Production of a Biocatalyst of Pseudomonas putida CECT5279 for

Energy & Fuels 2004, 18, 851-857

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Production of a Biocatalyst of Pseudomonas putida CECT5279 for Dibenzothiophene (DBT) Biodesulfurization for Different Media Compositions Ana B. Martin, Almudena Alcon, Victoria E. Santos, and Felix Garcia-Ochoa* Departamento de Ingenieria Quimica, Facultad de Ciencas Quimicas, Universidad Complutense, 28040 Madrid, Spain Received October 23, 2003. Revised Manuscript Received March 2, 2004

The production of a biocatalyst of a genetically modified microorganism (GMO) (Pseudomonas putida CECT5279) that can desulfurize dibenzothiophene (DBT) has been studied. The biomass growth rate and the development of the sulfur-removal capability during microorganism growth have been measured and modeled. Different growth media and different carbon-glucose, carboncitrate, and carbon-glutamic acid sources, as well as different nitrogen-ammonium and/or nitrogen-glutamic acid sources, have been used, in addition to different glutamic acid concentrations (5, 10, 20, and 40 g/L), to study their influence on the growth rate and desulfurizing capability. Experimental results show clear differences both in growth and in the biodesulfurization capability developed by the cells, depending on the media composition. To quantify the desulfurization capability, a parameter has been defined: DBDS, which is the degree of biodesulfurization developed during growth. This parameter is useful not only to compare the results achieved under different media and conditions but also to compare different microorganisms, in regard to the desulfurization capability. Inside the experimental range studied, the best production media is composed of 20 g/L of glutamic acid, with 670 ppm of NH4+ as the respective carbon and nitrogen sources. A nonstructured kinetic model that describes the growth and desulfurizing capability development is proposed and applied by nonlinear simple response fitting to all the experiments that have been performed. The model can be used to describe all the experimental data with good statistical parameters.

Introduction Sulfur oxides emissionssmainly those produced by fossil fuels combustionsare the major cause of environmental problems such as acid rain. Governments are increasing their attention on the reduction of the legal limit of sulfur content in coal1 and oil fractions after refining. In the United States and the European Union, the mandatory maximum level for sulfur in gasoline in 2005 will be 50 ppm. By 2008, the mandatory maximum sulfur level in gasoline will be 10 ppm.2-4 The conventional hydrodesulfurization process is conducted under severe conditions, such as extremely high temperature and pressure, and is not effective for the degradation of heterocyclic sulfur compounds such as alkylated dibenzothiophene (DBT). There are reports of microorganisms that can metabolize DBT via a hydrocarbon degradative pathway * Author to whom correspondence should be addressed. E-mail address: [email protected]. (1) Dusuroy, T.; O ¨ zbas Bozdemir, T.; Yu¨rum, Y. Rev. Process. Chem. Eng. 1999, 2, 39-52. (2) Regulatory Impact AnalysissControl of Air Pollution from New Motor Vehicles: Tier 2 Motor Vehicle Emissions Standards and Gasoline Sulfur Control Requirements, USEPA Technical Report No. EPA420-R-99-023, 1999. (3) Phase II RFG. Report on Performance Testing, USEPA Technical Report No. EPA420-R-99-025, 1999. (4) Oldfield, C.; Wood, N. T.; Gilbert, F. D.; Murray, D.; Faure, R. Antonie van Leeuwenhoek 1998, 74, 119-132.

(with the destruction of carbon-carbon bonds);4 however, a small number of microorganisms, mainly belonging to the genuses Rhodococcus, Bacillus, Corynebacterium, and Anthrobacter, have been shown to be able to remove sulfur from DBT via sulfur-specific pathways, selectively cleaving sulfur from DBT without ring destruction and therefore maintaining the fuel energy content.5-7 Most of the research over the last 10 years has been focused on the metabolism of sulfur heterocycles, particularly dibenzothiophenes, by Rhodococcus strains and other relatively closely species. R. erythropolis IGTS8 is the most usual and characterized bacteria that can perform desulfurization reactions by means of the 4S desulfurization pathway. This bacterium can desulfurize many types of organic sulfur molecules, including DBT. The most-used microorganisms belong to genus Rhodococcus, such as Rhodococcus sp. strain SY1;8 R. erythropolis D-1,9 R. erythropolis H-2,10 R. erythropolis N1(5) Gallardo, M. E.; Fernańdez, A.; Lorenzo, MD.; Garcıá, J. L.; Dıáz, E. J. Bacteriol. 1997, 179, 7156-7160. (6) Ohshiro, T.; Izumi, K. Biosci., Biotechnol., Biochem. 1999, 63 (1), 1-9. (7) Setti, L.; Farinelli, P.; Di Martino, P.; Frassineti, S.; Lanzarini, G.; Pifferi, P. G. Appl. Microbiol. Biotechnol. 1999, 52, 111-117. (8) Omori, T.; Monna, L.; Saiki, Y.; Kodama, T. Appl. Environ. Microbiol. 1992, 58, 911-915. (9) Izumi, Y.; Oshiro, T.; Ogino, H.; Hine, Y.; Shimao, M. Appl. Environ. Microbiol. 1994, 60, 223-226.

