5%) positive/negative values represents higher/lower expression levels in the hydrolysate media compared to standard medium. Values are indicated for samples collected during mid-log (ML) and late-log (LL) growth phases. C. thermocellum uses the hydrogenase-mediated pathway for production of molecular hydrogen to dispose the excess reducing equivalents generated during carbohydrate catabolism [12,28]. In the process, the Ech hydrogenase complex pump H+/Na+ ions across

the cell membrane and create proton gradients for powering ATP synthesis by mTOR activation ATP synthase (ATPase) [12]. The PM has a mutation in the non-coding region 127 bp upstream of the F-type ATP synthase operon (Cthe_2602 – Cthe_2609) which may lead to an increase in the expression of this gene cluster in the PM compared to the WT in standard medium (Table 3) [17]. The PM also increases the expression of 4 and 8 genes in the Ech hydrogenase complex (Cthe_3013-3024) compared to the WT in standard and Populus hydrolysate media (Table 3). The effect of the increased expression of the ATPase and Ech-type hydrogenases on the electron flux in the cell is unknown at the time [17]. However, analysis of the

H2 production rate of PM and WT in 0% and 10% v/v Populus hydrolysate media shows no significant difference [17]. In addition, regardless of the strain or growth medium, the five other hydrogen producing complexes in C. thermocellum are expressed at levels between 4 and 50 times greater than the Ech-type hydrogenases (data buy Tanespimycin not shown) [12]. Collectively these results argue against the increased activity of Ech-type hydrogenase complex significantly changing the electron flux in the PM. Another possibility for this change in gene expression could be electron bifurcation which was recently found in anaerobic microbes. For example, Acetobacterium woodii employs a sodium-motive ferredoxin: NAD+-oxidoreductase

(Rnf complex) that couples the exergonic electron flow from reduced ferredoxin to NAD+ to establish a transmembrane electrochemical Na+ gradient that then drives the synthesis of ATP via a well characterized Na+ F1F0- 3-mercaptopyruvate sulfurtransferase ATP synthase [29]. The data showed that the complex was reduced by the [FeFe]- hydrogenase of A. woodii and reduction of one was strictly dependent on the presence of the other electron acceptor [29]. Clostridium kluyveri have also been shown to catalyze acetyl-CoA and ferredoxin-dependent formation of H2 from NADH [30]. Table 3 Fold change in gene expression involved in cellular redox     PM vs. WT 0 PM vs. WT 10 PM 0 vs. 10 PM 0 vs. 17.5 WT 0 vs. 10     ML LL ML LL ML LL ML LL ML LL Redox transcriptional repressor Cthe_0422 Redox-sensing transcriptional repressor rex 1.13 −1.08 7.01 5.53 1.04 −1.02 −1.04 −1.11 −5.96 −6.08 Ech-type hydrogenases Cthe_3013 hydrogenase expression/formation Angiogenesis inhibitor protein HypE 1.39 1.19 3.42 2.34 −1.90 −2.24 1.30 −1.14 1.37 −1.03 Cthe_3016 [NiFe] hydrogenase maturation protein HypF 2.34 2.