Energy & Fuels 2006, 20, 1565-1571
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Oxygen-Uptake and Mass-Transfer Rates on the Growth of Pseudomonas putida CECT5279: Influence on Biodesulfurization (BDS) Capability Emilio Gomez, Victoria E. Santos, Almudena Alcon, Ana B. Martin, and Felix Garcia-Ochoa* Departamento Ingenieria Quimica, Facultad Quimicas, UniVersidad Complutense, 28040 Madrid, Spain ReceiVed NoVember 8, 2005. ReVised Manuscript ReceiVed March 27, 2006
Oxygen-uptake and oxygen mass-transfer rates in cultures of Pseudomonas putida [a genetically modified organism (GMO) able to desulfurize dibenzothiophene and its derivatives] have been determined in a stirred tank bioreactor under different transport conditions. The oxygen-uptake rate has been measured by applying a modified dynamic method using a step of pure oxygen, because of the very low oxygen concentration in the broth. A kinetic model is proposed, obtaining the kinetic parameter values: specific maintenance and yield coefficients. Volumetric mass-transfer coefficient, kLa, has been determined, in a wide interval of hydrodynamic conditions, and predicted using a theoretical model. An increase of the transport rate is detected with the increase of the biomass and oxygen-uptake rate, which is described by a biological enhancement factor, E, which is also calculated by a theoretical model. Experimental values of kLa and E are compared with those predicted by the models with good agreement. Influences of mass-transfer conditions have been observed in growth and desulfurization capability of the cells. The oxygen-dissolved concentration is calculated from the estimations of the oxygen-transfer rate and from the oxygen-uptake rate; the models proposed are able to reasonably predict the experimental change of the oxygen concentration with the time course of the process.
1. Introduction The permitted level of sulfur in diesel oils is quickly being reduced because of sulfur oxide problems. Thus, biocatalyst productions for biodesulfurization (BDS) are of increasing interest.1-8 The most employed microorganisms belong to genus Rhodococcus, Pseudomonas, Gordona, and BreVibacterium. However, nowadays, it has been recognized that the use of genetically modified microorganisms (GMOs) might increase the BDS yield.9-12 * To whom correspondence should be addressed. E-mail: fgochoa@ quim.ucm.es. (1) Omori, T.; Monna, L.; Saiki, Y.; Kodama, T. Desulphurization of dibenzothiophene by Corynebacterium sp. strain SY-1. Appl. EnViron. Microbiol. 1992, 58, 911-915. (2) Izumi, Y.; Oshiro, T.; Ogino, H.; Hine, Y.; Shimao, M. Selective desulphurization of dibenzothiophene by Rhodococcus erythropolis D-1. Appl. EnViron. Microbiol. 1994, 60, 223-226. (3) Ohshiro, T.; Kambayashi, Y.; Hine, Y.; Izumi, Y. Involvement of flavin coenzyme in dibenzothiophene degrading enzyme system from Rhodococcus erythropolis D-1. Biosci. Biotechnol. Biochem. 1995, 59, 1349-1351. (4) Wang, P.; Krawiec, S. Kinetic analyses of desulphurization of dibenzothiophene by Rhodococcus erythropolis in batch and fed-batch cultures. Appl. EnViron. Microbiol. 1996, 61, 1670-1675. (5) Martin, A. B.; Alcon, A.; Santos, V. E.; Garcia-Ochoa, F. Production of a biocatalyst of Pseudomonas putida CECT5279 for DBT biodesulfurization: Influence of the operational conditions. Energy Fuels 2005, 19, 775-782. (6) Rhee, S. K.; Chang, J. H.; Chang, Y. K.; Chang, H. N. Desulfurization ofdibenzothiophene and diesels oils by a newly isolated Gordona strain CYKS1. Appl. EnViron. Microbiol. 1998, 62, 2327-2331. (7) Kishimoto, M.; Inui, M.; Omasa, T.; Katakura, Y.; Suga, K.; Okumura, K. Efficient production of desulfurizing cells with the aid of expert system. Biochem. Eng. J. 2000, 5, 143-147. (8) Matsui, T.; Hirasawa, K.; Konishi, J.; Tanaka, Y.; Maruhashi, K.; Kurane, K. Microbial desulphurization of alkylated dibenzothiophene and alkylated benzothiophene by recombinant Rhodococcus sp. Strain T09. Appl. Microbiol. Biotechnol. 2001, 56, 196-200.
The bacterium Pseudomonas putida CECT5279 is a GMO able to remove the sulfur atom from dibenzothiophene (DBT) in a selective way, because it transforms the DBT molecule into 2-hydroxibifenyl (HBP) and sulfate by means of a nondestructive pathway. This GMO was built to increase the desulfurizing capability of the wild bacteria of Rhodococcus erythropolis IGTS8 by means of the addition of genes as described elsewhere.13 As in many aerobic fermentation processes, bioreactor productivity is often influenced by the oxygen supply and the liquid-phase resistance usually controls the overall oxygentransfer rate (OTR). Thus, the efficiency of the BDS process depends upon an adequate gas-liquid contact, and the OTR from the gas to the liquid phase has a decisive importance in both the growth of the microorganism and BDS capability developed by the cells. Previous studies have been carried out to determine the influence of media composition and of the operational conditions in both the growth rate of P. putida CECT5279 (biomass production rate) and also the desulfurization capability of the cells produced.14,15 Nevertheless, there is (9) Denome, S. A.; Olson, E. S.; Young, K. D. Identification and cloning of genes involved in specific desulphurization of dibenzothiophene by Rhodococcus sp. strain IGTS8. Appl. EnViron. Microbiol. 1993, 59, 28372843. (10) Denome, S. A.; Oldfield, C.; Nash, L. J.; Young, K. D. Characterization of the desulphurization genes from Rhodococcus sp. strain IGTS8. J. Bacteriol. 1994, 176, 6707-6716. (11) Monticello, D. Biodesulphurization and the upgrading of petroleum distillates. Curr. Opin. Biotechnol. 2000, 11, 540-546. (12) Maghsoudi, S.; Vossoughi, M.; Kheirolomoom, A.; Tanaka, E.; Katoh, S. Biodesulphurization of hydrocarbons and diesel fuels by Rhodococcus sp. strain P32C1. Biochem. Eng. J. 2001, 8, 151-156. (13) Gallardo, M. E.; Fernandez, A.; Lorenzo, V. D.; Garcia, J. L.; Diaz, E. Designing recombinant Pseudomonas strains to enhance biodesulphurization. J. Bacteriol. 1997, 179, 7156-7160.
10.1021/ef050362y CCC: $33.50 © 2006 American Chemical Society Published on Web 05/02/2006
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no information about the kinetics, metabolic parameters of oxygen uptake, and its influence on the OTR. The knowledge of mass-transfer parameters of P. putida is essential to design, optimize, and model the BDS industrial bioreactor. Because P. putida is a strict aerobe microorganism, its growth and metabolism are expected to be affected by different oxygen conditions. Thus, important influences on the parameters of the growth kinetic model (µ and CXmax) have been observed when different dissolved oxygen conditions were used.15 Also, P. putida presents a behavior that has not been observed in other microorganisms in the time course of the batch fermentation. In a typical batch culture, the dissolved oxygen concentration decreases in the media along with the growth time, until the microorganism reaches the stationary phase and then the dissolved oxygen concentration increases. This evolution can be observed in many cultures of bacteria,16 yeast, and fungi.17,18 However, in a culture of P. putida, the dissolved oxygen concentration decreases dramatically in a few minutes, approaches 0, and does not increase even when the microorganism reaches the stationary growth phase or the oxygen masstransfer conditions are improved (by increasing the gas flow rate or stirrer speed, in a tank bioreactor). Under these conditions, it is impossible to measure the oxygen-uptake rate (OUR) and volumetric mass-transfer coefficient (kLa) during fermentation by the classic dynamic method; because of this fact, a modified dynamic method has been employed in this paper. Although correlations for kLa are extensive in the literature, there are still considerable problems concerning the accuracy of the estimated values and their validity in different bioreactors or different scales and also under different operational conditions. These facts are due to strong influences of a lot of geometrical, physicochemical, and biological aspects. In recent years, predictive models have been proposed for column, airlift, and tank bioreactors.19-22 In this paper, a predictive model for stirred tank bioreactors has been used for estimation of kLa, which is able to take into account the geometrical