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36,11 R. erythropolis KA2-5-1,12 Rhodococcus sp. strain T09,13 and Rhodococcus sp. strain P32C1.14 Other microorganisms that have been used belong to other genuses, such as Pseudomonas,15 Gordona,16,17 and Brevibacterium.18 Mesophilic and thermophilic microorganisms have also been studied.19-23 Over the past few years, it has been recognized that the use of a genetically modified microorganism (GMO) might increase the biodesulfurization (BDS) yield.24-27 In these cases, the object is to increase the desulfurizing capability of the wild bacteria of R. erythropolis IGTS8 by means of the addition of genes through genetic engineering techniques, that is to say, to improve a microorganism that already had desulfurizing capability. Nevertheless, there are not many studies regarding the development of GMOs that are capable of DBT desulfurization from bacteria that do not possess desulfurizing capability. Thus, other works have been conducted to construct optimized strains for commercial use. The first patent on the incorporation of the desulfurization genes into a Pseudomonas bacterium was issued in 1999 in the United States.28 Another patent was performed on the incorporation of a flavin reductase into an artificial operon to collect all the genes required for BDS into a single transcript.29 The microorganism used in this work has been a GMOsin particular, Pseudomonas putida CECT5979.30 This bacteria carries the genes dszABC from R. erythropolis IGTS8, and the gene hpaC from E. coli W.31 (10) Ohshiro, T.; Hirata, T.; Izumi, Y. Appl. Microbiol. Biotechnol. 1995, 44, 249-252. (11) Wang, P.; Krawiec, S. Appl. Environ. Microbiol. 1996, 61, 16701675. (12) Kishimoto, M.; Inui, M.; Omasa, T.; Katakura, Y.; Suga, K.; Okumura, K. Biochem. Eng. J. 2000, 5, 143-147. (13) Matsui, T.; Hirasawa, K.; Konishi, J.; Tanaka, Y.; Maruhashi, K.; Kurane, K. Appl. Microbiol. Biotechnol. 2001, 56, 196-200. (14) Maghsoudi, S.; Kheirolomoom, A.; Vossoughi, M.; Tanaka, E.; Katoh, S. Biochem. Eng. J. 2000, 5, 11-16. (15) Constantı´, M.; Bordons, A.; Giralt, J. Lett. Appl. Microbiol. 1994, 18, 107-111. (16) Rhee, S. K.; Chang, J. H.; Chang, Y. K.; Chang, H. N. Appl. Environ. Microbiol. 1998, 62, 2327-2331. (17) Gilbert, S. C.; Morton, J.; Buchanan, S.; Oldfield, C.; McRoberts, A. Microbiology 1998, 144, 2545-2553. (18) Van Affender, M.; Schacht, S.; Klein, J.; Tru¨per, H. G. Arch. Microbiol. 1990, 153, 324-328. (19) Konishi, J.; Ishii, Y.; Onaka, T.; Okumura, K.; Suzuki, M. Appl. Environ. Microbiol. 1997, 63, 3164-3169. (20) Kargi, F.; Robinson, J. H. Biotechnol. Bioeng. 1984, 16, 687690. (21) Kirimura, K.; Furuya, T.; Nishii, Y.; Ishii, Y.; Kino, K.; Usami, S. J. J. Biosci. Bioeng. 2001, 91 (3), 262-266. (22) Li, F. L.; Xu, P.; Ma, C. Q.; Luo, L. L.; Wang, X. S. FEMS Microbiol. Lett. 2003, 233 (2), 301-307. (23) Furuya, T.; Ishii, Y.; Nada, K.; Kino, K.; Kirimura, K. FEMS Microbiol. Lett. 2003, 221 (1), 137-142. (24) Denome, S. A.; Oldfield, C.; Nash, L. J.; Young, K. D. J. Bacteriol. 1994, 176, 6707-6716. (25) Denome, S. A.; Ison, E. S.; Young, K. D. Appl. Environ. Microbiol. 1993, 59, 2837-2843. (26) Maghsoudi, S.; Vossoughi, M.; Kheirolomoom, K.; Tanaka, E.; Katoh, S. Biochem. Eng. J. 2001, 8, 151-156. (27) Monticello, D. Curr. Opin. Biotechnol. 2000, 11, 540-546. (28) 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. (29) Squires, C. H.; Ji, W.; Xi, L.; Ortego, B. C.; Pogrebinsky, O. S.; Gray, K. A.; Childs, J. D. Method of Desulfurization of Fossil Fuel with Flavoprotein, U.S. Patent No. 5,985,650, November 16, 1999. (30) Garcıá-Ochoa, F.; Garcıá-Calvo, E.; Garcıá, J. L. Spanish Patent No. P200301677, 2003. (31) Galań, B.; Dıáz, E.; Garcıá, J. L. Environ. Microbiol. 2000, 2 (6), 687-694.

