Mercury methylation by planktonic and biofilm cultures of Desulfovibrio desulfuricans

While biofilms are now known to be the predominant form of microbial growth in nature, very little is yet known about their role in environmental mercury (Hg) methylation. Findings of Hg methylation in periphyton communities have indicated the importance of investigating how environmental biofilms affect Hg methylation, as periphyton can be the base of the food webs in aquatic ecosystems. Chemical speciation influences the microbial uptake and methylation of inorganic Hg by planktonic cultures of sulfate-reducing bacteria; however, the effect of speciation on Hg methylation by biofilm cultures of these organisms has previously not been studied. In the present study, Hg methylation rates in biofilm and planktonic cultures of two isolates of Desulfovibrio desulfuricans from a coastal wetland were compared. Notably, the specific Hg methylation rate found was approximately an order of magnitude higher (0.0018 vs. 0.0002 attomol cell(-1) day(-1)) in biofilm cells than in planktonic cells, suggesting an important role for environmental biofilms in Hg methylation. To investigate the role of chemical speciation of Hg, experiments were conducted at two levels of sulfide. Both biofilm and planktonic cultures produced methylmercury at roughly twice the rate at low sulfide, when HgS(0)(aq), rather than HgHS2-, was the dominant Hg species. This indicates that the presence of a biofilm does not alter the relative availability of the dominant Hg species in sulfidic medium, in accordance with our previous studies of Hg uptake by Escherichia coli along a chloride gradient.     

The influence of sulfide on solid-phase mercury bioavailability for methylation by pure cultures of Desulfobulbus propionicus (1pr3)

To help understand the mechanism and control of Hg uptake in Hg-methylating bacteria, we investigated the effect of sulfide on Hg methylation by pure cultures of the sulfate-reducing bacterium Desulfobulbus propionicus (1pr3). Our previous research in natural sediments has suggested that Hg methylation occurs most rapidly when sulfide concentrations favor formation of neutral dissolved Hg-S species. In this study, the chemical speciation of Hg in culture media was manipulated by growing D. propionicus across a range of sulfide concentrations, with inorganic Hg (HgI) added in the form of ground ores. A solid-phase, rather than a dissolved source of Hg, was used to simulate the controls on Hg partitioning between solid and aqueous phases found in natural sediments. Methylmercury (MeHg) production by cultures was not related to the absolute solid-phase concentration of Hg in the ores, and it was only weakly related to the dissolved HgI concentration in the medium. However, MeHg production was linearly related to the calculated concentration of the dominant neutral complex in solution, HgS degrees. Furthermore, the diffusive membrane permeability of HgS degrees, as estimated from its octanol-water partitioning coefficient, was found to be sufficient to support MeHg production by cells. The present paper expands on our previous work by providing experimental support of our hypothesis that sulfide influences methylation by affecting the speciation of dissolved HgI and its uptake via passive diffusion.     

Effect of pH on mercury uptake by an aquatic bacterium: implications for Hg cycling

We studied the effect of increasing hydrogen ion (H+) concentration on the uptake of mercury (Hg(II)) by an aquatic bacterium. Even small changes in pH (7.3-6.3) resulted in large increases in Hg(II) uptake, in defined media. The increased rate of bioaccumulation was directly proportional to the concentration of H+ and could not be explained by assuming that the source of Hg to the bacteria was diffusion of neutrally charged species such as HgCl2. Thus, pH appeared to affect a facilitated mechanism by which Hg(II) is taken up by the cells. Lowering the pH of Hg solutions mixed together with natural dissolved organic carbon, or with whole lake water, also increased bacterial uptake of Hg(II). These findings have several potential implications for mercury cycling, including effects on elemental mercury production, mercury sedimentation, and microbial methylation of Hg(II), and could be part of the explanation for the observed positive correlation between lake acidity and methyl mercury levels in fish.     

