ASSESSMENT OF MERCURY (II) SPECIES BIOAVAILABILITY USING A BIOLUMINESCENT BACTERIAL BIOSENSOR

AUTOR(ES)
DATA DE PUBLICAÇÃO

2003

RESUMO

Mercury and its compounds are highly toxic, especially methylmercury - a potent neurotoxin. It has caused a significant number of human fatalities in several accidents around the world. Due to its wide dispersion through the atmosphere, mercury is considered a global pollutant, being deposited even in remote pristine aquatic systems, where it is biomagnified through the food chain. Although significant advances in our understanding of Hg biogeochemistry have been made in the last 30 years, a key issue is not well understood yet: How does the environmental speciation of Hg(II) control its bioavailability to methylating bacteria? This gap is in part due to the inability of traditional chemical speciation methods to accurately assess bioavailability. New techniques have been developed to address this critical issue. Among them, the biosensors show great potential. They offer a fast and inexpensive alternative for the detection of specific biologically important compounds. The objective of this research was to investigate the bioavailability of various Hg(II) species in laboratory and natural solutions using a mercury bioluminescent bacterial biosensor. We used an E.coli strain that was genetically altered to produce firefly luciferase in proportion to its exposure to bioavailable Hg(II). Computer modeling of the Hg(II) solution speciation was used to interpret the observed Hg(II) uptake by the biosensor. In the first part of the research, an analytical protocol was developed and optimized. The detection limit of the method (0.7 pM) is superior to most of the other reported Hg(II) biosensors and adequate to analyze mercury bioavailability at natural levels. The biosensor showed high specificity for Hg(II) and good reproducibility. The feasibility of using the Hg biosensor approach to determine the bioavailable Hg(II) concentrations in natural samples was assessed. Environmental samples from two Florida lakes and from the Everglades were analyzed. The experiments were successful, showing a dose-response relationship between Hg(II) added to the natural samples and the light produced by the biosensor. In the natural samples tested, the response to added Hg(II) seemed to be controlled by natural organic ligand complexation. However, the results also reinforced the need for careful chemical characterization of the samples and the use of a constitutive control strain to avoid bias in the results. In addition, a model was developed that uses the biosensor response to Hg(II) additions to calculate the total concentration of natural ligands that can bind Hg(II) and the apparent equilibrium constant for the Hg(II)-ligand complexation. In the second phase of the research, Hg(II) speciation was manipulated by the addition of inorganic and organic ligands and the bioavailabilities of the species formed were assessed. The chloride titration results suggested that neutral Hg(II) chloride complexes were more bioavailable than anionic Hg(II) chloride complexes. The two relevant Hg(II) neutral species in oxic solutions, Hg(OH)20 and HgCl20, showed similar bioavailability. These data agree with other studies performed with a different mercury biosensor, and support the hypothesis that neutral Hg(II) species may play an important role in bacterial Hg(II) uptake in the environment. The addition of EDTA decreased the biosensors response in both synthetic solutions and natural samples. This reduction was proportional to the total EDTA concentration, suggesting that the HgEDTA complexes had lower bioavailability. However, the combined experimental data revealed that the relationship between EDTA added and Hg(II) bioavailability was more complex than expected. Considering the Hg(II) and EDTA concentrations used in the experiments and the modeled speciation, as well as kinetic experiment data, it seems that the bacteria were able to incorporate part of the Hg(II) initially complexed by EDTA. On the other hand, EDTA addition appears to reduce the permeability of the bacterial cells to bioavailable Hg(II) species. Further studies are necessary to elucidate the roles EDTA may have in Hg(II) bioavailability to bacteria. In the last part of this study, we performed kinetic experiments to investigate the Hg(II) uptake process. The experimental data agreed with the threshold kinetic model proposed for the mer operon/Mer R protein response to Hg(II). The half-saturation constant for the enzymatic reaction was estimated, and was consistent for two different exposure times, however the maximum apparent Hg(II) uptake rates varied according to the exposure time. Longer exposure times resulted in lower apparent Hg(II) uptake rates, even when the Hg biosensor response was higher. The Hg biosensor response displayed non-linear increases to both increasing exposure times and Hg(II) concentrations. According to our kinetic results, both non-linear behaviors are probably due to the saturation of the mer operon/Mer R protein with Hg(II), which can cause the saturation of luciferase synthesis. The analysis of the Hg biosensor cells for the their total Hg(II) concentration during a kinetic assay revealed no evidence of saturation of the Hg(II) uptake process. The kinetic results agree with our previous titration data and support the hypothesis that the diffusion of neutral Hg(II) complexes is an important Hg(II) uptake mechanism for bacterial cells.

ASSUNTO(S)

mercury bioluminescente oceanografia quimica biodisponibilidade mercurio bioluminescent bacterial biosensor bactéria biosensor especiação química

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