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There are some precedents in the literature about P. putida growth in a basal medium with yeast extract.15,31,32 Some studies have been performed using bacteria of the Pseudomonas genus in which glucose is used as a carbon source; however, there are no antecedents that include comparative studies about media composition and its influence on desulfurizing capability. The purpose of this work is to study the influence of the growth media composition on both the growth rate of P. putida CECT5279sthe biomass production rates and the desulfurization capability of the cells produced. Therefore, different carbon and nitrogen sources have been studied in this work; also, their initial concentrations have been changed, measuring the biomass concentration with time. Afterward, a kinetic model that can fit different growth curves, taking into account the composition of the media, is proposed. Thus, the growth rate in different media composition can be compared through the values of the usual parameters used to describe biomass growth, such as the specific growth rate (µ) and the maximum biomass concentration reached (Cmax X ). Also, biomass yields on the different substrates (YSX, YNX) have been calculated, evaluating the possibilities of the different sources. Moreover, the desulfurization capability of the cells (XBDS) at different growth times has been calculated, using a desulfurization test of DBT, taking into account the concentration of 2-hydroxybiphenyle (HBP) formed, which is the last and only compound without sulfur from the 4S route. Also, a model that involves the development of the desulfurization capability of the cells during growth is proposed. A parameter that considers the maximum desulfurizing capability of cells obtained during the growth cycle is defined; this parameter involves the biomass concentration attained, their BDS capability, and the time needed to attain this concentration (DBDS). This parameter is used to show the best medium composition to obtain biodesulfurizing cells of P. putida CECT5279, and it could be used to compare the results of different microorganisms produced under different operational media and conditions. 2. Materials and Experimental Procedure 2.1. Microorganism. The bacterium was Pseudomonas putida CECT 5279, supplied by Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Spain. Cultures were maintained on concentrated stock with glycerol in a saline serum (10%) solution. 2.2. Materials. D(+)-Glucose was obtained from Riedel-de Hae¨n, and glutamic acid and citrate were obtained from Panreac. Dibenzothiphene (DBT) was obtained from Aldrich, 2-hydroxybiphenyle (HBP) was obtained from Fluka, and Hepes buffer was obtained from Sigma Chemical. Deionized water was used to prepare all media and stock solutions, unless indicated otherwise. 2.3. Media. For inoculum buildup, a complex medium was used (LB, Luria-Bertani). For the optimization of the growth media, P. putida was cultured in a standard medium (BSM) that had the following composition: NaH2PO4‚H2O, 4 g/L; K2HPO4‚3H2O, 4 g/L; MgCl‚6H2O, 0.0245 g/L; CaCl2‚2H2O, 0.001 g/L; Cl3Fe‚ 6H2O, 0.001 g/L; MgSO4, 2 mM; and glycerol, 2%. (32) Foght, J. M.; Westlake, D. W. S. Can. J. Microbiol. 1998, 34, 1135-1141.

