. Theoretical currents are also compared with experimental data, which are obtained with a range of mercury-coated platinum microelectrodes having different RG and h/a values. The measurements are performed by using cyclic voltammetry at 1 mVs(-1), in aqueous solutions containing Ru(NH3)6-Cl3 as electroactive species. A good agreement (within 3%) between theoretical and experimental steady-state currents is found. Finally, SECM operating in the feedback mode is used to assess the validity of the shape parameters found by voltammetry for sphere cap microelectrodes, whose insulating shields are of a thickness comparable to the electrode radius.