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Process Biochemistry 43 (2008) 775–778

Contents l is ts ava i lab le at ScienceDirec t

Process Biochemistry

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Short communication

Production of alginate by Azotobacter vinelandii in a stirred fermentorsimulating the evolution of power input observed in shake flasks

C. Pena *, M. Millan, E. Galindo

Departamento de Ingenierıa Celular y Biocatalisis, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico,

Apdo. Post. 510-3 Cuernavaca, 62250 Morelos, Mexico

R T I C L E I N F O

rticle history:

eceived 6 December 2007

eceived in revised form 7 February 2008

ccepted 13 February 2008

eywords:

lginate

olecular mass

ower input

caling-up

A B S T R A C T

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.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Alginates form an important family of biopolymers of bothtechnological and scientific interest. These polymers are linearpolysaccharides, 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 brownalgae and are used for a variety of applications, mainly in the foodand pharmaceutical industries [2]. Increasingly new applicationsare being discovered for these polymers; an example of this is theiruse as a source of soluble fiber [3]. Alginates can also be producedby bacteria, such as Azotobacter vinelandii and many of theirphysicochemical characteristics are similar to those of algae,therefore, they can be used for the same applications as algalalginates, as well as in other more sophisticated contexts [4].

Several studies have been carried out regarding bacterialalginate production in fermentors [4]. These studies havedescribed alginate production by A. vinelandii both in batch andfed-batch or continuous cultures; however, there are very fewreports covering aspects referring to the scale-up of the process[5,6].

The understanding of operational parameters involved inscaling-up of alginate production is important, as alginatesproduced 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.

E-mail address: carlosf@ibt.unam.mx (C. Pena).

359-5113/$ – see front matter � 2008 Elsevier Ltd. All rights reserved.

oi:10.1016/j.procbio.2008.02.013

to 1900 kDa and viscosities of up to 520 mPa s, for brothscontaining about 5 kg m�3 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 massand viscosity of the broths were considerably lower, obtainingalginates with a molecular mass of less than 680 kDa andviscosities lower than 100 mPa s for an alginate concentration ofaround 5.0 kg m�3 [5,8].

The scale-up from shake flasks to fermentor is troublesome andpoorly understood, mainly because of the lack of knowledgeconcerning the influence of the operation conditions on masstransfer, hydrodynamics and power input. Thanks to the devel-opment of a clever system to measure the volumetric powerconsumption in shake flasks [9,10], it has been possible tocharacterize (on line) the changes of power input in cultureshaving low and high viscosities, as in the case of alginatefermentation. In this context, Pena et al. [11] have studied boththe evolution of the specific power consumption and oxygentransfer rate, occurring in shake flasks, in cultures of A. vinelandii.These studies revealed that power consumption increasedexponentially during the course of the fermentation (up to1.4 kW m�3) due to an increase in the viscosity of the culturebroth. At the end of the fermentation, when the viscosity andalginate concentration reached a maximum, a slight drop in thepower consumption was observed [11].

Taking these data as a starting point, in the present study, ascale-up strategy – based in simulating the evolution of the actualpower input observed in shake flasks – was evaluated, trying to

Fig. 2. Changes in the agitation rate in the 14 L fermentor to simulate the power input

profile from the cultures conducted in shake flasks (inset, reproduced from [11]).

C. Pena et al. / Process Biochemistry 43 (2008) 775–778776

reproduce the mean molecular mass of the alginates obtained inshake flasks, in a stirred fermentor culture.

2. Materials and methods

2.1. Microorganism

Experiments were carried out using the wild type A. vinelandii ATCC-9046. This

was maintained by monthly subculture on Burk’s agar slopes and storage at 4 8C.

2.2. Inoculum preparation

A. vinelandii was grown in a modified Burk’s medium, which has been previously

described [7]. Cultures were carried out in 500 mL Erlenmeyer flasks during 72 h,

containing 100 mL of culture medium at 200 rpm and 29 8C in an orbital incubator

shaker (shaking amplitude of 2.5 cm, New Brunswick Scientific Co., Model G 25).

After 24 h, 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.

2.3. 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 0.8 VVM. pH and dissolved oxygen tension were left free. Dissolved

oxygen tension (DOT) was measured with a polarographic oxygen probe (Ingold),

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 [5,8].

Samples of 20 mL were withdrawn for analytical measurements. The results from

duplicate experiments were highly reproducible, resulting in standard deviations of

10% or less.

2.4. Power drawn estimation and measurement

Power drawn measurements were carried out in an accurate air bearing

dynamometer described elsewhere [12]. The vessel used was made up of acrylic, the

tank had a diameter of 0.205 m and a working volume of 10 L. All determinations

were made using the same configuration described previously for the bioreactor

used for A. vinelandii cultures. 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 0.8 VVM. Culture

broths were obtained from a 14 L bioreactor containing 10 L of Burk medium under

the conditions previously described by Reyes et al. [6]. Fig. 1 shows the results

obtained for different apparent viscosities (measured at 12 s�1). As expected, an

exponential increase was observed as a result of increasing the agitation rate.

2.5. 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

of A. vinelandii as determined experimentally by Pena et al. [11]. Using a 500 mL

Erlenmeyer flask, containing 100 mL of culture medium, the power consumption

during alginate production increased exponentially from 0.18 to 1.4 kW m�3 during

the first 40 h of culture and it was practically constant during the rest of the

fermentation [11]. The exponential profile of the power input (from 0.2 to

1.2 kW m�3) was simulated along the fermentation in a bench fermentor,

manipulating the agitation rate from 250 to 515 rpm (Fig. 2). These values were

estimated according to the data reported in Fig. 1.

Fig. 1. 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. (&)

1 mPa s; (!) 280 mPa s; (*) 720 mPa s.

2.6. Analytical methods

2.6.1. Biomass, alginate concentration and viscosity

Biomass and alginate concentrations were determined gravimetrically as

described previously [7]. Specific growth rate (m) was calculated using the logistic

model reported previously by Klimek and Ollis [13]. Culture broth viscosity was

measured using a cone/plate viscosimeter (Wells-Brookfield LVT, Series 82198).

Determinations were made at room temperature (25 8C) at 6 rpm using cone CP-52

which, according to the instrument manufacturer, corresponds to a shear rate of

12 s�1.

2.6.2. 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, Waters), using a HPLC system with a differential refractometer detector

(Waters, 410). Further details of the technique are reported elsewhere [7,11].

3. Results and discussion

3.1. Biomass growth, alginate production and mean molecular mass

The power input profile in the stirred fermentor reproducedwell the behavior observed in shake flasks, in terms of the specificgrowth rate (m) and the alginate concentration (Table 1). Thespecific growth rate was of 0.13 � 0.02 h�1, compared to0.1 � 0.01 h�1 observed in shake flasks. Alginate concentration of3.6 � 0.3 kg m�3 was obtained in the cultures conducted simulatingthe power input profile (Table 1). This value was very close to thatobtained in the cultures developed in shake flasks (4.0 � 0.5 kg m�3).

In a previous study [6], the initial power input was used ascriterion in order to scale-up (from shake flasks to fermentor) thealginate production. That study revealed that when an initialpower drawn of 0.27 kW m�3 was applied, a specific growth rate of0.16 h�1 was obtained in a stirred fermentor, which was verydifferent to that obtained in shake flasks (0.09 h�1). It is importantto point out that the study by Reyes et al. [6] was based on thetheoretical analysis of power consumption in shake flasks. Thepower input in shake flasks was estimated from extrapolation ofdata reported by Buchs et al. [9,10]. The actual profile of powerconsumption (experimentally measured) was not known at thetime. This information is very important as the changes in powerinput affect the oxygen transfer rate (OTR), which in turn affectsthe molecular characteristics of alginate [14].

The molecular mass distributions and the mean molecularmass (MMM) of the alginate produced by A. vinelandii during thefermentation are shown in Fig. 3 and Table 1. By simulating in astirred bioreactor the evolution of power input, as that occurringin shake flask, it was possible to produce alginates having a verysimilar distributions and mean molecular mass to thoseobtained in shake flasks. Thus, applying the exponential P/Vprofile during cultivation, an alginate having 1700 kDa wasobtained; whereas, in the shake flasks, the MMM of the alginatewas of 1800 kDa.

Table 1Specific growth rate (m) of Azotobacter vinelandii, alginate concentration and molecular mass, in shake flasks and stirred bioreactor cultures, conducted under similar

conditions of evolution of power input

Strategy Specific growth rate (m) (h�1) Alginate concentration (kg m�3) Mean molecular mass (kDa)

Exponential profile of power

input in stirred fermentor

0.13 � 0.02 3.6 � 0.3 1700 � 200

Shake flasksa 0.10 � 0.01 4.0 � 0.5 1800 � 150

a Taken from Pena et al. [7,11].

C. Pena et al. / Process Biochemistry 43 (2008) 775–778 777

It is important to point out that the maximal molecular masswas obtained during the stationary growth phase in both cases.However, in the cultures conducted in fermentor the MMMmax ofalginate was reached after 30 h of cultivation, whereas in thecultures from shake flasks it was obtained at 72 h (Fig. 3).

3.2. Dissolved oxygen tension and OTR

Fig. 4 shows the evolution of the dissolved oxygen tension in theculture developed in the fermentor. As dissolved oxygen tensionwas not measured during the cultures in shake flasks Fig. 4b showsthe evolution of oxygen transfer rate in shake flasks (taken fromPena et al. [11]). As it is explained by the authors [11], the regionbetween 10 and 62 h is a characteristic sign of an oxygen limitationand the decrease of OTR after 62 h of fermentation suggests asubstrate limitation. Therefore, the cultures in shake flasks werelimited by oxygen at least during 52 h. In the case of the cultures

Fig. 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

the shake flasks at 72 h of culture (taken from Pena et al. [7]).

Fig. 4. Profiles of dissolved oxygen tension (DOT) in the bioreactor of 14 L

simulating the power input from shake flasks, and its comparison with the oxygen

transfer rate (OTR) determined in shake flasks. (a) Exponential profile of the power

input; (b) OTR in shake flasks (taken from Pena et al. [11]).

conducted in fermentor, applying the exponential P/V profile, thedissolved oxygen tension was close to zero after the second hour ofcultivation (Fig. 4b) and remained at that value for 52 h.

It is clear from these data that in cultures in which anexponential profile of P/V was applied, the oxygen limitation,estimated by means of the DOT measurements (oxygen close tozero) would be very similar to that estimated (by means of theOTR) in the shake flasks. Therefore, it is possible that thesimilarity in the mean molecular mass between the alginateproduced in the fermentor (applying an exponential profile ofpower input) with respect to the polymer obtained from thecultures conducted in shake flasks could be due to the fact thatin both conditions, the oxygen limitation occurred under verysimilar conditions.

Our results are in agreement with a previous paper by Dıaz-Barrera et al. [14], reporting that alginates with high molecularmass (1560 kDa) were synthesized at low OTRmax (and thereforelonger period of oxygen limitation) with respect to the polymerisolated (220 kDa) from the cultures at high OTRmax. The meanmolecular mass of the alginate produced by A. vinelandii isdetermined by oxygen limitation conditions of the culture, whichin turn can be established by means of manipulating the powerinput profile during fermentation.

There are several reports in the literature regarding the scaling-up of processes from shake flasks to stirred tank [6,15–21].However, to our knowledge, this is the first time that a strategy ofscaling-up, simulating the evolution of actual power inputoccurring in shake flasks, is used for producing high molecularmass alginates.

Overall, our results showed that simulating the evolution of thepower input (measured experimentally in shake flasks) in 14 Lfermentors, allowed us to reproduce the mean molecular mass andmolecular mass distributions of the alginate obtained in shakeflasks, a situation that had not been possible to achieve using othercriteria (i.e., initial power input [6]).

Acknowledgements

Financial support of DGAPA-UNAM (grant IN230407) is grate-fully acknowledged. The authors thank Dr. M.S. Cordova-Aguilarand MSc. Martın Patino-Vera for analysis of power input andrheological properties of the broths and J.M. Hurtado for computersupport.

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