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During the first century BCE, a devastating fire destroyed a Roman villa on Elba Island (Tuscany, Italy) but 500 non-carbonized apple seeds were recovered from within four amphorae buried 1.5 m underground level. Most of the seeds had deteriorated, but 63 showed intact integument and embryo tissues suggesting they were unable to germinate. The seeds were coated in a clay-like organic layer baked into place during the fire covering a caramelized amorphous matrix generated by the transformation of sugars. The seeds inside this shield were protected from physicochemical and biological deterioration, even though fungal hyphae and bacteria were identified inside the embryos by DAPI staining. Some microbial strains were retrieved from the seed tissue, including the ascomycete Aspergillus awamori and bacteria of the genera Bacillus and Variovorax. In contrast, the protective layer around the seed yielded Enterobacteriaceae such as the genera Citrobacter and Salmonella, along with Pseudomonas and Streptomyces, and the fungi Thermothelomyces thermophila and Humicola fuscoatra, which are renowned for their resistance to heat. PCR-DGGE analysis showed no correspondence between 16S rRNA gene sequences isolated from the protective layer covering the seed and from within the seed tissues. Despite the presence of a diverse microbial population, the protective coat on the ancient seeds prevented the deterioration of about 20% of seeds, allowing a comparison to the present-day seeds of Malus sylvestris, the botanical species which the archaeological finds represents.

Aims: Preparation of the new metals-polymeric composite, Metx-EPS (I), to be used as a green catalyst in water or in two-phase aqueous conditions. Study Design: Recovery and valorization of polymetallic wastes to obtain directly new catalysts using a microorganism to explore their application in removal of difficult and dangerous chemical pollutants present in aqueous environment. Place and Duration of Study: Dipartimento di Scienze Molecolari e Nanosistemi, Università Ca’ Foscari Venezia, Venezia Mestre, Italy; University of Nova Gorica, Nova Gorica, Slovenia, Institut Jozef Stefan, Ljubljana, Slovenia and Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, between February 2018 and January 2019. Methodology: For the preparation of Metx-EPS (I), the metals source was an exhausted catalytic converter that was grinded and treated with an acidic solution at 20-25°C. After filtration, the solution was concentrated, neutralized and added to a broth of Klebsiella oxytoca DSM 29614 to produce (I) where metals are embedded in a peculiar polysaccharide structure. The composite was easily recovered from the fermentation broth and purified. The process protocol was verified many times and was shown to be reproducible satisfactorily. The % recovery of metals, originally present in the converter, was good as determined by atomic absorption. The morphology and the chemical state of main metals in (I) were investigated by X-ray absorption spectroscopy methods (XANES and EXAFS). No metallic alloy seems to be evident. Results: As first application of (I) as catalyst, the hydrodechlorination treatment of polychlorinated biphenyls (PCBs) was studied in water/methanol. A significant removal of higher chlorinated congeners was observed working at 1MPA of hydrogen and 60°C. This result improves significantly and surprisingly the methodology, previously studied by us using mono- or bi-metals embedded in the same polysaccharide moiety, indicating that positive synergies among the different metals were operating. Conclusion: The preparation of this new polymetallic species embedded in a polysaccharide moiety starting from spent catalytic converters represents an alternative valorisation of metallic wastes. Moreover, a synergic effect was exerted by the different metals when the catalyst Metx-EPS (I) was used in the hydrodechlorination treatment of polychlorinated biphenyls (PCBs) in water/methanol. Finally, a promising preliminary proof of concept for the removal of polychlorinated aromatic pollutants even in contaminated aqueous sites was carried out.

Truffles are highly valuable ectomycorrhizal fungi that grow naturally in alkaline, calcareous soils. Iron deficiency chlorosis is a common problem in truffle (Tuber spp.) cultivation due to the high quantity of lime added to increase the pH of acidic soils. In this work, the effects of ferric hydroxide nanoparticles embedded in an exopolysaccharide (Fe-EPS NPs), extracted from cultures of Klebsiella oxytoca DSM 29614, were investigated on Quercus robur seedlings under greenhouse conditions. The plants were inoculated with Tuber borchii (the bianchetto truffle) and were cultivated with and without iron nanoparticle additions and compared with non-inoculated control plants. The seedlings were grown in limed soil in order to induce iron deficiency. Low doses of Fe-EPS NPs had a beneficial effect on the growth of the plants inoculated with T. borchii, increasing their height and reducing their leaf chlorosis 5 months after the first Fe-EPS NP treatment. Moreover, Fe-EPS NP treatments significantly increased the level of T. borchii mycorrhizal colonization and the ectomycorrhizal mantle thickness. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) applied to cross sections of mycorrhizas showed that Fe accumulated in the fungal mantle and apparently was slowly released serving as a resilient reservoir of iron for the plant. The results suggest that the application of Fe-EPS NPs is a promising technique in the production of Tuber mycorrhized plants in the nursery and could have future applications in the field.

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.

