Studies of metal affinity interactions in metal recovery and bioremediation

Abstract

The primary goal of this dissertation project has been the study of metal affinity interactions in metal recovery and bioremediation. During the first part of this research a mathematical model that describes the affinity partitioning of metal ions in aqueous two-phase systems was derived. The model has been used to calculate complex formation constants between metal ions in solution and affinity ligands, satisfactorily describing their partition behavior. Simulation using this model shows the great effect that pH has on the partitioning of metal ions suggesting better conditions for the separation. Work on metal affinity interactions has led to the pursuit of characterization studies of metal uptake by microorganisms of relevance in bioremediation. The methanotrophic bacterium M. trichosporium 0B3b a mutant culture (PP358) that expresses soluble methane monooxygenase (sMMO) independent of the external copper concentration have been the subject of this research. Knowledge of substances and/or mechanisms that are involved in the copper uptake by M. trichosporium 0B3b will greatly facilitate application of this or like species to the bioremediation of hazardous waste. Specifically, the role of an extracellular copper-binding biochelator (CBL) in copper uptake by Methylosinus trichosporium 0B3b has been investigated. Experiments included the identification and physical characterization of the biochelator and elucidation of the environmental factors that affect its production. The biochelator is apparently an aromatic, low-molecular weight, hydrophobic molecule with high affinity and selectivity for copper. Results indicate that the mutation in PP358 is unrelated to possible defects in biochelator functionality and strongly suggest that the CBL is directly involved in the copper acquisition mechanism of this methanotroph. Finally, an existing colorimetric method currently used in the qualitative determination of sMMO has been modified and improved to provide additional quantitative information. Until now, the instability of one of the products of the reaction on which the current method is based has precluded the effective use of the assay as a quantitative tool. Stabilization of the compound of interest has been achieved, allowing the successful quantification of sMMO activity from M. trichosporium 0B3b and propane monooxygenase activity from the propane oxidizer M. vaccae JOB5.