Assessment of Mercury (II) Species Bioavailability Using a Bioluminescent Bacterial Biosensor

• Other Authors: Barrocas, Paulo Rubens Guimarães (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biology
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-1114

The objective of this research was to investigate the bioavailability of various Hg(II) species in laboratory and natural solutions using a bioluminescent bacterial biosensor. The Hg(II) bacterial biosensor is a genetically engineered E. coli strain, which produces firefly luciferase in proportion to its exposure to bioavailable Hg(II). The amount of luciferase inside the cells is assessed through its bioluminescent reaction with luciferin. A new analytical protocol for the use of bacterial bioassays to study the bioavailability of trace elements and relate it to their modeled chemical speciation was developed. The biosensor showed high specificity for Hg(II) and good reproducibility. 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 feasibility of using this new biosensor method to analyze natural samples was assessed. An approach for estimating conditional equilibrium constants for the complexation of Hg(II) by natural ligands is presented. The influence of inorganic and organic ligands on the Hg(II) speciation and bioavailability was investigated. Chloride titration results suggested that neutral HgCl20 and Hg(OH)20 complexes were more bioavailable than anionic Hg(II) chloride complexes. The addition of EDTA decreased the biosensor's response in both synthetic solutions and natural samples. This reduction was proportional to the total EDTA concentration. Kinetic experiments were performed to evaluate the Hg(II) uptake process by the bacterial biosensor. The experimental data agreed with the ultrasensitive kinetic model proposed for the Mer R protein response to Hg(II). The half-saturation constant for the enzymatic reaction was estimated. The Hg biosensor response displayed non-linear increases to both increasing exposure times and Hg(II) concentrations. According to the kinetic results, both non-linear behaviors are probably due to the saturation of the mer operon/Mer R protein with Hg(II). The analysis of the biosensor cells for their total Hg(II) concentration during a kinetic assay revealed no evidence for saturation of the Hg(II) uptake process. The kinetic results agree with the titration data and support the hypothesis that the diffusion of neutral Hg(II) complexes is an important Hg(II) uptake mechanism for bacterial cells. === A Dissertation Submitted to the Department of Oceanography in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. === Spring Semester, 2004. === November 25, 2003. === Bioluminescent, Biosensor, Bioavailability, Mercury, Chemical Speciation === Includes bibliographical references. === William M. Landing, Professor Directing Dissertation; Robert H. Reeves, Outside Committee Member; Lita M. Proctor, Committee Member; Joel E. Kostka, Committee Member; Jeffrey P. Chanton, Committee Member.