Extremely Thermophilic Geobacillus LC300: Genetic Identification, Growth Characteristics, Metabolic Modeling, and Scale-Up

Cordova, Lauren
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University of Delaware
A new thermophilic microorganism, Geobacillus LC300 was discovered with interesting characteristics. This organism grows optimally at 72 oC and has a fast growth rate (1.5 hr-1) utilizing xylose as the sole carbon source. Thermophilic organisms and more specifically, Geobacillus LC300, are of interest in biotechnology for the production of low-boiling fuels and thermostable enzymes. In order to effectively produce these compounds, metabolism must be understood and subsequently manipulated. In this work, a thermophilic strain, identified as Geobacillus LC300, was examined to determine the genomic content and growth characteristics for metabolic model reconstruction and metabolic flux analysis. First, Geobacillus LC300 was isolated through plating on agar plates and the genome sequenced using PacBio. The organism was cultured in 10 mL custom mini-bioreactors designed for thermophiles at 72 oC. The maximum growth rate of 1.5 hr-1 was observed with xylose as the sole carbon source. From the genome sequence, central carbon metabolism and amino acid biosynthesis pathways were annotated. Major metabolic pathways such as glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle were complete. These complete reactions were used to reconstruct a metabolic model which included 73 reactions with a lumped biomass reaction. The biomass composition was experimentally determined for accurate metabolic modeling. The reconstructed Geobacillus LC300 model was then validated with 13C-labeling experiments and analyzed through13C-metabolic flux analysis for growth on xylose. A 13C-labeling experiment was also completed with parallel glucose tracers to detect any major differences between glucose and xylose metabolism on central carbon metabolism. As expected, upper glycolysis is more active under glucose consumption compared to xylose. Likewise, the pentose phosphate pathway has higher flux during growth on xylose. Finally, scale-up analysis enabled high cell densities of Geobacillus LC300 which is industrially relevant. Increasing reactor control and feed components (glucose, nitrogen, vitamins, minerals, and yeast extract) led to higher cell densities. A fed-batch experiment with glucose reached a maximum OD600 (cell density) of 22, while growth on xylose reached a maximum of 25. This extensive analysis provides a genomic, metabolic and physical understanding of Geobacillus LC300 to create a strong foundation for future metabolic engineering and use in biotechnology applications.
Research Subject Categories::TECHNOLOGY::Chemical engineering , Chemical Engineering , Geobacillus LC300