Production of P. putida CECT5279 for DBT BDS

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Table 1. Experiments Performed in This Work to Realize the Study the Growth Medium for P. putida Strain CECT5279 Carbon Source type

concentration (gS/L)

nitrogen source, NH4+ (ppm)

YCX (gS/gX)

YNX (gN/gX)

max DBDS (%D gX h-1 L-1)

1 2 3 4 5 6

glucose citrate glutamic acid glutamic acid glutamic acid glutamic acid

20 20 20 5 10 40

670 670 670 670 670 670

2.03 2.89 1.57 1.11 1.46 2.75

0.124 0.150 0.168 0.147 0.187 0.270

1.14 9.81 21.65 4.86 13.15 14.80

7 8 9 10

glutamic acid glutamic acid glutamic acid glutamic acid

5 10 20 40

1.10 1.80 1.98 3.25

0.106 0.170 0.182 0.313

5.11 10.10 15.69 13.91

run

The sources of added carbon (glucose, citrate, and glutamic acid) and nitrogen (NH4Cl) were dependent on the experiment. 2.4. Inoculum Buildup. Inocula were always prepared by means of a standarized method to obtain comparative experimental results: a 250-mL Erlenmeyer flask that contained 50 mL of LB with tetracycline (25 µg/mL) was inoculated using 100 µL of bacteria from a stock (10% glycerol) solution. This culture was incubated in an orbital shaker for 24 h. Afterward, a culture was used to inoculate 250-mL Erlenmeyer flasks that contained 50 mL of LB medium with tetracycline (25 µg/mL), which were incubated for 4 h in an orbital shaker. In all cases, the initial biomass concentration was fixed at 0.1 g/L (measured by OD600). 2.5. Growth Procedure. The contents of these last Erlenmeyer flasks were inoculated into 250-mL Erlenmeyer flaks that contained BSM with tetracycline (25 µg/mL), with a fixed biomass initial concentration of 0.1 g/L in all the runs. The operational conditions used were a temperature of 30 °C and a stirring speed of 220 rpm. Several samples were withdrawn during the growth steps and were conserved at -18 °C after centrifugation and resuspended in 5 mL of a glycerol/NaCl (50/ 50) solution. These samples were then used for BDS capability determination of the resting cells. 2.6. Desulfurization Assays with Resting Cells. The cells, which were prepared as indicated previously, were placed in a 250-mL Erlenmeyer flask that contained 40 mL of a Hepes buffer (50 mM, pH 8.0) of DBT, which is the model compound to be desulfurized. In all cases, the cell concentration was 0.7 g/L. Afterward, Erlenmeyer flasks were incubated at 30 °C and 220 rpm. Aliquot samples were collected at different time intervals. The samples were diluted with an equal volume of acetonitrile and vortexed extensively to solubilize any waterinsoluble intermediates. The cells were removed by centrifugation at 14 000g for 5 min at room temperature, and supernatants were analyzed by reserved-phase high-performance liquid chromatography (HPLC), as discussed later in this work. 2.7. Analytical Methods. 2.7.1. Biomass. The evolution of biomass concentration was determined by following the OD600, using a spectrophotometer (Shimadzu, model UV 1603). 2.7.2. Nitrogen Source. The ammonium concentration was monitored with a selective ammonia electrode (Orion, model 95-12). 2.7.3. Glucose. The glucose concentration was measured by means of HPLC, using a Kontron model Sedex-45 lightscattering detector, with a gain value of 6, an air pressure of 1.9 atm, and a temperature of 28 °C. The column used was a Nucleosil-NH2 column, 250 mm long × 4.6 mm in diameter with 5-µm particles. The mobile phase was an acetonitrile: water mixture (75:25), using a flow of 1 L/min. 2.7.4. Glutamic Acid and Citrate. Both the glutamic acid and citrate were measured with an enzymatic kit from Boehringer-Mannheim (reference materials 139092 and 130084, respectively). 2.7.5. Dibenzothiphene and 2-Hydroxybiphenyle. HPLC-UV spectroscopy was used to analyze the compounds of the 4S route. To analyze DBT and HBP, a C-8 column (Supelcosil,

3-µm particles; column was 150 mm long × 4.6 mm in diameter) was used, using acetonitrile:water (50:50) as the mobile phase (1 mL/min). Peaks were monitored at 240 nm.

