Rheological characterization of culture broth containing the exopolysaccharide PS-EDIV from Sphingomonas pituitosa

Biochemical Engineering Journal 47 (2009) 116–121

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Rheological characterization of culture broth containing the exopolysaccharide PS-EDIV from Sphingomonas pituitosa b ˜ Ellen Schultheis a , Michael A. Dreger a , José M. Munoz-Villegas , José I. Escalante c , a,∗ a Ezequiel Franco-Lara , Bernd Nörtemann a b c

Institute of Biochemical Engineering, Technische Universität Braunschweig, 38106 Braunschweig, Germany Chemical Engineering Department, University of Guadalajara, Blvd. M.G. Barragán 1451, CP 44430, Guadalajara, Mexico Chemistry Department, University of Guadalajara, Blvd. M.G. Barragán 1451, CP 44430, Guadalajara, Mexico

a r t i c l e

i n f o

Article history: Received 19 December 2008 Received in revised form 11 May 2009 Accepted 20 July 2009

Keywords: Sphingan Biorheology Thixotropic behavior Viscoelasticity

a b s t r a c t Sphingomonas pituitosa excretes the capsular exopolysaccharide PS-EDIV into the culture broth augmenting considerably its fluid viscosity. Since this change particularly affects key processes like mixing and transport during the microbial production, this work was aimed at the rheological characterization of the polymer-containing culture broth of S. pituitosa. The study included investigations on basic properties of the culture broth, but also on the dependence of the biomass–polymer-solution properties on different physicochemical post-cultivation treatment steps like variations of temperature, pH-value or concentration of salts. The essential result is the characterization of the viscoelastic behavior of the culture broth, which was more gel-like than sol-like and exhibited slight elastic properties. This rheological behavior showed that the PS-EDIV culture broth formed non-Newtonian fluids, indicating that it is a pseudoplastic biopolymer, with yield stress appearance and exhibits thixotropic properties. Rheograms were fitted to the Herschel–Bulkley model. The amplitude sweep revealed a deformation of 21% as the limiting value of the linear viscoelastic interval. Furthermore, the PS-EDIV culture broth showed a high viscosity which was strongly influenced by salt type and concentration but weakly influenced by temperature and pH-value within the investigated experimental boundaries. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Bacterial polysaccharides are of great importance in industry due to their wide application range as food additives [1–4], in pharmaceuticals [5], cosmetics [6], for enhanced oil recovery [7–10] and in many other industrial sectors as thickeners, stabilizers and gelling agents [11]. However, prior to their application they have to be well characterized in several aspects. In particular, an efficient and economic process design for the production, separation and purification of the polysaccharide requires an adequate knowledge about the characteristics of the crude polymer, i.e. of the polymer-containing culture broth. This includes estimations about the influence of process variables like temperature, changes in the pH-value and the addition of salts especially on the rheological behavior of the broth. In general, polymeric solutions may show shear-thinning and sometimes thixotropic behavior, which is mainly dependent on the shear rate. Additionally, polysaccharides often show viscoelasticity. For these reasons, not every measuring method is equally suitable

∗ Corresponding author. E-mail address: [email protected] (E. Franco-Lara). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.07.012

to describe the rheological properties, and, therefore, each polymer sample has to be analyzed individually. Different polymer properties require different measurement methods and instruments. In this work, rotational and oscillatory measurements were the methods of choice. Data were interpreted using the model of Herschel–Bulkley  = 0 + K ˙ n , where  is the shear stress,  0 is the yield point, K describes the consistency factor, ˙ is the shear rate and n represents the flow index. Based on Hooke’s law in its complex form G* = (t)/(t), the storage modulus G = (/)cos ı and the loss modulus G = (/)sin ı can be derived from oscillatory measurements. The storage modulus G can be interpreted as a fraction of the stored deformation energy which is developed during the shear process and subsequently affects the reversible deformation. While G represents the elastic part of the polymer solution the loss modulus G describes its viscous part. This includes the lost energy that is dissipated during the shear process by irreversible modification of the sample’s constitution and thermal dissipation. The factor ◦ ı represents the  phase difference (0–90 ). The complex viscosity is ˙ described as ∗ = (t)/(t). In the linear viscoelastic interval (LVE interval) the complex viscosity is equivalent with the shear viscosity [12]. In oscillatory measurements the first step is to determine the LVE interval employing an amplitude sweep. The sample can be further char-

