title: Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824.
abstract: 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.
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Mahendra P
authors.initials: MP
authors.lastname: Raut
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Narciso
authors.initials: N
authors.lastname: Couto
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Trong K
authors.initials: TK
authors.lastname: Pham
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Evans
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK ; Chaire de Bioinformatique, LGBA, Conservatoire National Des Arts Et Métiers, 75003 Paris, France.
authors.forename: Josselin
authors.initials: J
authors.lastname: Noirel
authors.affiliation: The ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK ; School of Chemical Engineering and Advanced Materials, Faculty of Science, Agriculture & Engineering, Newcastle University, Newcastle upon Tyne, NE1 7RU UK.
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
keywords: Biofuel production; Cellobiose; Clostridium acetobutylicum; Fermentative end products; Lignin; Metabolic changes; Quantitative proteomics; iTRAQ