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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.

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.

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.

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.

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.

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.

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).

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.

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.

Klebsiella oxytoca BAS-10 is a Gram-negative micro-organism capable of growing on high concentrations of heavy metals. This bacterium produces large amounts of an iron-binding exopolysaccharide that, in the presence of metallic cations, precipitates as a dense gel. The primary and secondary structure of the repeating unit of such polysaccharide has been characterised by chemical and spectroscopic methods, resulting in the following heptasaccharide: 2)-α-Rha-(13)-β-Gal-(12)-α-Rha-(14)-β-GlcA-[β-GlcA-(14)]-(13)-α-Rha-(13)-α-Rha-(1. The absolute configurations for the Rha units are L, while those for the GlcA and Gal residues are D. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

The value of iron ore is adversely affected by phosphorus in concentrations over 0.03% by weight. The present research concerns the use of metabolic products of aPenicillium-like fungus to leach insoluble phosphates (hydroxyapatite) from ores. Ion chromatography was used to measure metabolism of glucose into acidic fragments. The rate and products of glucose degradation depended on both the chemical composition of the growth medium (buffered or not) and incubation conditions (shaken or quiescent). The principal products were identified as oxalic acid and isomers of propylene dicarboxylic acid, mainly itaconic acid. Continued, slow metabolism of itaconic acid generates more oxalic acid. Aliphatic acids were not detected. Both iron ore phosphate and calcium phosphate were partially solubilized by either the spent broth or aqueous oxalic acid. Solubilization of ore phosphorus was greatly assisted by hydrochloric acid added to the spent broth in small increments. The data suggest biological alternatives to costly leaching procedures that use only mineral acids.

Contaminated soil from a historical industrial site and containing sulfide ore ashes and aromatic hydrocarbons underwent sequential leaching by 0.5 M citrate and microbial treatments. Heavy metals leaching was with the following efficiency scale: Cu (58.7%) > Pb (55.1%) > Zn (44.5%) > Cd (42.9%) > Cr (26.4%) > Ni (17.7%) > Co (14.0%) > As (12.4%) > Fe (5.3%) > Hg (1.1%) and was accompanied by concomitant removal of organic contaminants (about 13%). Leached metals were concentrated into an iron gel, produced during ferric citrate fermentation by the metal-resistant strain BAS-10 of Klebsiella oxytoca. Concomitantly, the acidic leached soil was bioaugmented with Allescheriella sp. DABAC 1, Stachybotrys sp. DABAC 3, Phlebia sp. DABAC 9, Pleurotus pulmonarius CBS 664.97, and Botryosphaeria rhodina DABAC P82. B. rhodina was most effective, leading to a significant depletion of the most abundant contaminants, including 7-H-benz[DE]anthracene-7-one, 9,10-anthracene dione and dichloroaniline isomers, and to a marked detoxification as assessed by the mortality test with the Collembola Folsomia candida Willem. The overall degradation activities of B. rhodina and P. pulmonarius appeared to be significantly enhanced by the preliminary metal removal.

Klebsiella oxytoca BAS-10 is a Gram-negative micro-organism capable of growing on high concentrations of heavy metals. This bacterium produces large amounts of an iron-binding exopolysaccharide that, in the presence of metallic cations, precipitates as a dense gel. The primary and secondary structure of the repeating unit of such polysaccharide has been characterised by chemical and spectroscopic methods, resulting in the following heptasaccharide: 2)-α-Rha-(13)-β-Gal-(12)-α-Rha-(14)-β-GlcA-[β-GlcA-(14)]-(13)-α-Rha-(13)-α-Rha-(1. The absolute configurations for the Rha units are L, while those for the GlcA and Gal residues are D. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Massive mucilage events occur in the Northern Adriatic Sea presumably by cytoplasmic excretions from deteriorated diatoms. During three such events in the summer of 1991, 1997 and 1998 the presence of reduced sulfur species (RSS) was determined in samples of macroaggregate using electrochemical methods (in-phase alternating current (AC) and linear sweep voltammetry). The detected levels of sulfur, expressed as equivalent to sulfide concentrations, were about 200 nM. In the same mucilage samples, concentrations of organic matter were determined in the range from 60 to 600 mg/l of total organic carbon (TOC). The physico-chemical properties of organic matter in the macroaggregates correspond to those of polysaccharides of very high molecular mass. Scanning confocal laser microscopy (SCLM) and fluorescent molecular probes for sugars (the lectins concanvaline A (Con-A)) showed the transformation of polysaccharide polymer structure resulting in the formation of very stable filaments and layers after the treatment of mucilage samples with sodium sulfide. Commercial polysaccharides of bacterial and algal origin (xanthan, carrageenans types I and II, dextran-T-500) have been used to simulate macroaggregate formation under laboratory conditions after treatment with sodium sulfide. Raman spectroscopy indicated that for all model polysaccharides used, sulfide interaction occurred, as evidenced by visible change of the O–H stretching region in the vibration spectra of the water molecules. Our data suggest that the aggregated polysaccharides from the Adriatic Sea are: (1) structurally affected by addition of sulfide as was the case for most of the model polysaccharides, and (2) the stabilizing effect of sulfide on the aggregated polysaccharides is due to the formation of sulfur-organic compounds.

