Trichloroethylene (TCE) is a toxic pollutant that has become a widespread problem by seeping into groundwater across the developed world. Clean-up of sites contaminated with TCE is extremely difficult due to the absence of an efficient and cost-effective method for clean-up. Bioremediation efforts include a variety of potential microbial candidates with various metabolic capabilities as clean up options of contaminated sites. Cupriavidus necator, a soil bacterium was found to possess the ability to degrade TCE via a phenol-dependent pathway. Previous research by Ayoubi and Harker (1998) created a strain (MM02) capable of constitutive TCE degradation but the underlying genetic alteration causing constitutive production of the phenol hydroxylase pathway (PHL) and TCE breakdown was poorly characterized. We attempted to gain further understanding of the alterations that occurred in the PHL pathway to cause TCE to break down and replicate constitutive TCE degradation in a new strain with reduced foreign elements that may be introduced into the environment. Strain MM02 possessing this constitutive degradation activity and strain MM01were sequenced and compared to discover the source of this variation. A 210 base-pair deletion in the beginning of the PHL operon was identified and is likely the cause of this altered activity. The new strain of C. necator (MM14) was created using traditional bacterial mating methods and included a cleanly introduced kanamycin resistance gene and its associated promoter which could drive constitutive expression of the PHL pathway. The TCE degradation abilities of strains MM01, MM02, and MM14 were evaluated through the TCE degradation assay and gas chromatography. We had difficulty accurately measuring the concentration of TCE due to its volatile nature and dramatically altered the method ultimately reducing variation and capturing TCE concentrations in assays. When accurate readings were obtained, none of the strains measured exhibited quantifiable TCE degradation activity when compared to controls. Our results showed .08% of the degradation by strain MM02 measured previously (P. J. Ayoubi, 1997). Based on our findings, we were unable to replicate the TCE degradation caused my MM02 and our genetically modified strain also failed to breakdown TCE.

Effluents of industrial wastewaters from oil refineries, paper mills, dyes, ceramic factories, resins, textiles and plastic contain high concentrations of aromatic compounds, which are toxic to organisms. Degradation of these compounds to tolerant limits before releasing them into the environment is an urgent requirement. Candida tropicalis yeast is an important representative of eucaryotic microorganisms that are able to utilize phenol. During the first phase of phenol biodegradation, cytoplasmatic NADPH-dependent phenol hydroxylase of C. tropicatis oxidizes phenol to catechol. Catechol is in the second phase of biodegradative process oxidized to cis,cis-muconic acid by the reaction catalyzed with catechol-1,2-dioxygenase. In this diploma thesis we investigated the effect of the heavy metal ions on NADPH-dependent phenol hydroxylase and catechol-1,2-dioxygenase of C. tropicalis. Phenol hydroxylase was inhibited by Cu2+ and Pb2+ ions. Catechol dioxygenase was inhibited by all substances containing heavy metal ions (Fe2+ , Mn2+ , Cd2+ , Cu2+ and Pb2+ ), which were tested in this work. The most effective inhibition was produced by Pb2+ followed by Mn2+ , Cd2+ Fe2+ and Cu2+ ions. The higher sensitivity of catechol-1,2-dioxygenase to heavy metal ions might follow from the presence of histidine residue...