ORIGINAL PAPER L. D. Collins á A. J. Daugulis Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999 Abstract A two-phase organic/aqueous reactor con®g- uration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompat- ibility, and good predicted partition coecients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two- phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermenta- tion of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volu- metric degradation rate of 0.115 g l )1 h )1 . This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH. Introduction Benzene, toluene and p-xylene are common hydrocar- bon constituents produced by petrochemical companies in vast amounts each year (Chin et al. 1996). These xenobiotics are released into the environment through petroleum spills, leaks and discharge of industrial eu- ents. The presence of these compounds in the environ- ment poses signi®cant risks to indigenous biological populations because of their toxicity. Benzene, toluene and p-xylene compounds have been classi®ed as priority substances by Environment Canada, indicating that they may be harmful to the environment or constitute a danger to human health (Environment Canada, 1995). In addition, benzene has been identi®ed as a known carcinogen. These aromatic compounds are water-solu- ble and highly mobile, and are therefore likely to reach downstream drinking water sources. Various biological remediation schemes have been investigated for the treatment of soil, water and indus- trial euents containing inhibitory compounds such as benzene, toluene and p-xylene (Kelly et al. 1996; Lee et al. 1993, 1994; Choi et al. 1992; Ottengra et al. 1986). Biological treatment methods have traditionally been limited by the toxicity of these compounds, and the correspondingly low concentrations of the substrates to which the microbes must be exposed (Singleton 1994). In order for bioremediation to become a valid treatment option, the issue of substrate inhibition must be ad- dressed. To this end, a two-phase reactor scheme has been examined. Two-phase bioreactor systems have previously been used in extractive fermentation (Barton and Daugulis 1992; Jones et al. 1993; Daugulis et al. 1994). The im- miscible organic phase in these fermentations is used to remove the inhibitory end-product selectively from the aqueous phase as it is produced. This concept can be extrapolated to controlled delivery of a toxic substrate. In this scheme, the inhibitory substrate is loaded into the immiscible organic phase and allowed to partition into the aqueous phase at much lower concentrations. The overall concentration of the inhibitory substrate in the system is very high, but the concentration of substrate in the aqueous phase can be maintained well below inhib- itory levels (Vermue et al. 1993). This reactor con®guration has been used successfully to degrade pentachlorophenol (Munro and Daugulis 1997a, b) and phenol in batch and fed-batch modes Appl Microbiol Biotechnol (1999) 52: 354±359 Ó Springer-Verlag 1999 L. D. Collins á A. J. Daugulis (&) Department of Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada e-mail: daugulis@chee.queensu.ca Tel.: +1-613-533-2784 Fax: +1-613-533-6637