Divalent base cations hamper Hg(II) uptake

Despite the alarming trends of declining base cation concentrations in boreal lakes, no studies have attempted to predict the consequences of this decline on the geochemical cycle of mercury, a top priority contaminant worldwide. In this study, we used a whole-cell gram-negative bioreporter to evaluate the direction and magnitude of changes in net accumulation of Hg(II) by bacteria in response to changing base cation concentrations. We show that regardless of the speciation of Hg(II) in solution, increasing divalent base cation concentrations decrease net Hg(II) accumulation by the bioreporter, suggesting a protective effect of these cations. Our work suggests that the complexity of the cell wall of gram-negative bacteria must be considered when modeling Hg uptake pathways; we propose that base divalent cations contribute to hamper net Hg(II) accumulation by decreasing outer membrane permeability and, therefore, the passive diffusion of Hg(II) species to the periplasmic space. This work points to an unsuspected and likely harmful consequence of a delay in recovering from acidification in boreal lakes, in that uptake of Hg(II) by bacteria is not only enhanced by the reduced pH but can also be enhanced by a decline in base cation levels.     

Role of speciation in organotin toxicity to the yeast Candida maltosa

Assessment of organometal pollution requires an understanding of the various processes that influence the bioavailability and toxicity of the contaminant. Organotins may exist as both cationic species and neutral hydroxides in aqueous solution, with the formation of chloride species in the presence of Cl-. Although these species have different chemical properties, there is very little information on the influence of speciation on organotin and microbial cell interactions. Tributyltin (TBT) and triphenyltin (TPT) interactions with the yeast Candida maltosa were investigated between pH 3.5 and 7.5 and in up to 0.5 M NaCl at pH 5.5. Toxicity increased with both pH and NaCl concentration and the mechanisms of interaction depended on the species present in solution. TBT and TPT interacted by different mechanisms, as evidenced by action on membrane fluidity. Furthermore, there was a strong correlation between toxicity and overall octanol-water distribution ratio (D(OW)) of organotin compounds. Triorganotin cations are less toxic than triorganotin hydroxides, which are in turn less toxic than triorganotin chlorides. These findings underline the importance of speciation effects on organotin interactions in the environment.     

Formation of nanocolloidal metacinnabar in mercury-DOM-sulfide systems

Direct determination of mercury (Hg) speciation in sulfide-containing environments is confounded by low mercury concentrations and poor analytical sensitivity. Here we report the results of experiments designed to assess mercury speciation at environmentally relevant ratios of mercury to dissolved organic matter (DOM) (i.e., <4 nmol Hg (mg DOM)(-1)) by combining solid phase extraction using C(18) resin with extended X-ray absorption fine structure (EXAFS) spectroscopy. Aqueous Hg(II) and a DOM isolate were equilibrated in the presence and absence of 100 μM total sulfide. In the absence of sulfide, mercury adsorption to the resin increased as the Hg:DOM ratio decreased and as the strength of Hg-DOM binding increased. EXAFS analysis indicated that in the absence of sulfide, mercury bonds with an average of 2.4 ± 0.2 sulfur atoms with a bond length typical of mercury-organic thiol ligands (2.35 ?). In the presence of sulfide, mercury showed greater affinity for the C(18) resin, and its chromatographic behavior was independent of Hg:DOM ratio. EXAFS analysis showed mercury-sulfur bonds with a longer interatomic distance (2.51-2.53 ?) similar to the mercury-sulfur bond distance in metacinnabar (2.53 ?) regardless of the Hg:DOM ratio. For all samples containing sulfide, the sulfur coordination number was below the ideal four-coordinate structure of metacinnabar. At a low Hg:DOM ratio where strong binding DOM sites may control mercury speciation (1.9 nmol mg(-1)) mercury was coordinated by 2.3 ± 0.2 sulfur atoms, and the coordination number rose with increasing Hg:DOM ratio. The less-than-ideal coordination numbers indicate metacinnabar-like species on the nanometer scale, and the positive correlation between Hg:DOM ratio and sulfur coordination number suggests progressively increasing particle size or crystalline order with increasing abundance of mercury with respect to DOM. In DOM-containing sulfidic systems nanocolloidal metacinnabar-like species may form, and these species need to be considered when addressing mercury biogeochemistry.     

