Process Biochemistry 43 (2008) 775–778

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Production of alginate by Azotobacter vinelandii in a stirred fermentor simulating the evolution of power input observed in shake flasks
´ n, E. Galindo ˜ a *, M. Milla C. Pen
´lisis, Instituto de Biotecnologı´a, Universidad Nacional Auto ´noma de Me ´xico, Departamento de Ingenierı´a Celular y Biocata Apdo. Post. 510-3 Cuernavaca, 62250 Morelos, Mexico



Article history: Received 6 December 2007 Received in revised form 7 February 2008 Accepted 13 February 2008 Keywords: Alginate Molecular mass Power input Scaling-up

By simulating the evolution of the actual power input observed in shake flasks it was possible to produce, in a fermentor, alginates having a very similar mean molecular mass (1700 kDa) to that obtained in the cultures developed in shake flasks (1800 kDa). Similar profiles of dissolved oxygen tension and oxygen transfer rate could be the reason. ß 2008 Elsevier Ltd. All rights reserved.

1. Introduction Alginates form an important family of biopolymers of both technological and scientific interest. These polymers are linear polysaccharides, which are composed of variable amounts of (1-4)b-D-mannuronic acid and its epimer, a-L-guluronic acid [1]. Currently, commercial alginates are extracted from marine brown algae and are used for a variety of applications, mainly in the food and pharmaceutical industries [2]. Increasingly new applications are being discovered for these polymers; an example of this is their use as a source of soluble fiber [3]. Alginates can also be produced by bacteria, such as Azotobacter vinelandii and many of their physicochemical characteristics are similar to those of algae, therefore, they can be used for the same applications as algal alginates, as well as in other more sophisticated contexts [4]. Several studies have been carried out regarding bacterial alginate production in fermentors [4]. These studies have described alginate production by A. vinelandii both in batch and fed-batch or continuous cultures; however, there are very few reports covering aspects referring to the scale-up of the process [5,6]. The understanding of operational parameters involved in scaling-up of alginate production is important, as alginates produced by A. vinelandii in shake flasks can reach values of up

* Corresponding author. Tel.: +52 777 329 16 17; fax: +52 777 317 23 88. ˜ a). E-mail address: (C. Pen 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.02.013

to 1900 kDa and viscosities of up to 520 mPa s, for broths containing about 5 kg mÀ3 of alginate [7]. On the other hand, when the process has been translated to laboratory fermentors (1 L), in which pH and DOT were kept constant, the molecular mass and viscosity of the broths were considerably lower, obtaining alginates with a molecular mass of less than 680 kDa and viscosities lower than 100 mPa s for an alginate concentration of around 5.0 kg mÀ3 [5,8]. The scale-up from shake flasks to fermentor is troublesome and poorly understood, mainly because of the lack of knowledge concerning the influence of the operation conditions on mass transfer, hydrodynamics and power input. Thanks to the development of a clever system to measure the volumetric power consumption in shake flasks [9,10], it has been possible to characterize (on line) the changes of power input in cultures having low and high viscosities, as in the case of alginate ˜ a et al. [11] have studied both fermentation. In this context, Pen the evolution of the specific power consumption and oxygen transfer rate, occurring in shake flasks, in cultures of A. vinelandii. These studies revealed that power consumption increased exponentially during the course of the fermentation (up to 1.4 kW mÀ3) due to an increase in the viscosity of the culture broth. At the end of the fermentation, when the viscosity and alginate concentration reached a maximum, a slight drop in the power consumption was observed [11]. Taking these data as a starting point, in the present study, a scale-up strategy – based in simulating the evolution of the actual power input observed in shake flasks – was evaluated, trying to

