Co-fermentation of mixed sugars to produce butanol is an attractive route in sucrochemical production chains. Herein, high-level mixed sugars from sugarcane bagasse hemicellulosic hydrolysate (HH) and molasses (SCM) were investigated as potential substrates for acetone-butanol-ethanol (ABE) production in batch fermentation by Clostridium saccharoperbutylacetonicum DSM 14923. HH produced after hydrothermal pretreatment was concentrated 5-fold and was called concentrated hemicellulosic hydrolysate (CHH). The fermentation media that were investigated consisted solely of CHH and SCM and three CHH-to-SCM ratios were used to provide 30 g/L initial sugar concentrations and furan derivatives lower than 0.1 g/L. For CHH50/SCM50 and CHH75/SCM25, diluting CHH to concentrations of 15 g/L sugars and 22.5 g/L sugars, respectively, which were supplemented with SCM and nutrients, suffered growth inhibition as a function of the concentration of undissociated acid in the medium. The best-performing medium, CHH25/SCM75 (7.5 g/L and 22.5 g/L sugars from CHH and SCM, respectively and about 0.017 g/L furan derivatives), showed 97 % sugar consumption, and in the pH range of 5.5–6.5, undissociated acetic acid was not an inhibitory molecular form to C. saccharoperbutylacetonicum. After 30 h of fermentation, 7.8 and 9.8 g/L butanol and ABE were produced, respectively, which yielded 0.28 and 0.36 g/g. Based on our findings, C. saccharoperbutylacetonicum exhibits its potential and effective application for renewable butanol production by co-fermenting mixed sugars.

Robust strains are essential towards success of n-butanol production from lignocellulosic feedstock. To find a suitable strain to convert a non-detoxified hemicellulosic hydrolysate of sugarcane bagasse, we first assessed the performance of four wild-type butanol-producing Clostridium strains (C. acetobutylicum DSM 6228, C. beijerinckii DSM 6422, C. saccharobutylicum DSM 13864, and C. saccharoperbutylacetonicum DSM 14923)in batch fermentations containing either xylose or glucose at 30 g L⁻¹ as sole carbon sources. C. saccharoperbutylacetonicum was selected after achieving butanol yields as high as 0.31 g g⁻¹ on glucose and 0.25 g g⁻¹ on xylose. In a 48-h fermentation containing a mixture of sugars (93% xylose and 7% glucose)that mimicked the hydrolysate, C. saccharoperbutylacetonicum delivered the highest butanol concentration (14.5 g L⁻¹)when the initial sugar concentration was 50 g L⁻¹. Moreover, the selected strain achieved the highest butanol yield (0.29 g g⁻¹)on xylose-rich media reported so far. Meanwhile, C. saccharoperbutylacetonicum produced 5.8 g butanol L⁻¹ (0.22 g g⁻¹ butanol yield)when fermenting a non-detoxified sugarcane bagasse hemicellulosic hydrolysate enriched with xylose (30 g total sugars L⁻¹). Although sugars were not exhausted (4.7 g residual sugars L⁻¹)even after 72 h because of the presence of lignocellulose-derived microbial inhibitors, these results show that C. saccharoperbutylacetonicum is a robust wild-type strain. This microorganism with high butanol tolerance and yield on xylose can, therefore, serve as the basis for the development of improved biocatalysts for production of butanol from non-detoxified sugarcane bagasse hemicellulosic hydrolysate.

Aqueous two-phase systems (ATPS) can be applied to enzymatic reactions that are affected by product inhibition. In the biorefinery context, sugars inhibit the cellulolytic enzymes in charge of converting the biomass. Here, we present a strategy to select an ATPS (formed by polymer and salt) that can separate sugar and enzymes. This automated and miniaturized method is able to determine phase diagrams and partition coefficients of solutes in these. Tailored approaches to quantify the solutes are presented, taking into account the limitations of techniques that can be applied with ATPS due to the interference of phase forming components with the analytics. The developed high-throughput (HT) platform identifies suitable phase forming components and the tie line of operation. This fast methodology proposes to screen up to six different polymer-salt systems in eight days and supplies the results to understand the influence of sugar and protein concentrations on their partition coefficients.