Evaluation of mercury toxicity as a predictor of mercury bioavailability

Many studies on bioavailability of toxic metals have made the assumption that observation of toxicity is evidence thatthe metal was taken into the cells (i.e., was "bioavailable"). A second assumption is that results at the high concentrations necessary for toxic effect are applicable to the lower concentrations more commonly found in the environment. These assumptions were specifically tested for mercury (Hg(II)) toxicity (at concentrations of 0.25-50 nM Hg) and uptake (at lower concentrations of 0.005-0.015 nM Hg) in the aquatic bacterium, V. anguillarum. Toxicity was measured as reduction in levels of constitutively expressed bioluminescence in V. anguillarum pRB27. Hg(II) uptake was measured using the Hg(II)-inducible mer-lux operon in V. anguillarum pRB28. In experiments where the predominant Hg species was changed from HgCl2 to Hg(OH)2 or Hg(NH3)2(2+), toxicity results accurately predicted that there would be no effect of the dominant species on Hg(II) uptake at lower HgT concentrations. However, toxicity tests with these same ligands failed to predict that there would be an effect on Hg(II) uptake when conditions were changed from aerobic to anaerobic. Toxicity tests also failed to predict the effect of 5 mM histidine additions on Hg(II) uptake, as histidine addition protected cells completely from Hg toxicity under both aerobic and anaerobic conditions, at concentrations up to 50 nM Hg, but did not prevent Hg(II) uptake. Uptake occurred at low HgT concentrations (0.01 nM) at the same rate when histidine was added under aerobic conditions and was substantially increased under anaerobic conditions. Thus, toxicity assays for Hg under a variety of conditions were not always a reliable predictor of the effects of those conditions on Hg(II) uptake into the cell.     

Decrease in net mercury methylation rates following iron amendment to anoxic wetland sediment slurries

The rate of mercury methylation in anoxic wetland sediments is affected by the concentration of bioavailable complexes between Hg and sulfide. Previous research with pure bacterial cultures has shown that addition of ferrous iron reduces the net rate of mercury methylation by decreasing the concentration of dissolved sulfide. To assess the possibility of using this approach to decrease net mercury methylation in restored and constructed wetlands, laboratory experiments were conducted by adding Hg(II) and Fe(II) to sediments collected from six sites in five estuarine wetlands. Addition of 30 mM (0.07 mmol g(-1) or 3.9 mg g(-1)) Fe(II) decreased net mercury methylation relative to that of unamended controls by a factor of 2.1-6.6. In all cases, the observed decrease in net mercury methylation was accompanied by a decrease in the concentrations of sulfide and filterable mercury in the water overlying the sediments. When iron was added to one of the sediment samples at doses that were small relative to the concentration of sulfide present, net mercury methylation either increased slightly or was unaffected. Comparison of the results to speciation model predictions suggests that dissolved reduced sulfur-containing species play a role in the formation of uncharged, bioavailable Hg complexes. Although further research is needed to determine the long-term effect of iron amendment, these results suggest that iron addition decreases mercury methylation in authentic wetland sediments.     

Field,laboratory, and X-ray absorption spectroscopic studies of mercury accumulation by water hyacinths

We have studied water hyacinth (Eichhornia crassipes), a non-native nuisance plant found in the in San Francisco Bay Delta region, for its potential to phytoremediate mercury. Mercury is a common contaminant in San Francisco Bay Area waters because of gold mining activities. In this study, speciation of mercury in hyacinth roots and shoots, rates of mercury uptake by hyacinths in the laboratory, and mercury levels near the Big Break Region in the Delta were studied. In the speciation studies, Hg L3 edge X-ray absorption spectroscopic analysis of Hg model compounds and water hyacinth roots and shoots revealed that Hg was initially bound ionically to oxygen ligands in roots, most likely to carboxylate groups, and was bound covalently to sulfur groups in shoots. In laboratory uptake studies, we found that water hyacinths grown in 1 ppm Hg and one-quarter strength Hoagland's solution accumulated a maximum of 0.20 ppm in shoots and 16.0 ppm in roots, both reaching maximum concentrations after approximately 16 days. Mercury concentrations were found to be 0.26 +/- 0.20 ppm in the water and 0.86 +/- 1.70 ppm in sediment at Big Break. It was proposed that water hyacinths have the potential to phytoremediate mercury in the water at Big Break if the current herbicide treatments are replaced by physical removal.     

