One-way ANOVA analysis was conducted with the 6 hr samples as the control at a False Discovery Rate of
2% (P-value < 0.01) to identify differentially expressed genes of statistical significance. Genes significantly up- or down-regulated in at least one time-point comparison were analyzed in TIGR MeV 4.5 software  to identify similar temporal trends in gene expression using average-linkage hierarchical or K-means clustering methods. Results and Discussion In this study, we investigated the global changes in gene expression associated with fermentation of crystalline cellulose by the anaerobic bacterium Clostridium thermocellum. In order to achieve this, we conducted duplicate selleck screening library 2 L batch fermentations of C. thermocellum on
5 g/L of crystalline cellulose (Avicel®) and took a series of six time-point samples ranging from early-exponential to late-stationary phase of cell growth. Cell growth was monitored based on total cellular protein content in the solid pellet fraction which continued to increase until ~12 h when cells entered stationary phase (Figure 1). No visible residual Avicel® was found at the end of the fermentation. Metabolite analysis revealed an inversion of acetate-to-ethanol molar ratios over the course of the fermentation, with higher molar levels of acetate than ethanol in the beginning of the fermentation, but the ratio decreased to ~0.7 towards the end of the selleck inhibitor fermentation (Figure 1). Unlike earlier reports, no detectable levels of lactate
or formate were identified in the fermentations, possibly due to differences in culture conditions. For instance, while lactate and formate were readily detected in batch experiments Resminostat using Balch tubes with no pH control [19, 20], they were formed at very low rates in controlled fermentations in bioreactors with pH control . Moreover, these metabolites may not have been detected in this study possibly due to differences in the detection method (refractive index vs conductivity detector) used in HPLC measurements. Figure 1 Fermentation growth and metabolite production plots. Pellet protein-based growth and metabolite curves for duplicate Clostridium thermocellum ATCC 27405 fermentations on 5 g/L crystalline cellulose (Avicel®). Arrows in the upper panel indicate culture sampling points for microarray-based gene expression analysis. Acetate and ethanol data in the lower panel are shown in closed and open symbols, respectively. Total RNA was extracted from the cell pellets and the reverse transcribed cDNA was hybridized to oligo-arrays containing duplicated probes representing ~90% of the annotated ORFs in C. thermocellum ATCC27405 genome. Dual-channel dye swap experimental design was used to analyze the time-course of gene expression during cellulose fermentation using the 6 hr sample as the reference, to which all other samples were selleckchem compared.