The PCBs are known recalcitrant and toxic pollutants and significant values of contamination could be found in water. PCBs can be hydrodechlorinated using Pd-based catalysts and hydrogen, but the research to identify more efficient heterogeneous catalysts, able to work in an aqueous phase, less sensible to deactivation and easily removable at the end of the treatment, remains a considerable interesting goal. A strain of Klebsiella oxytoca, DSM 29614, known to produce a specific exopolysaccaride (EPS), was grown in different media with sodium citrate or with sodium citrate plus ferric citrate, as sole energy and carbon sources under anaerobic conditions. The cultures were amended with 50 mg of palladium as Pd(NO3)2 to generate Pd-EPS (Pd content 13%) or FePd-EPS (Pd content 8.4%; Fe 7.4%) species which were secreted from the cells, isolated by treatment with a cold ethanol solution (70%) and dried under vacuum as powders. The catalytic ability of these mono- and bi-metallic species was tested in the hydrodechlorination reaction of the Aroclor 1260 PCBs mixture under aqueous biphasic conditions. Here we demonstrate that the degree of PCBs hydrodechlorination is dependent by the nature of catalyst and of base used to neutralize HCl produced, the bimetallic species being more active and an organic base resulting more effective. Working with a substrate/catalyst 8/1 molar ratio, at 3 MPa H2 and 60°C in 20 h a significant removal of highly chl orinated PCBs was obtained under the best conditions. The result seems promising for remediation of groundwater contaminated with PCBs.

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPSaer) and anaerobic conditions (AgNPs-EPSanaer). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPSaer as the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPSaer mode of action. In particular, AgNPs-EPSaer induced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag+ accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPSaer toxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation.

Background Klebsiella oxytoca DSM 29614 - isolated from acid mine drainages - grows anaerobically using Fe(III)-citrate as sole carbon and energy source, unlike other enterobacteria and K. oxytoca clinical isolates. The DSM 29614 strain is multi metal resistant and produces metal nanoparticles that are embedded in its very peculiar capsular exopolysaccharide. These metal nanoparticles were effective as antimicrobial and anticancer compounds, chemical catalysts and nano-fertilizers. Results The DSM 29614 strain genome was sequenced and analysed by a combination of in silico procedures. Comparative genomics, performed between 85 K. oxytoca representatives and K. oxytoca DSM 29614, revealed that this bacterial group has an open pangenome, characterized by a very small core genome (1009 genes, about 2%), a high fraction of unique (43,808 genes, about 87%) and accessory genes (5559 genes, about 11%). Proteins belonging to COG categories “Carbohydrate transport and metabolism” (G), “Amino acid transport and metabolism” (E), “Coenzyme transport and metabolism” (H), “Inorganic ion transport and metabolism” (P), and “membrane biogenesis-related proteins” (M) are particularly abundant in the predicted proteome of DSM 29614 strain. The results of a protein functional enrichment analysis - based on a previous proteomic analysis – revealed metabolic optimization during Fe(III)-citrate anaerobic utilization. In this growth condition, the observed high levels of Fe(II) may be due to different flavin metal reductases and siderophores as inferred form genome analysis. The presence of genes responsible for the synthesis of exopolysaccharide and for the tolerance to heavy metals was highlighted too. The inferred genomic insights were confirmed by a set of phenotypic tests showing specific metabolic capability in terms of i) Fe2+ and exopolysaccharide production and ii) phosphatase activity involved in precipitation of metal ion-phosphate salts. Conclusion The K. oxytoca DSM 29614 unique capabilities of using Fe(III)-citrate as sole carbon and energy source in anaerobiosis and tolerating diverse metals coincides with the presence at the genomic level of specific genes that can support i) energy metabolism optimization, ii) cell protection by the biosynthesis of a peculiar exopolysaccharide armour entrapping metal ions and iii) general and metal-specific detoxifying activities by different proteins and metabolites. Electronic supplementary material The online version of this article (10.1186/s12866-018-1330-5) contains supplementary material, which is available to authorized users.

During primary colonization of rock substrates by plants, mineral weathering is strongly accelerated under plant roots, but little is known on how it affects soil ecosystem development before plant establishment. Here we show that rock mineral weathering mediated by chemolithoautotrophic bacteria is associated to plant community formation in sites recently released by permanent glacier ice cover in the Midtre Lovénbreen glacier moraine (78 degrees 53'N), Svalbard. Increased soil fertility fosters growth of prokaryotes and plants at the boundary between sites of intense bacterial mediated chemolithotrophic iron-sulfur oxidation and pH decrease, and the common moraine substrate where carbon and nitrogen are fixed by cyanobacteria. Microbial iron oxidizing activity determines acidity and corresponding fertility gradients, where water retention, cation exchange capacity and nutrient availability are increased. This fertilization is enabled by abundant mineral nutrients and reduced forms of iron and sulfur in pyrite minerals within a conglomerate type of moraine rock. Such an interaction between microorganisms and moraine minerals determines a peculiar, not yet described model for soil genesis and plant ecosystem formation with potential past and present analogues in other harsh environments with similar geochemical settings.

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii (“bianchetto” truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This “green” method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.

Three red seaweeds from the Venice Lagoon

In the last decades, the presence of high As levels in groundwaters poses a serious limitation to the use of this resources for drinking purposes in several parts of the world. Treatment of As-rich waters selected iron oxides filters as more effective, low cost and selective technology. Green and biologically-driven pathways to synthetize new nanostructured iron oxy-hydroxides are becoming always more attractive. We tested the suitability of FeOOH nanoparticles (9–15 nm) produced by Klebsiella oxytoca strain DSM 29614 and encapsulated in EPS gel structure to treat arsenic rich water. Different gel:water volume ratios were tested to treat 5000 μg As/L solution. 20% FeEPS solution was able to remove 95% of As(V) while in 5% solution removal was reduced to 60%. Arsenic adsorption was very fast and follows pseudo-2nd order kinetic with maximum adsorption capacity reached at about 30 min. Adsorption followed Langmuir model for As(V) with qmax = 31.8 mgAs/gFe and BET for As(III) with 8 mgAs/gFe for the first layer in 10% FeEPS solution. FeEPS dried into powder showed noticeable removal only after 2 h, hence not suitable for drinking water treatment. Treatment of natural As levels in mimicked groundwaters showed 87–95% As(V) and 45–61% As(III) removal after 5 min. FeEPS gel immobilized onto bivalve shell debris was used in packed-bed filters. It retained 49.8 mgAs/gFe from 150 μg/L As(V) spiked groundwater before reaching breakthrough at 8000 BVs. Biologically produced FeEPS gel showed good potentialities as eco-friendly material to remove As from contaminated groundwater.

