Manipulation of the acetylation degree of Azotobacter vinelandii alginate by supplementing the culture medium with 3-(N-morpholino)-propane-sulfonic acid

Authors

  • C. Peña,

    1. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México
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  • L. Hernández,

    1. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México
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  • E. Galindo

    1. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México
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C. Peña, Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Post. 510-3, Cuernavaca, 62250, Morelos, México. E-mail: carlosf@ibt.unam.mx

Abstract

Aims:  The aim of this study was to characterize the influence of 3-(N-morpholino)-propane-sulfonic acid (MOPS) on alginate production by Azotobacter vinelandii and its chemical composition (particularly its acetylation degree), as well as on the rheological behaviour of alginate-reconstituted solutions.

Methods and Results:  Cultures were grown in 500-ml flasks containing 90 ml of medium supplemented with MOPS in concentrations ranging from 0 to 13·6 mmol l−1. The acetylation degree of the alginate was significantly influenced by the MOPS concentration, obtaining an alginate with an acetylation degree of 1·4% when 13·6 mmol l−1 of MOPS was added to the medium. This value was twice as high as that obtained when no MOPS was used. The higher acetylation of the polymer resulted in higher viscosity of alginate solutions, having a more pronounced pseudoplastic behaviour.

Conclusions:  MOPS added to the culture medium determines the acetyl content of the alginate and thus, the physico-chemical properties of the polymer.

Significance and Impact of the Study:  These changes in the functional properties of the polymer can be very valuable in specific applications of alginate in the food and pharmaceutical fields.

Introduction

Microbial polysaccharides are gaining acceptance in a variety of applications (Sutherland 1998) as suspending and gelling agents and as viscosifiers. Amongst them, microbial alginate (Rehm and Valla 1997) is of particular interest because it has the potential to substitute alginates obtained from marine algae (the current commercial method to produce them). Alginates are copolymers of β-d-mannuronic acid and its C-5 epimer, α-l-guluronic acid. These polymers are produced by bacteria of the genera Pseudomonas and Azotobacter (Gacesa 1998), the latter being the most promising because of its nonpathogenic nature.

Alginate produced by Azotobacter vinelandii bears the closest resemblance to algal alginate (the commercial product), although unlike the latter, it contains O-acetyl groups (Sutherland 1998). Bacterial alginates are exclusively acetylated in the d-mannuronic acid residues and the majority of these residues are mono-O-acetylated, but a few are 2,3-di-O-acetylated (Gacesa 1998).

It has been shown that the acetylation of seaweed alginate increases the viscosity and decreases the affinity of these polymers for calcium ions (Skjåk-Braek et al. 1989). Specific acetylation of alginates may improve the functional properties of alginate, expanding the commercial potential of this polysaccharide.

Alginate can be acetylated using chemical methods, based on the reaction of this polymer with pyridine/acetic anhydride mixture (Skjåk-Braek et al. 1989). The main inconvenience of this method is that it is highly unspecific. On the other hand, previous studies (Lee and Day 1995) have demonstrated the possibility of carrying out the specific acetylation of alginate using immobilized cells of Pseudomonas syringae. However, it would be very convenient to explore new ways to increase the acetylation degree during the biosynthesis of alginate, rather than acetylating the alginate after it has been produced. As far as we know, there are no previous papers documenting this possibility for alginates synthesized by A. vinelandii.

It has been reported that the components of the culture medium play an important role in determining alginate production by using A. vinelandii (Horan et al. 1981; Clementi et al. 1995). One of these components is the 3-(N-morpholino)-propane-sulfonic acid (MOPS), which is generally added to the medium in order to keep the pH constant, during the cultivation of the bacteria (Clementi et al. 1995; Peña et al. 1997). In this work, we report the study of the influence of MOPS on alginate production by A. vinelandii and its chemical composition (particularly its acetylation degree). Also, the rheological behaviour of alginate-reconstituted solutions prepared with the polymer obtained from cultures conducted at different MOPS concentrations was studied. This was made as a part of a strategy to determine the influence of a number of culture medium components on the molecular characteristics of the alginate.

