Tools for improving and understanding microbial performance in biorenewable applications

The production of biorenewable fuels and chemicals is attractive because biomass is diverse, cheap, and abundant. Chemicals and fuels derived from renewable feedstocks can contribute to mitigation of climate change and improved rural economies. To compete with the petroleum industry, the biological processes involved in biorenewable fuel and chemical production need to be efficient. Here we engineer microorganisms and their environment for improved performance in biorenewable applications. In Chapters 1-4, we focus on lignin valorization. Lignin is a component of biomass that is underutilized, but abundant in renewable aromatics. We developed an emulsion formulation composed of Tween®20, Span®80, and a lignin-rich fraction of pyrolyzed biomass which enables P. putida KT2440 to grow in a lignin-rich fraction of pyrolytic bio-oil. This type of emulsion could be applied for microbial conversion of lignin-rich feedstocks to valuable products. To determine more about the specific compounds that are being utilized by the P. putida KT2440 and other lignin utilizing microorganisms, we developed a unique disk diffusion assay (Diffusive Inhibition with Substrate Consumption) which allowed for simultaneous quantification of inhibition and utilization. The DISC assay could be useful for quickly and easily screening lignin monomers or other monomers when little is known about their toxicity or microbial degradability. In Chapters 5, we focus on improving the robustness of biological membranes. Microorganism growth and production can be inhibited by both biomass feedstocks and the biorenewable products. One component of that inhibition is due to membrane damage. There is still little known about the biomolecular effects of alcohols which are attractive biorenewable chemicals. Here we utilized artificial biological membranes to characterize the membrane damage caused by alcohols. These experimental results are being used to inform an in silico model of a yeast plasma membrane which can be used to inform design strategies for more robust membranes. We observed increased fluidity and leakage in artificial phospholipid bilayers when treated with alcohols. Also, altering just 5% of the phospholipid head groups from phosphocholine to phosphoethanolamine somewhat improved the leakiness and fluidity. Therefore, small alterations in the phospholipid composition could be helpful in creating more robust membranes