a b s t r a c t A palladium species bound to an exopolysaccharide (Pd-EPS) was obtained from alive bacterial cells of Klebsiella oxytoca BAS-10 grown in static mode in the presence of Pd(NO3)2. Pd-EPS, after isolation and purification, was used as catalyst in the aqueous biphasic hydrogenation either of unfunctionalyzed olefins, as styrene, 1-octene and 1,3-diisopropenylbenzene, or of some ,-unsaturated aldehydes. The catalytic system was very active under mild reaction conditions and its activity was maintained in some recycle experiments. Even more efficient was the "activated Pd-EPS", obtained by a pre-treatment of Pd- EPS with 1 MPa of H2 at 30 ◦ C for 21 h; while Pd-EPS originally contains only Pd(II), as demonstrated by XPS measurements, the activated catalyst shows the presence of both Pd(II) and Pd(0) in the ratio 1.9/1. The two catalytic systems show different structures at TEM observations evidencing the transformation of electron ultradense nanoaggregates (Pd-EPS) into jagged microaggregates ("activated Pd-EPS"). © 2012 Elsevier B.V. All rights reserved.

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.

A closed chlor-alkali plant (CAP) discharged Hg for decades into the Aussa River, which flows into Marano Lagoon, resulting in the large-scale pollution of the lagoon. In order to get information on the role of bacteria as detoxifying agents, analyses of anions togetherwith the determination of total Hg (Hg-T) and methylmercury (MeHg) in the superficial part (0-1 cm) of sediments were conducted at four stations in the Aussa River. In addition,measurements of biopolymeric carbon (BPC) as a sum of the carbon equivalent of proteins (PRT), lipids (LIP), and carbohydrates (CHO) were performed to correlate with bacterial biomass such as the number of aerobic heterotrophic cultivable bacteria and their percentage of Hg-resistant bacteria. All these parameters were used to assess the bioavailable Hg fraction in sediments and the potential detoxification activity of bacteria. In addition, fifteen isolates were characterized by a combination of molecular techniques, which permitted their assignment into six different genera. Four out of fifteen were Gram negative with two strains of Stenotrophomonas maltophilia, one Enterobacter sp., and one strain of Brevibacterium frigoritolerans. The remaining strains (11) were Gram positive belonging to the genera Bacillus and Staphylococcus. We found merA genes in only a few isolates. Mercury volatilization from added HgCl2 and the presence of plasmids with the merA gene were also used to confirm Hg reductase activity. We found the highest number of aerobic heterotrophic Hg-resistant bacteria (one order magnitude higher) and the highest number of Hg-resistant species (11 species out of 15) at the confluence of the River Aussa and Banduzzi’s channel, which transport Hg from the CAP, suggesting that Hg is strongly detoxified [reduced to Hg(0)] at this location.

In arctic glacier moraines, bioweathering primed by microbial iron oxidizers creates fertility gradients that accelerate soil development and plant establishment. With the aim of investigating the change of bacterial diversity in a pyrite-weathered gradient, we analyzed the composition of the bacterial communities involved in the process by sequencing 16S rRNA gene libraries from different biological soil crusts (BSC). Bacterial communities in three BSC of different morphology, located within 1 m distance downstream a pyritic conglomerate rock, were significantly diverse. The glacier moraine surrounding the weathered site showed wide phylogenetic diversity and high evenness with 15 represented bacterial classes, dominated by Alphaproteobacteria and pioneer Cyanobacteria colonizers. The bioweathered area showed the lowest diversity indexes and only nine bacterial families, largely dominated by Acidobacteriaceae and Acetobacteraceae typical of acidic environments, in accordance with the low pH of the BSC. In the weathered BSC, iron-oxidizing bacteria were cultivated, with counts decreasing along with the increase of distance from the rock, and nutrient release from the rock was revealed by environmental scanning electron microscopy-energy dispersive X-ray analyses. The vegetated area showed the presence of Actinomycetales, Verrucomicrobiales, Gemmatimonadales, Burkholderiales, and Rhizobiales, denoting a bacterial community typical of developed soils and indicating that the lithoid substrate of the bare moraine was here subjected to an accelerated colonization, driven by iron-oxidizing activity.

Shallow marine sediments were collected from seven stations (three of which located at Gerlache Inlet, two at Tethys Bay, one at Adelie Cove and one just beneath the Italian Research Base) along the Terra Nova Bay coast (Ross Sea, Antarctica). Their chemical, biochemical and microbiological properties were studied in order to provide further insights in the knowledge of this Antarctic benthic ecosystem. Overall, the organic carbon (OC) represented the major fraction of total carbon (TC) and displayed concentrations similar to or slightly lower than those previously measured in Antarctic bottom sediments. The biopolymeric carbon within OC ranged from 4.1% to 19.9% and showed a wide trophic range (65–834 μg g −1 d.w.). Proteins (PRT) represented on average the main biochemical class contributing to labile organic carbon, followed by lipids (LIP) and carbohydrates (CHO). The activity of aminopeptidase, β - d -glucosidase, alkaline phosphatase and esterase was checked, giving the highest values at Tethys Bay and at the deepest water sediments. The principal component analysis, which was computed considering physical, chemical (elemental and biochemical sedimentary composition) and microbiological parameters (including bacterial abundance, ectoenzymatic activities, T-RFs richness and diversity indices), allowed to obtain two main clusters (“Tethys Bay” and “other stations”). Based on data obtained, two representative 16S rRNA clone libraries using samples from Tethys Bay and Gerlache Inlet were constructed. The sequences of 171 clones were compared to those available in public databases to determine their approximate phylogenetic affiliations. Both aerobic and anaerobic bacteria were disclosed, with the majority of them affiliated with the Gamma - and Deltaproteobacteria , Bacteroidetes and Acidobacteria . The occurrence of strictly anaerobic bacteria suggests that sediments might also undergo anoxic conditions that, in turn, could favor the accumulation of PRT in respect to CHO, assuming that fermentation of amino acids is slower than that of sugars from decomposing polysaccharides.

