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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

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi , or a mesophile, Thiobacillus ferrooxidans . Maximum rates of coal depyritization were also greater with M. sedula , although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula . Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.

A strain of broad-spectrum, mercury-resistant Pseudomonas putida FB1 was used to remove mercury as the gaseous element (Hg(0)) from a continuous axenic culture, fed with a synthetic medium containing 1 mg Hg l(-1) as HgCl2. Mercury determinations were performed in steady-state cultures using various culture fractions [whole culture, filtered supernatant, bacterial cells (dry wt), recovery trap liquid] in order to determine the removal efficiency at different dilution rates (from 0.1 to 3.0 day(-1)). The removal efficiency ranged from 99.2% to 99.8%, and the residual Hg was maintained below 5 μ l(-1) (the maximum allowable concentration of Hg in liquid wastes according to Italian law) at a dilution rate of 1.0 day(-1), corresponding to a Hg flux of 40 μg l(-1) h(-1). Hg accumulation by cell biomass was negligible for dilution rates under 1.0 day(-1). A progressive accumulation of Hg, both in the liquid phase and in cells, occurred at a higher dilution rate (3.0 day(-1); close to washout), corresponding to a Hg concentration of 25 μg g(-1) (dry wt). The estimated Km and Vmax for Hg reduction were 0.241 mg l(-1) and 9.5 mg g(-1) h(-1), respectively. In batch experiments maximum Hg removal occurred at the optimum growth temperature (28°C) of P. putida. The maximum recovery of Hg in the liquid trap was 78%.

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

The hydroxylation of phenol with aqueous H2O2 to afford catechol and hydroquinone was studied in a biphasic reaction medium, as well as in pure water, in the presence of a bio-generated iron(III) catalyst. This catalyst was purified from a culture of Klebsiella oxytoca BAS-10 growing under anaerobic conditions, with Fe(III)-citrate as the energy and carbon source. The overproduction of an exopolymer (EPS) encrusted bacterial cells. The EPS, binding Fe3+, (Fe-EPS), was extracted and studied before and after a reaction with phenol. Some of the reaction’s parameters, such as temperature, pH, and molar ratio between reagents and catalyst, were investigated to identify the best compromise between conversion and selectivity. The results could be useful either from a synthetic point of view or to support the biodegradation of aromatic substrates.

We report a novel aqueous derivatization of methylmercury chloride by NaBH 4 to mehtymercury hydride (CH 3 HgH), a volatile and unexpectedly stable species which we determined has a half-life of approximately 2 h. The analytical apparatus consisted of a purge and trap (PT) unit linked to a gas chromatograph (GC), in line with a Fourier transform infrared spectrometer (FTIR). The sample was purged with nitrogen, and volatile compounds were concentrated in a cold trap

Painted surfaces in the Chapel of the Holy Nail in the medieval ex-hospital of Siena show five areas of oxidation and blackening. Most were treated with Paraloid B72 acrylic resin 40 years ago. To study deterioration of the painted surfaces by scanning microscopy with X-ray dispersion microanalysis, fragments obtained from the five areas were analyzed directly and after hydration. Hydrated fragments treated with Paraloid showed a compact, shiny, impermeable surface. The acrylic resin forms a layer that inhibits interaction of the painting with the environment, including transpiration and gaseous exchange. Element composition of paint layers of different colours revealed gold, iron (oxides in ochre) and lead (oxide) tempered with lime and/or gypsum. A few microbial cells were found on all fragments. To study biological deterioration in the five areas, fragments were hydrated and incubated in minimal culture medium without added carbon source to select bacteria capable of using carbon sources in the medieval fresco. Analysis did not reveal any spores or hyphae, and excluded physical and mechanical damage. Bacteria of the genus Bacillus were isolated only from untreated samples. Paraloid resin forms an inert film over the mural, preventing access to microbes able to use nutrient sources in the painted layer. The screening of 16S rRNA libraries from enrichment cultures showed wide phylogenetic diversity. Forty-four percent of the clones retrieved from the clone library were affiliated with the order Firmicutes, confirming the prevalence of aerobic spore-formers among the colonizing microflora. Firmicutes therefore presumably produced extracellular material which made water available to other bacteria which may have converted thiosulphate in the medieval tempera to sulphur globules. Sulphur is a known oxidant of metallic pigments, in this case aluminium, which may explain the blackening of the untreated sample.

