Four Carbon Oxychemicals from Renewable Resources

Butanol, butyric acid and butyraldehyde are important 4-carbon oxychemicals typically generated from petro-chemical sources. All have significant markets in the food industry either for direct use as flavorings or as chemical feedstocks for generating butyric acid and butyraldehyde derived flavour compounds. Strong consumer sentiment against the consumption of petro-chemical derived products yields a demand for butyrate and butyraldehyde generated through all-natural methods. The bacterial fermentation production of butyric acid as well as bioconversion of butanol to butyraldehyde by yeast is presented in this work as a means of producing these products naturally. This thesis demonstrates the fermentation production of butyric acid with the Gram-positive anaerobic bacteria Clostridia tyrobutyricum. The organism consumes monomeric hexoses and pentoses to generate the carboxylic acids lactate, acetate and butyrate. The fermentations undertaken in this thesis were performed with either glucose or xylose as the primary carbon source in minimal media. Butyric acid studies were performed under anaerobic conditions as batch fermentations with lag, log and stationary phase growth being monitored by the optical density of the fermentation broth. Samples were drawn throughout the fermentations and HPLC analysis was performed to determine sugar consumption and butyric acid production over time. Another element expounded in this thesis is the potential use of the economical and renewable resource hot water extracted (HWE) hemicellose as a substrate for Clostridial fermentation. HWE hemicellose is produced as a waste stream from the pulp and paper industry and is converted to fermentable xylose with the concomitant release of acetic acid from the acetyl groups on the xylan backbone. With the presence of such a high concentration of acetic acid, microbial inhibition occurs and the productivity of xylose fermentation to butyric acid is diminished with the increased lag phase. C. tyrobutyricum xylose fermentation studies were performed with synthetic media challenging the fermentation with up to 26.3 g/L acetic acid to gain an understanding of the effects of acetic acid inhibition. Once the acetic acid induced lag phase growth was characterized this work was furthered by adapting a strain of C. tyrobutyricum to 26.3 g/L acetic acid conditions and demonstrating that this pre-adaptation could drastically reduce the acetic acid induced lag phase of a batch fermentation. From this set of studies, it is noted that the presence of acetic acid in the media increases carbon efficiency of the fermentation as during stationary growth C. tyrobutyricum re-uptakes free acetic acid from the environment and converts it into butyric acid. This thesis also demonstrates the bioconversion of butanol to butyraldehyde by the methylotrophic yeast Pichia pastoris. P. pastoris were grown to high cell densities under glycerol feed and then induced to produce the endogenous alcohol oxidase (AOX) enzyme by beginning the culture on methanol consumption after a short starvation period. AOX converts short chain aliphatic alcohols to the corresponding aldehyde with the utilization of oxygen. The AOX enzyme is inhibited by the final product butyraldehyde so studies were performed utilizing alternative amine based pH buffering systems which also aid the bioconversion by binding free butyraldehyde as a Schiff-base. By binding the butyraldehyde longer bioconversions were observed. For these conversions, AOX activity was monitored with an absorbance based enzyme assay and the butanol substrate and butyraldehyde product were determined by gas chromatography.