Biochemical Engineering Journal 66 (2012) 52–60 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journa l h o me pa ge: www.elsevier.com/locate/bej Regular article Extended kinetic model for DBT desulfurization using Pseudomonas Putida CECT5279 in resting cells J. Calzada, A. Alcon, V.E. Santos ∗ , F. Garcia-Ochoa Departamento de Ingeniería Química, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain a r t i c l e i n f o Article history: Received 7 December 2011 Received in revised form 10 April 2012 Accepted 27 April 2012 Available online xxx Keywords: Biodesulfurization Pseudomonas putida CECT5279 Kinetic modeling Dibenzothiophene desulfurization 4S route Enzyme activity decay a b s t r a c t Dibenzothiophene desulfurization by Pseudomonas putida CECT5279, genetically modified microorgan- ism, in resting cells is studied. In previous works, operational conditions were established and a kinetic model describing the four serial reactions was proposed. Later studies showed the existence of two char- acteristic growth times of this bacterium, 5 and 23 h, offering maximum activities in the desulfinase and the monooxygenase enzymes of this route. The combination of cells collected at 5 and 23 h of growth time was proved to be a very effective biocatalyst for desulfurization in resting cells. In this work, the previously proposed kinetic model is extended and applied to these cells with different ages. Moreover, other extension is considered, taking into account the activity loss of the enzymes involved in 4S route, and the influence of biomass concentration employed. These extensions are of considerable importance in order to scale-up the process. The kinetic model developed is able to fit the experimental results for resting cell operation with cells of different ages, in different concentration taking into account the enzyme deactivation. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Sulfur oxide emissions are responsible for many well-known problems on human health, environment and materials. More restrictive legal limitations about sulfur content in fossil fuels have been imposed in European Union [1], United States [2] and other countries such as Japan and Canada [3]. Many efforts are focused on reaching such low limits by developing different proposed tech- nologies [4]. Hydrodesulfurization (HDS) is the most extensively employed method. However, some aromatic sulfur-compounds, such as 4- and 4,6-alkyldibenzothiophene DBT, and polyaromatic sulfur compounds, show resistance to be completely removed. Biodesulfurization (BDS) is one of the emerging technologies proposed to solve these problems. BDS consists of the employ of microorganisms, their enzymes or cellular extracts as catalysts in order to remove sulfur present in fuels [5–9]. BDS has been pre- sented as a complementary technique which, added to a previous HDS process. Among its advantages, BDS offers a high selectivity through the employ of microbial enzymatic systems with the ability of reducing the generation of undesirable byproducts [5,6,10], and selective routes to avoid C C bond breakdown helping to maintain the final properties of the fuel [3,5,6,11,12]. ∗ Corresponding author. Fax: +34 913944179. E-mail address: vesantos@quim.ucm.es (V.E. Santos). Due to abundance of some aromatic sulfur in fossil fuels [5,13] (particularly in heavier oil distillates [14]) and their special resis- tance to be removed by conventional HDS processes [5,6,14], dibenzothiophene (DBT) and its alkylated forms are usually cho- sen as model compounds in desulfurization studies. 4S route is an oxidative and non-destructive metabolic pathway, carried out by Rhodococcus erythropolis IGTS8, which belongs to this kind of selective routes [2,3,9,15–17]. This route is formed by four serial reactions make up this route through the transformation of DBT into a free sulfur molecule, 2,2 ′ -hydroxybiphenil (HBP) [18,19]. 4S route is catalyzed by two monoxygenases, DszC and DszA, and one desulfinase, DszB [17,20]. The third enzyme involved in 4S route, desulfinase DszB, catalyses the last step of 4S route, which involves the conversion of HBPS into the final product, HBP [18,20,21]. This route has been found in other wild type microoganisms such as Pantoea agglomerans [22] or Lysinibacillus sphaericus [23]. One of the aspects to be improved in BDS in order to be developed as an industrial scale process involves obtaining bet- ter biocatalysts for desulfurization [3,6,8,9,24–26]. Pseudomonas putida CECT 5279 is the biocatalyst employed in this work. It is a genetically modified bacterium with the ability of expressing the 4S pathway due to carrying the genes dszABC from Rhodococcus ery- thtropolis IGTS8, and a flavin-oxydo-reductase from Escherichia coli (hpaC) [27,28]. Previous works focused on desulfurization of DBT using P. putida CECT5279 studied the influence of the medium composition and the conditional operations on the desulfurization capabilities of this bacterium. A maximum in DBT conversion is 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.04.018