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.

A strain of Klebsiella oxytoca BAS-10, known to produce a specific exopolysaccharide (EPS), when grown aerobically in static mode in the presence of Pd(NO3)2, generated the species Pd-EPS that was used as catalyst precursor in the aqueous biphasic treatment of some nitrocompounds with hydrogen. Nitrobenzene was hydrogenated to aniline with almost quantitative yields and the catalyst, embedded in the aqueous phase, was used with success and with near the same efficiency in three recycling experiments. In the case of 1-iodo-4-nitrobenzene only nitrobenzene was obtained while the unsaturated nitro compound β-methyl-β-nitrostyrene afforded both the corresponding oxime and the saturated nitro derivative.

Klebsiella oxytocaBAS-10 ferments citrate to acetic acid and CO2, and secretes a specific exopolysaccharide (EPS), which is able to bind different metallic species. These biomaterials may be used for different biotechnological purposes, including applications as innovative green biogenerated catalysts. In production of biogenerated Pd species, the Fe(III) as ferric citrate is added to anaerobic culture ofK. oxytocaBAS-10, in the presence of palladium species, to increase the EPS secretion and improve Pd-EPS yield. In this process, bi-metallic (FePd-EPS) biomaterials were produced for the first time. The morphology of bi-metallic EPS, and the chemical state of the two metals in the FePd-EPS, are investigated by transmission electron microscopy, Fourier transform infra-red spectroscopy, micro-X-ray fluorescence, and X-ray absorption spectroscopy methods (XANES and EXAFS), and compared with mono-metallic Pd-EPS and Fe-EPS complexes. Iron in FePd-EPS is in the mineralized form of iron oxides/hydroxides, predominantly in the form of Fe3+, with a small amount of Fe2+in the structure, most probably a mixture of different nano-crystalline iron oxides and hydroxides, as in mono-metallic Fe-EPS. Palladium is found as Pd(0) in the form of metallic nanoparticles with face-centred cubic structure in both bi-metallic (FePd-EPS) and mono-metallic (Pd-EPS) species. In bi-metallic species, Pd and Fe nanoparticles agglomerate in larger clusters, but they remain spatially separated. The catalytic ability of bi-metallic species (FePd-EPS) in a hydrodechlorination reaction is improved in comparison with mono-metallic Pd-EPS.

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk− mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.

Three areas of the 18th century fresco by Sebastiano Conca in the apse of the church (Santissima Annunziata, Siena, Italy) showed signs of deterioration. Twelve small fragments with a total weight of about 15 mg were detached from each of three damaged areas of the fresco and analysed. Element composition of specimens was determined by scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX). Some fragments were incubated in mineral medium without organic carbon. Samples from the lower part of the fresco (area A) produced colonies of heterotrophic aerobic bacteria on the carbon available in this medium. Fragments from two other two areas (areas B and C) did not produce any microorganisms. Two strains were isolated from area A and identified as Kocuria erythromyxa strain CV1 and Sphingomonas echinoides strain CV2 by sequencing gene 16S rDNA. When fresco fragments were soaked in mineral medium without carbon source, Sphigomonas echinoides grew rapidly. This strain produced a visible biofilm on the fresco fragments. The mucilage consisted mainly of extracellular polymeric substances (EPS) detected in situ by confocal scanning laser microscopy (CSLM) with Con-A fluorescent lectin and confirmed by transmission electron microscopy (TEM) observations. Ultrastructural analysis of sections of the microorganisms by TEM showed that only strain CV2 adhered to the substrate and caused biological deterioration of the fresco.

A medieval fresco at the Chapel of the Holy nail, Siena was analysed to determine microbiological causes of deterioration of the painted surface or fresco micro-layer. Fragments of fresco a few mm wide were sampled under sterile conditions and some were incubated in mineral medium to isolate microbial species growing on endogenous carbon sources. Quiescent spores of fungi were detected. After 18 h fungal hyphae grew from turgid spores and pH dropped from 5.5 to 5.0, suggesting organic acid production during growth. Light microscopy (LM) and transmission electron microscopy (TEM) were used to study the ultrastructure of the fungi. Molecular studies involving sequencing 18S rDNA led to identification of Penicillium crysogenum. Element concentrations in the painted layer were analysed by scanning electron microscopy (SEM) with energy dispersive X-ray microanalysis of the fresco micro-layer before and after incubation of fragments. The main finding was that total carbon decreased from 26% to 13% while copper increased from 5% to 28%. The high concentration of copper and the acid pH inhibiting bacterial proliferation, indicated P. crysogenum as the main agent of deterioration. Hypotheses are formulated with regard to the high concentrations of copper useful for conservation of fresco substrates.