Materials and methods

Micro-organism, culture medium and fermentation system

Azotobacter vinelandii ATCC9046 was cultured as previously described (Peña et al. 1997). Azotobacter vinelandii was grown in liquid medium with the following composition (in g l−1): sucrose 20; yeast extract (Difco, Sparks, MD, USA) 3; K2HPO4 0·66; KH2PO4 0·16; CaSO4 0·05; NaCl 0·2; MgSO4·7H2O 0·2; Na2MoO4·2H2O 0·0029; FeSO4·7H2O 0·027. The pH was adjusted to 7·2 with a concentrated NaOH solution. To avoid precipitation during autoclaving, the solutions of FeSO4·7H2O and Na2MoO4·2H2O were separated from the other components during sterilization (121°C, 35 min). The cultures were grown in a rotary shaker (Model G 25; New Brunswick Scientific Co., New Brunswick, NJ, USA) at 200 rpm and 29°C, up to an absorbance (measured at 540 nm) of 0·15 (dilution 1 : 50). Ten millilitres of this inoculum were transferred to 500-ml flasks containing 90 ml of the medium and were cultivated, under the same conditions, for 72 h. Under these conditions, the cells were grown under oxygen limitation (Peña et al. 1997). In order to evaluate the effect of MOPS concentration, the medium was supplemented with MOPS in concentrations ranging from 0 to 13·6 mmol l−1. In all the broths, the ionic strength was measured with a conductimeter (Cole Palmer 1500) and showed an average value of 2·2 ± 0·2 mOhms.

All experiments were conducted in triplicate and the results presented are the average of the independent runs. The data were analysed by an one-way analysis of variance (anova). A confidence level of 95% was considered.

Analytical determinations

Biomass was determined by dry weight and a correlation was established with absorbance at 540 nm (Peña et al. 2000). Alginate was measured as follows. A 10-ml sample of the culture broth was mixed with 1 ml Na4EDTA (0·1 M) and 1 ml of NaCl (1·0 M) and then centrifuged at 15 500 g. The supernatant was added to 30 ml of propan-2-ol and the mixture was shaken vigorously. After 10 min, the resultant precipitate (total solids) was dried and the alginic acid content was measured according the method reported by Blumenkrantz and Asboe-Hansen (1973), which is described later.

Purity of the alginate

The purity was estimated by spetrophotometry according the method reported by Blumenkrantz and Asboe-Hansen (1973). A 1·2 ml of sodium tetraborate solution (0·0125 M in sulphuric acid) was added to 0·2 mL of samples of alginate isolated by precipitation. Tubes were cooled and subsequently introduced into a boiling water batch for 5 min. Samples were cooled and 20 μl of m-hydroxybiphenol solution [(0·15% (w/v) in 0·5% (w/v) NaOH] was added. Tubes were shaken and left to stand for 5 min. After this time absorbance was measured at 520 nm. Mannuronic acid and commercial algae alginate (Sigma, Sigma-Aldrich, St Louis MO, USA) were used as standards. In all the polymers isolated using 30 ml of propan-2-ol, the alginic acid percentage was around 60% (alginic acid in grams/total solids).

Mean molecular mass and G/M ratio

The mean molecular mass of alginate was measured by gel filtration chromatography coupled to an HPLC system as detailed before (Peña et al. 1997, 2000). The ratio of l-guluronic acid and d-mannuronic acid (G/M ratio) was estimated by colorimetric reaction of carbazole according to the method reported by Knutson and Jeanes (1968).

Purification and reconstitution of the bacterial alginate

Total solids isolated initially by precipitation with 30 ml of propan-2-ol, having a purity of 60% (alginic acid in grams/total solids), were reprecipitated with one volume of propan-2-ol without agitation. Previously, the cells were separated by centrifugation at 15 500 g for 20 min. The cake obtained was dried in an oven at 60°C for 24 h and later milled in a mortar with a pestle. The purity of these powders was around 90% (alginic acid in grams/total solids). The purified alginate was reconstituted in distilled water at a concentration of 1·8 g l−1 and the ionic strength was adjusted at 2·2 mOhms using concentrated solutions of NaCl. These conditions were selected to mimic the conditions (alginate concentration and ionic strength) occurring in the actual fermentation.

Viscosity measurement

Viscosity of the reconstituted bacterial alginate solutions (having a concentration of 1·8 g l−1) was measured using a cone/plate viscosimeter (Wells-Brookfield LVT, Series 82198) at a constant temperature of 22°C. The dependency of the viscosity on the shear rate was described by the Ostwald-de Waele model (power law), which has been previously used to characterize the rheological behaviour of algal alginates (Mancini et al. 1996).

image

where η is the viscosity (Pa s), K is the consistency index (Pa sm) and m is the flow behaviour index (−).

Acetyl determination

The O-acetyl content of the various alginates (expressed as ratio in percentage of grams of acetyl/grams of alginic acid) was determined as described elsewhere (McComb and McCready 1957), using β-d-glucose penta-acetate (Sigma) as standard. This method is based on the colorimetric determination of the hydroxamic acid, which is formed by the reaction with hydroxylamine in alcoholic solution. This method is highly specific for the secondary acetyl groups 2′ and 3′ positions of hexuronate residues.

Results and discussion

Influence of MOPS on alginate production

Independently of the MOPS concentration added to the medium, the pH of the four conditions tested was 5·55 ± 0·22 at the end of the fermentation (Table 1). On the other hand, there were no significant differences (α = 0·05) in the final biomass and alginate concentration for all the MOPS concentrations tested. For all the conditions, the final biomass was 4·95 ± 0·25 g l−1 and the final purified alginate concentration was 1·8 ± 0·2 g l−1 (Table 1).

Table 1.   Influence of MOPS concentration on final values* of pH, biomass, alginate, mean molecular mass and G/M ratio
pH 5·55 ± 0·22
  1. *, 72 h of cultivation.

Biomass (g l−1)4·95 ± 0·25
Purified alginate (g l−1)1·8 ± 0·20
MMM (kDa)1430 ± 25
G/M ratio0·78 ± 0·09

Our results are in general agreement with a previous report (Horan et al. 1981) that found that the use of MOPS in a concentration of 50 mmol l−1 did not affect alginate production, with respect to the control experiment, in which no MOPS was used. In contrast, other authors (Clementi et al. 1995) have proposed that MOPS influenced alginate production. These authors found that by adding MOPS to the medium (at a concentration of 50 mmol l−1), it was possible to control the pH in the range of 7·5–6·5 in cultures conducted in shake flasks. These authors reported that due to a better control of pH, alginate concentration increased 2·5-fold with respect to the control (with no MOPS added). The contradicting observations concerning the influence of MOPS on alginate production might be the result of differences in the medium composition used, as Horan et al. (1981) used a phosphate-limited medium, whereas Clementi et al. (1995) conducted their experiments using a medium with excess of phosphate. In these two latter papers, no data was provided regarding the molecular characteristics of the polymer.

Influence of MOPS in the culture medium on the rheological behaviour of alginate solutions

Figure 1 shows the rheological behaviour of the reconstituted bacterial purified alginate solutions obtained under the different concentrations of MOPS present in the culture medium. All alginate solutions exhibited a pseudoplastic behaviour, with decreasing apparent viscosity (η) and with increasing shear rate (inline image). It is interesting to point out that the highest viscosity was obtained with alginate isolated from the cultures conducted using the highest MOPS concentration (13·6 mmol l−1). The rheological properties were described using the power-law model. The higher the MOPS concentration in the broth, the higher the consistency index (K), obtaining a value of 0·290 Pa sm for alginate recovered from cultures using 13·6 mmol l−1 (Table 2). Additionally, the flow behaviour index (m), showed a decrement with increasing MOPS concentration, having a minimal value of 0·55 for alginate obtained from the cultures with 13·6 mmol l−1 MOPS (Table 2).

