Characterisation of Clostridium saccharoperbutylacetonicum for biotechnological applications

Clostridium saccharoperbutylacetonicum is an anaerobe that is growing in importance for biotechnological applications. In recent years, significant strides have been made in improving the genetic tractability and in understanding C. saccharoperbutylacetonicum fermentative biology. Despite this, multiple facets of the species’ underlying biology are not well-characterised. In particular, there is little published work examining C. saccharoperbutylacetonicum using either forward or reverse genetics approaches. Particularly, the understanding of sporulation, germination, and quorum sensing in the species is severely lacking.
This thesis aimed to establish a pipeline for transposon insertion site sequencing, a forward genetics approach, in C. saccharoperbutylacetonicum to determine its essential genome. Whilst we were not ultimately successful, we did identify 677 open reading frames that are possibly essential to growth. We believe this list, along with a partial list of non-essential open reading frames, provides invaluable data with which to aid future C. saccharoperbutylacetonicum work.
We also examined sporulation and germination. These processes are a key to the life cycle of the species in its natural habitat. Sporulation is also intimately tied to the solventogenic stationary phase of vegetative cells that is key to biotechnological applications. Here we characterised for the first time the morphology and ultrastructure of C. saccharoperbutylacetonicum sporulating cultures. In addition, we screened and identified effective germinants and germination inhibitors to allow for control over those processes.
Finally, quorum sensing, the process by which bacteria sense population density, is likely extremely important in high density cell contexts. However, it has been little studied in C. saccharoperbutylacetonicum, despite its industrial relevance and routine fermentation. We present the first work partially characterising the accessory gene regulator quorum sensing system in C. saccharoperbutylacetonicum. We showed the importance of the system to both solventogenesis and sporulation and highlight pathways to a better understanding of this crucial but complex process.