Ionic-liquid-tolerant enzymes from an adapted Bacillus paralicheniformis for one-pot conversion of lignocellulosic substrates to ethanol

A newly isolated thermophilic cellulase-producing Bacillus paralicheniformis was subjected to adaptive laboratory evolution in the presence of increasing concentration (from 1 to 5% w w−1) of an otherwise toxic ionic liquid (either 1-ethyl-3-methylimidazolium chloride, or 1-butyl-3-methylimidazolium chloride) over 200-days to obtain strains that produced ionic-liquid-stable enzymes. The ability of the ionic-liquid-adapted strains to grow and produce cellulose hydrolyzing enzymes was proven using several different untreated agroindustrial lignocellulosic substrates. The cellulase of the adapted isolate retained more than 99% of its initial activity after incubation (12 h, ∼25 °C) with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]; 50% w w−1 concentration of ionic liquid). In contrast, the wildtype cellulase retained < 9% of its initial activity after an equivalent exposure to a lesser concentration (15% w w−1) of the ionic liquid. Using untreated sugarcane bagasse as the substrate, the optimal conditions for producing the ionic-liquid-stable enzymes (cellulase, xylanase, β-glucosidase) by the adapted bacterium were 48 °C and an initial pH ∼6. The ionic-liquid-adapted crude enzymes were used in a one-pot process (at 50 °C, 48 h) to release glucose from the ionic-liquid-pretreated wheat straw (pretreatment at 90 °C, 1 h, using 10% (w w−1) wheat straw in [Emim][Ac]), in the presence of the residual ionic liquid. At 48 h of enzymolysis, the glucose concentration exceeded 50 g L−1. This glucose mixed with the residual ionic liquid was fermented (24 h, 37 °C) to ethanol using the conventional yeast Saccharomyces cerevisiae. The ethanol yield on glucose was nearly 56% of the theoretical maximum, despite the yeast not having been adapted to withstand the toxic ionic liquid.