Methylation of mercury by bacteria exposed to dissolved, nanoparticulate, and microparticulate mercuric sulfides

The production of the neurotoxic methylmercury in the environment is partly controlled by the bioavailability of inorganic divalent mercury (Hg(II)) to anaerobic bacteria that methylate Hg(II). In sediment porewater, Hg(II) associates with sulfides and natural organic matter to form chemical species that include organic-coated mercury sulfide nanoparticles as reaction intermediates of heterogeneous mineral precipitation. Here, we exposed two strains of sulfate-reducing bacteria to three forms of inorganic mercury: dissolved Hg and sulfide, nanoparticulate HgS, and microparticulate HgS. The bacteria cultures exposed to HgS nanoparticles methylated mercury at a rate slower than cultures exposed to dissolved forms of mercury. However, net methylmercury production in cultures exposed to nanoparticles was 6 times greater than in cultures treated with microscale particles, even when normalized to specific surface area. Furthermore, the methylation potential of HgS nanoparticles decreased with storage time of the nanoparticles in their original stock solution. In bacteria cultures amended with nano-HgS from a 16 h-old nanoparticle stock, 6-10% of total mercury was converted to methylmercury after one day. In contrast, 2-4% was methylated in cultures amended with nano-HgS that was aged for 3 days or 1 week. The methylation of mercury derived from nanoparticles (in contrast to the larger particles) would not be predicted by equilibrium speciation of mercury in the aqueous phase (<0.2 μm) and was possibly caused by the disordered structure of nanoparticles that facilitated release of chemically labile mercury species immediately adjacent to cell surfaces. Our results add new dimensions to the mechanistic understanding of mercury methylation potential by demonstrating that bioavailability is related to the geochemical intermediates of rate-limited mercury sulfide precipitation reactions. These findings could help explain observations that the "aging" of mercury in sediments reduces its methylation potential and provide a basis for assessing and remediating methylmercury hotspots in the environment.     

Accumulation of inorganic and methylmercury by freshwater phytoplankton in two contrasting water bodies

Phytoplankton concentrate mercury from their aqueous surroundings and represent the primary entry point for Hg in aquatic food webs. We used 203Hg to compare the uptake of inorganic mercury, Hg(II), and methylmercury, MeHg, in four phytoplankton species (a diatom, a chlorophyte, a cryptophyte, and a cyanobacterium) in two waters containing different concentrations of dissolved organic carbon (DOC). At steady state, volume concentration factors (VCFs) for Hg(II) in the four species were similar and ranged from 0.5 to 5 x 10(4) for both water types, whereas VCFs for MeHg exceeded those for Hg(II) and ranged from 1.3 to 14.6 x 10(5). The VCFs for MeHg in the three eukaryotic cells in the high DOC water were 2-2.6 times greater than those in the low DOC water, but the VCFs for the prokaryote were similar in both waters. Higher cell surface area to volume ratios correlated with increased MeHg concentrations but not with Hg(II). In both water types, VCFs of Hg(II) were similar for living and heat-killed cells, but the VCFs of MeHg were 1.5-5.0 times greater in living cells, suggesting an active uptake component for MeHg. Hg(II) and MeHg were entirely bound to cell surfaces of the dead cells, whereas 59-64% of the MeHg and 9-16% of the Hg(II) in living cells entered the cytoplasm.     