and physicochemical influences; moreover, the increase of the transfer rate by oxygen consumption by the cells is also considered through an estimated enhancement factor.23 Thus, the present work reports a detailed study of both the OUR and OTR on the growth and BDS capability of the (14) Martin, A. B.; Alcon, A.; Santos, V. E.; Garcia-Ochoa, F. Production of a biocatalyst of Pseudomonas putida CECT5279 for dibenzothiophene biodesulphurization for different media compositions. Energy Fuels 2004, 18, 851-857. (15) Martin, A. B.; Alcon, A.; Santos, V. E.; Garcia-Ochoa, F. Production of a biocatalyst of Pseudomonas putida CECT5279 for DBT biodesulphurization: Influence of the operational conditions. Energy Fuels, in press. (16) Garcia-Ochoa, F.; Gomez, E.; Santos, V. E. Oxygen transfer and uptake rates during xanthan gum production. Enzymol. Microbiol. Technol. 2000, 27, 680-690. (17) Koutinas, A. A.; Wang, R.; Kookos, I. K.; Webb, C. Kinetic parameters of Aspergillus awamori in submerge cultivations on whole wheat flour under oxygen limiting conditions. Biochem. Eng. J. 2003, 16, 2334. (18) Scha¨fer, S.; Schrader, J.; Sell, D. Oxygen uptake rate measurements to monitor the activity of terpene transforming fungi. Process Biochem. 2004, 39, 2221-2228. (19) Kawase, Y.; Halard B.; Moo-Young, M. Theoretical prediction of volumetric mass transfer coefficients in bubble columns for Newtonian and non-Newtonian fluids. Chem. Eng. Sci. 1987, 42, 1609-1617. (20) Garcia-Calvo, E. Fluid dynamic of airlift reactors: Two-phase friction factors. AIChE J. 1992, 38, 1662-1666. (21) Tobajas, M.; Garcia-Calvo, E.; Siegel, M. H.; Apitz, S. E. Hydrodynamics and mass transfer prediction in a three-phase airlift reactor for marine sediment biotreatment. Chem. Eng. Sci. 1999, 54, 5347-5354. (22) Garcia-Ochoa, F.; Gomez, E. Theoretical prediction of gas-liquid mass transfer coefficient, specific area and hold-up in sparged stirred tanks. Chem. Eng. Sci. 2004, 59, 2489-2501.
Gomez et al.
cultures of a GMO, P. putida CECT5279, under oxygen-limiting conditions. Measured and model-predicted values of both the OUR and OTR under different operational conditions are compared, with very good agreement. 2. Theoretical Background The OTR in a bioprocess, NO2, can be expressed as
NO2 ) aJO2 ) aEJ0 ) kGa(pG - pi) ) EkLa(Ci - CL) (1) where J is the molar flux, a is the interfacial area, kG and kL are the mass-transfer coefficients, and E is the biological enhancement factor. Considering the overall volumetric mass-transfer coefficients, it can be written
NO2 ) KGa(pG - p*) ) KLa(C* - CL)
(2)
1 1 1 ) + KLa HkGa EkLa
(3)
being
It can be observed that the overall volumetric mass-transfer coefficient in the presence of a biochemical reaction, KLa, is a lumped parameter comprising the resistance to mass transport of oxygen because of gas- and liquid-phase resistances and also because of the oxygen consumption, which can be expressed by a biological enhancement factor, E. When the fact that oxygen is only slightly soluble in water is taken into account, the gas phase resistance can usually be neglected and the overall resistance to transport can be written as
KLa ) EkLa
(4)
Although the biological enhancement factor can be experimentally evaluated, as the ratio between the volumetric masstransfer coefficient in the presence of a biochemical reaction and that under inert conditions, it frequently is assumed to be equal to 1. When a biochemical reaction does not take place, E ) 1 and the overall mass transfer will be denoted kLa and the flux, J0. For kLa determination, a theoretical model based on the Higbie penetration theory has been used.22 According to this model, the mass-transfer coefficient, for a Newtonian media can be calculated as
kL )
2 F 1/4 xDL µ xπ
( )
(5)
The specific interfacial area, a, is a function of the hydrodynamic conditions, the physicochemical properties, and the geometrical vessel parameters. It can be calculated from the average bubble size, db, and the gas holdup, φ, assuming spherical bubbles, by the following equation
a)
6φ db
(6)
where both hydrodynamics parameters, db and φ, can be estimated as described in a previous work22 using equations given in the literature.24,25 (23) Garcia-Ochoa, F.; Gomez, E. Prediction of gas-liquid mass transfer coefficient in sparged tank bioreactors. Biotechnol. Bioeng., in press.
