Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

Spatially oscillating activity and microbial succession of mercury-reducing biofilms in a technical-scale bioremediation system

Mercury-contaminated chemical wastewater of a mercury cell chloralkali plant was cleaned on site by a technical-scale bioremediation system. Microbial mercury reduction of soluble Hg(II) to precipitating Hg(0) decreased the mercury load of the wastewater during its flow through the bioremediation system by up to 99%. The system consisted of a packed-bed bioreactor, where most of the wastewater's mercury load was retained, and an activated carbon filter, where residual mercury was removed from the bioreactor effluent by both physical adsorption and biological reduction. In response to the oscillation of the mercury concentration in the bioreactor inflow, the zone of maximum mercury reduction oscillated regularly between the lower and the upper bioreactor horizons or the carbon filter. At low mercury concentrations, maximum mercury reduction occurred near the inflow at the bottom of the bioreactor. At high concentrations, the zone of maximum activity moved to the upper horizons. The composition of the bioreactor and carbon filter biofilms was investigated by 16S-23S ribosomal DNA intergenic spacer polymorphism analysis. Analysis of spatial biofilm variation showed an increasing microbial diversity along a gradient of decreasing mercury concentrations. Temporal analysis of the bioreactor community revealed a stable abundance of two prevalent strains and a succession of several invading mercury-resistant strains which was driven by the selection pressure of high mercury concentrations. In the activated carbon filter, a lower selection pressure permitted a steady increase in diversity during 240 days of operation and the establishment of one mercury-sensitive invader.     

细菌抗汞分子机制与进化的研究及应用

微生物中存在一类抗汞细菌,操纵子Mer中的MerRTPA参与细菌抗汞的调控、转运及还原。汞通过MerTP所表达的蛋白由细胞外转运至细胞内,经还原酶MerA将其还原为毒性小的可挥发的金属汞。细菌抗汞基因的形成有着古老的起源,基因间的整合、转移进化形成了Mer操纵子结构与功能的多样性。抗汞细菌对汞的吸附具有高选择性及专一性,可利用此特性对汞污染环境进行修复,也可作为分子遗传操作中稳定的抗性筛选标记。     

Spatially Oscillating Activity and Microbial Succession of Mercury-Reducing Biofilms in a Technical-Scale Bioremediation System

Mercury-contaminated chemical wastewater of a mercury cell chloralkali plant was cleaned on site by a technical-scale bioremediation system. Microbial mercury reduction of soluble Hg(II) to precipitating Hg(0) decreased the mercury load of the wastewater during its flow through the bioremediation system by up to 99%. The system consisted of a packed-bed bioreactor, where most of the wastewater's mercury load was retained, and an activated carbon filter, where residual mercury was removed from the bioreactor effluent by both physical adsorption and biological reduction. In response to the oscillation of the mercury concentration in the bioreactor inflow, the zone of maximum mercury reduction oscillated regularly between the lower and the upper bioreactor horizons or the carbon filter. At low mercury concentrations, maximum mercury reduction occurred near the inflow at the bottom of the bioreactor. At high concentrations, the zone of maximum activity moved to the upper horizons. The composition of the bioreactor and carbon filter biofilms was investigated by 16S-23S ribosomal DNA intergenic spacer polymorphism analysis. Analysis of spatial biofilm variation showed an increasing microbial diversity along a gradient of decreasing mercury concentrations. Temporal analysis of the bioreactor community revealed a stable abundance of two prevalent strains and a succession of several invading mercury-resistant strains which was driven by the selection pressure of high mercury concentrations. In the activated carbon filter, a lower selection pressure permitted a steady increase in diversity during 240 days of operation and the establishment of one mercury-sensitive invader.     

Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene

INTRODUCTIONEnvironmental pollution is an increasing prob-lem both fOr developing and developed countries.Mercury, both in organic and ionic fOrm, is one of themost hazardous pollutants among the heavy met-als[l]and its accumuIation in human body results ininactivation of metabolic enzymes and structuralproteins[2, 3] giving rise to serious health problems(Minamatasyndrome).Usually mercury pollution is caused by indus-trial and agricultural activities, releasing mercuryinto air, water an…     

Volatilization of mercury by immobilized mercury-resistant bacterial cells

Highly toxic mercury compounds may come into the environment through the use of mercury compounds as disinfectants for hospital and household purposes, Hg catalyst in industries, burning of coal and petroleum products, mercury-based pesticides and fungicides used in agriculture, and seed dressings. Toxic effects of mercury can be counteracted by microbial cells through the enzymes mercuric reductase and organomercurial lyase. Immobilized mercury-resistant bacterial cells of Azotobacter chroococcum could effectively volatilize mercury from mercury-containing buffer and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells, as immobilized cells can be reused. Immobilized cells continuously volatilized mercury from mercury-containing buffer after four consecutive 24 h cycles. The storage stability of immobilized cells was much better than that of the native cells.     