Strains DBVPG 6662 and DBVPG 6739 of Rhodosporidium toruloides , a basidiomycete yeast, grew on thiosulfate as a sulfur source and glucose (2 g liter −1 or 10.75 mM) as a carbon source. DBVPG 6662 has a defective sulfate transport system, whereas DBVPG 6739 barely grew on sulfate. They were compared for the ability to use dibenzothiophene (DBT) and related organic sulfur compounds as sulfur sources. In the presence of glucose as a carbon source and DBT as a sulfur source, strain DBVPG 6662 grew better than DBVPG 6739. In the presence of thiosulfate as a sulfur source, the two yeast strains did not use DBT, DBT-sulfone, benzenesulfonic acid, biphenyl, and fluorene. When the two strains were grown in the presence of glucose, strain DBVPG 6662 transformed 27% of the DBT present (10 μM) at a rate of 0.023 μmol liter −1 h −1 in 36 h. Traces of 2,2′-dihydroxylated biphenyl were transiently accumulated under these conditions. When the same strain was grown on glucose in the presence of a higher concentration of DBT (0.5 g liter −1 ), mainly in an insoluble form, the whole surface of the DBT crystals was colonized by a thick mycelium. This adherent structure was imaged by confocal microscopy with fluorescent concanavalin A, a lectin that specifically binds glucose and mannose residues. When DBVPG 6662 was grown on glucose in the presence of a commercial emulsion of bitumen, i.e., orimulsion, 68% of the benzo- and dibenzothiophenes and DBTs was removed after 15 days of incubation. The fungus adhered by hyphae to orimulsion droplets. When cultivated in the presence of commercial emulsifier-free fuel oil containing alkylated benzothiophenes and DBTs and having a composition similar to that of orimulsion, strain DBVPG 6662 removed only 11% of the total organic sulfur that occurs in the medium and did not adhere to the oil droplets. These results indicate that strain DBVPG 6662 is able to utilize the organic sulfur of DBT and a large variety of thiophenic compounds that occur extensively in commercial fuel oils by physically adhering to the organic sulfur source.

The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C12-C28) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (μm2) per aggregate was found. Each cell presented an average surface of 1.21 μm2. Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred.

Objective: The aim of this study was to quantify the efficacy of three different surgical techniques for entropion repair in a 3-year follow-up study: (1) the Fox procedure, (2) everting sutures and (3) a modified technique of lower lid retractor plication. Methods: We included in our study 32 eyelids of 32 consecutive patients with involutional lower lid entropion; 10 lids underwent the Fox procedure, 13 the everting sutures and 9 the modified retractor plication. We evaluated the pre- and postoperative horizontal lid laxity (HLL), the pre- and postoperative lower lid excursion (LLE) and the number of recurrences in each patient group. Statistical analysis was performed with the non-parametric Mann-Whitney test and the exact Fisher’s test where appropriate. Results: Only the modified retractor plication technique showed statistically significant differences in HLL and LLE (p < 0.05). In the group treated with this technique, there was a lower incidence of entropion recurrence (p < 0.05). The everting suture technique showed a statistically significant improvement of the LLE only (p < 0.05). The Fox procedure did not show any statistically significant improvement of the studied parameters. Conclusions: The modified retractor plication technique gave the best results in terms of improvement of HLL and LLE and a lower incidence of entropion recurrence compared to the two other techniques studied.

The role of sulfur species in the formation and stability of mucus aggregates in the Adriatic Sea was studied. Natural aggregates were compared with a mixed diatom culture. A sample from the sea bottom collected from the northern Adriatic Sea was cultivated and transferred to f/2 medium to isolate benthic diatoms. Diatom cultures were incubated at room temperature and irradiated with dim light (30 to 40 mmol m-2 s-1). Subcultured samples were incubated at room temperature under anaerobic conditions for up to 1 mo, with different concentrations of sulfide (10-4 and 10-3 mol l-1). In diatom cultures, interactions between sulfide and organic matter were investigated and followed for 10 d through: (1) formation of organosulfur species ; (2) changes in surfactant activity and (3) degradation of pennate diatoms followed by cell lysis and release of large amount of polysaccharides. A large increase in dissolved organic carbon (DOC) concentration was recorded in the ambient water of the sulfide-treated samples. A DOC concentration of 7.17 mg l-1 was determined in the ambient water of untreated diatom cultures increasing to 40 mg l-1 in sulfide-treated samples. Simultaneously, in the same sulfide-treated samples (both ambient water and aggregates of enriched diatom cultures), surfactant activity was observed to increase by 2 orders of magnitude. The concentration of surface-active substances (SAS, expressed as Triton-X-100 conc.) increased from 0.35 mg l-1 in the aggregate and 0.59 mg l-1 in the ambient water of the untreated diatom cultures to 20 mg l-1 in sulfide-incubated samples. Epifluorescence microscopy observations showed that diatoms decomposed under sulfide treatment and released a large amount of polysaccharides, as revealed by a specific molecular probe for glucose and mannose, i.e. Con-A lectin conjugated by fluorescein. These comparative experiments indicated that one of the factors triggering mucilage formation in the Adriatic Sea could be massive diatom lysis in bottom or anoxic microzones where, due to microbial activity, reduced sulfur species are prominent and have an important role in the formation and stability of mucilage development.

Three different strains of filamentous cyanobacteria, Tychonema, Limnothrix, and Pseudoanabaena, were selected among the fastest growing taxa collected in the salt marshes of Venice Lagoon and were grown in laboratory for growth rate determination and biochemical characterization of chlorophyll-a, total proteins, total carbohydrates, and exopolysaccharides. Experiments were carried out both in liquid medium and two different substrates: artificial plant protection fabric and ground indigenous shells. Cyanobacterial behavior was recorded to better understand colonization of natural and new artificial marshes.