Most environmental pollutants are synthetic organic compounds that did not occur on earth at biologically significant concentrations before their industrial synthesis started around the turn of the century. Nevertheless, due to their remarkable genetic flexibility and physiological diversity, microorganisms have evolved to grow at the expense of these compounds. Mutations in structural genes, rearrangement of DNA segments, and exchange of genetic information has led to the evolvement of strains with novel metabolic activities. Such activities proliferate and lead to the adaptation of a microbiological community toward toxic or recalcitrant compounds. These principles can be illustrated with examples of the adaptation to chloroalkanes, chloroaromatics, and heavy metals.

The transformation of MeHg under anaerobic conditions in axenic cultures of Desulfovibrio desulfuricans strain LS is compared to that in anoxic marine sediments contaminated by Hg of industrial origin. MeHg was added to cultures of D. desulfuricans strain LS and incubated at 28°C for two weeks. Significant amounts of dimethylmercury and metacinnabar were produced. These two Hg compounds were formed from the slow decomposition of the intermediate dimethylmercury-sulfide. Other collateral compounds, such as methane and ionic Hg, were also detected during the MeHg degradation process. On the other hand a sample of fresh sediment (1.5 g d.w.) was spiked with 10 μg of MeHg and 2 mmoles.ml−1 of pyruvate, as carbon source for sulfate-reducing bacteria. After 9 days of incubation at 28°C, significant amounts of dimethylmercury were produced. A lower content of this volatile species was found in a sub-sample of sediment supplemented with sodium molybdate, which is a strong inhibitor of sulfate-reducing activity. A kinetic study showed the disappearance of monomethylmercury from the sediment and the formation of dimethylmercury over the incubation period. The environmental significance of dimethylmercury and dimethylmercury-sulfide in the natural biogeochemical cycle of Hg is discussed.

Several mercury‐resistant bacteria were isolated from sites in Italy that surrounded natural mercury deposits. Bacterial strains resistant to 5 mg mercuric chloride per liter reduced Hg2+ to elemental mercury by an inducible process. One bacterial strain was resistant to methylmercuric chloride and produced elemental mercury when incubated with several organomercurial compounds. No methylmercury was detected in bacterial cultures growing in the presence of 2 mg mercuric chloride per liter. Mercury reduction is the major mercury transformation in mercury‐resistant bacteria isolated from these environments.

Three sediment cores were sampled from the continental shelf of the Tyrrhenian Sea close to two areas contaminated by mercury from two different sources: the cinnabar deposits of southern Tuscany, and the outlet of a chlor-alkali plant at Rosignano Solvay (Livorno). The accumulation of mercury in marine sediment cores is influenced mainly by the high carbonate content of both natural and industrial origin. The grain-size of sediment particles plays an important role in the distribution of mercury in one core (CB 7), in which the highest concentration is associated with the coarse fraction, indicating that the abundance of organic matter in sediment, except at the top of of the lack of variability in the abundance of organic matter in the sediment, except at the top of the core, its influence on mercury distribution could not be determined. We have tentatively calculated the sedimentation rate (0.82 cm year −1 ) in one core (RO 14) by means of the peaks of the carbonates and mercury concentrations combined with data supplied by the chemical company. The results show that in both areas over the last decade mercury concentrations have decreased significantly.

This paper investigates mercury contamination in recent marine sediment of the Tyrrhenian continental shelf near the mouths of three rivers draining an area with cinnabar deposits (M. Amiata, Italy). By means of chemical leaching, two fractions of the total mercury were distinguished: a ‘non-leachable’ fraction, consisting of mercury held in relatively stable forms and a ‘leachable’ fraction, composed of forms that are more weakly bound to the sediments. The two Hg fractions are correlated to the organic matter content, the distribution of which is strictly dependent on the sediment surface area. In order to eliminate the effects of grain size variations among the samples, the Hg concentrations were normalized to the unit of surface area (ng/m2). Following this procedure, the ‘non-leachable’ Hg was found to be concentrated mostly in a belt along the coast, while the ‘leachable’ Hg fraction was shown to accumulate largely in the zones with high depositional dynamics near the mouths of the rivers. Hypotheses to account for the discrepancy between the low concentrations of ‘leachable’ Hg in the sediment of the outer continental shelf and the high concentrations in the fauna of the same area, reported in previous papers, are presented.

Two strains of Desulfovibrio desulfuricans , one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of Clostridium pasteurianum , a H 2 producer, and with the broad-spectrum mercury-resistant Pseudomonas putida strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH 3 HgCl resistance of D. desulfuricans strains was 10 times that of P. putida FB-1 and 100 times that of C. pasteurianum . The methylmercury resistance of D. desulfuricans was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH 3 ) 2 Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH 3 HgCl. This was compared with a 1-liter culture of D. desulfuricans LS spiked with 100 mg of HgCl 2 . In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH 3 Hg) 2 S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The D. desulfuricans strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter -1 ) or low (0.09-g · liter -1 ) sulfate contents. Methane was produced from CH 3 HgCl at a lower rate than by the broad-spectrum Hg-resistant P. putida strain FB-1.