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Perchlorate reduction using Azospira oryzae enzymes in synthetic vesicles

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Title: Perchlorate reduction using Azospira oryzae enzymes in synthetic vesicles
Author(s): Hutchison, Justin
Advisor(s): Zilles, Julie L.
Department / Program: Civil & Environmental Eng
Discipline: Environ Engr in Civil Engr
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: M.S.
Genre: Thesis
Subject(s): Synthetic Vesicles Perchorate Metabolism Biological Perchlorate Treatment Azospira oryzae
Abstract: The combination of manmade materials and biological components to form perchlorate-reducing vesicles may address several of the limitations associated with current perchlorate treatment technologies. The vesicles have a membrane comprised of the synthetic polymer, PMOXA-PDMS-PMOXA (ABA), which is stable over several months and is able to incorporate membrane proteins such as the outer membrane pores (OmpF). As the vesicles are formed, they can encapsulate perchlorate-reducing enzymes such as perchlorate reductase (Pcr) and chlorite dismutase (Cld). To investigate the use of the vesicles in the treatment of perchlorate, polymer vesicles were formed with and without OmpF using the film-hydration method. Lipid vesicles were also tested. The size and size distribution of the vesicles was determined using dynamic light scattering and transmission electron microscopy. The perchlorate-reducing activity of the vesicles was determined using a colorimetric methyl viologen assay. The activity of the enzymes was assessed in oxygen and oxygen free environments, in 10% glycerol, and in the presence of protease and protease inhibitors. The activity of the enzymes toward similar anions such as nitrate and sulfate were determined with a UV-absorbance NADH assay and endpoint analysis using capillary electrophoresis. The lipid and polymer vesicles demonstrated similar abilities to reduce perchlorate. Perchlorate-reducing activity was higher in vesicles with OmpF versus vesicles without OmpF. The activity of the enzymes showed no deterioration in the presence of oxygen versus non-oxygen environments. Addition of 10% glycerol proved to be an effective stabilizing agent for enzyme activity. Protease treatment of the enzymes resulted in partial enzyme inactivation. Protease inhibitors afforded no protective effects and even decreased activity. Enzymes had activity with nitrate but not sulfate. Perchlorate and nitrate competed for limited electron donor; however, the enzymes’ affinity for perchlorate was much higher than nitrate. The polymer and lipid vesicles demonstrate perchlorate-reducing capabilities in drinking water relevant conditions. With optimization, the vesicles show potential to compete with current treatment technologies for perchlorate. More broadly, the application of the treatment technology could be applied to any type of contaminant in which there are microbial degrading enzymes. Enzymes are also not limited to a specific set. Multiple contaminant-degrading enzymes could be encapsulated at various ratios to achieve flexible treatment. The flexibility of this
Issue Date: 2012-09-18
URI: http://hdl.handle.net/2142/34559
Rights Information: Copyright 2012 Justin Hutchison
Date Available in IDEALS: 2012-09-18
Date Deposited: 2012-08
 

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