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For the estimation of the biological enhancement factor, a model proposed in a previous work has been used.23 In this model, the presence of serial layers of adsorbed surfactants and microorganisms directly adjacent to the gas-liquid interface because of their surface activities is assumed, followed by a stagnant liquid layer to account for the oxygen-transfer resistance in the liquid-phase based on the film theory. The different layer resistances are taken into account by the diffusion coefficient (Di) inside the layer and the layer thickness (zi). According to this, the biological enhancement factor, E, can be calculated by
[
E) 1+
qO2CXmz2m 2Dm(C* - CL)
(
2z2L
zLDm
)
1+2 + + zmDL 3z2 m
qO2CXiz2L
1
][ ] zL/DL
3 Dm(C* - CL)
∑i zi/Di
(7)
The results obtained are a combination of two mechanisms: (i) the biological enhancement, because of the respiration of interfacial cells, and (ii) the physical blocking, resulting from the semipermeable nature of cell bodies. Therefore, E can take values smaller than, equal to, or bigger than 1, depending on the operational conditions. Thus, the volumetric mass-transfer coefficient in bioreactors can be predicted considering theoretical equations for kL and a (eqs 5 and 6) coupled to the estimation of the biological enhancement factor, E, by eq 7. In aerobic processes, the OUR is the sum of the oxygen consumption for cell maintenance and cell growth, according to
OUR ) mO2CX + YOX
dCX dt
(8)
where YOX is the cell yield for oxygen consumption necessary for growth and mO2 is the coefficient of oxygen consumption for maintenance. When the growth kinetic model for this microorganism is considered, previously determined,15 the biomass growth rate can be expressed according to
(
)
CX dCX ) µmaxCX 1 dt CXmax
and the gene hpaC from Escherichia coli W.26 Cultures were maintained on concentrated stock with glycerol in saline serum (10%) solution. Detailed information about media, inoculum preparation, and desulfurization assays are given in previous works.14,15 3.2. Experimental Procedure. A 2 L BIOSTAT B (Braun Biotech) fermentor was used for bacteria culture. Agitation was provided with a two-disk turbine stirrer type with a ratio of stirrer/ tank diameters (D/T) of 0.37. Each turbine was made by four blades having 1 mm of thickness. A 4 cm diameter ring type gas sparger was employed, with holes of 0.5 mm in diameter. The oxygendissolved concentration was measure using a sterilizable oxygen sensor (from Ingold) interfaced to a computer. The surface tension was determined by a tensiometer based on the ring method. The buildup of inoculums, media composition, and experimental procedure for microorganism growth has been described in a previous work.15 The temperature was maintained at 30 °C. The gas flow rate during fermentation was 2 L/min (1 vvm), and the stirrer speed was 200-500 rpm. 3.3. OUR and OTR Measurements. In batch fermentations, the mass balance for the dissolved oxygen in the well-mixed liquid phase can be established as dC ) KLa(C* - C) - qO2CX dt
(11)
where dC/dt is the accumulation of oxygen in the liquid phase, the first term on the right-hand side of eq 11 is the OTR, and the second term is the OUR. The experimental determination of OUR and KLa can be carried out applying the dynamic method in two cycles desorptionabsorption.16 This method requires interruption of air flow and a high-medium oxygen concentration dissolved in the broth. However, in cultures of P. putida, the oxygen dissolved is practically 0 after a short time of fermentation. Because of this fact, a modified dynamic method has been used. The modification consists of the introduction of pure oxygen in the gas stream; instead, to cut the air-flow inlet, a stream of pure oxygen is fed to the bioreactor. The evolution of the oxygen-dissolved concentration in the transition between two pseudo-steady states allows for the OUR and KLa calculations. The oxygen concentration change is the result of both physical oxygen absorption (OTR) and oxygen consumption by microorganisms (OUR). Figure 1 is a typical representation of results obtained during an experiment using dynamic absorption of pure oxygen for KLa and OUR determination. Assuming that qO2, CX, C*, and KLa are constants during the measurement time, eq 11 can be expressed as dC ) c - KLaC dt
(9)
(12)
with c being a constant given by
The OUR can be described by the following equation
(
OUR ) mO2CX + YOXµmaxCX 1 -
)
CX CXmax
c ) KLaC* - qO2CX
(13)
(10)
where µmax is the specific growth of the microorganism and CXmax is the maximum biomass concentration reached.