3. Experimental Results and Discussion To study the influence of medium composition on P. putida CECT5279 growth, 10 experiments have been performed, where the type of source (for carbon and nitrogen) and the initial concentration (for glutamic acid) were each varied. The conditions of the experiments performed in this work are given in Table 1. Experimental results of biomass concentration (CX) evolution with time are shown in Figures 1-3 as data points, together with the desulfurizing capability attained by the cells (XBDS, DBDS). For all the experiments conducted, the yields of substrates (carbon and nitrogen sources) into biomass have been calculated, according to eq 1 (below), and the values obtained are also given in Table 1. When glutamic acid is used as the carbon source, the yield on nitrogen into biomass is obtained by means of nitrogen mass balance in the system when biomass reaches the stationary phase. Thus, the different yields were calculated according to

YjX )

Cj0 - CXj max Cmax - CX0 X

(j ) C,N)

(1)

In addition, two parameters have been defined to calculate the desulfurizing capability of the cells: (i) the percentage of desulfurization, measured as HBP conversion (XBDS), according to eq 2:

XBDS )

CHBP′3 × 100 CDBT′0

(2)

(where CDBT′0 is the concentration of DBT initially used to perform the standard BDS resting cell assay, CHBP′3 is the HBP concentration obtained after a 3-h resting cell assay, and XBDS is the percentage of BDS reached by the cells in the standard test of resting cells assay) and (ii) the desulfurizing development grade during growth (DBDS), according to eq 3,

DBDS )

XBDSCX tG

(3)

to quantify the desulfurizing capability, taking into account all the variables that influence it (desulfurization percentage obtained at each time by resting cells, together with the biomass concentration reached). In

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Figure 1. Evolution and model prediction of (a) biomass concentration, (b) percentage of desulfurizing, and (c) desulfurizing development grade obtained in Runs 1-3.

eq 3, tG is the time of growth needed to reach a biomass concentration and CX is the percentage of BDS attained by the cells in the standard test of resting cells assay. 3.1. Carbon Source Influence. Runs 1-3 have been conducted to study the influence of the nature of carbon source (three types of carbon sources have been checked: glucose, citrate, and glutamic acid). All these experiments have been conducted using an initial carbon source concentration of 20 g/L and ammonium chloride (NH4Cl) as the nitrogen source (670 ppm NH4+). Experimental resultssbiomass concentration, percentage of desulfurization, and BDS degree during growthsare shown in Figure 1. Figure 1a shows that the best results are obtained when glutamic acid is used as the carbon source. The maximum biomass concentration attained in the stationary phase is higher when the growth was performed with glutamic acid, where a ) 4.70 g/L was attained. The final value of Cmax X concentration obtained with glucose and citrate is smaller than that obtained with glutamic acid. In runs 1-3, the growth rates are quite similar. In Table 1, the values obtained for the yields of the different substrates into biomass (YCX and YNX) are

Martin et al.

shown for each one of the experiments. The parameter YCX obtained with citrate is higher than those obtained for the other two carbon sources that have been studied. The values obtained for YNX with citrate and glutamic acid are similar to and higher than that obtained using glucose as a carbon source. Figure 1b also shows the evolution of the percentage of desulfurization of P. putida (XBDS) when different carbon sources are used for growth. This parameter attains its highest value (64%) when glutamic acid is used as a carbon source. When growth is performed with citrate, the value of XBDS is lower (38%), and even lower for glucose (5%). The DBDS values are given for the different runs in Table 1. The maximum value of DBDS is 21.65, which is obtained when glutamic acid is used as a carbon source for the growth. With the other two carbon sources that have been checked, the value of DBDS is lower, reaching 1.14 for glucose and 9.81 for citrate. Runs 3-6 have been performed to study the influence of the initial concentration of glutamic acid (four initial concentrations of glutamic acid have been checked: 5, 10, 20, and 40 g/L). All these experiments have been performed using 670 ppm of NH4Cl as a nitrogen source. As can be observed, the initial concentration of glutamic acid has a clear influence in the desulfurizing capability of the cells, but not in the growth of the microorganism. The growth rate and the maximum biomass concentration attained at the stationary phase are very similar when the initial concentration of glutamic acid is 5, 10, and 20 g/L (Figure 2a); however, those values are lower when the initial concentration of glutamic acid is 40 g/L. The values of YCX and YNX are higher when 40 g/L of glutamic acid is used as the initial concentration of the carbon source; these values are always lower than and similar to the rest of the initial concentrations of glutamic acid. The influence of the initial concentration of glutamic acid on the desulfurizing capability (see Figure 2b) is noticeable; the maximum value of XBDS is ∼60% with initial glutamic acid concentrations of 20 and 40 g/L, but the difference between them is the time needed to reach the maximum value. Thus, when the initial concentration of glutamic acid is 20 g/L, the maximum value of XBDS requires 9 h of growth, whereas when the initial concentration of glutamic acid used is 40 g/L, this time is ∼16 h. When the initial concentration of glutamic acid is 5 and 10 g/L, the maximum values of XBDS are 15% and 44%, respectively. The maximum value of DBDS is 21.65, which is obtained when the initial concentration of glutamic acid used is 20 g/L. When the growth is conducted with initial concentrations of glutamic acid of 10 and 40 g/L, the value of DBDS is lower (13.15 and 14.80, respectively) and even lower for 5 g/L (4.86). 3.2. Nitrogen Source Influence. To study the influence of nitrogen source, it is necessary to consider that glutamic acid can be used as both carbon and nitrogen sources of growth, making it possible to compare the results obtained in runs performed using glutamic acid (as the carbon and nitrogen source) with or without the addition of NH4Cl (as a complementary nitrogen source). Figures 2 and 3 show the results obtained in different runs with ammonium addition