Fig.11]. in a stirred fermentor culture.2. 2. All determinations were made using the same configuration described previously for the bioreactor used for A. That study revealed that when an initial power drawn of 0. Model G 25).18 to 1. 3. Cultures were carried out in 500 mL Erlenmeyer flasks during 72 h.27 kW mÀ3 was applied. [6] was based on the theoretical analysis of power consumption in shake flasks.09 hÀ1). the tank had a diameter of 0. In a previous study [6].1 Æ 0.4. This was maintained by monthly subculture on Burk’s agar slopes and storage at 4 8C.13 Æ 0. which was very different to that obtained in shake flasks (0.205 m and a working volume of 10 L. in the shake flasks. Pen reproduce the mean molecular mass of the alginates obtained in shake flasks. Fig. The exponential profile of the power input (from 0. The vessel used was made up of acrylic. vinelandii was grown in a modified Burk’s medium.6.1. corresponds to a shear rate of 12 sÀ1. vinelandii ATCC-9046. Dissolved oxygen tension (DOT) was measured with a polarographic oxygen probe (Ingold).8 VVM. compared to 0. vinelandii as determined experimentally by Pen Erlenmeyer flask. [11].2 kW mÀ3) was simulated along the fermentation in a bench fermentor.3 kg mÀ3 was obtained in the cultures conducted simulating the power input profile (Table 1). / Process Biochemistry 43 (2008) 775–778 C.6. and its signal was amplified and acquired by a Macintosh II SI computer via a Mac Adios II A/D & D/A interface board (GW Instruments) as detailed elsewhere [5. This value was very close to that obtained in the cultures developed in shake flasks (4. The power input in shake flasks was estimated from extrapolation of ¨ chs et al. 1. Microorganism Experiments were carried out using the wild type A. After 24 h. Waters).8]. Series 82198). 2. which in turn affects the molecular characteristics of alginate [14]. Analytical methods 2. Results and discussion 3. Alginate concentration of 3. Inoculum preparation A. (*) 720 mPa s. Mean molecular mass The mean molecular mass of alginate was estimated by gel permeation chromatography (GPC) with a serial set of ultrahydrogel columns (UG 500 and Linear. according to the instrument manufacturer.2 to 1.3. Culture broth viscosity was measured using a cone/plate viscosimeter (Wells-Brookfield LVT. (&) 1 mPa s. containing 100 mL of culture medium. 1. The molecular mass distributions and the mean molecular mass (MMM) of the alginate produced by A. pH and dissolved oxygen tension were left free. 2. an exponential increase was observed as a result of increasing the agitation rate. The specific growth rate was of 0. resulting in standard deviations of 10% or less. Further details of the technique are reported elsewhere [7. a specific growth rate of 0. Samples of 20 mL were withdrawn for analytical measurements. Using a 500 mL of A. it was possible to produce alginates having a very similar distributions and mean molecular mass to those obtained in shake flasks. whereas. Power drawn estimation and measurement Power drawn measurements were carried out in an accurate air bearing dynamometer described elsewhere [12]. the cells were transferred to shake flasks of 2000 mL containing 400 mL of Burk’s medium and incubated during 24 h under the same conditions previously described. 2).02 hÀ1. 2. manipulating the agitation rate from 250 to 515 rpm (Fig. [9. Thus. 2. Biomass growth.8 VVM. the initial power input was used as criterion in order to scale-up (from shake flasks to fermentor) the alginate production. applying the exponential P/V profile during cultivation. alginate concentration and viscosity Biomass and alginate concentrations were determined gravimetrically as described previously [7]. Specific power input (P/V) in a 14 L fermentor as a function of the agitation rate for bacterial alginate solutions having different apparent viscosities.0 Æ 0.01 hÀ1 observed in shake flasks.6. the MMM of the alginate was of 1800 kDa.5 cm. These values were estimated according to the data reported in Fig. Materials and methods 2.1. [6]. Bioreactor cultures The cultures in the bioreactor were performed in a 14 L stirred tank equipped with three Rushton turbines with an initial working volume of 10 L and using an aeration rate of 0. alginate production and mean molecular mass The power input profile in the stirred fermentor reproduced well the behavior observed in shake flasks. 2.. Simulating of power input in the fermentor The strategy was based on the information generated in shake flasks in terms of the changes of the specific power consumption during alginate-producing cultures ˜ a et al. The results from duplicate experiments were highly reproducible. in terms of the specific growth rate (m) and the alginate concentration (Table 1). reproduced from [11]). the power consumption during alginate production increased exponentially from 0. Biomass.4 kW mÀ3 during the first 40 h of culture and it was practically constant during the rest of the fermentation [11]. vinelandii cultures. using a HPLC system with a differential refractometer detector (Waters. Measurements were performed at 29 8C using culture broths containing alginate with viscosities in a range from 1 to 720 mPa s and varing the agitation rate from 100 to 600 rpm and an aeration rate of 0. 3 and Table 1. .776 ˜a et al. an alginate having 1700 kDa was obtained. As expected. Changes in the agitation rate in the 14 L fermentor to simulate the power input profile from the cultures conducted in shake flasks (inset. This information is very important as the changes in power input affect the oxygen transfer rate (OTR).6 Æ 0.5. Specific growth rate (m) was calculated using the logistic model reported previously by Klimek and Ollis [13]. Determinations were made at room temperature (25 8C) at 6 rpm using cone CP-52 which. It is important to point out that the study by Reyes et al. 410). which has been previously described [7]. (!) 280 mPa s. New Brunswick Scientific Co. 2.1. 1 shows the results obtained for different apparent viscosities (measured at 12 sÀ1). containing 100 mL of culture medium at 200 rpm and 29 8C in an orbital incubator shaker (shaking amplitude of 2.10].2. By simulating in a stirred bioreactor the evolution of power input. The actual profile of power data reported by Bu consumption (experimentally measured) was not known at the time. 2.5 kg mÀ3). Fig. Culture broths were obtained from a 14 L bioreactor containing 10 L of Burk medium under the conditions previously described by Reyes et al.16 hÀ1 was obtained in a stirred fermentor. vinelandii during the fermentation are shown in Fig. as that occurring in shake flask.

