Student Research

Quantification of ABE Fermentation Products by 1D and 2D NMR

Butanol is a gasoline substitute and naturally derived, making the production of butanol of increasing importance. Butanol is produced through acetone, butanol, ethanol (ABE) fermentation pathway in some bacteria, including the Clostridium species. Because of the increasing interest in butanol production, understanding the metabolic flux through the ABE pathway is important for optimizing the recovery of the biofuel. Global profiling of the ABE pathway intermediates is difficult due to the volatile nature of the metabolites and their structural similarity. Our goal is to develop a procedure for the quantification of butanol and butyric acid using Nuclear Magnetic Resonance Spectroscopy (NMR). Due to the complexity of the spectra, traditional 1H-NMR is insufficient for quantification, so we developed 2D approaches for quantification that involve J-resolved spectroscopy (JRES) and Total Correlation Spectroscopy (TOCSY) through the application of a calibration curve. We created a calibration curve with standards and then tested the correlation of the expected concentration of an analyte with the known concentration, and found that they were in high agreement (R2 = 0.97). Additionally, the 1H and TOCSY data for butyric acid had a %RSD of 0.45 and 23, respectively. For butanol, the 1H and TOCSY data had a %RSD of 0.36 and 64%, respectively. The 2D-JRES approach is currently being carried out. Although the 1H approach was the most accurate, we are working on improving the 2D approaches so that butanol and butyric acid can be quantified in a complex sample.