Enzyme and Microbial Technology 53 (2013) 143–151 Contents lists available at SciVerse ScienceDirect Enzyme and Microbial Technology jou rn al hom epage: www.elsevier.com/locate/emt In situ extractive fermentation for the production of hexanoic acid from galactitol by Clostridium sp. BS-1 Byoung Seung Jeon a , Chuloo Moon c , Byung-Chun Kim d , Hyunook Kim e , Youngsoon Um c,∗∗,1 , Byoung-In Sang a,b,∗,1 a Department of Chemical Engineering, Hanyang University, 17 Hangdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea b Department of Fuel Cells and Hydrogen Technology, Hanyang University, 17 Hangdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea c Clean Energy Research Center, Korea Institute and Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea d Energy Materials and Process, BK 21, Hanyang University, 17 Hangdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea e Department of Environmental Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Republic of Korea a r t i c l e i n f o Article history: Received 30 November 2012 Received in revised form 14 February 2013 Accepted 18 February 2013 Keywords: Hexanoic acid Clostridium sp. BS-1 Fractional factorial experimental design Box–Behnken experimental design In situ extractive fermentation Galactitol a b s t r a c t Clostridium sp. BS-1 produces hexanoic acid as a metabolite using galactitol and enhanced hexanoic acid production was obtained by in situ extractive fermentation with Clostridium sp. BS-1 under an optimized medium composition. For medium optimization, five ingredients were selected as variables, and among them yeast extract, tryptone, and sodium butyrate were selected as significant variables according to a fractional factorial experimental design, a steepest ascent experimental design, and a Box–Behnken experimental design. The optimized medium had the following compositions in modified Clostridium ace- tobutyricum (mCAB) medium: 15.5 g L -1 of yeast extract, 10.13 g L -1 of tryptone, 0.04 g L -1 of FeSO 4 ·7H 2 O, 0.85 g L -1 of sodium acetate, and 6.47 g L -1 of sodium butyrate. The predicted concentration of hexanoic acid with the optimized medium was 6.98 g L -1 , and this was validated experimentally by producing 6.96 g L -1 of hexanoic acid with Clostridium sp. BS-1 under the optimized conditions. In situ extractive fermentation for hexanoic acid removal was then applied in a batch culture system with the optimized medium and 10% (v/v) alamine 336 in oleyl alcohol as an extractive solvent. The pH of the culture in the extractive fermentation was maintained at 5.4–5.6 by an acid balance between production and retrieval by extraction. During a 16 day culture, the hexanoic acid concentration in the solvent increased to 32 g L -1 while it was maintained in a range of 1–2 g L -1 in the medium. The maximum rate of hexanoic acid production was 0.34 g L -1 h -1 in in situ extractive fermentation. © 2013 Published by Elsevier Inc. 1. Introduction Fossil fuel depletion and global warming have heightened the need for the development of alternative energy and chemical mate- rials [1,2]. One such alternative is biological conversion of biomass carbohydrates into biofuels and biomaterials. Seaweed (Ceylon moss) has a high content of carbohydrates, galactose (23%) and glucose (20%), and thus its potential as a biomass resource is com- parable to that of land plants [3]. One of the major constituents of seaweed, d- and l-galactose, could be converted to galactitol by an enzymatic reaction catalyzed by aldose reductase or a hydro- genation reaction with a chemical catalyst. Both form of d- and ∗ Corresponding author. Tel.: +82 2 958 5819; fax: +82 2 958 6858. ∗∗ Corresponding author at: Department of Chemical Engineering, Hanyang University, 17 Hangdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea. Tel.: +82 2 2220 2328; fax: +82 2 2220 4716. E-mail addresses: yum@kist.re.kr (Y. Um), biosang@hanyang.ac.kr (B.-I. Sang). 1 Both authors contributed equally as corresponding authors to this work. l-galactose could be utilized for the production of bioenergy or bio- chemicals, if a microorganism utilized galactitol. Recently, a strict anaerobic bacteria metabolizing galactitol was reported, and this anaerobe produced volatile fatty acids including hexanoic acid as a metabolic end product [4]. Hexanoic acid is a saturated fatty acid that has six carbons and one carboxylic group, and is a light colorless or yellow oily liquid with an acrid odor [5]. It is often found in oils and animal fats and has been used in diverse industrial applications such as perfumes, medicine, food additives, lubricating grease, tobacco flavor, rubber, and dyes [6,7]. In addition, hexanoic acid can be converted into other useful materials such as hexyl hexanoate and hexanol via esterification and hydrogenation [8,9]. Several bacterial species have been reported as hexanoic acid producers, and diverse approaches are being applied for the production of hexanoic acid by bacterial fermentation. For uti- lization of cellulose in hexanoic acid production, Clostridium kluyveri, producing hexanoic acid from ethanol plus either acetate or succinate, was co-cultured with ruminal cellulolytic bacteria, 0141-0229/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.enzmictec.2013.02.008