Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains Seohyoung Kim a , Eunhee Seol a , You-Kwan Oh b , G.Y. Wang c , Sunghoon Park a, * a Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735, Republic of Korea b Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-543, Republic of Korea c Department of Oceanography, University of Hawaii at Manoa Honolulu, HI 96822, USA article info Article history: Received 3 August 2008 Received in revised form 13 May 2009 Accepted 14 May 2009 Available online 12 June 2009 Keywords: H 2 production yield Glucose fermentation Metabolic engineering of Escherichia coli Carbon and energy balance Metabolic flux analysis abstract Escherichia coli can produce H 2 from glucose via formate hydrogen lyase (FHL). In order to improve the H 2 production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H 2 production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H 2 production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase ( frdAB) further improved the H 2 yield; 1.80 mol/mol glucose under high H 2 pressure or 2.11 mol/mol glucose under reduced H 2 pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/ L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading to H 2 production by FHL, not to the pentose phosphate pathway. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. 1. Introduction Hydrogen is a highly relevant energy carrier and its conver- sion to heat or power is simple and clean. When it is com- busted with oxygen, only water is formed and no pollutants are generated or emitted [1]. In addition, H 2 can be produced from a variety of different feedstocks including fossil resources, biomass and water [1,2]. When fossil fuel and water are used as a feedstock, energy-intensive thermochemical and electrochemical methods are required for H 2 production. In comparison, biological methods that use biomass as a feedstock are less energy-intensive and considered to be neutral for CO 2 emissions [3]. Among the available biological methods, microbial fermentation of organic carbon sources has been extensively studied. Glucose is a monomer of cellulose, the most abun- dant biomass in the world and a good source for H 2 production by many microorganisms. However, low yield has been a major obstacle for the hydrogen production through microbial fermentation of glucose. For example, Clostridium species can use 1 mol of glucose to produce at the most 4 mol of H 2 while Escherichia coli can only produce at the most 2 mol of H 2 from the same amount of glucose [4–9]. As of now, the low production yields, ranging from 1 to 2 mol H 2 /mol glucose, have commonly been reported with actual glucose fermentations. * Corresponding author. Tel.: þ82 51 510 2395; fax: þ82 51 510 2716. E-mail address: parksh@pusan.ac.kr (S. Park). Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/he 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.05.053 international journal of hydrogen energy 34 (2009) 7417–7427