Importance of dissolved neutral mercury sulfides for methyl mercury production in contaminated sediments

Biotic transformation of inorganic mercury, Hg(II), to mono methyl mercury (MeHg) is proposed to be largely controlled by passive uptake of neutral Hg complexes by sulfate reducing bacteria (SRB). In this study, the chemical speciation of Hg(II) in seven locally contaminated sediments covering environments such as (i) brackish water, (ii) low-productivity freshwater, and, (iii) high-productivity freshwater was related to potential Hg methylation rates, determined by incubation at 23 degrees C for 48 h under N2(g), and to total MeHg concentrations in sediments. Pore water speciation was modeled considering Hg complexes with halides, organic thiols [Hg(SR)2(aq), associated to dissolved organic matter], monosulfides, and bisulfides. The sum of neutral mercury sulfides [Hg(SH)20(aq)] and [HgS0(aq)] was significantly, positively (p < 0.001, n = 20) correlated to the specific methylation rate constant (Km, day(-1)) at depths of 5-100 cm in two brackish water sediments. Total Hg, total mercury sulfides or Hg(SR)2(aq) in pore water gave no significant relationships with Km. In two subsets of freshwater sediments, neutral mercury sulfides were positively correlated to total Hg in pore water, and therefore, total Hg also gave significant relationships with Km. The sum of [Hg(SH)20(aq)] and [HgS0(aq)] was significantly, positively correlated to total sediment MeHg (microg kg-1) in brackish waters (p < 0.001, n = 23), in southern, high-productivity freshwaters (p < 0.001, n = 20), as well as in northern, low-productivity freshwater (p = 0.048, n = 6). The slopes (b, b') of the relationships Km (day-1) = a + b([Hg(SH)20(aq)] + [HgS0(aq)]) and MeHg (microg kg-1) = a' + b'([Hg(SH)20(aq)] + [HgS0(aq)]) showed an inverse relationship with the C/N ratio, supposedly reflecting differences in primary production and energy-rich organic matter availability among sites. We conclude that concentrations of neutral inorganic mercury sulfide species, together with the availability of energy-rich organic matter, largely control Hg methylation rates in contaminated sediments. Furthermore, Hg(SH)20(aq) is suggested to be the dominant species taken up by MeHg producing bacteria in organic-rich sediments without formation of HgS(s).     

Acute toxicity of mercury to Daphnia magna under different conditions

We investigated the variations of acute toxicity of mercury (Hg) in Daphnia magna under different temperatures, population origins, body sizes, and Hg pre-exposures. We measured Hg concentrations in the water and in the surviving daphnids, and used the subcellular fractionation approach to determine Hg in the metal-sensitive fraction (MSF) to predict Hg toxicity. The 24-h median lethal concentrations and 24-h lethal body burden were 12-55 microg L(-1) and 10-26 mg kg(-1) wet wt, respectively. High Hg tolerance accompanied by reduced Hg uptake occurred in the daphnids under extreme conditions (low temperature and high pre-exposure to Hg). Correlating Hg levels in different compartments and daphnid survival resulted in the following order of sequence: aqueous Hg > whole body Hg > Hg in the MSF. However, the threshold lethal concentration of Hg (concentration causing 1% mortality) based on the concentration of Hg in the MSF was the best indicator of Hg toxicity. Therefore, the subcellular fractionation approach is less useful in explaining acute toxicity than is sub-lethal Hg toxicity. The number of Hg binding sites in the animals varied under different conditions but the affinity of the transporter to Hg generally decreased as the animals' tolerance increased. Mercury tolerance under different conditions could be enhanced by reducing the Hg uptake, enhancing the intrinsic tolerance, and/or increasing the detoxification activity.     

