Quantification of butanol produced by C. pasteuranium and isolated by L-A-lecithin liposomes using quantitative proton nuclear magnetic resonance spectroscopy (qNMR)
Date of Award
Master of Science in Engineering (MSE)
Chemical Engineering and Material Sciences
Butanol., Proton magnetic resonance spectroscopy., Biochemical engineering.
A cleaner and more efficient option to ethanol in the alternative fuel industry is n-butanol. The focus of this study was to establish that quantitative Nuclear Magnetic Resonance spectroscopy (qNMR) is a viable method to determine levels of n-butanol as it is produced by Clostridium pasteurianum in the fermentation of glycerol. Extractive fermentation using L-α-Lecithin liposomes is proposed to separate n-butanol, hence the spectroscopic behavior of n-butanol in the presence of liposomes was studied and partition coefficients were determined. Additionally, qNMR was used to quantify glycerol and other fermentation products during the fermentation of C. pasteurianum using a bioreactor. Two different methods were used to determine n-butanol concentrations: Vnmrj 4.2 software and 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) used as internal standard. The calibration curves for butanol-water and butanol-liposome-water were linear and yielded accurate butanol concentrations between 0.05 g/L and 8 g/L n-butanol. Partition coefficients for butanol into liposomes were determined by separating liposomes from the mixture and determining the butanol concentration in the supernatant. Liposomes were separated using the following techniques: centrifugation, centrifugal filters, Sephadex spin columns, and PVDF membrane syringe filters. By measuring the filtrate/supernatant’s concentration, the overall average partition coefficient was determined to be 72 with a standard deviation of 42. After removing outlier concentration values the partition coefficient was corrected to 66 with a standard deviation of 18.9. The butanol Kp value for L-α-Lecithin liposomes is about half of that for dimyristoylphoshatidylcholine (DMPC) liposomes (Kp= 120) and close to that for dioleoylphosphatidylcholine (DOPC) liposomes (Kp= 62). The low cost of L-α-Lecithin ($ 0.08/g for lecithin compared to $ 170.00/g for DMPC and $ 190.00/g for DOPC ) makes this lipid an affordable option for large scale butanol extraction in a bioreactor. Quantitative NMR spectroscopy was used to quantify glycerol and n-butanol in steady-state fermentations performed in the bioreactor and compared to data obtained by HPLC analysis. Glycerol and butanol concentrations determined by qNMR differed from HPLC data by an average of 10%, but with matching trends. In conclusion, qNMR is a viable option for measuring the concentration of n-butanol produced in a bioreactor and can be used in future extractive fermentations.
Viegas, Luke X., "Quantification of butanol produced by C. pasteuranium and isolated by L-A-lecithin liposomes using quantitative proton nuclear magnetic resonance spectroscopy (qNMR)" (2019). Theses. 283.