An iron-poor stream of nearly neutral pH polluted by mine tailings has been investigated for a natural phenomenon responsible for the polishing of heavy metals in mine wastewaters. A white mineralized mat, which was determined to be hydrozincite [Zn5(CO3)2(OH)6] by X-ray diffraction analysis, was observed in the stream sediments mainly in spring. The precipitate shows a total organic matter residue of 10% dry weight and contains high concentrations of Pb, Cd, Ni, Cu, and other metals. Scanning electron microscopy analysis suggests that hydrozincite is mainly of biological origin. Dormant photosynthetic microorganisms have been retrieved from 1-year-old dry hydrozincite. The autofluorescent microorganisms were imaged by a scanning confocal laser microscope. A photosynthetic filamentous bacterium, classified as Scytonema sp. strain ING-1, was found associated with microalga Chlorella sp. strain SA1. This microbial community is responsible for the natural polishing of heavy metals in the water stream by coprecipitation with hydrozincite. Abandoned mines present a high environmental hazard in all countries today. In several parts of the world where mining activities have shut down, the problem of the control and reclamation of polluted areas for new activities arises. The polishing of metals from a mining area is a difficult task. The transformation of metals into harmless species or their removal in a suitable recycled mineral form such as carbonates (1, 15) is a possible solution for the remediation of a mining area. Therefore, research in this field continues, with the isolation of new strains with more-successful mechanisms for the reduction of metal toxicity. At Ingurtosu (southwestern Sardinia, Italy) lead and zinc sulfide ore deposits were mined until 1968 and tailings were deposited along the Rio Naracauli creek. The MontevecchioIngurtosu deposit consists of galena-sphalerite veins in a quartz gangue containing iron, calcium, and magnesium carbonate minerals. Pyrite, chalcopyrite, barite, cerussite, and anglesite are the most commonly associated minerals (22). Previous studies in this area have shown that waters are highly polluted by heavy metals, in spite of their near-neutral pH (4‐6, 30).

The adhesion of a recently described species, Acinetobacter venetianus VE-C3 (F. Di Cello, M. Pepi, F. Baldi, and R. Fani, Res. Microbiol. 148:237–249, 1997), to diesel fuel (a mixture of C 12 to C 28 n -alkanes) and n -hexadecane was studied and compared to that of Acinetobacter sp. strain RAG-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. Oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains. The dropping-mercury electrode (DME) was used as an in situ adhesion sensor. In seawater, RAG-1 was hydrophobic, with an electrophoretic mobility (μ) of −0.38 × 10 −8 m 2 V −1 s −1 and zeta potential (ζ) of −4.9 mV, while VE-C3 was hydrophilic, with μ of −0.81 × 10 −8 m 2 V −1 s −1 and ζ of −10.5 mV. The microbial adhesion to hydrocarbon (MATH) test showed that RAG-1 was always hydrophobic whereas the hydrophilic VE-C3 strain became hydrophobic only after exposure to n -alkanes. Adhesion of VE-C3 cells to diesel fuel was partly due to the production of capsular polysaccharides (CPS), which were stained with the lectin concanavalin A (ConA) conjugated to fluorescein isothiocyanate and observed in situ by confocal microscopy. The emulsan from RAG-1, which was negative to ConA, was stained with Nile Red fluorochrome instead. Confocal microscope observations at different times showed that VE-C3 underwent two types of adhesion: (i) cell-to-cell interactions, preceding the cell adhesion to the n -alkane, and (ii) incorporation of nanodroplets of n -alkane into the hydrophilic CPS to form a more hydrophobic polysaccharide– n -alkane matrix surrounding the cell wall. The incorporation of n -alkanes as nanodroplets into the CPS of VE-C3 cells might ensure the partitioning of the bulk apolar phase between the aqueous medium and the outer cell membrane and thus sustain a continuous growth rate over a prolonged period.

Summary Twenty-five bacterial strains isolated from the Venice lagoon and implicated in the degradation of n-alkanes, n-alkanols, n-alkanals and n-alkanoates were characterized in molecular and physiological terms. The isolates were grouped by amplified ribosomal DNA restriction analysis (ARDRA) into seven clusters, corresponding to seven species, six of which were identified on the basis of 16S rDNA sequencing. Genetic variability among strains was shown by random amplified polymorphic DNA (RAPD). Only strains of the new species Acinetobacter venetianus grew with n-alkanes (C10, C14 and C20) and their respective oxidation products as sole carbon sources. Strains of the other three species identified thrived on n-alkane oxidation products (n-alkanols, n-alkanals, n-alkanoates). The other three species were not able to grow on any of the substrates tested. Analysis of plasmid content showed that only A. venetianus strains harboured plasmids. These plasmids contained sequences homologous to the Pseudomonas oleovorans alkBFGH genes.

Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids). The strain SC-1 was resistant up to 16 mm of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mm and 4 mm vanadate, respectively. The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy. The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mm of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg cells dry weight) was found in the biomass. The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.

