(488h) In situ Partitioning Measurements of Multiple Fermentation End-Products into Lipid Membranes

Product inhibition during fermentation is driven in part by solvent partitioning into bacterial lipid membranes. Solvent partitioning leads to membrane fluidization, or a reduction in lipid ordering, that alters transmembrane permeability and the function of membrane proteins. Membrane engineering continues to be used to enhance bacterial resistance to membrane fluidization and create robust bacteria for biofuel production. A key aspect to membrane engineering is understanding how solvent partitioning varies with lipid composition in realistic, complex environments. We present a new approach for measuring the partitioning behavior of metabolic end-products produced during butanol fermentation using quantitative nuclear magnetic resonance (qNMR) spectroscopy. Butanol is a viable alternative fuel to gasoline; however, it is lipophilic and inhibits fermentation at low concentrations. The qNMR technique facilitates rapid in situ measurements of multiple species partitioning simultaneously and can be performed as a function of temperature and solution chemistry. This presents clear advantages over calorimetric techniques. Butanol, ethanol, 1,3-propanediol, acetic acid, and butyric acid have been examined individually and in mixtures as a function of lipid tail saturation using mixtures of dipalmitoylphosphatidylcholine (DPPC, saturated) and dioleoylphosphatidylcholine (DOPC, unsaturated), as well as reconstituted membrane extracts from Clostridium pastuerianum. qNMR and dynamic light scattering results show that butanol partitioning is minimally affected by the presence of other metabolic end-products and membranes swell to accommodate butanol based on their ability to interdigitate at high butanol concentrations. The qNMR technique should be broadly applicable to fermentation processes and can enable rapid screening of membrane partitioning.