Characterization of molecular and metabolic phenotypes of Clostridium syntrophic co-culture

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University of Delaware
Historically, Clostridium species were used in Acetone-Butanol-Ethanol (ABE) fermentation. However, the ABE fermentation processes face competition from petroleum based processes, thus fermentation processes need to evolve to produce a higher valued chemical efficiently. Clostridium species are known to metabolize a range of sugar molecules and produce valuable chemicals such as hexanol and octanol. To achieve this goal, utilization of co-culture allows each species of bacteria to specialize in one task and avoids complex metabolism engineering. In this thesis, optimization of the bioreactor performance of syntrophic Clostridium co-culture for medium chain alcohol or fatty acid production utilizing Clostridium kluvyeri and Clostridium saccharolyticum. And the characterization of the molecular interactions and localization of proteins of interest related to cell-to-cell interaction was investigated. In the bioreactor investigation, the optimized C. kluyveri monoculture yielded 173 mM of hexanoate. In comparison the co-culture yielded 123 mM of hexanote. The result exhibits promising industrial value for Clostridium co-culture, and the insufficient supply of ethanol from C. saccharolyticum could be the major drawback for this co-culture system, which may be improved by incorporating a third species into the system such as Clostridium ljungdahlii. Direct cell-to-cell material exchange between Clostridium acetobutylicum and C. ljungdahlii has been previously shown by our lab 1 This phenomenon greatly improves the productivity and carbon utilization in the co-culture, thus understanding the mechanism of cell-to-cell material exchange is critical to improve the performance of the co-culture and bring more industrial and economical significance. It is possible that C. ljundahlii could be actively seeking CO2 rich source and therefore fused with C. acetobutylicum to consume CO2 waste produced by C. acetobutylicum. To further investigate this phenomenon, the localization pattern of the proteins of interest such as carbonic anhydrase (CA) and methyl-accepting chemotaxis protein (MCP) could be helpful. These proteins may be involved in CO2 metabolism and CO2 sensing respectively. To visualize the localization of these proteins, Halotag was fused with the two proteins of interest. The preliminary testing of the fluorescence of both MCP Halotag and CA-Halotag under the microscope in Escherichia coli and under the flow cytometry in C. ljungdahlii shows the strong fluorescent intensity of the fluorescent fused protein. Visualizing the proteins in C. ljungdahlii under the microscopy, uniform membrane localization of MCP-Halotag and uniform cytoplasmic localization were observed. Future experiment of fluorescent imaging C. acetobutylicum and C. ljungdahlii utilizing the MCP-Halotag and CA-Halotag could provide more information about the proteins involved in the cell-to-cell direct material exchange.
Clostridium, Proteins, Co-culture