Figure 2. Evolution and model prediction of (a) biomass concentration, (b) percentage of desulfurizing, and (c) desulfurizing development grade obtained in the experiments performed to study the concentration of glutamic acid with ammonia (Runs 3-6).

(runs 3-6) and without ammonium addition (runs 7-10), respectively. As can be observed, the presence of NH4 + has an influence on the desulfurizing capability of the cells but not on the growth of the microorganism. The growth rate and the maximum biomass concentration attained at the stationary phase are similar in the experiments conducted with or without NH4Cl. The values of the yields of substrates into biomass (YCX and YNX) are quite similar in the experiments that have been performed with or without ammonium in the media; these values are always higher when the initial concentration of glutamic acid is 40 g/L. The influence of the presence of ammonium on the desulfurizing capability (see Figures 2 and 3) is noticeable when the initial concentration of glutamic acid is 20 or 40 g/L. The maximum value of XBDS is ∼60% when NH4Cl is added to the medium, whereas this value decreases to a value of 40%-50% if an inorganic nitrogen source is not added. When the initial concentration is 5 or 10 g/L, the absence or presence of NH4Cl


Figure 3. Evolution and model prediction of (a) biomass concentration, (b) percentage of desulfurizing, and (c) desulfurizing development grade obtained in the experiments performed to study the concentration of glutamic acid without ammonia (Runs 7-10).

in the media does not influence the desulfurizing capability. The parameter Dmax BDS attains its maximum values 21.65swhen the initial concentration of glutamic acid is 20 g/L and ammonium is added to the media; however, this value decreases to 15.69 when the initial concentration of glutamic acid is the same but NH4Cl is not added to the media. 4. Kinetic Modeling. Growth Kinetic Model Experimental data of biomass concentration with time are fitted to the following kinetic model:

(

CX dCX ) µCX 1 - max dt C X

)

(4)

Because the data are integral (biomass concentration change with time), these data must be differentiated or the equation (eq 4) must be integrated. Integration of the model introduces much less error than the data

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Table 2. Parameters Obtained by Fitting of Experimental Data to eq 4 µ

(gX/L) Cmax X

(h-1)

95% confidence level

run

parameter value

Student’s t-test

parameter value

Student’s t-test

Fischer’s F-test

SSRa

Student’s t-test

Fischer’s F-test

1 2 3 4 5 6 7 8 9 10

0.36 ( 0.03 0.35 ( 0.03 0.34 ( 0.05 0.35 ( 0.06 0.34 ( 0.01 0.31 ( 0.02 0.36 ( 0.06 0.36 ( 0.04 0.35 ( 0.07 0.30 ( 0.05

30.15 31.42 13.88 13.25 14.56 18.20 17.45 18.58 12.77 15.90

3.84 ( 0.08 3.63 ( 0.07 4.70 ( 0.25 4.25 ( 0.22 4.54 ( 0.24 3.48 ( 0.10 4.79 ( 0.19 4.72 ( 0.17 4.46 ( 0.27 3.66 ( 0.15