Reduction of oxidized mercury species by dicarboxylic acids (C2-C4): kinetic and product studies

Mercury is an environmental contaminant of global concern. The reduction of oxidized mercury species (Hg(II)) by organic acids to elemental mercury (Hg0) is significant for understanding the cycling of mercury between the atmosphere and aqueous systems. This study focused on the reduction of Hg(II) by small, semivolatile dicarboxylic acids (C2-C4). The reaction kinetics was studied using cold vapor atomic fluorescence spectroscopy (CVAFS), and the products of the reaction were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR) spectrometry. The effects of light, dissolved oxygen and chloride ion on reaction rates were also investigated. The highest reaction rates were observed in systems free of both oxygen and chloride ion with the second-order apparent rate constants of 1.2 x 10(4), 4.9 x 10(3), and 2.8 x 10(3) (L x mol(-1) x s(-1)) for oxalic, malonic, and succinic acids at pH 3.0 and T = 296 +/- 2 K, respectively. The photoreduction of Hg(II) was mediated by the complexes formed between Hg" and dicarboxylic acids, and the identified products were Hg0, hydroxycarboxylic acids and monocarboxylic acids. Our results also indicated that the presence of chloride ion significantly reduced the reduction rate by competing with the complexation of Hg" with dicarboxylic acids, while dissolved oxygen retarded the production of Hg0 by involving in the reoxidation of reduced Hg species to Hg(II). Based on our experimental results, a tentative mechanism is proposed and the potential environmental implications are discussed.     

Volatilization and recovery of mercury from mercury wastewater produced in the course of laboratory work using Acidithiobacillus ferrooxidans SUG 2-2 cells

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans SUG 2-2 is markedly resistant to mercuric chloride and can volatilize mercury (Hg0) from mercuric ion (Hg2+) under acidic conditions. To develop a microbial technique to volatilize and recover mercury from acidic and organic compound-containing mercury wastewater, which is usually produced in the course of everyday laboratory work in Okayama University, the effects of organic and inorganic chemicals on the mercury volatilization activity of A. ferrooxidans cells were studied. Among 55 chemicals tested, the mercury volatilization from a reaction mixture (pH 2.5) containing resting cells of SUG 2-2 (1 mg of protein) and mercury chloride (14 nmol) was strongly inhibited by AgNO3 (0.05 mM), K2CrO7 (1.0 mM), cysteine (1.0 mM), trichloroethylene (1 microM), and commercially produced detergents (0.05%). However, the strong inhibition by trichloroethylene and detergents was not observed when these organic compounds were chemically decomposed using Fenton's method before the treatment of the wastewater with SUG 2-2 cells. When 20 ml of water acidified with sulfuric acid (pH 2.5) containing ferrous sulfate (3%), diluted mercury wastewater (17.5 nmol of Hg2+) and SUG 2-2 cells (0.05 mg of protein) were incubated for 10 d at 30 degrees C, 47% of the total mercury in the wastewater was volatilized and recovered into a trapping reagent for metal mercury. However, when the organic compounds in the mercury wastewater were decomposed using Fenton's method and then treated with A. ferrooxidans cells, approximately 100% of the total mercury in the wastewater was volatilized and recovered.     

Simulation of the chemical fate and bioavailability of liquid elemental mercury drops from gold mining in Amazonian freshwater systems

Elemental mercury (Hg(o)) for gold amalgamation is the main process applied by artisanal gold miners in South America, leading to important discharges into freshwater ecosystems. Through a 28-day experimental approach based on indoor microcosms, we simulated the chemical fate and bioavailability of Hg(o) droplets in the presence or absence of sediment collected from a typical forest creek that is unaffected by gold mining activities. Our results clearly showed significant mercury transfers in the water column in both the dissolved gaseous Hg(o) and oxidized (Hg(II)) forms, with a marked effect of the presence of sediment. After 28 days, Hg total (HgT) concentration in the water column was 25 times higher in sediment-free units (108 +/- 17 vs. 4 +/- 0.4 nM). Methylmercury (MeHg) determinations in the dissolved fraction showed a significant increase only in the presence of sediment after 7 and 14 days. Zebrafish (Danio rerio) were used as indicators for mercury bioavailability. The HgT determinations in four organs revealed significant accumulation levels as early as 7 days exposure, with marked differences in favor of fish collected from the sediment-free units. Significant MeHg increases were observed in the four organs only when sediment was present. Genomic tools applied to estimate sulfate-reducing bacteria communities showed mercury impacts on their diversity and distribution in the different compartments (water, sediment, biofilm, fish gut).     

