Abstract
BackgroundClostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis.ResultsIn this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume.ConclusionThis study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.
Highlights
Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol
This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors
C. thermocellum growth response to ethanol and extracellular metabolite profiles Culture turbidity, as measured by OD600 units, for the ethanol-treatment fermentors prior to ethanol shock was similar to the control fermentors (0.59 ± 0.02 vs 0.55 ± 0.02, respectively) (Figure 1)
Summary
Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that produces large extracellular enyzme complexes, called cellulosomes. It can rapidly solubilize biomass and it is a candidate microorganism for converting biomass into lignocellulosic ethanol via a process termed consolidated bioprocessing (CBP) Increased ethanol tolerance and productivity are not necessarily linked, with the highest concentrations of ethanol produced reported as ≤ 30 g/L [12]. The role of compatible solutes such as trehalose, amino acids, such as proline [14,15], in ethanol tolerance and specific genes (e.g. [16,17,18]) have been investigated in a variety of microorganisms
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