Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824

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

Evaluation of Methods for the Analysis of Untreated and Processed Lignocellulosic Biomasses

The overall efficiency of the transformation of lignocellulosic materials to usable products as chemicals and fuels must be governed by adequate analysis of products before and after treatments. Using some promising technologies, lignocelluloses which are biomasses from marine plant and trees, grains, food and non-food crops, and woodbased can give products as fuel alcohol and other chemicals. Various methods of transformation from feedstock to valuable end products are discussed in the scientific literature. Therefore, yields must justify methods used for biomass transformations. As a result, adequate compositional analysis of these processing stages is needed. In this chapter, standard common methods such as gravimetric, chromatography, spectroscopic and their variations for analysis on both untreated and treated lignocelluloses are highlighted. The ease of the use and challenges with recommendations to their applicability to quantifying lignocelluloses fractionations for reproducibility and to be representative are discussed. With biomass technology, virtually all and even more products that can be produced from fossil energy can also be produced from biomass energy. Adequate analysis is therefore necessary