Biotechnology Letters 26: 1607–1612, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 1607 Ethanol production from H 2 and CO 2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1 S. Sakai 1 , Y. Nakashimada 1 , H. Yoshimoto 1 , S. Watanabe 2 , H. Okada 2 & N. Nishio 1,∗ 1 Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8530, Japan 2 Research and Development Center, Cosmo Oil Co. Ltd., Gongendo 1134-2, Satte, Saitama 340-0193, Japan ∗ Author for correspondence (Fax: +81-82-424-7046; E-mail: nnishio@hiroshima-u.ac.jp) Received 24 June 2004; Revisions requested 20 July 2004; Revisions received 24 August 2004; Accepted 24 August 2004 Key words: alcohol dehydrogenase, ethanol production, H 2 and CO 2 , intracellular pyridine nucleotide pool, Moorella sp. Abstract The thermophilic bacterium, Moorella sp. HUC22-1, newly isolated from a mud sample, produced ethanol from H 2 and CO 2 during growth at 55 ◦ C. In batch cultures in serum bottles, 1.5 mM ethanol was produced from 270 mM H 2 and 130 mM CO 2 after 156 h, whereas less than 1 mM ethanol was produced from 23 mM fructose after 33 h. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were higher in cells grown with H 2 and CO 2 than those grown with fructose. The NADH/NAD + and NADPH/NADP + ratios in cells grown with H 2 and CO 2 were also higher than those in cells grown with fructose. When the culture pH was controlled at 5 with H 2 and CO 2 in a fermenter, ethanol production was 3.7-fold higher than that in a pH-uncontrolled culture after 220 h. Introduction Most anaerobic acetogenic bacteria (acetogens) gain energy by acetate production from H 2 and CO 2 with ATP synthesis (Drake et al. 2002). Although these bacteria usually produce only acetate as an end- product, several species of mesophilic acetogens, such as Clostridium ljungdahlii and Acetobacterium woodii, can produce ethanol (Buschhorn et al. 1989, Tanner et al. 1993). On the other hand, some studies of ethanol metabolism in thermophilicacetogens have been reported; for example, some strains of Moorella thermoacetica can use ethanol when nitrate is dissim- ilated (Fröstl et al. 1996). Resting cells and crude extracts of M. thermoacetica formed 14 C-labeled eth- anol from [5- 14 C]methyltetrahydrofolate, which is an intermediate in the acetyl-CoA pathway (White et al. 1987), and reduced many carboxylic acids to the corresponding alcohols in the presence of electron donors, such as CO and formate, together with ar- tificial electron mediators, such as various viologens and cobalt sepulchrate (Simon et al. 1987). These re- ports suggest that some thermophilic acetogens also have an ethanol-producing pathway. To our know- ledge, however, direct evidence of ethanol production by thermophilic acetogens and information about re- lated enzymes during growth have not been reported to date. In this paper, we report that a thermophilic bacterium, Moorella sp. HUC22-1, isolated from a mud sample, produces ethanol together with acetate when grown on H 2 and CO 2 during growth. Materials and methods Organism and medium composition Moorella sp. HUC22-1, newly isolated from a mud sample collected from underground hot water in Chiba, Japan, was used in this study. For the enrichment cultures, ATCC 1754 PETC medium (http://www.atcc.org) was used as the basal medium. For the pure cultures, the basal medium had cysteine · HCl · H 2 O at 0.3 g l −1 , and Na 2 S · 9H 2 O was