102.9 105.3 41.57 40.52 41.50 63.30 53.23 64.96 37.37 55.75

8251 7989 1814 2345 2653 2233 3370 3945 1638 2651

8.4 × 10-3 7.2 × 10-3 7.6 × 10-3 1.5 × 10-2 7.2 × 10-3 5.5 × 10-2 8.6 × 10-3 8.3 × 10-3 1.1 × 10-2 7.6 × 10-3

2.17 2.18 2.18 2.23 2.18 2.14 2.23 2.23 2.20 2.16

3.49 3.49 3.49 3.71 3.49 3.39 3.71 3.71 3.59 3.41

a

Sum-of-squares residual.

differentiation.33 Therefore, eq 4 has been integrated, with the initial condition: t ) 0 ∴CX ) CX0, which is known, based on the inoculum procedure used, yielding

CX )

CX0 exp(µt)[1 - exp(µt)] 1 - (CX0/Cmax X )[1 - exp(µt)]

(5)

The fitting has been conducted by means of nonlinear regression, using the Fischer and Student statistical tests to evaluate the quality of fitting. Moreover, the sum-of-squares residual (SSR), calculated according to eq 6, has been used as a representative of the fitting: N

2 (CXexp - Cthe ∑ X ) i)1 i

SSR )

i

N

(6)

This model can be fit to all the experiments performed in this work, and the fitting results are summarized in Table 2, showing very good statistical parameter values in all cases. For runs 1, 2, and 3, which were performed with different carbon sources, the specific growth rates are very similar (0.34-0.36 h-1; see Table 2). The parameter is clearly greater when growth is performed with Cmax X glutamic acid (4.70 g/L) and lesser with glucose (3.84 g/L) and citrate (3.63 g/L). The prediction of biomass concentration changes with time in this model is very similar to the experimental data, as can be seen in Figure 1, where the model predictions are given by solid lines. The specific growth rate and the maximum concentration of biomass attained in the stationary phase of growth are quite similar in all the experiments that have been conducted with or without NH4Cl in the media, except when the initial concentration of glutamic are acid is 40 g/L. In this case, the values of µ and Cmax X less than those in the other runs. 5. Biodesulfurization Model A model that describes the development during growth of the desulfurizing capability has been used, considering XBDS as an associated product of growth. The equation considered is a modification of that of (33) Garcia-Ochoa, F.; Romero, A.; Santos, V. E. Int. Chem. Eng. 1992, 32 (3), 538-551.

Luedeking-Piret,34 as follows:

dXBDS dCX )R - βCX dt dt

(7)

with the boundary conditions t ) 2.5 h and XBDS ) 0, which means that a delay time must be taken into account in the development of the desulfurization capability of the cells. Experimental data are fitted to eq 7 using a fourthorder Runge-Kutta algorithm to integrate it, coupled to a simple-response nonlinear algorithm.35 The model can be fit to all the runs that have been conducted, showing good results for statistical parameters (Student’s t-test and Fischer’s F-test), as shown in Table 3. The prediction of DBDS values can be performed using eq 3 but introducing theoretical values for both the percentage of BDS capability (XBDS) and biomass concentration (CX). Figures 1-3 show the model predictions for both variables (XBDS and DBDS) as solid lines, yielding good reproductions of the experimental data. The value of the fitting parameter R, if the production of desulfurizing capability is considered to be associated with growth, is highest when glutamic acid is used as the carbon source. The tendency for this ability to decrease during the stationary growth phase (indicated by the parameter β) is greater when glucose is used as the carbon source. The presence of ammonium in the media has a clear influence when the initial concentration of glutamic acid is 20 g/L. With this initial concentration of carbon source, the parameter R is equal to 33.72 when NH4Cl is added to the media and it decreases to 15.42 when ammonium is not added to the media. In runs 3-10, the value of the parameter β is approximately zero, which means that the desulfurizing capability remains constant during the stationary growth phase, except when the experiment is conducted with an initial concentration of glutamic acid of 20 g/L and NH4Cl is added to the media; in this case, the value of β is 1.77, which means that the desulfurizing capability decreases during the stationary growth phase. 6. Conclusions In all the experiments that have been conducted, the capability to eliminate sulfur from dibenzothiophene (DBT) has been observed to attain a maximum value (34) Luedeking, R.; Piret, E. L. J. Biochem. Microbiol. Technol. Eng. 1959, 1, 393-412. (35) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 11, 433-441.



