Exploring alternative feedstocks and engineering butanol tolerance to optimise biofuel production by Clostridium saccharoperbutylacetonicum

Acetone-butanol-ethanol (ABE) fermentation is an established industrial process that uses Clostridium bacteria for the conversion of plant-derived 'feedstocks' into solvents (acetone, butanol and ethanol) that can be used as biofuels. These solventogenic clostridial strains are naturally adapted to access energy/carbon from complex sugars found in common feedstocks made from corn and rice, but it is of interest to explore a variety of waste biomass to provide a stable supply of feedstock for industrial biofuel production. It was of particular interest to investigate microalgae as a feedstock for ABE fermentation: microalgae are currently used by project partners Algaecytes® to produce Omega 3, which results in large amounts of low-value spent biomass following product extraction. In this study, commercially-available Chlorella vulgaris was used as well as a Eustigmatophyceae proprietary strain obtained from the project partner. Feedstocks were processed in a variety of ways and fermentations were performed in serum bottles and 500 mL fermenters to optimise optical detection of clostridial growth and solvent production. The highest solvent yield of 3.27 g/L (acetone: 0.40 g/L; butanol: 1.40 g/L; ethanol: 1.47 g/L) was achieved with non-autoclaved and non-centrifuged 10 % Eustigmatophyceae spent biomass supplemented with 1 % glucose, whereas a 10 % feedstock of C. vulgaris supplemented with 1% glucose had a lower yield of 1.20 g/L (acetone: 0.20 g/L; butanol: 1.00 g/L). These yields are significantly lower than those obtained with industrial feedstocks (in excess of 20 g/L) where butanol toxicity becomes limiting, so further work will be necessary to refine the use of algal biomass as a feedstock.

In addition to investigation of alternative feedstocks, there is clear biotechnological value in producing a Clostridium strain with increased butanol tolerance. Previously, overexpression of the FocA formate transporter has been shown to enhance butanol tolerance in Escherichia coli. Cloning/overexpression of E. coli focA and the clostridium homologue fdhC were done, with the aim of generating a butanol-tolerant strain of C. saccharoperbutylacetonicum. This work has the potential to generate higher solvent yields that could improve process economics for industrial biofuel production by ABE fermentation.