Biochemical Engineering Journal 12 (2002) 3741

Broth rheology of Beta vulgaris cultures growing in an air lift bioreactor

Marco Jurez Snchez b , Antonio Jimnez-Aparicio a, , Gustavo Gutirrez Lpez b , Gabriela Trejo Tapia a , Mario Rodrguez-Monroy a
b a Departamento de Biotecnologia, Centro de Desarrollo de Productos Bioticos-IPN, P.O. Box 24, Yautepec, 62731 Morelos, Mexico Escuela Nacional de Ciencias Biologicas-IPN, Carpio y Plan de Ayala, s/n Col. Santo Tomas, Mexico 11340, Distrito Federal, Mexico

Received 5 June 2001; accepted after revision 27 February 2002

Abstract Cell cultures of Beta vulgaris were developed in an air lift bioreactor of 10 dm3 . Culture broth rheology exhibited non-Newtonian, shear thinning characteristics. The pseudoplasticity of the broth was governed by the presence of the cells as well as by the proteins secreted by the cells in the medium. The accumulation of extracellular proteins produced an increase in the viscosity and a change in the rheological properties of the cell-free medium. This phenomena may be a response of the cells to hydrodynamic stress. The accumulation of extracellular proteins and the change in the rheology of cell-free medium were discussed with respect to those data reported in literature obtained in shake asks and stirred tank bioreactor. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Air lift bioreactor; Rheology; Hydrodynamic stress; Extracellular proteins

1. Introduction Plant cell culture has been considered as an alternative to produce secondary metabolites [1]. The plant cell cultures of Beta vulgaris produce betalains; these compounds represent a particular interest because they can be used as natural dyes in food and drug industries [25]. Cell suspension cultures of B. vulgaris have been grown in shake asks [2,3,5], and in different types of laboratory bioreactors: air lift of 10 dm3 [6], a bubble-free TaylorCouette of 2.5 dm3 [7], uidized bed of 5 and 50 dm3 [8], and stirred tank of 2 dm3 [2]. In order to scale-up plant cell cultures in bioreactors, it is necessary to know the rheology of the cultures. This property is very important to determine the bioreactor power requirement and mass transfer characteristics [9]. However, there are few reports on the subject. Doran [9] and Kieran et al. [10] had published a summary of relevant studies in relation to the rheology of plant cell suspension cultures. They mentioned that the majority of suspension cultures showed non-Newtonian characteristics. The broths presented a pseudoplastic behavior, which was associated with: the biomass concentration, cellular aggregation and cell morphology [2,1113].

Corresponding author. Tel.: +52-735-394-2020; fax: +52-735-394-1896. E-mail address: aaparici@ipn.mx (A. Jim nez-Aparicio). e

On the other hand, the rheological behavior of cell-free medium has been reported as Newtonian as well as non-Newtonian; this behavior will depend on cellular species and culture conditions used to grow the cells. For example, Tanaka [14] used shake ask to grow Cudrania tricuspidata, Vinca rosae and Nicotiana tabacum. This author reported that the ltrated mediums exhibited a Newtonian behavior with viscosity values near to that of water. Similar results were obtained with ltrated mediums of B. vulgaris [2], Papaver somniferum [12], Glycine max [12] and Perilla frutescens [15]. In addition, Trejo Tapia et al. [13] indicated that the ltered medium of Solanum chrysotrichum grown in a stirred tank had Newtonian behavior. Nevertheless, Rodrguez-Monroy and Galindo [2] reported that the ltrated obtained from B. vulgaris culture developed in a stirred tank, exhibited pseudoplastic behavior. They associated this behavior with a high accumulation of extracellular proteins and polysaccharides. Recently, Rodrguez-Monroy and Galindo [16] reported that a glycoprotein of 110 kDa was the main component of the extracellular accumulated compounds. Similarly, the cultures of N. tabacum grown in a 30 dm3 fermentor accumulated an extracellular glycoprotein, which increased the viscosity of the ltered medium from 0.9 to 2.2 MPa s during cultivation [17,18]. Meijer et al. [19] reported that the supernatants of the samples of N. tabacum growing in a stirred tank at two tip speeds (35 and 236 cm s1 ), were increasingly viscous at day 6 of the culture, but the viscosity was not evaluated. Rodrguez-Monroy

1369-703X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 6 9 - 7 0 3 X ( 0 2 ) 0 0 0 4 3 - 8