Dark oxidation of dissolved gaseous mercury in polar ice mimics

The low-temperature chemistry associated with environmentally available mercury has recently attracted considerable scientific interest due to the discovery of systemic gas-phase mercury depletion events (MDEs) which occur periodically at the poles. However, the fate of the mercury once it enters the snowpack is not fully understood, even its chemical speciation has yet to be well characterized. An issue that is of particular concern in frozen environments is the transformation of elemental mercury (Hg(0)) to more bioavailable oxidized forms, which can then be methylated by biotic and abiotic processes. The resulting methyl mercury species produced can bioaccumulate through the food chain and the health effects of this on humans and mammals have been well-documented. During the current study, a novel set of "freeze-induced" pathways, which can potentially affect the reactivity of dissolved gaseous mercury (DGM) were followed. The experiments were performed using environmentally relevant cosolutes at appropriate concentration levels and temperatures. Evidence is thereby presented that due to rate accelerations associated with the operation of the freeze-concentration effect, DGM is oxidized to Hg(2+) ions when frozen in the presence of a variety of materials including hydrogen peroxide, nitrous acid and the sulfuric acid/O(2) couple.     

Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization

Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5μg of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67g/l of ethanol) 8.7×10(4)CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5days) of alcoholic fermentation.     

Metal speciation dynamics and bioavailability: Zn(II) and Cd(II) uptake by mussel (Mytilus edulis) and carp (Cyprinus carpio)

In the analysis of metal biouptake from complexing environments, both chemical speciation and biological uptake characteristics have to be taken into account. The commonly used free ion activity model is based on equilibrium speciation and implies that diffusion of the bioactive free metal toward the organism is not rate-limiting. In the presence of complexes, however, sufficiently labile species might contribute to the biouptake via preceding dissociation. Coupling of the ensuing diffusional mass transfer flux of metal with the biouptake flux of free metal, the supposedly bioactive species, shows under which conditions labile metal complexes can contribute to the uptake. The goal of the present paper is to apply this type of analysis to experimental data on metal uptake by mussel (Mytilus edulis) and carp (Cyprinus carpio) in complexing environments. These biosystems have fairly well-characterized uptake parameters, but the uptake fluxes cannot be fully explained by considering equilibrium speciation only. For Zn(II) uptake by mussel, evidence was found for diffusional limitation at low concentrations, whereas for Cd(III) uptake by carp, diffusion is not limiting at all. The analysis provides an example of how a more comprehensive treatment of complex systems can be applied to real experimental data.     

Antagonistic interaction of mercury and selenium in a marine fish is dependent on their chemical species

It is well-known that selenium (Se) shows protective effects against mercury (Hg) bioaccumulation and toxicity, but the underlying effects of Se chemical species, concentration, and administration method are poorly known. In this study, we conducted laboratory studies on a marine fish Terapon jurbua to explain why Hg accumulation is reduced in the presence of Se observed in field studies. When Se and Hg were administrated concurrently in the fish diets, different Se species including selenite, selenate, seleno-dl-cystine (SeCys), and seleno-dl-methionine (SeMet) affected Hg bioaccumulation differently. At high concentration in fish diet (20 μg g(-1) normally), selenate and SeCys significantly reduced the dietary Hg(II) assimilation efficiency (AE) from 38% to 26%. After the fish were pre-exposed to dietary selenite or SeMet (7 μg g(-1) normally) for 22 days with significantly elevated Se body concentrations, the Hg(II) AEs were pronouncedly reduced (from 41% to 15-26%), whereas the dissolved uptake rate constant and elimination rate constant were less affected. In contrast to Hg(II), all the MeHg biokinetic parameters remained relatively constant whether Se was administrated simultaneously with the fish diet or when the fish were pre-exposed to Se with elevated body concentrations. Basic biokinetic measurements thus revealed that Se had direct interaction with Hg(II) during dietary assimilation rather than with MeHg and that different Se species had variable effects on Hg assimilation.