Inhibition of Clostridium thermocellum metabolism and growth under high-solids loading fermentation-relevant conditions

In addition to fermentable sugars, the cell walls of lignocellulosic biomass also contain many diverse compounds that can be potent microbial inhibitors. In consolidated bioprocessing platforms, biofuel producing microbes are therefore forced to both simultaneously convert fermentable sugars to ethanol and also deal with the toxic effects of these inhibitors. These challenges are further exacerbated when lignocellulose is subjected to chemical or physical pre-treatments, which promote inhibitor release. High-solids loading fermentations are particularly prone to sub-optimal fermentation conditions as the increased levels of biomass facilitate the accumulation of inhibitory molecules at lethal concentrations. As such, a study was undertaken to investigate the toxic effects of a broad range of compounds on the growth and fermentation capabilities of Clostridium thermocellum under high-solids loading fermentation-relevant conditions. Investigated compounds included aliphatic acids, aromatic acids, aldehydes, alcohols, glycolaldehyde and Kraft lignin at either reported or empirically determined concentrations. Of those tested, glycolaldehyde and Kraft lignin were particularly strong inhibitors at concentrations as low as 0.18 g/L or 3 g/L respectively, and resulted in the complete inhibition of growth. To better identify the most toxic components of Kraft lignin, samples were fractionated via size-exclusion chromatography, lyophilized and reconstituted in growth medium at normalized concentrations. While twenty fractions were collected, only a small subset of four consecutive fractions was inhibitory to C. thermocellum growth at concentrations as low as 2 g/L. Mass-spec analyses was performed to identify the constituent compounds in these fractions and their potential contribution to the observed growth inhibition was assessed.