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and Galindo [2] and Wongsamuth and Doran [20] proposed that the accumulation of extracellular compounds should be associated with a response of the plant cells to high hydrodynamic stress, present in the stirred tanks. Air lift bioreactors represent an alternative to stirred tank to grow plant cells [1,8,21]. These bioreactors are considered as systems with low stress conditions, due to the low relative velocities between the liquid and the bubbles in current ow [1,8]. Considering that the hydrodynamic stress generated in air lift fermentor is lower than that present in stirred tanks. The objective of this work was to evaluate the growth of B. vulgaris carried out with an air lift bioreactor (10 dm3 ), considering the betalain production, the accumulation of extracellular protein and to characterize the rheological behavior of the broth as well as that of the ltered medium. 2. Material and methods 2.1. Cell cultures Cell suspension cultures of B. vulgaris L. var. crosby Egyptian were obtained according to the methodology reported by Rodrguez-Monroy and Galindo [2]. The cells were grown on Gamborgs (B5 ) medium [22], supplemented with sucrose (30 kg m3 ), 2,4 dichlorophenoxy-acetic acid (9.0 108 M) and kinetin (9.2 107 M). The pH of the medium was adjusted to 5.5 with KOH (0.1 N) prior to sterilization. Cell suspension cultures were subcultivated every 15 days in 500 cm3 Erlenmeyer asks with 100 cm3 of medium. The agitation conditions were 100 min1 in an orbital shaker (Lab-line) at 26 2 C. In order to obtain a homogeneous culture, in every subculture, cellular aggregates having a size larger than 250 m were eliminated with a sieve. The inocula produced in 500 cm3 shake asks were used in fermentor cultures. 2.2. Bioreactor cultures The air lift bioreactor (10 dm3 ) used in this work has the dimensions shown in Fig. 1. The bioreactor was aerated at 2 dm3 min1 (0.28 vvm) with a sintered sparger of 3.5 cm diameter. These conditions correspond to an initial KL a value of 19 h1 and supercial air velocity in the riser (ugr ) of 0.180 m s1 . The pH and dissolved oxygen probes (Phoenix electrodes) were localized inside the draft tube. Temperature was maintained at 26 2 C. The fermentation vessel (containing 6.0 dm3 ) was inoculated with 1.0 dm3 of 13-day-old suspension culture grown on B5 medium. Every 3 days, a sample of 30 cm3 was removed from the vessel for its analysis. 2.3. Analytical methods Biomass. Dry weight (DW) was determined by ltration of 3 cm3 aliquots through a lter paper of known DW.

Fig. 1. Dimensions of air lift bioreactor (m).

The cells were dried to constant weight (70 C, 1 day). Biomass concentrations reported were the average of two independent runs. Betalains. They were evaluated spectrophotometrically according to the methodology reported previously by Rodrguez et al. [23] and the protein by the Bradford assay [24]. Viability. It was estimated using the methodology reported by Rodrguez-Monroy and Galindo [2]. The mem brane integrity was measured by Evans blue dye exclusion test and uorescein diacetate staining. A Nikon microscope (Alphaphot-2 YS2) was used for counting about 700 cells (10). In general, single cells or small groups of cells were easily observed and counted. Apparent viscosity measurement. The viscosity of the whole broth and the ltrate was measured using a Haake viscometer (Rotovisco RV20), equipped with a double gap system (NV). The range of shear rate was from 27 to 2700 s1 at 25 C. The rheological behavior was adjusted to the Ostwald de Weale model (power law): = K n where is the shear stress (Pa), the shear rate (s1 ), K the consistency index (Pa sn ) and n the ow behavior index (dimensionless). In order to determine the values of n and K, a plot of log vs. log was obtained; the slope of the line represents n and the intercept represents log K.

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3. Results and discussion Fig. 2A shows the growth prole of B. vulgaris cells. Maximum cell concentration was 230 g fresh weight dm3 , which corresponds to 9.8 g DW dm3 . Specic growth rate was 0.24 per day. The production of betalains was of 1 mg g1 DW of cells during the cell growth phase (Fig. 2B). Nevertheless, the accumulation of betalains reached a maximum of 9.3 mg g1 DW of cells in the stationary phase. The biomass yield obtained in this work was similar to that reported by other studies. For example, in shake ask the biomass yield was 1012 g DW dm3 [2,3,5], while in a stirred tank the cultures achieved 10 g DW dm3 [2], and with a uidized bed bioreactor 918 g DW dm3 was produced [8]. This comparison shows that the maximal biomass yield of B. vulgaris cultures might be independent of the system used to grow the cells. However, the betalains yields showed wide variability (4.235.0 mg dm3 ). These results suggest that the mixing and aeration pattern present in each bioreactor may be affecting betalains biosynthesis. Further, the mechanisms of that effect are not evident, further research is necessary in order to understand the possible relation. Kinetical proles of betalains production and cell growth showed that the production of betalains occurred after 12

Fig. 3. Cell viability ( ) and extracellular protein concentration ( ) of B. vulgaris cultures developed in an air lift bioreactor.