Bioleaching of a pyrite ore containing high concentrations of cobalt (0.1%) and zinc (0.065%) was affected by small amounts of calcitic gangue (from 0.01 to 1.01%). Results from an air-lift percolator and from Erlenmeyer flask experiments show that a small percentage of calcite raises the pH and arrests the growth of the acidophilic bacterium Thiobacillus ferrooxidans. In percolator experiments, when calcite is completely removed by the continuous addition of small quantities of acid, and the pH of the liquor becomes acid, the micro-organism begins to grow and to bio-oxidize the pyrite ore. The growth of T. ferrooxidans shows different lag phase spans (from 13 to 190 days) depending on carbonate dissolution. The metals Fe, Zn and Co are released into the leaching solution together at different rates after a lag-time which depends on calcite concentrations in pyrite gangue. Metal ratios in the mineral bulk are different from those in the liquor, Zn dissolving 5 times more readily than Co. Bioleaching rates for metal removal from pyrite are higher in percolator (for Fe, from 5 to 15 mg/l/h) than in flask experiments (from 0.5 to 2 mg/l/h), but the lag phases are shorter (from 2 to 65 days). The differences between the two systems are related to calcite dissolution and gypsum precipitation.

Bacteria are resistant to Hg compounds by virtue of two specific enzymes: mercuric reductase and organomercurial lyase. We investigate these specific enzyme systems 1) to determine McHg in biological samples by its enzymatic transformation to the respective hydrocarbon and 2) as an environmental index of Hg pollution in geothermal areas, by studying the distribution of the percentage of Hg-resistant bacteria. The first application is based on the enzymatic conversion of McHg to CH4. by whole cells of the Pseudomonas putida strain FBI. A 1 ml aliquot of 0.01N aqueous solution of thiosulphate containing MeHg, extracted from a biological sample by a conventional procedure was mixed with a dense (1 mg cells/mL dw) culture of FBI strain in a microreaction vessel. After a suitable period of incubation (from 4 to 18 hours), methane was assayed in the headspace by gas chromatograph equipped with flame ionization detector. In the second application, Hg-resistant bacteria (MRB) isolated from mosses collected in a geothermal area (Travale) in Tuscany showed almost the same distribution pattern as total Hg in briophytes. Mosses and MRB are both suitable bioindicators for mapping contaminated areas. The finding of Hg-resistant strains depends on the availability of the metal for cell accumulation and interaction at molecular level in the cytoplasm to produce mercuric reductase. MRB/g dw is therefore an indirect measure of the enzyme in this terrestrial geothermal environment.

A new method is suggested for determining methylmercury in biological samples. The determination is based on the use of whole cells of a broad-spectrum mercury-resistant bacterium, Pseudomonas putida, strain FB1, which is induced to produce the enzymes organomercurial lyase and mercuric reductase. These biocatalysts convert organomercurials to elemental mercury and their derivative hydrocarbons. The extraction procedure for methylmercury in biological samples follows the conventional toluene extraction method, which is retained until methylmercury is concentrated in an aqueous phase containing 0.01 M Na{sub 2}S{sub 2}O{sub 3}. This solution is mixed with bacterial cells in exponential growth phase and then incubated in microreaction vessels. A complete biological degradation of methylmercury to methane occurs. The efficiency of derivatization depends on the organomercurial concentration, the incubation time, and the cell density. The detection limit is 15 ng of methylmercury in 1 g of biological tissue and the coefficient of variation is 1.9% in 10 replicate samples with 100 ng/mL.

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)

A Cr(VI)-resistant yeast, designated strain DBVPG 6502, was isolated from a sewage treatment plant receiving wastes from tannery industries in Italy. The strain was tentatively identified as a species of Candida based on morphological and physiological analyses. This strain was highly resistant to Cr(VI) when compared with eight other yeast species, growing at Cr(VI) concentrations of up to 500 micrograms/ml (10 mM). This resistance was constitutive. The Cr(VI)-resistant yeast did not reduce Cr(VI) to Cr(III) species under aerobic conditions. The yeast showed very little accumulation of Cr(VI). Consequently, the mechanism of resistance of the yeast to Cr(VI) appears to involve reduced accumulation of Cr, as has been shown in Cr(VI)-resistant bacteria.

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.

