Journal of Biotechnology 96 (2002) 281 – 289 Membrane-facilitated bioproduction of 3-methylcatechol in an octanol/water two-phase system Leonie E. Hu ¨ sken a,b, *, Mirjam Oomes a , Karin Schroe ¨n a , Johannes Tramper a , Jan A.M. de Bont c , Rik Beeftink a a Food and Bioprocess Engineering Group, Department of Agrotechnology and Food Sciences, Wageningen Uniersity, PO Box 8129, 6700 EV Wageningen, The Netherlands b Department of Agrotechnology and Food Sciences, Diision of Industrial Microbiology, Wageningen Uniersity, PO Box 8129, 6700 EV Wageningen, The Netherlands c TNO MEP, PO Box 342, 7300 AH Apeldoorn, The Netherlands Received 5 November 2001; received in revised form 15 February 2002; accepted 6 March 2002 Abstract Bioproduction of 3-methylcatechol from toluene by Pseudomonas putida MC2 was studied in the presence of an additional 1-octanol phase. This solvent was used to supply the substrate and extract the product, in order to keep the aqueous concentrations low. A hollow-fibre membrane kept the octanol and aqueous phase separated to prevent phase toxicity towards the bacterium. Volumetric production rates increased approximately 40% as compared to two-phase 3-methylcatechol production with direct phase contact. Preliminary investigations on downstream process- ing of 3-methylcatechol showed that 1 M of sodium hydroxide selectively extracted the disodium salt of 3-methylcat- echol into an aqueous phase. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Pseudomonas putida ; Membrane bioreactor; 3-Methylcatechol production; Organic/aqueous two-phase system www.elsevier.com/locate/jbiotec 1. Introduction Catechol and its derivatives are important chemicals that are used in the manufacture of synthetic flavours such as vanillin (Shirai, 1986) and are used as precursors for pharmaceutical production (Ennis and Ghazal, 1992; van Schar- renburg and Frankena, 1996). They are difficult to synthesize chemically (Held et al., 1999; van Scharrenburg and Frankena, 1996). Some microorganisms are known to produce catechols (Held et al., 1999; Robinson et al., 1992; Haigler and Spain, 1991). As a reference system, we have studied the conversion of toluene to 3-methylcatechol by Pseudomonas putida MC2 in aqueous media (Hu ¨ sken et al., 2001a, 2002). The product is not converted or consumed by these cells. For the production of 3-methylcatechol, growing cells are needed, but in single-phase me- dia growth stopped early due to the rising product * Corresponding author. Tel.: +31-317-482-884; fax: +31- 317-482-237. E-mail address: leonie.husken@algemeen.pk.wau.nl (L.E. Hu ¨ sken). 0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0168-1656(02)00045-7