Species Diversity Improves the Efficiency of Mercury-Reducing Biofilms under Changing Environmental Conditions

Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant Pseudomonas putida strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater. The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency). Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped. In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater. Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations. The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis. Mercury-mediated selective pressure decreased the number of prevalent strains. Microbial diversity was completely restored after easing of the selective pressure. Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.     

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.     

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.     

Pilot plant for bioremediation of mercury-containing industrial wastewater

Mercury is an extremely toxic pollutant that is currently being emitted mainly by low level industrial sources. It is distributed globally through the atmosphere, from where it precipitates onto the surface of the Earth, enters aquatic organisms, accumulates in fish and finally affects the health of human populations. Microbes have evolved a mechanism for mercury detoxification [mercury resistance operon ( mer)] based on intracellular reduction of Hg(2+) to non-toxic Hg(0) by the mercuric reductase enzyme and subsequent diffusional loss of Hg(0) from the cell. It was shown that Hg(0) produced by microbial detoxification can be retained quantitatively in packed bed bioreactors, in which biofilms of mercury-resistant bacteria are grown on porous carrier material. This review describes operation of this system on a technical, fully automated, scale, and its operation at a chloralkali electrolysis factory. It was shown to work with high efficiency under fluctuating mercury concentrations and to be robust against transiently toxic conditions. The gradient of mercury concentration in the technical scale system exerted a strong selective pressure on the microbial community, which resulted in a succession of mercury-resistant strains at high mercury concentrations and an increase in phylogenetic and functional diversity at low mercury concentrations. Clean-up of mercury-containing wastewater by mercury-resistant microbes is a simple, environmentally friendly and cost-effective alternative to current treatment technologies.     

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.     

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.     

Long-term performance of bioreactors cleaning mercury-contaminated wastewater and their response to temperature and mercury stress and mechanical perturbation

The long-term performance of bioreactors retaining mercury from contaminated industrial wastewater was analyzed at the laboratory scale, and its response to mechanical perturbations (gas bubbles and shaking) as well as to physical (increased temperature and hydraulic load) and chemical stresses (increased mercury concentration) likely to occur during on site operation was studied. Two packed-bed bioreactors with 80-cm(3) lava chips as biofilm carrier were inoculated with nine Hg(II)-resistant natural isolates of alpha- and gamma-proteobacteria. Chloralkali wastewater containing ionic mercury (3.0 to 9.7 mg/L Hg(2+)), amended with sucrose and yeast extract, flowed through the bioreactors at 160 mL/h. During the 16-month investigation the bioreactors showed no sign of depleted performance in terms of mercury-retaining capacity. After 16 months, both bioreactors still retained 96% of the mercury load. The performance of the bioreactors was sensitive to mechanical perturbations (e.g., sheer forces of gas bubbles). Shifts to higher Hg(2+) inflow concentrations initially decreased the mercury retention efficacy slightly. However, the bioreactors could adapt to Hg(2+) concentrations of up to 7.6 mg/L within several days. Old biofilms were less affected than the younger ones. The performance of the bioreactors was not affected by an increase in temperature up to 41 degrees C and an increased volumetric load (up to 240 mL/h). The bioreactors regained activity spontaneously after the stress had stopped. Recovery could be accelerated by increased nutrient concentration, although this may lead to blocking of the packed bed.     

Long-Term Stability of Mercury-Reducing Microbial Biofilm Communities Analyzed by 16S-23S rDNA Interspacer Region Polymorphism

The composition of mercury-reducing communities in two bioreactors retaining Hg(II) from chloralkali electrolysis wastewater for 485 days was analyzed based on effluent community DNA. Packed bed bioreactors with lava chips as carrier of the biofilm were inoculated with nine Hg(II)-resistant isolates that belonged to the alpha and gamma subdivisions of the proteobacteria. A rapid DNA-fingerprinting method was applied, using the intergenic spacer region (ISR) of the 16S-23S rDNA for analysis of the community composition. This allowed discrimination of the inoculum strains down to subspecies level. A merA specific PCR permitted the discrimination of the community's merA genes. During the 485 days of operation, the bioreactors were exposed to various physical stresses (mixing, gas bubbles, temperature increase up to 41°C, increased flow velocity) and repeated high mercury inflow concentrations, resulting in reduced bioreactor performance and decreased culturable cell numbers in the reactor effluent. Nevertheless, the composition of the microbial community remained rather stable throughout the investigated time period. Of the inoculum strains, two could be detected throughout, whereas three were sometimes present with varying periods of nondetection. Two inoculum strains were only detected within the first month. Two strains of gamma-proteobacteria that were able to reduce ionic mercury invaded the bioreactor community. They did not outcompete established strains and had no negative effect on the Hg(II)-retention activity of the bioreactors. The community comprised diverse merA genes. The abundance of merA genes matched the abundance of their respective strains as confirmed by ISR community analysis. The continuously high selection pressure for mercury resistance maintained a stable and highly active mercury-reducing microbial community within the bioreactors.     

The diversity of mercury reductases among mercury-resistant bacteria

Two immunologically non-cross-reactive types of mercury reductases were found among Gram-negative and two among Gram-positive mercury-resistant environmental bacteria. Mercury reductases were further discriminated by 'spur' formation immunodiffusion tests. Immunologically indistinguishable mercury reductases were found among strains belonging to phylogenetically distant genera. This suggests a horizontal transfer of mercury resistance genes between these strains.     

Microbial characterisation of activated sludge in jet-loop bioreactors treating winery wastewaters

Jet-loop reactors (JLR) used as biological waste treatment processes introduce an additional selective pressure on the natural microbial flora of the incoming effluent. Several high-performing microbial inocula were tested for winery wastewater treatment and the microbial composition was analysed. A microbial consortium was enriched and selected for use with a new type of aerobic JLR. The reactor was operated continuously for more than 1 year using winery wastewaters collected in different seasons. Chemical oxygen demand (COD) removal efficiency was on average greater than 80%, with retention times of 0.8–1 day. Microbial populations were sampled for characterisation after 6 months and at the end of the study. Isolates were identified at genus and/or species level. Almost all isolates belonged to the genera Pseudomonas and Bacillus. Saccharomyces cerevisiae was also found but no filamentous fungi. These results show that a highly adapted population develops in JLRs treating winery effluents as compared to other bioreactors. Aerobic JLRs impose a stringent selective criterion on the composition of the microbial biomass.     

Biodegradation of dimethyl phthalate with high removal rates in a packed-bed reactor

Biological treatment of a dimethyl phthalate (DMP)-containing waste stream was evaluated in packed-bed bioreactors using an acclimated mixed bacterial culture. The passive immobilization start-up strategy was successful in the development of a stable biofilm on the packing material in the reactor. Nutrient supplementation significantly improved the removal efficiency. High removal rates with 100% efficiencies of DMP removal were achieved up to the phthalate-loading rate of 560 g/m³ h.     

Fast kinetics of fe oxidation in packed-bed reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe oxidized per liter per h (1,400 mmol of Fe oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria

Abstract: The microorganisms used for the mercury retention experiments were natural isolates and genetically engineered bacteria. All mercury-resistant strains contained the merA gene. Column experiments with these strains were carried out by immobilizing them on different support materials. To obtain kinetic data of the reductase activity for whole cells and the crude extract, batch experiments were carried out under different conditions.     

Fast Kinetics of Fe2+ Oxidation in Packed-Bed Reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe²⁺ oxidized per liter per h (1,400 mmol of Fe²⁺ oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.     

Structure and species composition of mercury-reducing biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Structure and Species Composition of Mercury-Reducing Biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Effects of Cinnabar on Pyrite Oxidation by Thiobacillus ferrooxidans and Cinnabar Mobilization by a Mercury-Resistant Strain

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.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.     

Volatilization of Mercury by Immobilized Bacteria (Klebsiella pneumoniae) in Different Support by Using Fluidized Bed Bioreactor

Klebsiella pneumoniae, a mercury-resistant bacterial strain able to reduce ionic mercury to metallic mercury, was isolated from wastewater of Casablanca. This strain exhibits high minimal inhibition concentrations for heavy metals such as mercury 2400 μM, lead 8000 μM, silver 2400 μM, and cadmium 1000 μM. This bacterium was immobilized in alginate, polyacrylamide, vermiculite, and cooper beech and was used for removing mercury from a synthetic water polluted by mercury by using a fluidized bead bioreactor. Immobilized bacterial cells of Klebsiella pneumoniae could effectively volatilize mercury and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells. The highest cleanup and volatilization rates were obtained when Klebsiella pneumoniae was entrapped in alginate beads, with a cleanup rate of 100% and a volatilization rate of 89%. Immobilized cells in alginate continuously volatilized mercury even after 10 days without loss of activity. Received: 21 February 2001 / Accepted: 13 March 2001