This study reports the assessment of a novel process combining sequential treatments of a historically contaminated soil from the ACNA site (Cengio, Savona, Italy), a decommissioned industrial area. The soil was leached with 0.5 M citric acid leading to a removal of metals in the following order: Pb (74.2%) > Cu (72.6%) > Zn (40.2%) > Ni (55.7%) > Cd (41.5%) > Cr > (21.7%) Co > (19%) Fe (8.2%) with a concomitant low removal of organic contaminants (12%). The leachate was then incubated with the metal-resistant Klebsiella oxytoca strain BAS-10, capable of using residual citrate to produce an iron gel that co-precipitated metals. Concomitantly, the leached solid waste was bioaugmented with the autochthonous isolate of Allescheriella sp. DABAC 1 leading to a complete degradation of several organic contaminants, including trichlorobenzene, naphtalene, dichloroaniline and pentachloroaniline. The overall removals of organic contaminants by the fungus were 44.3% and 63.8% in non-leached and leached soil.

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.

A set of 21 Antarctic marine bacteria isolated from the Ross Sea and able to utilise diesel fuel as the sole carbon and energy source was characterised. Isolates were analysed by amplified 16S rDNA restriction analysis using the enzyme Alu I, resulting in two different groups corresponding to different bacterial species. These species were assigned to the genera Rhodococcus and Alcaligenes , on the basis of 16S rDNA sequencing. This low degree of inter-specific biodiversity was parallel to a low intra-specific biodiversity, as shown by Random Amplified Polymorphic DNA analysis. Then, a 550-bp DNA fragment coding for the inner region of alkane mono-oxygenase was PCR-amplified from the genome of each strain. The phylogenetic analysis of the sequence of the putative AlkB protein coded for by the amplified DNA fragment revealed that these alkB genes were very likely inherited by horizontal gene transfer. Lastly, the analysis of the biodegradation ability of four strains revealed two different strategies of hydrocarbon uptake, mediated either by bio-surfactants and peculiar of Rhodococcus isolates, or by membrane modifications and shown by Alcaligenes isolates. In order to understand the interrelationships between hydrocarbon-degrading isolates, the dynamics of two strains, belonging to Rhodococcus and Alcaligenes , grown together in a co-culture was also followed over a seventeen days period.

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg(-1) polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very Close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564 32 mg O-2 l(-1) d(-1) and SB1 oxidised biphenyl at 426 25 mg O-2 l(-1) d(-1) in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring. (c) 2006 Elsevier Ltd. All rights reserved.

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)

A sensor responsive to Hg2+ (pDL20) was constructed by fusing part of a narrow spectrum mer operon, including its regulatory elements, to promoterless lux genes. This was compared with another mer::lux fusion (pRB28) in order to estimate the available Hg in moss, a conventional bioindicator used for surveying Hg pollution in terrestrial environments. Hg(II) was measured as the relative luminescence unit (RLU) emitted by E. coli cells carrying either pRB28 or pDL20. The linearity ranges of standard curves were measured by spiking HgCl2 at different nanomole levels in a phosphate buffered solution (PBS). The level of correlation between RLU and spiked HgCl2 depended on the incubation time of E. coli cells: the correlation factors (R2) of the regression lines were highly significant only after 150 min of incubation. pDL20 detected Hg(II) concentrations in the linear range between 0.05 nM and 0.5 nM and was approximately 27 times more sensitive than pRB28. The latter was less sensitive and showed a different range of linearity, from 20 nM to 200 nM. Measurements of bioavailable Hg were performed in buffered solutions leached from moss. Concentrations of Hg(II) were determined by external standard addition of HgCl2. pDL20 was found to be more reliable than pRB28 in the estimation of very low concentrations of bioavailable Hg (II). Both sensors were unable to determine Hg(0) emitted by geothermal activities.

