Appl Microbiol Biotechnol (2003) 61:77–81 DOI 10.1007/s00253-002-1203-5 ORIGINAL PAPER E. S. Gilbert · A. W. Walker · J. D. Keasling A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion Received: 8 May 2002 / Revised: 6 November 2002 / Accepted: 15 November 2002 / Published online: 16 January 2003  Springer-Verlag 2003 Abstract A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophe- nol mineralization. The co-culture effectively hydrolyzed 500 mM parathion (146 mg l 1 ) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium mem- bers. Parathion hydrolysis by E. coli SD2 followed Michaelis–Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibi- tion kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 mM p-nitrophenol (83 mg l 1 ) and P. putida KT2440 pSB337 inhibited by 1,000 mM p-nitrophenol (139 mg l 1 ). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construc- tion of degradative consortia may prove beneficial. Introduction Successful detoxification of recalcitrant organic chemi- cals may require the concerted effort of multispecies bacterial consortia. There are several recent examples of multispecies consortia isolated by enrichment culture that degrade structurally diverse organic compounds, includ- ing endosulfan (Sutherland et al. 2000), chloronitroben- zene (Park et al. 1999), 1,3-dichloropropene (Katsivela et al. 1999), atrazine (Alvey and Crowley 1996), and nitrate esters (Ramos et al. 1996). However, it may not be possible to isolate a natural bacterial consortium capable of degrading some organic compounds. Some of the factors that can prevent the enrichment of a degradative consortium include natural infrequency of an essential degradative gene (Shapir et al. 1998), production of recalcitrant intermediates (Van Hylckama Vlieg and Janssen 2001), molecular structural features of the target compound that limit its degradability (e.g. polyhalogenat- ed compounds; Wackett et al. 1994), poor bioavailability (e.g. 5-ring polycyclic aromatic hydrocarbons; Bastiaens et al. 2000), and the natural dominance of a nonproduc- tive metabolic pathway (Oh and Bartha 1997). An alternative strategy to isolating natural degradative consortia is to combine bacteria with complementary metabolic pathways into functional assemblages. This approach has been used for biological degradation of several recalcitrant compounds, including 4,4’-dichloro- biphenyl (Adriaens et al., 1989), chlorinated dibenzofu- rans (Arfmann et al. 1997; Wittich et al. 1999), 9- fluorenone (Casellas et al. 1998), and mixtures of volatile organic hydrocarbons (Komukai-Nakamura et al. 1996; Oh and Bartha 1997). The use of multiple organisms with distinct metabolic capabilities may be advantageous in some cases over introducing the requisite degradative genes into a single microorganism. This is particularly true when biochemical intermediates inhibit the activity of one or more steps in the catabolic pathway, as was reported for aerobic polychlorinated biphenyl metabolism (Adams et al. 1992). E. S. Gilbert ( ) ) Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30303 USA e-mail: esgilbert@gsu.edu Tel.: +1-404-4639917 Fax: +1-404-6512509 A. W. Walker Sandia Laboratories, Livermore, Calif., USA J. D. Keasling Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA