BUTYRIC ACID PRODUCTION FROM RENEWABLE RESOURCESBy Adam Marschall JarosAbstract Butyric acid is globally marketed with uses ranging from flavor additive to cattle feed enhancer to chemical feedstock for construction materials. Naturally found in milks, butter and numerous cheeses, butyric acid imparts a buttery flavor to food products. The manufacture and sale of butyric acid as a food flavoring for human consumption is subject to market demand for all-natural products. The "all-natural" labeling requirements preclude the use of synthetic or petro-chemical sourced compounds as a component of the product. In order for producers to cater to the demand for all-natural products alternative methods for generating butyric acid must be developed and evaluated.The fermentation production of butyric acid is an all-natural method utilizing the anaerobic bacteria Clostridium tyrobutyricum from hydrolyzed lignocellulosic biomass, a renewable resource. The overall hypothesis of this dissertation is that real-world lignocellulosic hydrolysates are feasible fermentation substrates for generating butyric acid and challenges from using lignocellulosics, such as acetate inhibition, can be overcome. Hydrolysis of hemicellulose, a component of lignocellulose, through hot water extraction provides a low-cost source of xylose for the fermentation production of butyric acid but also yields acetate, a microbial inhibitor. This dissertation provides a characterization of the effects of acetate on the fermentation production of butyrate (dissociated form of butyric acid at fermentation pH) and reveals that the lowered productivity is due to an extended lag-phase in bacterial growth. However, once the acetate induced lag-phase is overcome, acetate (26.3 g/L) challenged batch fermentations of C. tyrobutyricum demonstrate higher butyrate yields (12.6%) owing to an acetate re-uptake metabolic mechanism. Selective adaptation of C. tyrobutyricum cultures to the presence of 26.3 g/L acetate developed tolerant cultures with high butyrate productivity rates compared with non-adapted C. tyrobutyricum cultures under comparable batch conditions. Enzyme activity for the metabolic enzyme acetate kinase (responsible for acetate production) was evaluated and reduced activity was observed equally in both acetate tolerant and control C. tyrobutyricum in the presence of acetate. Lignocellulosic hydrolysate material having undergone pretreatments of either ammonia-fiber expansion (AFEX), alkaline hydrogen peroxide (AHP) or extracted ammonia-fiber expansion (E-AFEX) was obtained for fermentation trials on C. tyrobutyricum and all were adequate as fermentation substrates. A 1-liter batch fermentation of AFEX hydrolysate was conducted and similar sugar consumption and butyrate production was observed compared with a control batch. The C. tyrobutyricum culture fermenting AFEX hydrolysate experienced half the specific growth rate during log-phase growth than the control batch. Taken together, these results indicate that renewable lignocellulosic biomass is a feasible fermentation substrate for the fermentation production of butyric acid.
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