The integration of eq 12 with the following boundary conditions t ) 0; C* ) C/0
C ) C L0
3. Materials and Methods
yields the following equation
3.1. Microorganism. P. putida CECT5279 from the Biological Research Centre (CIB-CSIC, Madrid, Spain) was used. This bacterium carries the genes dszABC from R. erythropolis IGTS8
CL ) C/0 -
(24) Kudrewizki, F.; Rabe, P. Model of the dissipation of mechanical energy in gassed stirred tanks. Chem. Eng. Sci. 1986, 41, 2247-2252. (25) Bhavaraju, S. M.; Russell, T. W. F.; Blanch, H. W. The design of gas sparged devices for viscous liquid systems. AIChE J. 1978, 24, 454466.
(
) (
qO2CX KL a
∴
t ) t1; C ) CL
- C/0 - CL0 -
)
qO2CX KLa
e-KLat
(14)
(15)
(26) Galan, B.; Diaz, E.; Fernandez, A.; Prieto, M. A.; Garcia-Ochoa, F.; Garcia-Calvo, E.; Garcia, J. L. Un procedimiento para desulfurar dibenzotiofeno utilizando como biocatalizador una cepa de Pseudomonas putida recombinante en la que se han introducido genes aislados de Rhodococcus erythropolis y Escherichia coli. Spanish Patent 2000/0661.
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Figure 2. Evolution of qO2 and OUR with the biomass concentration in the P. putida culture, for a stirrer speed of 200 rpm and superficial gas velocity of 2.5 × 10-3 m/s. Figure 1. Typical experimental response to a pure oxygen step for the measurement of the volumetric mass-transfer coefficient.
Equation 15 represents the evolution of the oxygen concentration in the liquid phase from a starting concentration CL0 to when the composition of the gas stream is modified changing air to pure oxygen. According to the above procedure, KLa and OUR values were determined during the growth process, at several cellular ages. A nonlinear regression technique27 was used for parameter determination. The measurements of OTR have been made using this modified dynamic technique also without biotransformation; that is, when OUR ) 0. In this case, the air flow was changed by pure oxygen flow and introduced downward into the vessel; the dissolved oxygen concentration was recorded with time. Now, the rate of concentration change in the liquid phase is given by dC ) kLa(C* - C) dt
(16)
The solution of eq 16 is rather straighforward, obtaining the physical volumetric oxygen mass transfer, kLa, without biotransformation, that is, under inert conditions. In all cases, the dissolved oxygen concentration was measured using an oxygen polarographic electrode from Ingold with a response time less than 20 s. It was assumed that the response of the oxygen electrode to the change in the oxygen concentration is sufficiently fast and does not affect the determination accuracy.
4. Results and Discussion The experimental measurements of OUR and OTR were carried out at different stages of the microorganism growth. Volumetric mass-transfer coefficient values have been determined in a wide interval of operational conditions during fermentation. The measurements have been carried out by changing the superficial gas velocity, VS, between 1.25 × 10-3 and 3.8 × 10-3 m s-1 and stirrer speed, N, from 100 to 400 rpm (from 1.67 to 6.7 s-1). 4.1. OUR during Growth. Figures 2 and 3 show the tendency of qO2 and OUR with the biomass concentration and time course of the P. putida culture, for a stirrer speed of 200 rpm and a gas flow of 2 L/min (1 vvm). As can be seen, both qO2 and OUR are influenced by the microorganism growth phase. The specific oxygen uptake, qO2, increases dramatically in the lag and first exponential stages of growth, until a maximum value is reached. First, qO2 increases mainly because of the increasing of biomass production and substrate consump(27) Marquardt, A. W. An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math. 1963, 11, 431-441.