Mercury chloride toxicity was investigated in two strains of Chlorella and in a strain of Scenedesmus isolated from polluted areas in Tuscany (Italy). No Hg resistance was found in the autotrophic microorganisms isolated, but Scenedesmus sp. strain AR-2489, isolated from the Arno river, was able to grow at concentrations of up to 5 micrograms ml-1 of Hg. This concentration was twice that which inhibited growth of the two Chlorella strains and Scenedesmus acutus 8M, the reference strain from a culture collection. Photosynthesizing cells of Scenedesmus sp. AR-2489 showed reduced Hg uptake, with the highest percentage of Hg removal from the medium. Loss of Hg was not due to Hg(0) volatilization, as shown by a comparison test with the broad-spectrum Hg-resistant Pseudomonas putida FB1. The metabolic differences between Scenedesmus sp. strain AR-2489 and Siacatus strain 8M were: (1) higher growth rate (doubling time of 6.0 h versus 10.6 h); (2) higher O2 production rate (maximum 2 mumol h-1 mg-1 dry weight); and (3) higher intracellular pH during growth. The latter was imaged with a green fluorescence molecular probe (BCEFC-AM) and observed by scanning confocal laser microscopy (SCLM). The distribution of red-autofluorescence chlorophyll-a showed that strain AR-2489 had a rougher and hence more extended specific chloroplast surface than strain 8M. Hg tolerance in strain AR-2489 was related to the rapid increase in dissolved O2 in the medium and in intracellular pH; this caused a loss of soluble mercury transformed to insoluble mercury hydroxide, which is thermodynamically more stable at alkaline pH in highly oxygenated systems.

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10μg/ml Hg (as HgCl2) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10μg/ml HgCl2 was diagnostic for Hg detoxification based on reduction.

The results are presented of an interlaboratory study on methylmercury (MeHg) in sediment carried out by a group of European laboratories within the framework of a project managed by the EC Standards, Measurements and Testing Programme (formerly BCR). The aim of this exercise was to evaluate the performance of current methods used for MeHg determination in sediment in order to improve the state-of-the-art prior to the certification of a candidate reference material. The paper describes the organization of the interlaboratory study, the preparation of the sediment material used, the techniques evaluated and the results obtained by the participating laboratories. The outcome of the collaborative project showed that certification could be contemplated, providing that certain analytical techniques were optimized, especially with regard to extraction methods.

High concentrations of total barium, ranging from 0.42 to 1.58 mg(middot)g(sup-1) (dry weight) were found in sludges of two sewage treatment plants near Florence, Italy. Barium concentrations in the suspended matter decreased as redox potential values changed from negative to positive. An anoxic sewage sludge sample was aerated, and 30% of the total barium was removed in 24 h. To demonstrate that barium was solubilized from barite by sulfate-reducing bacteria, a strain of Desulfovibrio desulfuricans was used to study the solubilization of barium from barite under laboratory conditions. During cell growth with different concentrations of barite from 0.01 to 0.3 g(middot)liter(sup-1) (the latter is the MIC) as the only source of sulfates in the cultures, the D. desulfuricans strain accumulated barium up to 0.58 (mu)g(middot)mg(sup-1) (dry weight). Three times the quantity of barium was dissolved by bacteria than in the uninoculated medium (control). The unexpectedly low concentration of soluble barium (1.2 mg of Ba(middot)liter(sup-1)) with respect to the quantity expected (109 mg of Ba(middot)liter(sup-1)), calculated on the basis of the free H(inf2)S evolved from the dissimilatory reduction of sulfate from barite, was probably due to the formation of other barium compounds, such as witherite (BaCO(inf3)) and the transient species barium sulfide (BaS). The D. desulfuricans strain, growing on barite, formed visible aggregates. Confocal microscopy analysis showed that aggregates consisted of bacteria and barite. After 3 days of incubation, several autofluorescent crystals surrounded by a dissolution halo were observed. The crystals were identified as BaS by comparison with the commercial compound.

The terrestrial biogeochemical cycle of mercury between humus and fungi was investigated in a mercury-contaminated former mining area of Germany. We used a variable volume extraction procedure and aqueous sodium boron tetraethyl derivatization to determine accurately and precisely methylmercury bioconcentration factors for fungi. The methylmercury and total mercury content for fungi carpophores ranged from 0.08 to 7.94 μg/g of Hg as methylmercury and from 6.2 to 144.0 μg/g of inorganic Hg, respectively. The levels in humus material were between 0.01 and 0.09 μg/g of Hg as methylmercury and 15.8 and 140.2 μg/g of inorganic Hg, respectively. The bioconcentration factors obtained are generally lower than 1 for total mercury and between 3.0 and 199.0 for methylmercury. By an in vitro methylation experiment with two axenic cultures of Coprinus comatus and Coprinus radians, we could show for the first time that these saprophytic macromycetes are able to methyl ate mercury.

The bioindex percentage of element-resistant microorganisms is suggested to study airborne pollution near geothermal plants. A new method of determining the percentage of element-resistant bacteria is described. The index was calculated as the percentage ratio of the metal-resistant colony-forming units grown on agar plates to the whole cultivable eterotrophic colony forming units. Element resistance was regarded as the ability of bacteria to grow on media amended with high concentrations of toxic compounds: HgCl2 (10 μg/ml), NaAsO2 (750 μg/ml) and H3BO3 (1000 μg/ml). Available elements induced activity of the element-resistance in epiphytic bacteria. The mosses from which the bacteria were isolated were collected in winter from two different areas near Siena: ten sites in the geothermal area around Radicondoli and five sites in an unpolluted part of the Chianti region. Percentages of element-resistant bacteria were found to be directly correlated with concentrations of acid-leachable mercury, arsenic and boron. The percentages of mercury- and arsenic-resistant bacteria were significantly higher in the geothermal area, especially in the GEO-6 station. High concentrations of leachable boron were found in the geothermal area and in a wooded patch surrounded by vinyards in the Chianti region. Boron-resistant bacteria responded only to boron from geothermal emissions.

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.

