- Aravind Arunachalam, Chemical Engineering, University of Delaware
- Eleftherios Papoutsakis, Chemical and Biochemical Engineering, University of Delaware
Isopropanol is a valuable biofuel and antiseptic chemical used in various industries. Currently, this alcohol is produced through petrochemical synthesis, resulting in CO2 emission and other pollutants. A more environment-friendly method involves the syntrophic interaction between two Clostridia species: Clostridium acetobutylicum (Cac) and Clostridium ljungdahlii (Clj). In a two-species consortium, acetone produced by Cac is converted to isopropanol by Clj. Electron flow limits metabolite production as essential reactions depend on the gain/loss of electrons. These electrons are delivered to enzymes via cofactors. Manipulation of the cofactor ratio redirects metabolite flux towards specific metabolites, allowing for the overproduction of desired compounds, such as acetone or isopropanol. Since unique electron balances govern both bacteria, two different engineering approaches need to be employed for Cac and Clj. Hydrogen produced by Cac’s NADPH-dependent hydA is a necessary gas required for CO2-uptake by the native Wood-Ljungdahl Pathway and is used as an electron source in Clj. Three Cac strains differing in the overexpression of three cofactor ratio-manipulating genes (gapN, pos5, hydA) were constructed. The overexpression of gapN–hydA resulted in the highest H2 production, while the overexpression of gapN–pos5 resulted in high ethanol titers, confirming ethanol production via three NADPH-dependent chromosomal butanol dehydrogenases. A method to manipulate Clj’s electron balance is through nitrate, which can be reduced to the weak base ammonium. This pathway is especially interesting because it may allow for: 1.) fine control over consortium population ratios through nitrogen balancing and 2.) metabolic pH control. Simple batch addition of nitrate to Cac–Clj coculture has been shown to substantially affect metabolite flux. The second aspect is a promising method to regulate the Cac population and, consequently, the culture pH. This project scaled down and validated a nitrate-quantification assay needed for future nitrate-based experimentation.