Figure 3. Evolution of qO2 and OUR during the time course of the P. putida culture, for a stirrer speed of 200 rpm and superficial gas velocity of 2.5 × 10-3 m/s.
tion rates. When the substrate consumption and biomass production rates decrease, at higher fermentation times, qO2 also decreases. On the other hand, OUR values present an increase during the lag stage and especially during the exponential growth stage; afterward, the values of OUR decrease slowly to a practically constant value during the stationary growth stage. Similar tendencies in OUR and qO2 values have been found for other bioprocesses.16,28,29 A maximum value of OUR in the P. putida culture was obtained in the middle of the exponential phase, in all of the experiments carried out, with values ranging from 3.0 to 3.5 × 10-6 mol of O2/(L s), depending upon the biomass concentration obtained during the experiment; the maximum value of specific OUR, qO2, was around 5 × 10-6 mol of O2/(g s), which is reached at the end of the lag stage. The parameters of the oxygen consumption rate for maintenance, mO2, and growth yield, YOX, have been determined by the nonlinear regression of eq 10 to experimental data for different runs. The results are summarized in Table 1, showing very good statistical parameters. It can observed that mO2 and YOX take values of 5.16 × 10-7 mol of O2/(g of X s) and 5.26 × 102 mol of O2/g of X, respectively. Both values are bigger than those obtained for other microorganisms with desulfurization capability.30 A comparison between model predictions and experimental data is presented in Figures 2 and 3; good (28) Pinches, A.; Pallent, L. J. Rate and yield relationships in the production of xanthan gum by batch fermentations using complex and chemically defined growth media. Biotechnol. Bioeng. 1989, 28, 14841496. (29) Calik, P.; Vural, H.; Ozdamar, T. H. Bioprocess parameters and oxygen transfer effects in the growth of Pseudomonas dacunhae for L-alanine production. Chem. Eng. J. 1997, 65, 109-116.
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Table 1. Values of Parameters mO2 and YOX Obtained by Fitting of Eq 10 to Experimental Data by Nonlinear Regression
value Student t Fischer F SSR 95% confidence Student t Fischer F level
mO2 [mol of O2/ (g of X s)] × 107
YOX (mol of O2/ g of X) × 102
5.16 ( 0.67 16
5.26 ( 0.71 15
579 5.73 × 10-13 2.18 3.88
Table 2. Parameter Values Employed in This Paper for the Theoretical Estimation of the Volumetric Mass-Transfer Coefficient, kLa, and the Biological Enhancement Factor, E parameter
transport
biochemical system operational conditions
symbol
value
µ [kg/(m s)] σ (N/m) F (kg/m3) C* (mol/m3) DL (m2/s) CXm (kg/m3) zm (m) Dm (m2/s) qO2 [mol of O2/(g of X s)] VS (m/s) N (rpm)
2 × 10-3 0.0475 1000 0.21 2.0 × 10-9 200 5.0 × 10-7 6.4 × 10-10 1.0 × 10-6-5 × 10-6 1.25 × 10-3-3.75 × 10-3 100-500
agreements between both, for different biomass concentrations and on the time course of fermentation, are obtained. 4.2. OTR Values. The OTR is proportional to the difference between the equilibrium concentration and the dynamic dissolved oxygen concentration. The proportional constant is the volumetric mass-transfer coefficient, KLa. This parameter is affected by a lot of factors, such as geometrical parameters of the bioreactor, the media properties, and the presence of the microorganism (morphology and oxygen consumption). It is well-known that OTR increases when the energy dissipated in the bioreactor is increased (usually by increasing N and/or VS). The influences of operational conditions on volumetric masstransfer coefficient values in inert conditions have widely been studied. Inert mass-transfer coefficient values, kLa, have been estimated by applying the model presented above,22 applying eqs 5 and 6 with the parameter values given in Table 2. In Figure 4, experimental and predicted values of kLa are represented as a function of the power input per unit volume, P/V, for different gas velocities, VS; as can be seen, the agreement between experimental and predicted values is very good over the range of conditions studied. It has been detected31-33 that OTR values change with the increasing of the biomass concentration. As commented above, OTR can be influenced by OUR, and therefore, the volumetric oxygen-transfer coefficient, KLa, can be modified by the presence of biomass, with this effect being taken into account by a biological enhancement factor, E. This factor has been experimentally evaluated, as the ratio of the experimental oxygen mass-transfer coefficient in the presence of the microorganism, (30) Santos, V. E.; Galdeano, C.; Gomez, E.; Alcon A.; Garcia-Ochoa, F. OUR measurements during R. erythropolis IGTS8 desulfurization biocatalyst production. ECB12. European Congress on Biotechnology. J. Biotechnol. 2005, 118, S58-S59. (31) Tsao, G. T. Simultaneous gas-liquid interfacial mass transfer and uptake by small particles. Biotechnol. Bioeng. 1969, 11, 1071-1087. (32) Merchuk, J. C.; Asenjo, J. A. Fundamentals of bioreactors design. In Bioreactors System Design; Asenjo, J. A., and Merchuk, J. C., Eds.; Plenum Press: New York, 1994; pp 191-193. (33) Galaction, A.-l.; Cascaval, D.; Oniscu, C.; Turnea, M. Prediction of oxygen mass transfer coefficients in stirred bioreactors for bacteria, yeasts and fungus broths. Biochem. Eng. J. 2004, 20, 85-94.