Functioning of the mercury resistance operon at extremely high Hg(II) loads in a chemostat: a proteome analysis

The transformation of extremely high concentrations of ionic mercury (up to 500 mg L(-1)) was investigated in a chemostat for two mercury-resistant Pseudomonas putida strains, the sediment isolate Spi3 carrying a regulated mercury resistance (mer) operon, and the genetically engineered strain KT2442Colon, two colonsmer73 expressing the mer operon constitutively. Both strains reduced Hg(II) with an efficiency of 99.9% even at the maximum load, but the concentration of particle bound mercury in the chemostat increased strongly. A proteome analysis using two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) showed constant expression of the MerA and MerB proteins in KT2442Colon, two colonsmer73 as expected, while in Spi3 expression of both proteins was strongly dependent on the Hg(II) concentration. The total cellular proteome of the two strains showed very little changes at high Hg(II) load. However, certain cellular responses of the two strains were identified, especially in membrane-related transport proteins. In Spi3, an up to 45-fold strong induction of a cation efflux transporter was observed, accompanied by a drastic downregulation (106-fold) of an outer membrane porin. In such a way, the cell complemented the highly specific mercury resistance mechanism with a general detoxification response. No indication of a higher demand on energy metabolism could be found for both strains.     

Diversity and characterization of mercury-resistant bacteria in snow, freshwater and sea-ice brine from the High Arctic

It is well-established that atmospheric deposition transports mercury from lower latitudes to the Arctic. The role of bacteria in the dynamics of the deposited mercury, however, is unknown. We characterized mercury-resistant bacteria from High Arctic snow, freshwater and sea-ice brine. Bacterial densities were 9.4 × 10(5), 5 × 10(5) and 0.9-3.1 × 10(3) cells mL(-1) in freshwater, brine and snow, respectively. Highest cultivability was observed in snow (11.9%), followed by freshwater (0.3%) and brine (0.03%). In snow, the mercury-resistant bacteria accounted for up to 31% of the culturable bacteria, but <2% in freshwater and brine. The resistant bacteria belonged to the Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. Resistance levels of most isolates were not temperature dependent. Of the resistant isolates, 25% reduced Hg(II) to Hg(0). No relation between resistance level, ability to reduce Hg(II) and phylogenetic group was observed. An estimation of the potential bacterial reduction of Hg(II) in snow suggested that it was important in the deeper snow layers where light attenuation inhibited photoreduction. Thus, by reducing Hg(II) to Hg(0), mercury-resistant bacteria may limit the supply of substrate for methylation processes and, hence, contribute to lowering the risk that methylmercury is being incorporated into the Arctic food chains.     

Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain

A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater.     

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg⁰, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.     

Removal of Mercury from Chloralkali Electrolysis Wastewater by a Mercury-Resistant Pseudomonas putida Strain

A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater.     

Development of microbial mercury detoxification processes using mercury-hyperresistant strain of Pseudomonas aeruginosa PU21

A mercury-hyperresistant strain of Pseudomonas aeruginosa PU21 harboring plasmid Rip64 was utilized to develop bioprocesses able to detoxify and recover soluble mercuric ions in aquatic systems. The kinetics of mercury detoxification was investigated to determine the parameters needed for the design of the bioprocesses. Batch, fed-batch, and continuous bioreactors were utilized to evaluate the efficiency and feasibility of each mode of operation. The results showed that the specific mercury detoxification rate was dependent on cell growth phases, as well as the initial mercury concentrations. Cells at the lag growth phase exhibited the best specific detoxification rate of approximately 1.1 x 10(-6) microg Hg/cell/h, and the rate was optimal at an initial mercury concentration of 8 mg/L. In batch operations with initial mercuric ions ranging from 2 to 10 mg/L, the mercuric ions added were rapidly volatilized from the media in less than 2-3 h. With periodic feeding of 3 or 5 mg Hg/L at fixed time intervals, the fed-batch processes had mercury removal efficiencies of 2.9 and 3.3 mg Hg/h/L, respectively. For continuous operations, the effluent cell concentration (Xe) was essentially invariant at 527 and 523 mg/L with the dilution rates (D) of 0.18 and 0.325 h-1, respectively. The increase in mercury feeding concentrations (Hgf) from 1.0 to 6.15 mg Hg2+/L did not affect the steady-state cell concentration (Xe) but forced the effluent mercury concentration (Hge) to increase. The decrease in the dilution rate, however, resulted in lower Hge values. It was also found that sequential mercury vapor absorption columns recovered over 80% of the Hg degrees released from the bioreactor while the residual mercury vapor was subsequently immobilized by an activated carbon trap in the down stream of the absorption column.     

