Short Communication Sitosterol bioconversion with resting cells in liquid polymer based systems Filipe Carvalho, Marco P.C. Marques, Carla C.C.R. de Carvalho, Joaquim M.S. Cabral, Pedro Fernandes * IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal article info Article history: Received 5 January 2009 Received in revised form 12 March 2009 Accepted 13 March 2009 Available online 11 April 2009 Keywords: Silicone oil 4-Androstene-3-17-dione Sitosterol Bioconversion Resting cells abstract The use of a biocompatible water-immiscible organic phase as a substrate and product pool has been acknowledged as an effective tool to overcome the low volumetric productivity of aqueous bioconversion systems involving hydrophobic compounds. The growing environmental and public health awareness is nevertheless leading to restrictions in the use of organic solvents in industrial processes, in order to ren- der these more environmentally friendly. Different approaches are hence being assessed for the design of alternative bioconversion media, involving the use of supercritical fluids, ionic liquids and natural oils and liquid polymers, among others. In this work, the use of liquid polymers as key components in the bioconversion media for a multi-step microbial bioconversion was assessed. The model system used was the selective cleavage of the side- chain of b-sitosterol by free resting cells of Mycobacterium sp. NRRL B-3805, a well established industrial multi-enzymatic process involving the use of nine catabolic enzymes in a fourteen-step metabolic path- way. High product yields were obtained when silicone B oil was used as substrate carrier/product pool, both in single oil and in oil:buffer two liquid phase system. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The use of organic solvents as substrate and/or product reser- voir, in order to improve the efficiency of bioconversion systems, is possibly the most widely chosen approach to overcome the tox- icity and/or low solubility of useful compounds (Heipieper et al., 2007; Kim et al., 2007). Most of the organic solvents commonly used as carriers present nevertheless some drawbacks, namely the damaging effects on microbial cells, along with their explosive and environmentally hazardous nature (Schmid et al., 1998; Déziel et al., 1999; Kim et al., 2007). Alternative approaches that retain the advantages of organic solvents, namely solubilization of hydro- phobic compounds to high concentration, while avoiding the drawbacks have thus been sought after and presented (Kim et al., 2007; Wang, 2007). One of these is based in the use of liquid polymers as components of the bioconversion media, either in polymer:polymer systems or in polymer:salt solution systems (Zijlstra et al., 1998). This strategy, which provides a mild environ- ment to the biocatalysts and is also environmentally friendly (Zijl- stra et al., 1998; Raghavarao et al., 2003), has been scarcely applied to sterol/steroid bioconversions, namely to D 1 -dehydrogenation of cortisol (Kaul and Mattiasson, 1986; Santos et al., 1991) and side- chain cleavage of cholesterol (Flygare and Larsson, 1989) and of phytosterols (Kutney et al., 2000; Stefanov et al., 2006). Flygare and Larsson (1989) performed cholesterol bioconversions using immobilized resting cells in a polymer:polymer:detergent media and an initial substrate concentration of only 1.0 g l À1 , whereas Kutney et al. (2000) and Stefanov et al. (2006) performed phytos- terol bioconversions using growing cells in a polymer:aqueous media system. In the present work the feasibility of using free rest- ing Mycobacterium sp. cells for sitosterol side-chain cleavage to 4- androstene-3,17-dione (AD), in a polymer based medium is ad- dressed. The phytosterol used was recovered from tall-oil (Dias et al., 2002) and cells were occasionally grown using waste glyc- erol recycled from a lab-scale biodiesel production systems as part of the carbon source. When a given bioconversion can be per- formed by either growing or resting cells, and provided cell viabil- ity is not compromised, in case this feature affects catalytic activity, the use of resting cells can be advantageous over growing cells. The former approach allows for a more focused evaluation of the bioconversion itself, given that the complex metabolic path- ways involved in microbial cell growth are not present and thus do not have to be taken into consideration, and there is no need for sterile environment, the whole leading to a simpler system (Mutafov et al., 1997; Wang et al., 2005; Cánovas et al., 2007). The use of a single liquid phase allows on the other hand higher volumetric productivities, and in the overall simplifies the design of reaction media, selection of operational conditions and down- stream processing. High product yields were observed for initial sitosterol concentrations of 5.0 g l À1 (12 mM) both in silicone oil media and in silicone oil:buffer two-phase systems. The poly(methylphenylsiloxane) oil, henceforth termed silicone B oil, 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.044 * Corresponding author. Tel.: +351 218419065; fax: +351 218419062. E-mail addresses: pedro.fernandes@ist.utl.pt, pedro.fernandes@mail.ist.utl.pt (P. Fernandes). Bioresource Technology 100 (2009) 4050–4053 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech