KREC: Differentiating Microbial Pathway and Membrane Adaptations for Enhanced Performance in Extreme Environments

Thermophilic anaerobic bacteria such Clostridium thermocellum have significant advantages relative to yeast in their ability to convert fibrous organic material to bioproducts. The ethanol adaptation of thermophilic bacteria addresses their relatively low ethanol tolerance, one of the technological barriers to efficient biomass conversion. This adaptation has been linked to the bacteria's general response to extreme environments (i.e., temperature, pressure, pH, and organic solvents). One of the major challenges in determining the mechanism of adaptation is that the global response elicited by ethanol stress involves the interplay of pathway effects and membrane effects. Our proposed work will quantify the metabolic pathway and membrane fluidity of wild-type and ethanol-adapted C. thermocellum in response to exogenous ethanol and controlled pressure environments. We will demonstrate the use of pressure as a highly tunable perturbation for metabolomics and provide a mechanistic view of cell adaptation to extreme environments on the basis of the resulting metabolic footprints. OBJECTIVES: The overall goal of this project is to quantify and differentiate the metabolic pathway flux and membrane fluidity changes of wild-type and ethanol-adapted C. thermocellum in response to controlled pressure and exogenous ethanol. This overall goal will be achieved by: quantifying metabolic pathway (Obj. 1) and membrane fluidity changes (Obj. 2) between the wild type and an ethanol-adapted strain of C. thermocellum as a function of hydrostatic pressure and exogenous ethanol; and correlate the pathway changes and membrane fluidity with environmental treatments to gain a mechanistic understanding of cellular adaptations in an ethanol-tolerant organism (Obj. 3) METHODS: Cultivations at hydrostatic pressure and in the presence of ethanol will be conducted for both wild-type and adapted C. thermocellum. Metabolic flux analyses are expected to reveal significant pathway changes based on the pressure and solvent effects we have observed previously. Differences due to treatments will be compared within and between the wild-type and adapted strains. Membrane fluidity will be determined in situ using fluorescent probe molecules embedded in the membrane bilayer. A principal component analysis will quantify the relationship between treatment, metabolic response and membrane fluidity measurements. The clustering of similar biological status treatments in principal component plots will be used to identify the underlying mechanisms of ethanol adaptation. POTENTIAL IMPACTS: This study will provide a rationale for the engineering and commercialization of microorganisms capable of degrading biomass and producing ethanol.