A bacterial strain ANT-3b was isolated at the sea-ice seawater interface from Terra Nova Bay station, Ross Sea, Antarctica. It was isolated on mineral medium supplemented with 2% diesel fuel as a sole carbon and energy source and grown routinely on 2% n-hexadecane. Analysis of 16S rRNA gene sequence indicates that the strain has 99.8% sequence similarity with Halomonas neptunia. The strain ANT-3b was grown in mineral medium supplemented with n-hexadecane between 4 and 20 degrees C, but not at 30 degrees C. The maximum degradation rate of the n-alkane was measured at 15 degrees C, with 5.6+/-1.7 mg O2 microg(-1) protein d(-1). The strain ANT-3b produced emulsifying compounds when grown on n-hexadecane, but not on mineral medium supplemented with D-fructose. A preliminary characterisation of the emulsifier was carried out. The lipid moiety contained a mixture of fatty acids with a following composition in molar ratio: caprylic acid 18.85, myristic acid 1.0, palmitic acid 9.68, palmitoleic acid 5.69 and oleic acid 1.26. The polysaccharide moiety also contained a mixture of sugars with the following molar ratio: mannose 1.71, galactose 1.00 and glucose 2.96. The molecular weight of the glycolipid component determined by gel permeation chromatography was in the 18 kDa range and contained smaller fragments, possibly oligomeric contaminants. Transmission electron microscopy showed contact between the glycolipid secreted by the strain and n-hexadecane broken down to nanodroplets at the water interface, to form a material with mesophase (liquid crystal) organisation.

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 (microm2) per aggregate was found. Each cell presented an average surface of 1.21 microm2. 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.

The enterobacterium Klebsiella oxytoca strain BAS-10 was isolated from sediments under an iron mat formed in a stream receiving leached waters from pyrite mine tailings. Under anaerobic laboratory conditions, BAS-10 fermented Fe(III)-citrate and Na-citrate giving CH(3)COOH and CO(2). In the presence of ferric citrate, BAS-10 secreted quantities of a thick gel containing glucose and/or mannose, if not other sugars. Sugar residues were observed in microbial aggregates using the sugar-specific concanavalin A lectin conjugated with fluorescein and imaged by a scanning confocal laser microscope. The gel bound Fe(III) which quickly precipitated. During fermentation, however, half the initial Fe(III) concentration was reduced to Fe(II) which did not bind to the gel and remained in solution. BAS-10 showed a high tolerance to heavy metals. Its growth was not inhibited by 1 mM Zn-, Pb- or Cd-acetate. These cations also co-precipitated with the iron gel, suggesting a possible application of this strain for abatement of toxic metals under anaerobic conditions.

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.

Acinetobacter strain RAG-1 (ATCC 31012) is an industrially important strain which has been extensively characterized with respect to its growth on hydrocarbons and its production of a high molecular mass bioemulsifier, emulsan. Although RAG-1 has been investigated in detail for specific biochemical characteristics, its taxonomic status is uncertain and it is usually referred to as A. lwoffii or A. calcoaceticus sensu lato. However, results obtained by restriction analysis of the amplified rDNA and subsequently substantiated by DNA-DNA hybridization, partial 16S rDNA nucleotide sequence comparison and biochemical characterization indicate that RAG-1 belongs to the genomic species recently described as ' A. venetianus '. Furthermore, these data confirm that ' A. venetianus ' constitutes a new and distinct genomic species within the genus Acinetobacter.

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.

Beneficial and detoxifying mechanisms in microorganisms are described in relation to their potential exploitation for metal and radionuclide bioremediation. Metal toxicity and essentiality are amphibolous aspects of metals in regards to their homeostatic and non homeostatic metabolism. Metals are energy sources, electron acceptors, and are essential for secondary metabolism Metals are effluxed by specific systems to reduce cytoplasmic content to subtoxic levels. Metals may be converted to harmless species by specific enzymes and by the intermediates or final products of microbiological metabolism. In certain circumstances metals are precipitated as colloids, or crystals may be formed; conversely they may be removed by bioleaching, or volatilized by methylation and hydriditization. Most of these microbiological processes can be exploited to reduce metal and radionuclide contamination. Various technologies ranging from different bioreactor configurations to in situ operations have been developed An attempt of radionuclide bioremediation in situ of soil at Chernobyl, Ukraine, is described.

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 μg ml− (0.2 mm) Cr(VI) as K2Cr2O7. DBVPG 6662 was inhibited by 100 μg ml− (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 μg mg− cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 μg ml− Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at μm levels increased tolerance up to 10 μg ml−, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.

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.

Wide variations were found in the rate of chemical and microbiological leaching of iron from pyritic materials from various sources. Thiobacillus ferrooxidans accelerated leaching of iron from all of the pyritic materials tested in shake flask suspensions at loadings of 0.4% (wt/vol) pulp density. The most chemically reactive pyrites exhibited the fastest bioleaching rates. However, at 2.0% pulp density, a delay in onset of bioleaching occurred with two of the pyrites derived from coal sources. T. ferrooxidans was unable to oxidize the most chemically reactive pyrite at 2.0% pulp density. No inhibition of pyrite oxidation by T. ferrooxidans occurred with mineral pyrite at 2.0% pulp density. Experiments with the most chemically reactive pyrite indicated that the leachates from the material were not inhibitory to iron oxidation by T. ferrooxidans .

