“Carbon Neutral Production of Isopropanol: An Evaluation of Performance of Genetically Engineered Clostridium acetobutylicum”

• Julia Cichowska, Chemical Engineering, University of Delaware

• Eleftherios Papoutsakis, Chemical & Biomolecular Engineering, University of Delaware

Climate change has a significant impact on agriculture, human health, and water resources. Its main cause are greenhouse gases (CO₂, CH₄, N₂O, etc.) from non-renewable fossil fuel use. For example, petroleum-based chemical manufacturing emits CO₂, which is equal to 76% of all greenhouse emissions. Microbial fermentation is a promising, carbon neutral alternative to this process. In this poster, I will present how clostridial fermentation can be used for production of isopropanol. Clostridium acetobutylicum (Cac) performs acetone-butanol-ethanol (ABE) fermentation, but loses about one-third of sugar carbons to CO₂ in this process. Luckly, Clostridium ljungdahlii (Clj), paired with Cac in a coculture, recycles the CO₂ and produce isopropanol using acetone from Cac. Enhancing this process involves integrating the acetone pathway into the Cac chromosome, knocking out genes for ethanol and butanol production, however all those modifications could also affect culture’s health. Therefore, precise analysis and evaluation of engineered Cac strains is important in development of the carbon neutral isopropanol production. Two experiments were conducted to check the impact of the specific genetic modifications on production rate, yield, and titer. In the first one, two different Cac strains (Cac Dhbd and Cac ∆hbd ∆bdhA::atoB-adc) were cocultured with Clj using serum bottles to evaluate isopropanol production and metabolic performance. The ∆bdhA::atoB-adc modification removed the gene responsible for ethanol production and added two new ones for acetone production. Based on measured optical density (OD) and High-Performance Liquid Chromatography (HPLC) analysis, the Cac ∆hbd ∆bdhA::atoB-adc strain produced isopropanol faster and in greater concentration. The second experiment involved monocultures of Cac ∆hbd and Cac ∆hbd ∆buk::ctfAB in Turbo Clostridial Growth Medium (TCGM) with 45mM and 30mM butyrate in the serum bottles. The ∆buk::ctfAB modification removed ability to synthesize butyrate kinase and added gene capable of converting acetate into acetone. The OD results based growth curves showed no significant differences between the strains except for the second replicate of Cac ∆hbd ∆buk::ctfAB that grew without a lag phase. The HPLC analysis of both cultures in TCGM with 45mM butyrate showed no differences in the concentrations of acetone.