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). Overall, there were 4 successful reproducible trails. 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. Once these tests work on known sample, they can be moved onto real samples from Dr. Wei-Jen Lin's lab from Biological Sciences.