Development of an automated repetitive batch fermentation system and understanding rates of cellulose utilization by cellulolytic microorganisms

It is clear that a sustainable energy future depends on shifting our energy sources to renewable forms. Biofuels, such as ethanol, produced from lignocellulosic biomass show great promise for providing renewable energy, especially for transportation energy. The recalcitrance of lignocellulosic materials has evolved as a defense mechanism against degradation by bacteria and fungi. This recalcitrance is the primary obstacle to allowing these widely available materials to be used in the commercial production of biofuels. Enrichment of environmental cellulolytic consortia has been used to discover cellulolytic microorganisms and provide understanding of the mechanisms and kinetics of cellulose degradation. Serial batch transfer experiments have also been used to improve both pure and mixed cultures toward this specific goal. An automated repetitive batch fermentation system was designed to allow for time-effective batch transfer experiments of thermophilic cellulolytic organisms. This system allows for these experiments to take place in a fermentor to take advantage of online measurement capabilities to control batch transfer timing as well as automated sample taking. This system was used to compare utilization of Avicel, a lignin-free microcrystalline cellulose, for a pure culture of Clostridium thermocellum and an environmental consortium obtained from horse manure compost. These two cultures exhibited very similar behavior, with both showing increasing rates over time, similar product formation profiles, and -- of particular note -- essentially the same rate of cellulose solubilization. Cellulose utilization as determined by wet chemistry methods was shown to correlate well with CO2 production, and similar carbon recovery was obtained for both the pure culture and mixed culture. It is anticipated that the automated batch system can be used to approach several important questions in the future, including the relative rates of solubilization of lignocellulosic substrates by pure cultures and mixed cultures, and the extent of pretreatment required by various microbial systems.