Table 3. Parameters Obtained by Fitting of Experimental Data to eq 7 R (%D L gX-1)

β (%D L gX-1 h-1)

95% confidence level

run parameter value Student’s t-test parameter value Student’s t-test Fischer’s F-test 1 2 3 4 5 6 7 8 9 10 a

4.03 ( 0.42 13.34 ( 1.57 33.72 ( 5.76 4.16 ( 0.16 13.30 ( 2.09 13.80 ( 2.44 8.19 ( 1.11 12.87 ( 2.52 15.42 ( 3.42 13.7 ( 1.95

6.32 15.22 9.96 8.61 5.31 10.51 12.21 9.15 19.16 6.65

3.98 ( 0.04 1.68 ( 0.35 1.77 ( 0.66 0 0.14 ( 0.01 0 0.62 ( 0.09 0.08 ( 0.05 0.15 ( 0.06 0

5.82 9.69 4.18

464 543 784 1041 123 1228 610 600 4192 560

5.51 8.21 6.07 19.07

SSRa 0.61 3.72 5.17 2.51 15.32 6.25 0.45 5.85 0.87 8.75

Student’s t-test Fischer’s F-test 2.57 2.57 2.44 2.57 2.57 2.44 2.57 2.57 2.57 2.44

5.79 5.79 5.14 5.79 5.79 5.14 5.79 5.79 5.79 5.14

Sum-of-squares residual.

at the end of the exponential phase of growth (between 9 and 15 h of growth time). The maximum biomass concentration of Pseudomonas putida is obtained when glutamic acid is used as the carbon source; however, the growth rates are very similar for the three carbon sources that have been checked. The presence or absence of NH4Cl in the media does not influence the growth of the microorganism, because the values of µ and Cmax are quite similar in the X experiments that have been performed with or without ammonium chloride (NH4Cl). Nevertheless, the best desulfurization capability of P. putida is obtained with an initial concentration of glutamic acid of 20 g/L and with 670 ppm of NH4Cl added to the media. This can be easily remarked by the value of the parameter DBDS, whose maximum values during the growth time in the different runs are given in Table 1. This parameter (DBDS, which indicates the desulfuration capability of the biomass as a whole) is useful not only to choose the optimal biocatalyst production conditions but also to compare such capabilities for different microorganisms. The kinetic growth model can predict all the experiments that have been conducted in this work with a very good fitting of the experimental results. The values of the kinetic parameters (µ and Cmax X , given in Table 2) are in agreement with the experimental observations. The proposed biodesulfurization model, considering the desulfuration capability to be associated with growth, can be fit to the experimental results of this capability, using two parameters, also with good reproducibility, yielding meaningful parameters that can involve all the influences (see Table 3). Acknowledgment. This work has been supported by MCyT-Plan Nacional de I+D-Programa de Procesos

y Productos Quimicos (under Contract No. PPQ20011361-C02-01). The grant support for two of the authors, by Comunidad Autonoma de Madrid (A. Alcon) and by Ministerio de Ciencia y Tecnologia (A. B. Martin), is gratefully recognized. Nomenclature Cj: concentration of compound j (gj/L) DBT: dibenzothiophene DBDS: degree of biodesulfurization development in growth (%D gX h-1 L-1) HBP: 2-hydroxybenzothiophene N: number of experimental data of each experiment SSR: sum-of-squares residual referenced to data number XBDS: percentage of biodesulfurization (%HBP), given by eq 2 YjX: yield of source j into biomass (gi/gX), defined by eq 1 t: time (h) R: parameter of eq 7 (%D L gX-1), kinetic of the production of desulfurization capability as associated to growth β: parameter of eq 7 (%D L gX-1), kinetic of the loss of desulfurization capability µ: specific growth rate (h-1) Subscripts C: refers to the carbon source DBT: refers to dibenzothiophene G: refers to the growth time HBP: refers to 2-hydroxybenzothiophene N: refers to the nitrogen source X: refers to biomass 0: refers to the initial value 3: refers to the value after 3 h Superscripts exp: refers to the experimental value max: refers to the maximum value of the parameter the: refers to the theoretical value EF030174C

Production of a Biocatalyst of Pseudomonas putida CECT5279 for

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