Two chromium(VI) resistant yeast strains (Candida sp. and Rhodosporidium sp.) were isolated from industrial wastes. Four different yeasts, three from the Industrial Yeast Collection and one of pharmaceutical origin, were also studied in relation to chromate toxicity and its alleviation by sulfur species. The growth of yeasts from industrial wastes was inhibited by 50% by high concentrations of Cr(VI): Candida sp. by 4 mM Cr(VI) and Rhodosporidium sp. by 10 mM Cr(VI) in Sabouraud Broth medium. The other Cr(VI)-sensitive yeasts were inhibited by 0.1 mM Cr(VI). The general mechanism of chromium resistance in Candida sp. and Rhodosporidium sp. was due to reduced uptake of chromium, but not to biological reduction from Cr(VI) to Cr(III). In Cr(VI)-sensitive yeasts, chromium was accumulated as much as 10-fold, as in Saccharomyces cerevisiae. Cr(VI) toxicity in Candida sp. was modulated from Cr(VI)-resistance to Cr(VI)-hypersensitivity depending on the addition of methionine, cysteine, sulfate and djenkolic acid. If Candida sp. was grown in the presence of S-amino acids, especially methionine, it was more resistant than if the sulfur source was sulfate. When sulfate transport was enhanced by addition of djenkolic acid, Candida sp. became hypersensitive. Rhosporidium sp. was always resistant to Cr(VI) because sulfate transport was inefficient and it assimilated sulfur as S-amino acids. Cr(VI)-sensitive yeasts required larger amounts of S-amino acids, especially methionine, to tolerate Cr(VI) toxicity. Cysteine was toxic for C.famata 6016 above 50 microM.

PURPOSE. Helicobacter pylori is a major pathogen etiologically associated with gastritis, peptic ulcer disease, gastric cancer, and primary gastric lymphoma. This study was conducted to investigate a possible association between Helicobacter pylori infection and blepharitis. Two hundred fifty consecutive patients with symptomatic blepharitis and 250 control subjects without blepharitis symptoms were evaluated. After exclusions, the blepharitis group consisted of 186 patients with blepharitis and a control group of 215 patients. METHODS. Blepharitis was diagnosed on the basis of findings in ophthalmic and dermatologic examinations. All patients underwent a 13 C-urea breath test (UBT) to detect H. pylori infection, and impression cytology was performed before and after eradication therapy. The follow-up period was 4 months ± 28 days. RESULTS. The blepharitis group showed an H. pylori infection prevalence of approximately 76.3% (UBT-positive group with blepharitis: n = 142 patients), compared with 42.3% of the control group (UBT-positive group with blepharitis [although asymptomatic]: n = 66 patients; UBT-positive group without blepharitis: n = 25 patients). Furthermore, we observed blepharitis in 30.6% (n = 66 patients) of UBT-positive control subjects and 13.4% (n = 29 patients) of UBT-negative control subjects. Impression cytology revealed that blepharitis was more severe in UBT-positive patients than in negative ones, and a clinical improvement in blepharitis was noted in approximately 50% of patients after H. pylori eradication. CONCLUSIONS. Even though possible sources of error in defining the association of two highly prevalent conditions must be considered, the data seem to validate an association between H. pylori infection and blepharitis, but may not be indicative of a causal association. Eradication of H. pylori improved ocular cytology results. It is possible that chronic blepharitis is an extradigestive manifestation of H. pylori infection.

The effect of cinnabar on pyrite oxidation by mercury-sensitive and mercury-resistant strains of Thiobacillus ferrooxidans was investigated by using percolation columns. Mercury-resistant strains oxidized pyrite in pyrite-cinnabar mixtures (1 and 10%, wt/wt), whereas a mercury-sensitive strain did not. Elemental mercury was produced by the mercury-resistant strains growing in the pyrite-cinnabar mixtures in percolation columns and in flasks containing cinnabar only. Manometric experiments showed that cinnabar had little effect on oxygen uptake of mercury-sensitive or mercury-resistant cells growing on ferrous sulfate, pyrite, or pyrite-ferrous sulfate mixtures. In addition, shake flask leaching experiments showed that cinnabar had little effect on pyrite oxidation at 1% (wt/wt) but inhibited growth of mercury-sensitive and mercury-resistant strains at 10%. Mercury-resistant strains were unable to grow on cinnabar as an energy source.