Figure 4. Experimental and predicted values of the inert volumetric mass-transfer coefficient as a function of the power input per unit volume for different superficial gas velocities.
Figure 5. Biological enhancement factor values, experimental and estimated from eq 7.
KLa, and the value measured in the medium in the absence of biomass under the same hydrodynamics condition, kLa. Biological enhancement factor values have been estimated from eq 7 using biological characteristic parameters resumed in Table 2. The values obtained are compared with the experimental values of E in Figure 5. As can be observed, again the agreement between experimental and predicted values is good. The biological enhancement factor, E, take values up to 1 for the biomass concentration up to 0.3 g/L, increasing until a value of 1.1, which means that, under these conditions, mass transfer is accompanied with a big OUR. A similar evolution of the biological enhancement factor has been described in papers by others.23,34,35 4.3. Influence of the Oxygen Concentration on the Growth and BDS Capability. The oxygen-dissolved concentration changes dependent upon the OTR from the air bubbles to liquid phase and the OUR for growth, maintenance, and also desulfurization development by the cells. In the P. putida culture, the dissolved oxygen concentration decreases to 0, as a result of its rapid uptake by the microorganism, because the growth and desulfurization capability of the bacteria is strongly influenced by the availability of oxygen in the fermentation broth. In Figure (34) Ju, L.-K.; Sundararajan, A. Model analysis of biological oxygen transfer enhancement in surface-aerated bioreactors. Biotechnol. Bioeng. 1992, 40, 1343-1352. (35) Calik, P.; Yilgo¨r, P.; Ayhan, P.; Demir, A. S. Oxygen transfer effects on recombinant benzaldehyde lyase production. Chem. Eng. Sci. 2004, 59, 5075-5083.
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be very bad for enzyme stability; therefore, an optimum value would be observed by coupling all of the phenomena. Finally, in Figure 6c, experimental oxygen concentration profiles and those values predicted by eq 11 under different agitations are shown. In this equation, the mass-transfer coefficient considered is in the presence of biotransformation. KLa values were estimated according to the model previously commented; constant values for the consumption parameters, mO2 and YOX, were assumed: 5.16 × 10-7 mol of O2/(g of X s) and 5.26 × 102 mol of O2/g of X, respectively. It can be observed that, in all cases, the dissolved oxygen concentration decreases dramatically in a few minutes and approaches 0, because of the very high oxygen uptake of cells. Obviously, the slopes decrease as the OTR is increased, by the increase in the stirrer speed. As can be seen, there is a very good agreement between the model prediction and the experimental values for the different stirrer speeds employed. Conclusions
Figure 6. (a) Biomass concentration on the course of the growth of P. putida. (b) Percentage of desulfurizing capability on the course of fermentation. (c) Experimental data and predicted values of the oxygendissolved concentration for different mass-transfer conditions during fermentation.
