Year of Publication

2004

Document Type

Dissertation

College

Engineering

Department

Chemical Engineering

First Advisor

Barbara Knutson

Abstract

Compressed and supercritical fluids, such as pressurized CO2, ethane, orpropane, provide a versatile and environmentally acceptable alternative to conventionalliquid organic solvents in bioprocessing applications – specifically in the areas ofproduct extraction, protein purification, microbial sterilization, and enzymatic and wholecellbiocatalysis. While their advantages have been well demonstrated, the effects ofcompressed and supercritical fluids on whole cells are largely unknown.Metabolic and structural perturbations of whole cells by compressed andsupercritical fluid solvents were examined. These perturbations exist as cell metabolismand membrane structure are influenced by pressure and the presence of a solventphase. Continuous cultures of Clostridium thermocellum (a model ethanol-producingthermophilic bacterium) were conducted under elevated hydrostatic and hyperbaricpressure to elucidate pressure- and solvent-effects on metabolism and growth.Fluorescence anisotropy was employed to study liposome fluidization due to thepresence of compressed and supercritical fluids and their partitioning/accumulation inthe phospholipid bilayer.Under elevated hydrostatic pressure (7.0 and 13.9 MPa; 333 K), significantchanges in product selectivity (towards ethanol) and growth were observed in C.thermocellum in conjunction with reduced maximum theoretical growth yields andincreased maintenance requirements. Similarly, metabolism and growth were greatlyinfluenced under hyperbaric pressure (1.8 and 7.0 MPa N2, ethane, and propane; 333K); however, severe inhibition was observed in the presence of supercritical ethane andliquid propane. These changes were attributed to mass-action effects on metabolicpathways, alterations in membrane fluidity, and the dominant role of phase toxicityassociated with compressed and supercritical fluids.Fluorescence anisotropy revealed fluidization and melting point depression ofdipalmitoylphosphatidylcholine liposomes in the presence of CO2, ethane, and propane(1.8 to 20.7 MPa; 295 to 333 K). The accumulation of these fluids within the bilayerupon pressurization and the ordering effects of pressure influenced liposome fluidity, themelting temperature, and the gel-fluid phase transition region. These resultsdemonstrate the disordering effects of compressed and supercritical fluids on biologicalmembranes and the ability to manipulate liposomes.

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