Background: Clostridium acetobutylicum has been a focus of research because of its ability to produce high-value
compounds that can be used as biofuels. Lignocellulose is a promising feedstock, but the lignin–cellulose–hemicellulose
biomass complex requires chemical pre-treatment to yield fermentable saccharides, including cellulose-derived
cellobiose, prior to bioproduction of acetone–butanol–ethanol (ABE) and hydrogen. Fermentation capability is
limited by lignin and thus process optimization requires knowledge of lignin inhibition. The effects of lignin on cellular
metabolism were evaluated for C. acetobutylicum grown on medium containing either cellobiose only or cellobiose
plus lignin. Microscopy, gas chromatography and 8-plex iTRAQ-based quantitative proteomic technologies were
applied to interrogate the effect of lignin on cellular morphology, fermentation and the proteome.
Results: Our results demonstrate that C. acetobutylicum has reduced performance for solvent production when
lignin is present in the medium. Medium supplemented with 1 g L−1
of lignin led to delay and decreased solvents
production (ethanol; 0.47 g L−1
for cellobiose and 0.27 g L−1
for cellobiose plus lignin and butanol; 0.13 g L−1
for cellobiose
and 0.04 g L−1
for cellobiose plus lignin) at 20 and 48 h, respectively, resulting in the accumulation of acetic
acid and butyric acid. Of 583 identified proteins (FDR < 1 %), 328 proteins were quantified with at least two unique
peptides. Up- or down-regulation of protein expression was determined by comparison of exponential and stationary
phases of cellobiose in the presence and absence of lignin. Of relevance, glycolysis and fermentative pathways were
mostly down-regulated, during exponential and stationary growth phases in presence of lignin. Moreover, proteins
involved in DNA repair, transcription/translation and GTP/ATP-dependent activities were also significantly affected
and these changes were associated with altered cell morphology.
Conclusions: This is the first comprehensive analysis of the cellular responses of C. acetobutylicum to lignin at metabolic
and physiological levels. These data will enable targeted metabolic engineering strategies to optimize biofuel
production from biomass by overcoming limitations imposed by the presence of lignin