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.

One-hundred-ninety-five specimens of higher fungi and their substrata collected in the Hg mining area of M. Amiata and around Siena (Central Italy), were analyzed for their total Hg content. Whereas wood decomposers and many species of mycorrhizal fungi accumulated the metal at a very low rate, some mycorrhizal species and all the humus decomposers may accumulate up to 100 ..mu..g.g/sup -1/ d.w. of Hg and in the least contaminated sites, up to 63 times as much Hg as the substratum. Instead in mineralized areas the concentration factor rarely exceeded 1. The methyl mercury content of 35 species (almost all edible), ranged between 0.01 and 3.7 ..mu..g.g/sup -1/ d.w. The possible significance of these findings and the toxicologic implications for local consumers are considered.

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.

Samples of soils and mosses were collected near a geothermal power plant, which is located in one of the most active geothermal fields of the world (Southern Tuscany). High concentrations of Hg (up to 1.8 μg g−1 d.w.) occur in mosses as far as 0.6 km from the geothermal plant, and the Hg uptake of mosses is unrelated to the species except for Bryum torquescens. The Hg in the soil was lower than in mosses, and the greatest concentrations in soil were near the Travale-22 well, which feeds the most powerful geothermal plant (30 MW) in the area. In addition, the concentrations of Hg detected in soil cores and in old bottom leaves of several specimens of Hypnum cupressiforme show that this element has been emitted into the air also in the past probably since the discovery of T-22 well in 1972.

Marine sediments are the ultimate recipient of mercury introduced into aquatic ecosystem either by man or by weathering. In the surficial sediments of the northern Tyrrhenian sea high concentrations of this metal were found in an area close to the effluent of a chlor-alkali plant and in the bays of La Spezia and Leghorn, which receive wastes from numerous industries and from port activities. The sediments of a much larger marine area off the mouths of the Ombrone, Albegna and Fiora rivers also contain high levels of Hg, part of which derives from the weathering of the rich cinnabar deposits of Mount Amiata and part from mining and processing the cinnabar.

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.

Marine surficial sediments, intertidal molluscs and reed leaves collected in the Bay of Follonica are slightly contaminated by Pb, Ni, Zn and Fe. The main pollution sources are the waste water and the atmospheric emission of industrial plants. Probably, the weathering of the pyrite deposits along the eastern coast of Elba Island and adsorption and coprecipitation with hydrous Fe-Mn oxides increase the trace metal concentrations in sediments from the Piombino Channel. Mussels and limpets are contaminated by Hg because of the higher background level of the northern Tyrrhenian Sea.

The distribution of the macrobenthic community, in relation to various physical and chemical characteristics of the sediment, was studied along the coast of Southern Tuscany (Central Tyrrhenian) in an area under the influence of the Ombrone River outflow. The zonation of the benthos was found to be primarily under the control of a gradient of water movement, as is reflected by variations in the sediment structure from the coast to the open sea. This correlation is somehow influenced by secondary gradients such as the degree of mixing of the sediment and the deposition of terrigenous materials along the river plume.

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.

We explore the possibility of tracing routes of dense waters toward and within the ocean abyss by the use of an extended set of observed physical and biochemical parameters. To this purpose, we employ mercury, isotopic oxygen, biopolymeric carbon and its constituents, together with indicators of microbial activity and bacterial diversity found in bottom waters of the Eastern Mediterranean. In this basin, which has been considered as a miniature global ocean, two competing sources of bottom water (one in the Adriatic and one in the Aegean seas) contribute to the ventilation of the local abyss. However, due to a recent substantial reduction of the differences in the physical characteristics of these two water masses it has become increasingly complex a water classification using the traditional approach with temperature, salinity and dissolved oxygen alone. Here, we show that an extended set of observed physical and biochemical parameters allows recognizing the existence of two different abyssal routes from the Adriatic source and one abyssal route from the Aegean source despite temperature and salinity of such two competing sources of abyssal water being virtually indistinguishable. Moreover, as the near-bottom development of exogenous bacterial communities transported by convectively-generated water masses in the abyss can provide a persistent trace of episodic events, intermittent flows like those generating abyssal waters in the Eastern Mediterranean basin may become detectable beyond the availability of concomitant measurements.