Biosorption of mercury by the inactivated cells of pseudomonas aeruginosa PU21 (Rip64)

Biomass of a mercury-resistant strain Pseudomonas aeruginosa PU21 (Rip64) and hydrogen-form cation exchange resin (AG 50W-X8) were investigated for their ability to adsorb mercury. The maximum adsorption capacity was approximately 180 mg Hg/g dry cell in deionized water and 400 mg Hg/g dry cell in sodium phosphate solution at pH 7.4, higher than the maximum mercury uptake capacity in the cation exchange resin (100 mg Hg/g dry resin in deionized water). The mercury selectivity of the biomass over sodium ions was evaluated when 50 mM and 150 mM of Na(+) were present. Biosorption of mercury was also examined in sodium phosphate solution andphosphate-buffered saline solution (pH 7.0), containing 50mM and 150 mM of Na(+), respectively. It was found that the presence of Na(+) did not severely affect the biosorption of Hg(2+), indicating a high mercury selectivity ofthe biomass over sodium ions. In contrast, the mercury uptake by the ion exchange resin was strongly inhibited by high sodium concentrations. The mercury biosorption was most favorable in sodium phosphate solution (pH 7.4), with a more than twofold increase in the maximum mercury uptake capacity. The pH was found to affect the adsorption of Hg(2+)bythe biomass and the optimal pH value was approximately 7.4. The adsorption of mercury on the biomass and the ion exchange resin appeared to follow theLangmuir or Freundlich adsorption isotherms. (c) 1994 John Wiley & Sons, Inc.     

Detoxification of Toxic Heavy Metals by Marine Bacteria Highly Resistant to Mercury

Pollution in industrial areas is a serious environmental concern, and interest in bacterial resistance to heavy metals is of practical significance. Mercury (Hg), Cadmium (Cd), and lead (Pb) are known to cause damage to living organisms, including human beings. Several marine bacteria highly resistant to mercury (BHRM) capable of growing at 25 ppm (mg L(-1)) or higher concentrations of mercury were tested during this study to evaluate their potential to detoxify Cd and Pb. Results indicate their potential of detoxification not only of Hg, but also Cd and Pb. Through biochemical and 16S rRNA gene sequence analyses, these bacteria were identified to belong to Alcaligenes faecalis (seven isolates), Bacillus pumilus (three isolates), Bacillus sp. (one isolate), Pseudomonas aeruginosa (one isolate), and Brevibacterium iodinium (one isolate). The mechanisms of heavy metal detoxification were through volatilization (for Hg), putative entrapment in the extracellular polymeric substance (for Hg, Cd and Pb) as revealed by the scanning electron microscopy and energy dispersive x-ray spectroscopy, and/or precipitation as sulfide (for Pb). These bacteria removed more than 70% of Cd and 98% of Pb within 72 and 96 h, respectively, from growth medium that had initial metal concentrations of 100 ppm. Their detoxification efficiency for Hg, Cd and Pb indicates good potential for application in bioremediation of toxic heavy metals.     

Mechanisms of mercury bioremediation

Mercury is one of the most toxic heavy metals, and has significant industrial and agricultural uses. These uses have led to severe localized mercury pollution. Mercury volatilization after its reduction to the metallic form by mercury-resistant bacteria has been reported as a mechanism for mercury bioremediation [Brunke, Deckwer, Frischmuth, Horn, Lunsdorf, Rhode, Rohricht, Timmis and Weppen (1993) FEMS Microbiol. Rev. 11, 145-152; von Canstein, Timmis, Deckwer and Wagner-Dobler (1999) Appl. Environ. Microbiol. 65, 5279-5284]. The reduction/volatilization system requires to be studied further, in order to eliminate the escape of the metallic mercury into the environment. Recently we have demonstrated three different mechanisms for mercury detoxification in one organism, Klebsiella pneumoniae M426, which may increase the capture efficiency of mercury.     

Metallothioneins in marine mammals.

