236 Introduction Molecular hydrogen as potential fuel and its production by bacteria during mixed-acid fermentation H 2 has a great potential as an ecologically-clean, renew- able and capable fuel. It is generating no toxic by-prod- ucts producing only water. H 2 has higher (~3-fold) energy content (~140 MJ/kg) than hydrocarbon fuels as oil (Momirlan & Veziroglu, 2005; Maeda et al., 2007c). he most of H 2 is now produced from the water by the process of steam reforming or as a by-product from petroleum reining or chemicals production (Das & Veziroglu, 2001). However, H 2 may be produced from agricultural products and organic wastes by bacteria during either fermenta- tive or photosynthetic processes. But fermentative H 2 production is more eicient than photosynthetic one. Signiicant reduction of energy costs is also important as these processes do not require heating or extensive electricity. Among H 2 -producing bacteria, Escherichia coli is the best-characterized bacterium, having established meta- bolic pathways and, importantly, there are many strains to manipulate genetically (Maeda et al., 2007a,b,c; 2008a,b; 2011; Sanchez-Torres et al., 2009; Hu & Wood, 2010). During fermentation, the oxidation of common sugar (glucose) proceeds via consequent biochemical pathways (Figure 1); at the stage of phosphoenolpyruvate, some intermediates may be used for succinic acid formation, whereas all other end products are formed from pyruvate (Clark, 1989; Bock & Sawers, 2006; Poladyan & Trchounian, REVIEW ARTICLE Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F 0 F 1 -ATPase Karen Trchounian 1 , Anna Poladyan 1 , Anait Vassilian 2 , and Armen Trchounian 1,3 1 Department of Biophysics, 2 Department of Ecology and Nature Protection, and 3 Department of Microbiology and Biotechnology of the Biology Faculty, Yerevan State University, Yerevan, Armenia Abstract Molecular hydrogen (H 2 ) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two diferent H 2 -evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH] out ) and the presence of formate. FHL is related with the F 0 F 1 -ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H 2 -oxidizing enzymes during glucose fermentation at neutral and low [pH] out . They operate in a reverse, H 2 -producing mode during glycerol fermentation at neutral [pH] out . Hyd-1 and Hyd-2 activity depends on F 0 F 1 . Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H 2 production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of diferent Hyd enzymes activity is an efective way to enhance H 2 production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H 2 production, reduced fuels and other chemicals with higher yields than those obtained by common sugars. Keywords: Bacteria, glucose and glycerol fermentation, formate, hydrogenases, gene expression and regulation, pH, F 0 F 1 -ATPase, bioenergy Address for Correspondence: Prof. A. Trchounian, Department of Microbiology and Biotechnology of the Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025, Yerevan, Armenia. Tel: 37410–570591. E-mail: Trchounian@ysu.am (Received 24 November 2011; revised 22 December 2011; accepted 04 January 2012) Critical Reviews in Biochemistry and Molecular Biology, 2012; 47(3): 236–249 © 2012 Informa Healthcare USA, Inc. ISSN 1040-9238 print/ISSN 1549-7798 online DOI: 10.3109/10409238.2012.655375 Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by University of Wisconsin Madison on 05/04/12 For personal use only.