whereas in the cultures from shake flasks it was obtained at 72 h (Fig. Therefore. Appl Microbiol Biotechnol 1997. alginate concentration and molecular mass. Atherton M. ´ n MA. Allen A. Overall. Deckwer WD. the cultures in shake flasks were limited by oxygen at least during 52 h. the shake flasks at 72 h of culture (taken from Pen conducted in fermentor. Taken from Pen It is important to point out that the maximal molecular mass was obtained during the stationary growth phase in both cases. 4. Hurtado for computer support.6 Æ 0. (a) Exponential profile of the power ˜ a et al. in the cultures conducted in fermentor the MMMmax of alginate was reached after 30 h of cultivation. As it is explained by the authors [11]. the region Pen between 10 and 62 h is a characteristic sign of an oxygen limitation and the decrease of OTR after 62 h of fermentation suggests a substrate limitation. 3).45: 497–510. input. [3] Brownlee I.3 4. The authors thank Dr. The mean molecular mass of the alginate produced by A. ´n ´n G. Zeng AP. Profiles of dissolved oxygen tension (DOT) in the bioreactor of 14 L simulating the power input from shake flasks. vinelandii is determined by oxygen limitation conditions of the culture. Pen strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii.5 Mean molecular mass (kDa) 1700 Æ 200 1800 Æ 150 ˜ a et al. reporting that alginates with high molecular mass (1560 kDa) were synthesized at low OTRmax (and therefore longer period of oxygen limitation) with respect to the polymer isolated (220 kDa) from the cultures at high OTRmax. allowed us to reproduce the mean molecular mass and molecular mass distributions of the alginate obtained in shake flasks. Molecular and bioengineering ˜ a C.S. In the case of the cultures Fig. [11]).. Alginate as a source of dietary fiber. the oxygen limitation occurred under very similar conditions. Therefore. Appl Microbiol Biotechnol 2001. Espı [4] Galindo E. applying the exponential P/V profile. M. the dissolved oxygen tension was close to zero after the second hour of cultivation (Fig. [14]. The effect of oscillating ˜ a C. alginate production and Fig. Microb Cell Fact 2007. to our knowledge. Dissolved oxygen tension and OTR Fig.0 Æ 0. estimated by means of the DOT measurements (oxygen close to zero) would be very similar to that estimated (by means of the OTR) in the shake flasks. However. simulating the evolution of actual power input occurring in shake flasks. It is clear from these data that in cultures in which an exponential profile of P/V was applied. Bacterial alginate: physiology. which in turn can be established by means of manipulating the power input profile during fermentation.15–21]. [2] Sabra W. Dettmar P. 3. ´azOur results are in agreement with a previous paper by Dı Barrera et al.56:315–25. this is the first time that a strategy of scaling-up. Valla S. 3. Molecular mass distribution (MMD) of the alginates obtained from the 14 L bioreactor (after 30 h of cultivation) simulating the power input profile and from ˜ a et al. References [1] Rhem B. in shake flasks and stirred bioreactor cultures. There are several reports in the literature regarding the scalingup of processes from shake flasks to stirred tank [6. Pen ´rez OT. product quality and process aspects. Havler M. Crit Rev Food Sci Nutr 2005. Martı rheological properties of the broths and J. conducted under similar conditions of evolution of power input Strategy Exponential profile of power input in stirred fermentor Shake flasksa a Specific growth rate (m) (hÀ1) 0.M.2.10 Æ 0. et al. [11]). Nu ˜ ez C.01 Alginate concentration (kg mÀ3) 3. [7]). Pearson J. Cordova-Aguilar ´n Patin ˜ o-Vera for analysis of power input and and MSc. 4b shows the evolution of oxygen transfer rate in shake flasks (taken from ˜ a et al.48:281–8. / Process Biochemistry 43 (2008) 775–778 C. Segura D. it is possible that the similarity in the mean molecular mass between the alginate produced in the fermentor (applying an exponential profile of power input) with respect to the polymer obtained from the cultures conducted in shake flasks could be due to the fact that in both conditions. is used for producing high molecular mass alginates. initial power input [6]).˜a et al. Ramı [5] Trujillo-Rolda dissolved tension upon the kinetics of growth. [7. However. (b) OTR in shake flasks (taken from Pen . Galindo E. 4b) and remained at that value for 52 h. Pen 777 Table 1 Specific growth rate (m) of Azotobacter vinelandii.13 Æ 0.11]. and its comparison with the oxygen transfer rate (OTR) determined in shake flasks. Bacterial alginate: biosynthesis and applications.e. Acknowledgements Financial support of DGAPA-UNAM (grant IN230407) is gratefully acknowledged. a situation that had not been possible to achieve using other criteria (i.02 0. As dissolved oxygen tension was not measured during the cultures in shake flasks Fig. our results showed that simulating the evolution of the power input (measured experimentally in shake flasks) in 14 L fermentors. 4 shows the evolution of the dissolved oxygen tension in the culture developed in the fermentor.6(7). the oxygen limitation.

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