Metallothioneins (MTs) have been detected in livers and kidneys of 10 marine mammals species (Pinnipeds and Odontocetes). Characterization of renal MTs of striped dolphin has shown that the protein has two isoforms (MT-1 and MT-2) with a molecular weight estimated around 6,800. MT concentrations also vary widely in marine mammals tissues (from 58 to 1,200 microg x g(-1) ww) underlying the numerous parameters involved: physiological status, pregnancy, age, diet. The participation of this protein in metal detoxification has been investigated since high levels of cadmium (Cd) and mercury (Hg) have been measured in livers and kidneys of marine mammals. It has been suggested that those animals can mitigate at least in part, the toxic effects of Cd and Hg through binding to MTs. The percentage of the cytosolic Cd bound to MTs can reach almost 100%. On the contrary, the percentage of hepatic and renal Hg bound to MT is very low (generally less than 10%) and this metal is mainly associated with selenium (HgSe) under a detoxified form in the insoluble fraction of the tissues. MTs appear to play a minor role in the binding and detoxification of Hg by marine mammals. On the contrary, close and dynamic interactions occur between Cd and MTs. Cytosolic MTs appear as a potential short term way of detoxification of Cd accumulated from diet. Long-term detoxification would imply a sequestration of the metal under a precipitated form (e.g. in lysosomes).     

Potential for mercury reduction by microbes in the high arctic

The contamination of polar regions due to the global distribution of anthropogenic pollutants is of great concern because it leads to the bioaccumulation of toxic substances, methylmercury among them, in Arctic food chains. Here we present the first evidence that microbes in the high Arctic possess and express diverse merA genes, which specify the reduction of ionic mercury [Hg(II)] to the volatile elemental form [Hg(0)]. The sampled microbial biomass, collected from microbial mats in a coastal lagoon and from the surface of marine macroalgae, was comprised of bacteria that were most closely related to psychrophiles that had previously been described in polar environments. We used a kinetic redox model, taking into consideration photoredox reactions as well as mer-mediated reduction, to assess if the potential for Hg(II) reduction by Arctic microbes can affect the toxicity and environmental mobility of mercury in the high Arctic. Results suggested that mer-mediated Hg(II) reduction could account for most of the Hg(0) that is produced in high Arctic waters. At the surface, with only 5% metabolically active cells, up to 68% of the mercury pool was resolved by the model as biogenic Hg(0). At a greater depth, because of incident light attenuation, the significance of photoredox transformations declined and merA-mediated activity could account for up to 90% of Hg(0) production. These findings highlight the importance of microbial redox transformations in the biogeochemical cycling, and thus the toxicity and mobility, of mercury in polar regions.     

Phytodetoxification of hazardous organomercurials by genetically engineered plants

Methylmercury is a highly toxic, organic derivative found in mercury-polluted wetlands and coastal sediments worldwide. Though commonly present at low concentrations in the substrate, methylmercury can biomagnify to concentrations that poison predatory animals and humans. In the interest of developing an in situ detoxification strategy, a model plant system was transformed with bacterial genes (merA for mercuric reductase and merB for organomercurial lyase) for an organic mercury detoxification pathway. Arabidopsis thaliana plants expressing both genes grow on 50-fold higher methylmercury concentrations than wild-type plants and up to 10-fold higher concentrations than plants that express merB alone. An in vivo assay demonstrated that both transgenes are required for plants to detoxify organic mercury by converting it to volatile and much less toxic elemental mercury.     

Tolerance to Various Toxicants by Marine Bacteria Highly Resistant to Mercury

Bacteria highly resistant to mercury isolated from seawater and sediment samples were tested for growth in the presence of different heavy metals, pesticides, phenol, formaldehyde, formic acid, and trichloroethane to investigate their potential for growth in the presence of a variety of toxic xenobiotics. We hypothesized that bacteria resistant to high concentrations of mercury would have potential capacities to tolerate or possibly degrade a variety of toxic materials and thus would be important in environmental pollution bioremediation. The mercury-resistant bacteria were found to belong to Pseudomonas, Proteus, Xanthomonas, Alteromonas, Aeromonas, and Enterobacteriaceae. All these environmental bacterial strains tolerant to mercury used in this study were capable of growth at a far higher concentration (50 ppm) of mercury than previously reported. Likewise, their ability to grow in the presence of toxic xenobiotics, either singly or in combination, was superior to that of bacteria incapable of growth in media containing 5 ppm mercury. Plasmid-curing assays done in this study ascertained that resistance to mercury antibiotics, and toxic xenobiotics is mediated by chromosomally borne genes and/or transposable elements rather than by plasmids.     

Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.