Date of Award
Doctor of Philosophy in Chemical Engineering
Geoffrey D. Bothun
During n-butanol fermentation, n-butanol partitions into microbial membranes result in destabilizing cellular lipid membranes by altering their lipid composition (generally the ratio of saturated to unsaturated lipids) whereas adapted microorganisms respond by altering the ratio of unsaturated to saturated lipids. The mechanism of how microbes achieve a high adaptation in response to n-butanol is barely known. This dissertation describes the role of unsaturated lipids and charged lipid composition in modulating n-butanol partition into membrane using model bacteria (i.e. lipid bilayer vesicles or liposomes and Langmuir monolayers) and those studies were compared with reconstituted membranes (Clostridium pasteurianum) that represented an original sample which was collected during batch fermentation as a function of different fermentation conditions.
Calorimetric, spectroscopic, Langmuir balance and chromatographic techniques were used to examine the effects of unsaturated lipid, charged lipid and n-butanol on membrane phase behavior, membrane packing, and membrane structure. The effects of n-butanol on heterogeneous membrane phase behavior was dependent on n-butanol concentration and which phase was continuous (saturated or unsaturated lipids). An increase of unsaturated lipid ratios increased n-butanol partitioning into the membranes due to "binding pocket" on acyl chain of unsaturated lipid and increased area per molecule resulting in enhancing membrane elasticity.
Heterogenous monolayer membrane of DPPC/DOPC with n-butanol was also examined using Langmuir balance trough and fluorescence microscope. Lipid phase behavior, lipid packing, and monolayer elasticity were evaluated by surface pressure-area (Π-A) analysis. This study shows that n-butanol partitioning in DPPC, DOPC domain and at DPPC/DOPC interface. n-Butanol partitioning into DPPC monolayers led to lipid expansion and decreased elasticity. Lipid expansion became greater when DOPC content increased. n-Butanol accumulation at equimolar DPPC/DOPC was amplified at the interface between coexisting liquid expanded (LE, DOPC-rich) phases and liquid condensed (LC, DPPC-rich) domains. The accumulation of n-butanol also reduced LE-LC line tension and changed the domain size and morphology of LC domains.
The integrity of charged lipid membrane was driven by electrostatic interactions between cations and negatively charged lipid headgroups and hydrophobic effects on lipid tails. However, above interdigitation concentration (0.13 M) of n-butanol, n-butanol partitioning into membrane transformed the gel phase to the interdigitated phase disregarding DPPG content and salt concentration. Increasing DPPG content in the DPPC/DPPG membrane and salts above 0.13 M of n-butanol concentration, aggregation/ fusion could be prevented and the transformation of LUV-SUVs could be observed. Increasing salt and DPPG concentration, screening electrostatic repulsion between PG headgroups was apparent to promote more rigid bilayer structures and reduced butanol partition.
Reconstituted membrane of C. pasteurianum have been examined to determine membrane composition, membrane phase behavior, and membrane fluidization using different techniques such as chromatographic, spectroscopic, and Langmuir balance. n-Butanol adaptated membrane was the result of lipid modification by increasing longer fatty acids and decreasing the amount of unsaturation and protein improvement that increased membrane rigidity that counter-acted the fluidizing effect of butanol.
Model and reconstituted membrane studies revealed that membrane rigidity and stability were promoted by decreasing unsaturated lipids, increasing the length of lipid tails and increasing charged lipid ratios in the electrolyte solution. The accumulation of n-butanol within membrane influenced membrane fluidity and membrane packing. These results demonstrate a fundamental link of the disordering effects of butanol and lipid compositions on cell membranes.
Kurniawan, Yogi, "The Effects of N-Butonol on the Model and Reconstituted Membrane of Clostridium Pasteurianum" (2013). Open Access Dissertations. Paper 44.