Fig. 2. Kinetic proles of B. vulgaris cell cultures developed in an air lift bioreactor: fresh weight ( ), DW ( ) (A) and betalain production ( ) (B).

days, presenting the higher accumulation in the stationary phase. These results indicated that the betalains production was not associated with cell growth. These kinetical proles are in agreement with that obtained with a uidized bed bioreactor [8]. However, they do not agree with the associated pattern reported by the B. vulgaris cultures carried out in shake asks [2,3,23,25], or in a stirred tank [2]. It is possible that the differences in aeration between both systems could be affecting the betalains yields and their kinetical prole. The cells growing in air lift and uidized bed bioreactor are in contact with a higher number of air bubbles than those cells growing in the stirred tank and shake asks. As a consequence, it may exist a stripping of key volatiles (e.g., dissolved carbon dioxide and ethylene). On this respect, Schlatmann et al. [26,27] using a gas recirculation bioreactor in cultures of Catharanthus roseus, conrmed the importance of dissolved gaseous compounds in the system performance. They concluded that the loss of an unidentied essential volatile was responsible for the reduced ajmalicine synthesis observed as a result of the scale-up from shake asks to an aerated bioreactor. Cell viability of B. vulgaris ranged from 70 to 80%, from the beginning of the culture until day 16; only in the stationary phase the cell viability dropped slightly to 66% (Fig. 3). Extracellular protein accumulation was not detected during the rst 7 days, nevertheless after 10 days, it had an essential increase. The maximum concentration was attained at day 16 (25 mg protein dm3 ) (Fig. 3). The high values of cell viability detected suggest that cell lysis was not present during the growth of these cells. These kinetic proles of viability and extracellular protein were very similar with those reported by the same cell line grown in shake asks and stirred tank [2]. However, the concentration of extracellular protein accumulated in the air lift was higher than that reported for the culture carried out in shake ask (12 mg dm3 ), while it was lower than that generated in a stirred tank (100 mg dm3 ) [2]. This effect is in agreement with the hypothesis suggested in other reports [1,2,20], which propose that the cells

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Fig. 4. Rheograms of B. vulgaris broths obtained from cultures developed in an air lift bioreactor. Rheological behavior of whole broths ( ) and cell-free medium ( ).

might be secreting proteins and polysaccharides as a response to a sublethal cell damage imposed by the hydrodynamic stress generated in bioreactors. In order to analyze this relationship, the power input was used as an indication of hydrodynamic stress intensity. The values obtained were: shake asks 11 W m3 , air lift 34 W m3 and stirred tank 313 W m3 . This result showed that the production of extracellular protein was proportional to power input used to grow B. vulgaris cells. Broth rheology of B. vulgaris cultures exhibited non-Newtonian behavior for all the samples (Fig. 4). This behavior was adjusted to the Ostwald de Weale model (power law) obtaining a satisfactory adjust (the regression coefcients were found to be in the range of 0.91.0, data not shown). Fig. 5A shows that the values of n ranged between 0.6 and 0.7 for the samples of 017 days, but in the samples of 19 and 20 days (stationary phase) n presented values of 0.5. Nevertheless, K presented values lower than 100 MPa sn during the rst 17 days, and then it increased considerably attaining a maximal of 300 MPa sn (Fig. 5A). The values of n and K obtained in this work were similar with those reported by B. vulgaris cultivated in shake ask and stirred tank [2]. The rheology of cell-free medium exhibited a Newtonian behavior in the samples obtained for 0 and 7 days, with a viscosity similar to that of water (Fig. 4). However, the samples obtained from the cultures of 1221 days presented a pseudoplastic behavior. This phenomena was evidenced by the evolution of n (Fig. 5B). At the beginning of the cultures, n was practically 1, and after 17 days (in samples from stationary phase), n presented values of 0.80.9. However, the K value did not present variation during the kinetic (Fig. 5B).

Fig. 5. Evolution of the ow behavior index (n, ) and consistency index (K, ) of B. vulgaris cultures grown in an air lift bioreactor: cell broth (A) and cell-free medium (B).

The change observed by the cell-free medium, from Newtonian to pseudoplastic was associated with the high concentrations of extracellular protein generated in the samples obtained in stationary phase (Fig. 3). These results are in

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agreement with the results obtained by Rodriguez-Monroy and Galindo [2] and Wongsamuth and Doran [20]. They proposed that the presence of extracellular protein in the cell-free medium is responsible for the increase of viscosity. The pseudoplastic properties exhibited by B. vulgaris broths are in agreement with that reported by the same specie grown in a stirred tank [2], and those reported by N. tabacum [17,18]. Non-Newtonian broth rheology of plant cell suspension cultures has several problems of mixing and gas dispersion [28,29], which should be solved in order to scale-up the process in air lift or stirred tanks.

4. Conclusion The results showed that the non-Newtonian, shear thinning characteristics of B. vulgaris broths were determined by the cell concentration as well as by the extracellular protein secreted by the cells. It was found that the hydrodynamic stress (estimated as the power input) in the air lift bioreactor was lower than that generated in the stirred tank. As a consequence, the level of protein produced in air lift was considerably lower than that reported in stirred tanks.

Acknowledgements Grants from the Instituto Politcnico Nacional (COFFA/ CGEPI-IPN) and Consejo Nacional de Ciencia y Tecnologa (Mxico) supported this work.

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