6, experimental values of the biomass concentration, CX, desulfurization capability, calculated as indicated in a previous paper14, and the change of the oxygen concentration versus the time course of fermentation for several mass-transport conditions (changing the stirrer speed) are shown. The biomass concentration versus the time course of fermentation is shown in Figure 6a for four different agitation rates. It can be observed that the maximum cell concentration obtained increases when the stirrer speed is increased, as a result of the decrease in the mass-transfer limitation. Figure 6b is represented by the desulfurizing capability of the cells in the time course of fermentation under the same transport conditions. It can be observed now that the maximum percentage of desulfurizing capability is very similar for different stirrer speeds, from 100 to 300 rpm, with approximately 80% being for 25 h of growth, although this capability decreases after 45 h, deeper in the case of work at 100 rpm. When the stirrer speed is 500 rpm, although the biomass concentration increases at the beginning, the percentage of desulfurization is much lower (about 40% for 18 h) and decreases dramatically after a few more hours. This fact must be related to the difference in the dissolved oxygen concentration caused by the increases of agitation, which could cause a faster growth, but the high oxygen concentration would
Bacteria P. putida CECT5279 is a GMO able to remove the sulfur atom from DBT in a selective way (4S route). The dissolved oxygen concentration affects the growth and BDS capability of the cells, with the result being the magnitude of two opposite rates, OTR and OUR. While a high oxygen concentration increases the biomass growth rate, the desulfurization capability of the cultured cells is decreased. Therefore, a macroscopic maximum of biocatalyst production is achieved at medium stirrer speeds, such as 200-300 rpm. In the P. putida culture, the dissolved oxygen concentration decreased dramatically in a few minutes and approached 0 under these conditions and the classical dynamic method is not applicable for volumetric mass-transfer and OUR determination. Therefore, a modified dynamic method has been applied, on the basis of the substitution of air flow by pure oxygen flow, that is, a step of oxygen. When this method is employed, reliable measurements of both, OUR and OTR, have been obtained. The OUR shows maximum values during the lag and first exponential stage of the growth. OUR has been modeled, and kinetic parameters, mO2 and YOX, have been determined. The OTR is affected by the presence of microorganisms. kLa values are affected by process variables, such as power input per unit volume, superficial gas velocity rate, and physical properties of the culture. When biotransformation is carried out, the values of KLa obtained are almost always greater than those obtained for kLa. Thus, the use of an enhancement factor, E, because of the presence of the microorganism, is necessary for an adequate description of the actual OTR. This is absolutely necessary for the design and simulation of bioreactors. A theoretical model for kLa and E predictions has been applied. Satisfactory results have been found when experimental and predicted values are compared. The model proposed is also able to predict reasonably the evolution of the oxygen concentration with the time course of fermentation under different OTR and OUR conditions. Acknowledgment. This work has been supported by MCyT (Plan Nacional de I+D, Programa de Procesos y Productos Quimicos, under contract numbers PPQ2001-1361-C02-01 and CTQ2004-06553-C02-01). The grant support for two of the authors by Comunidad Autonoma de Madrid (to A. A.) and by Ministerio de Ciencia y Tecnologia (to A. B. M.) is gratefully recognized.
Nomenclature c ) constant defined in eq 13 C ) concentration (mol m-3 or g/L)
OUR and OTR on the Growth of P. putida CECT5279 db ) bubble diameter (m) DBT ) dibenzothiophene E ) biological enhancement factor HBP ) 2-hidroxibenzothiophene J ) flux density molar (mol of O2 m-2 s) k ) consistency index in a power-law model (Pa sn) kL ) mass-transfer coefficient (m s-1) KLa ) volumetric oxygen mass-transfer coefficient in the presence of biotransformation (s-1) kLa ) volumetric oxygen mass-transfer coefficient in cell-free medium (s-1) mO2 ) dissolved oxygen consumption coefficient [mol of O2 (g of X)-1 s-1] N ) stirrer speed (s-1 or rpm) NO2 ) oxygen mass-transfer rate (mol of O2 L-1 s-1) OTR ) oxygen-transfer rate (mol of O2 L-1 s-1) OUR ) oxygen-uptake rate (mol of O2 L-1 s-1) P ) power input (W) qO2 ) specific oxygen-uptake rate [mol of O2 (g of X)-1 s-1] SSR ) sum of square residuals referred to the data number t ) time (s or h) V ) volume (m3) VS ) superficial gas velocity (m s-1) XBDS ) percentage of biodesulfurization (% HBP) YOX ) macroscopic specific yield of oxygen (mol of O2 kg-1 X)
Energy & Fuels, Vol. 20, No. 4, 2006 1571 z ) film thickness or distance from the gas-liquid interface (m) Greek Letters ) energy dissipation rate (W kg-1) φ ) gas holdup µ ) viscosity (kg m-1 s-1) or specific growth rate (s-1) σ ) interfacial tension (N m-1) Subscripts DBT ) referred to dibenzothiophene G ) relative to gas phase HBP ) referred to 2-hidroxibenzothiophene i ) relative to an interphase L ) relative to liquid phase m ) relative to cell monolayer max ) referred to maximum value O2 ) referred to dissolved oxygen X ) referred to biomass 0 ) referred to initial value Superscripts 0 ) referred to cell-free medium * ) referred to the saturation value EF050362Y