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acterized as sol- or gel-like. Gel character is often found in polysaccharides such as gellan [13] and rhamsan [14], which react gel-like under static conditions and exhibit very low viscosities at high shear rates. A bacterial exopolysaccharide showing such interesting rheological properties is the PS-EDIV produced by the bacterium Sphingomonas pituitosa DSM 13101 [15]. Early research in structure determination of PS-EDIV rendered a linear not cross-linked molecular structure that consists of glucose, rhamnose, glucuronic acid and 2-deoxy-glucuronic acid [16]. The knowledge of the structure of the polymer is crucial information for the development of an efficient process for production and purification of bacterial polysaccharides since it provides an indication of the possible rheological behavior. In the particular case, due to the uronic acids the exopolysaccharide PS-EDIV features a slight anionic character. For the characterization of the rheological properties of the PS-EDIV-containing culture broth rotational measurements were conducted under different temperature- and pH-conditions and at different concentrations of salts to investigate the influence of these parameters on viscosity. Moreover, oscillatory measurements were carried out to obtain information about the elastic and viscous properties of the biomass–polymer-solution. Finally, PS-EDIV culture broth displayed thixotropy, i.e. stirring makes them thinner, but leaving them to rest thereafter thickens them again [17]. This particular property adds considerable advantage to the practical use of biodegradable materials such as paints, adhesives and coatings.

2. Materials and methods The biomass–polymer-solutions used were obtained from cultivations of S. pituitosa DSM 13101 with sucrose medium containing 100 g/L sucrose, 12 g/L NaNO3 , 5 g/L FeCl3 , 1 g/L K2 HPO4 , 0.5 g/L KCl, 0.5 g/L MgSO4 ·7H2 O and 11.5 mL/L phosphate buffer (0.5 M, pH 7). The cultivation volume was 5 L in a stirred batch reactor with a three stage Intermig stirrer system. The cultivation settings were adjusted to a stirrer speed of 600 min−1 , an aeration rate of 0.5 L/L min and a cultivation time of 48 h. Biomass concentration was determined by measurement of optical density at 640 nm, and the exopolysaccharide concentration was detected gravimetrically after purification and drying of the polymer. Purification of the polymer was carried out twice by centrifugation at 15,000 min−1 for 30 min and precipitation with isopropanol in a ratio of 1:4. When necessary, a wash step and another precipitation were applied. The rheological measurements were performed using a rotational viscosimeter (type CS10, BOHLIN instruments, UK), which works according to the principle of Searle with a rotating cone (collector slope, 2◦ ; diameter, 60 mm) and a stationary plate. Since the concentration of the biomass and the exopolysaccharide in the culture broth of different cultivations was not always the same in the end of fermentation (48 h of cultivation), each series of rheological tests was always carried out with samples from the same cultivation. To analyze the influence of the pH-value on the rheological properties of the polymer-containing culture fluid, the pH was shifted to values between 2.0 and 12.0 by addition of 1 M HCl and 1 M NaOH, respectively. The rheological parameters in these rotational measurements were shear stress controlled in a range from 0.018 Pa to 50 Pa. The effect of temperature was determined by heating the samples during the rheological measurements within a range between 0.018 Pa and 30 Pa. To investigate the effect of salt addition, the samples were charged with different salts (KCl, NaCl, CaCl2 , MgCl2 or FeCl3 ) at different concentrations (0.31–5.25 mol/L). A possible gel formation of the PS-EDIV was investigated by the addition of different salts to culture samples after deacylation of the

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polymer. Deacylation was carried out by addition of 0.1 M NaOH, incubation at 50 ◦ C for 2 h and subsequent neutralization with concentrated HCl prior to water or salt addition. The shear stress in this experiment was varied between 0.018 Pa and 6 Pa. The oscillatory measurements were split into first an amplitude sweep (angular frequencies: 0.63 s−1 (0.1 Hz) to 93.75 s−1 (15 Hz); imposed shear stress: 0.018–90 Pa) and second a frequency sweep with given frequencies (0.03 s−1 (0.005 Hz) to 942 s−1 (150 Hz)) and imposition of either shear stress (2.5 Pa or alternatively 5 Pa) or a deformation (0.01% or 0.1%). All these measurements were performed at 20 ◦ C. 3. Results The factors affecting rheological behavior of culture broth with PS-EDIV studied were temperature, pH-value and salt type. The cultivation is described in detail in a former publication [18]. After 48 h the cultivation was stopped and the culture broth was stored at 5 ◦ C until starting rheological measurements. 3.1. Temperature and pH-value The dependence of PS-EDIV broth to temperature and pH variations is illustrated in Fig. 1 for two different PS-EDIV culture broth concentrations (cBTM = 1.7 g/L and cPS-EDIV = 2.5 g/L for temperature variation; cBTM = 3.95 g/L and cPS-EDIV = 1.18 g/L for pH variation). Considering a negligible effect of the biomass on the viscosity compared to that of the polysaccharide, the PS-EDIV culture broth was relatively stable to increases in temperature and was able to retain its apparent viscosity up to 60 ◦ C, whereby biomass can be actually considered heat unstable and partially lysed (data not shown). The apparent viscosities were approximately 4.9 Pa s with only 10–15% differences in the temperature range studied. It is important to notice that the PS-EDIV culture broth studied displayed a higher viscosity than that reported for xanthan or gellan gums [19]. Although a slight tendency for increasing viscosities at higher temperatures can be assumed, a significant influence on the culture broth viscosity is not obvious. Similarly to the results observed for the process parameter temperature, the viscosity (averaging 1.7 Pa s at a shear rate of 1 s−1 )

Fig. 1. Dependence of the viscosity of the biomass–polymer-solution (based on the model of Herschel–Bulkley) as function of temperature (shear rate of ˙ = 1 s−1 , cBTM = 1.7 g/L, cEPS = 2.5 g/L) and pH (shear rate of ˙ = 1 s−1 , cBTM = 3.95 g/L, cEPS = 1.18 g/L).

