Enhanced Butyric Acid Fermentation by Clostridium Tyrobutyricum Immobilized in a Fibrous-bed Bioreactor

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Abstract: Production and application of bio-based industrial products are increasingly important to the nation2s economic development. The goal of this work was to develop a novel bioprocess to economically produce butyric acid from low-value agricultural commodities. Butyric acid has many important applications in chemical, food, and pharmaceutical industries. Conventional fermentation technologies for butyric acid production are limited by low reactor productivity, product concentration, and yield. In this study, novel metabolic engineering approaches, at both molecular biology and process engineering levels, were developed for enhanced butyric acid production by Clostridium tyrobutyricum. First, a novel fibrous-bed bioreactor (FBB) was developed for fermentation of glucose and xylose to produce butyrate with high reactor productivity (>2.5 g/L/h), high butyrate concentration (58 g/L), and a butyrate yield of 0.47 g/g. Cells in the FBB were able to grow into high density (>70 g/L) and tolerate high butyrate concentration, and they were physiologically different from the original wild type. This was not achievable in conventional fermentation systems. In order to optimize cell immobilization in the FBB for stable long-term operation of this bioreactor, the factors controlling cell adhesion on fibers including cell age, pH, ionic strength, and composition of the media were studied. Methods to modify the fiber surface to facilitate the immobilization of productive cells and to remove non-productive cells were also developed. At the molecular biology level, integrational plasmid technology was used to knock out ack and pta genes to block the byproduct acetate formation and improve the fermentation ability of C. tyrobutyricum. Inactivation of ack or pta increased butyrate concentration and productivity significantly as compared to the wild-type. Overexpression of C. acetobutylicum buk and ptb genes involved in butyrate formation did not improve butyrate production due to increased sensitivity to butyrate inhibition. These studies demonstrated the feasibility of using genetic engineering technologies to develop mutant strains of C. tyrobutyricum and their potential in improving the butyrate fermentation. The new FBB and immobilization matrix design, and the mutant bacteria obtained in this work are important to the development of an economical bioprocess for butyric acid production from biomass.

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