Production of a Biocatalyst of Pseudomonas putida CECT5279 for DBT Biodesulfurization: Influence of the Operational Conditions Ana B. Martin, Almudena Alcon, Victoria E. Santos, and Felix Garcia-Ochoa* Dpto. Ingenieria Quimica, Facultad CC. Quimicas, Universidad Complutense, 28040 Madrid, Spain Received April 7, 2004 The influence of working conditions on the growth batch of Pseudomonas putida CECT5279 has been studied, in regard to both the growth rate and the desulfurization capability accumulated in the cells. These operational conditions include pH conditions (buffered and nonbuffered media, using different carbon sources (glucose, citrate, and glutamic acid)), operating temperatures (26- 32 °C), and different dissolved oxygen concentrations, due to different aeration conditions (different air flows, using enriched air, etc.). Pseudomonas putida CECT5979, which is a genetically modified microorganism (GMO), has the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl (HBP), desulfurizing the organic molecule. To get the best conditions to obtain desulfurizing cells, a parameter (D BDS ) that incorporates both biomass concentration and time to reach a particular percentage of desulfurizing capability (X BDS ) has been used. The optimum value of D BDS has been obtained under the following working conditions: temperature, 30 °C; nonbuffered medium with glutamic acid as the carbon source; and, in relation to the dissolved oxygen concentration, the best conditions for growth are not the same as those required to get the highest desulfurizing activity. A kinetic model based on a logistic equation has been applied to describe biomass concentration during P. putida CECT5979 growth. Kinetic model parameters (µ and C X max ) were obtained under several operating conditions. A model proposed in a previous work [Martin et al., Energy Fuels 2004, 18, 851-857] was applied to describe biodesulfurization capability evolution during growth. Predicted values of biomass concentration and biodesulfurizing capability percentage achieved by the cells can be obtained during bacteria growth, with values very similar to those found experimentally, in a wide interval of operating conditions. Introduction Recent regulations will drive sulfur levels in crude oil to <350 ppm and refiners are expected to get <10- 15 ppm sulfur soon (2005-2007). 2 Currently, petroleum refining is mainly based on the use of physicochemical processes and chemical catalysis operating under drastic conditions (high temperature and pressure). These processes are costly in energy and highly contaminating; 3 therefore, the application of a biodesulfurization (BDS) process after hydrodesulfur- ization (HDS), mainly for diesel oils, has attracted attention as a new ecotechnology to achieve more- efficient desulfurization. 4 This type of BDS is usually studied with model compounds, with dibenzothiophene (DBT) being the model compound used most often. A wider range of literature is available on the biotransformation of DBT by Rhodococcus erythropolis IGTS8, which is the bacteria more frequently used in this type of study. The genes involved in DBT desul- furization constitute the Dsz pathway, which is also called the 4S pathway. The enzymes and genes of metabolic pathways for desulfurization have been elu- cidated. 5,6 Genetic engineering has been used to improve the IGTS8 strain, in an effort to increase the DBT desul- furizing activity, allowing shorter residence times that are compatible with a commercial application. 7 There are some studies that are developing geneti- cally modified microorganisms (GMOs) capable of DBT desulfurization from bacteria unable to develop the 4S pathway. The first patent on the incorporation of the desulfurization genes into a Pseudomonas bacterium was issued in 1999 in the United States. 8 Another patent was awarded on the incorporation of a flavin reductase into an artificial operon to collect all the genes required for BDS into a single transcript. 9 (1) Martin, A. B.; Alcon, A.; Santos, V. E.; Garcia-Ochoa, F. Energy Fuels 2004, 18, 851-857. (2) Monticello, D. Curr. Opin. Biotechnol. 2000, 11, 540-546. (3) Le Borgne, S.; Quintero, R. Fuel Process. Technol. 2003, 81, 155- 169. (4) Kirimura, K.; Furuya, T.; Nishii, Y.; Ishii, Y.; Kino, K.; Usami, S. J. Biosci. Bioeng. 2001, 91 (3), 262-266. (5) Denome, S. A.; lson, E. S.; Young, K. D. Appl. Environ. Microbiol. 1993, 59, 2837-2843. (6) Oldfield, C.; Pogrebinski, O.; Simmonds, J.; Kulpa, C. F. Micro- biology 1997, 143, 2961-2973. (7) Pacheco, M. A.; Lange, E. A.; Pienkos, P. T.; Yu, L. Q.; Rouse, M. P.; Lin, Q.; Linguist, L. K. Presented at the Annual Meeting of the National Petrochemical and Refiners Association, San Antonio, TX, 1999, Paper AM-99-27. (8) Darzins, A.; Xi, L.; Childs, J. D.; Monticello, D. J.; Squires, C. H. Dsz Gene Expression in Pseudomonas Hosts, U.S. Patent No. 5,952,208, September 14, 1999. 775 Energy & Fuels 2005, 19, 775-782 10.1021/ef0400417 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/19/2005