Figure 1.

 Rheological behaviour of the reconstituted bacterial alginate solutions (1·8 g l−1) obtained under different concentrations of MOPS in the culture medium. (bsl00063) 0 mmol l−1, (bsl00001) 6·8 mmol l−1 and (bsl00066) 13·6 mmol l−1.

Table 2.   Rheological parameters (m and K) of reconstituted purified alginate solutions at 1·8 g l−1. The alginate was isolated from cultures of Azotobacter vinelandii conducted at different MOPS concentrations
MOPS concentration (mmol l−1)Flow behaviour index m (−)Consistency index K (Pa sm)
  1. ND, not determined.

00·800·070
3·40·75ND
6·80·700·107
13·60·550·290

Although it has been reported that MOPS can improve alginate concentration in shake flask cultures (Clementi et al. 1995), this is the first time that the influence of the MOPS content in the medium upon the rheology of alginate solutions is reported. As it will be shown next, these changes in the rheological behaviour of alginate solutions are closely related to the acetylation degree of the polymer.

Influence of MOPS in the culture medium on the chemical composition of alginate

As the viscosity of alginate solutions is strongly influenced by the molecular mass distribution (Martinsen et al. 1991) and, to a lesser extent, by the content and distribution of the two monomers in alginate molecule, both parameters were analysed in this work. There were no differences in the mean molecular mass (1430 ± 25 kDa) or in the G/M ratio (0·78 ± 0·09) for alginates obtained from the different culture conditions tested (Table 1). This suggests that the differences in the rheological behaviour of the polymers are due to differences in other chemical characteristics of the polymer rather than the molecular mass and/or the G/M ratio.

A previous report (Skjåk-Braek et al. 1989) has indicated that the random acetylation of seaweed alginate increased the viscosity of their solutions and decreased the affinity of these polymers for calcium ions. For this reason, the acetylation degree was quantified in alginates obtained by fermentation. Figure 2 shows the acetylation degree of alginates obtained under different MOPS concentrations present in the culture broth. MOPS turned out to have a strong influence on the acetyl content of alginate. The acetylation degree was twofold higher in alginate isolated from the cultures conducted with 13·6 mmol l−1 of MOPS, with respect to the acetylation degree measured in alginate derived from the fermentation without MOPS.

Figure 2.

 Aceylation degree per cent (grams of acetyl/grams of alginic acid) of the alginates obtained (at 72 h) from cultures conducted at different MOPS concentrations.

It is known that alginate acetylation occurs in the cellular periplasmic space, by means of the action of the acetylase enzyme, using acetyl-CoA as the source of acetyl (Vázquez et al. 1999). Therefore, the acetylation degree may reflect acetyl-CoA availability, which in turn may be regulated by the aeration of the culture and the pH. It is possible that MOPS could contribute to a better control of the pH in the periplasmic space and therefore the acetylase activity would be higher than that likely obtained when no MOPS was supplemented to the medium.

Our results have shown that MOPS, supplemented to the culture medium, determine importantly the acetyl content of the alginate and, in turn, the physico-chemical properties of this polymer. Although it is known that the acetylation degree may be critical for determining the rheological behaviour of alginate solutions (Skjåk-Braek et al. 1989), there were no previous reports regarding the influence of medium components, and particularly MOPS, on the acetyl content and on the rheological characteristics of alginate solutions obtained by cultivation of A. vinelandii.

Finally, it is important to point out that the specific acetylation of alginate, by simply manipulating the MOPS concentration in the culture medium (not being necessary a postfermentative step), is a convenient method that would considerably improve the properties of alginates, expanding the application potential of this polysaccharide.

Acknowledgements

This work was partially financed by Dirección General de Asuntos del Personal Académico – Universidad Nacional Autónoma de México (grants IN231305 and IX109304). The authors thank Ivette Pacheco Leyva for the purification and analysis of alginate and R. Ciria for his computer support.

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