A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5 mg l{sup −1} HgCl{sub 2} and incubated at 30 °C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam fleshmore » were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills. -- Highlights: ► We isolated Gram-positive and Gram-negative Hg resistant strains from soft tissues of Ruditapes philippinarum. ► We identify 14 mercury resistant strains by 16S rRNA gene sequences. ► Bacteria in siphon and gill tissues of clams were observed by TEM and identified with different FISH probes. ► Hg-reductase (MerA) activity in glass homogenized clam tissues was also determined.« less

A strain of Klebsiella oxytoca, isolated from acid pyrite-mine drainage, characteristically produces a ferric hydrogel, consisting of branched heptasaccharide repeating units exopolysaccharide (EPS), with metal content of 36 wt%. The high content of iron in the EPS matrix cannot be explained by a simple ferric ion bond to the sugar skeleton. The bio-generated Fe-EPS is investigated by X-ray absorption spectroscopy. Fe K-edge XANES analysis shows that iron is mostly in trivalent form, with a non-negligible amount of Fe(2+) in the structure. The Fe EXAFS results indicate that iron in the sample is in a mineralized form, prevalently in the form of nano-sized particles of iron oxides/hydroxides, most probably a mixture of different nano-crystalline forms. TEM shows that these nanoparticles are located in the interior of the EPS matrix, as in ferritin. The strain produces Fe-EPS to modulate Fe-ions uptake from the cytoplasm to avoid iron toxicity under anaerobic conditions. This microbial material is potentially applicable as iron regulator.

Background A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant. Results Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production. Conclusion Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

A sensitive and specific technique, gas chromatography/Fourier transform infrared spectroscopy (GC/FTIR), has been used to detect traces of methane from the biodegradation of methylmercury due to the detoxification process of Pseudomonas putida strain FB1, which was isolated from cinnabar mines in southern Tuscany (Italy). The cured strain FB4 was used together with uninoculated samples as controls for the experiment. The calibration curve of methane was linear up to 1.3 ..mu..g/mL when 0.3 mL of headspace was injected into the GC/FTIR. The detection limit of methane determination in the closed system was 10 ng/mL. The biodegradation of methylmercury was measured during the 130-min experiment by means of elemental mercury analysis, which was determined specifically in the headspace by atomic absorption spectroscopy (AAS), by methylmercury analysis, and by consideration also of the growth rate of the bacterial strains.

International audience; Benthic diatoms represent an important element of global nutritional productivity; to raise attention on their role, which is often neglected due to analytical difficulties, surface (1 cm top layer) coastal sediments from Gerlache inlet to Penguin Bay at Terra Nova Bay were collected and stored at -20°C. DNA amplification by real-time-PCR, based on diatom-specific oligonucleotide primers designed on small-subunit rRNA (SSU rRNA), was performed in addition to diatom conventional cell counting and spectrophotometric determination of photo-pigments. Moreover, cations and anions were determined in sediments with the aim to identify factors involved in the control of diatom abundance. Diatom distribution was found quite heterogeneous displaying significant differences from site to site. The salinity in sediments ranged from 45.1 at Gerlache inlet to 76.2 at Penguin Bay and it was correlated with cell abundance, biodiversity, amount of pigments and amplified DNA. The dominant species, , was associated to sediment salinity brines.

Iron and palladium binding an exopolysaccharide (EPS) were obtained and purified from cultures of bacterial cells of Klebsiella oxytoca BAS-10. The strain BAS-10 was able to grow under anaerobic conditions with Fe(III)-citrate as energy and carbon source, producing Fe(III)-EPS that was extracted and used as catalyst in the oxidation reaction of phenol with H(2)O(2). The same bacterial strain was cultivated anaerobically with Na-citrate and Pd(2)(NO)(3) was added during the exponential growth to afford a Pd-EPS, named Bio-Pd (A), that, after isolation and purification, was used as catalyst in the reductive dehalogenation of chlorobenzene as model reaction. For comparison other two palladium binding polysaccharides were prepared: (a) a second type Pd-EPS, named Bio-Pd (B), was obtained by an exchange reaction with Pd acetate starting from an iron-free EPS produced by strain BAS-10 growing on Na-citrate medium; (b) a third type of palladium, named Bio-Pd (C), bound to a different polysaccharide, was recovered after the same exchange reaction applied on glycolipid emulsan obtained from an aerobic culture of Acinetobacter venetianus RAG 1. The superiority of Bio-Pd (A), as catalyst, vs Bio-Pd (B) and (C) was demonstrated. This approach to use microorganisms to prepare metal bound polysaccharides is novel and permits to prepare metal species, sequestrated in aqueous phase that can be useful either as catalysts for synthetic applications or to support the microbial biotransformation of pollutants.

A new approach was used to determine the reaction products of methylcobalamin and ionic mercury: purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy (PT GCIFTIR). This technique simultaneously and specifically determines the spectrum of dimethylmercury (DMeHg) and methylmercury produced by the reaction. No interference from other known organic mercury species could be detected. The method is different from others because it does not require solvent extraction of the organomercurials from aqueous solution, but relies on immediate volatilization from the reaction vessel by addition of 100 pl of 10 m~ NaBH,,. The sample was purged with nitrogen for 10 min. The volatile species of mercury were trapped in a column at -120 "C, injected into the gas chromatograph and detected by FTIR. The efficiency of DMeHg and MeHg formation depended on different parameters: pH, temperature, reaction time, and the methylcobalamin/ionic mercury ratio. The initial reaction product was MeHg which was further transformed to DMeHg. The first methylation rate was two times faster than the second. MeHg formed first, reaching a maximum at higher temperatures (28 "C and 37 "C) and later decreasing as DMeHg formed. At lower temperatures (2OOC) the rate of MeHg formation was slower, being similar to the formation rate of DMeHg. Different species of inorganic mercury such as