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was fairly stable at high pH-values in the range of 6–12, but between pH 4 and 5.3, there is an abrupt increase of the viscosity (circa 90%) most probably due to increasing intermolecular association as a result of decreasing coulombic repulsion. 3.2. Gelation In many applications, several additives such as salts are also present in the polymer solution and can significantly influence the viscoelastic and structural properties, e.g. by deacylation and subsequent gelation. Therefore, a possible gel formation of the polysaccharide-containing culture broth was experimentally investigated by the addition of different salts. The apparent viscosity was determined at a shear rate of ˙ = 1 s−1 (Herschel–Bulkley model) and, due to hysteresis effects, with increasing and decreasing shear stress (Table 1). At relatively low salt concentrations (approximately 0.5 mol/L) the apparent viscosities of the culture broths were comparable to each other in the range of 0.9 ± 0.2 Pa s regardless of the type and valence of the cations applied. However, at higher salt concentrations, the apparent viscosity of the culture broths strongly increased with CaCl2 and, in particular, with FeCl3 . However, gelation itself was not observed in any experiment (visual observance). In the measurements carried out with decreasing shear stress, the apparent viscosity did not depend too much on the salt concentration and, in general, were significantly lower compared to those determined with increasing shear stress. An exception was observed with FeCl3 at higher concentrations (0.77 and 1.54 mol/L, respectively) where the apparent viscosities were found to be nearly the same at increasing and decreasing shear stresses, which indicates a stabilizing effect of FeCl3 on the polymer network not observed for other salts and concentrations. Fig. 2 depicts the apparent viscosity is plotted against the shear rate of deacylated PS-EDIV. Exemplarily, viscosities obtained after deacetylation with the addition of 10 mmol/L KCl, CaCl2 and simple re-dissolution in water of the biomass–polymer-solution are compared to the reference without deacetylation. Typical shearthinning curves can be observed for both salts without significant

Table 1 Apparent viscosities of not deacylated culture broths at a shear rate of ˙ = 1 s−1 (Herschel–Bulkley model) for different salt concentrations. Estimations were made with increasing and decreasing shear stress, cBTM = 1.7 g/L, cPS-EDIV = 2.6 g/L. Salt

Reference

csalt [g/L]

–

csalt [mol/L]

 [Pa s] at ˙ = 1 s−1 (increasing shear stress)

 [Pa s] at ˙ = 1 s−1 (decreasing shear stress)

–

1.091

0.517

KCl

25 50 100 150

0.34 0.67 1.34 2.01

1.119 1.148 0.993 1.201

0.363 0.305 0.325 0.427

NaCl

25 50 100 150

0.43 0.85 1.71 2.56

0.891 0.920 1.546 1.580

0.343 0.288 0.391 0.404

CaCl2

50 100 200 400

0.45 0.90 1.80 3.60

0.734 0.789 0.946 1.985

0.342 0.327 0.394 0.438

MgCl2

50 125 250 500

0.53 1.31 2.63 5.25

1.069 0.856 0.783 0.919

0.486 0.336 0.378 0.534

FeCl3

50 125 250

0.31 0.77 1.54

1.038 1.606 2.248

0.589 1.707 2.891

Fig. 2. Dependency of the viscosity of the deacylated biomass–polymer-solution on the shear rate. The flow index n is given for the non-deacylated sample (reference, black symbols) and for three deacylated samples including the biomass–polymersolution re-dissolved in water and the solution after addition of two different salts (grey symbols). cBTM = 1.4 g/L, cPS-EDIV = 2 g/L, cSalt = 10 mmol/L.

differences between them; the flow index n is nearly the same at 0.47. A difference in the flow index between the reference at n = 0.18 and the deacylated samples was observed. 3.3. Amplitude sweep The sol- or rather gel-like character of a PS-EDIV broth can be specified applying an amplitude sweep. The storage modulus G and the loss modulus G represent the elastic and the viscous part of the polymer-containing culture broth. G and G were estimated as a function of the deformation at frequencies of 1.6, 7.5 and 15 Hz (Fig. 3) and, at final shear stresses of 50 Pa and 90 Pa (Fig. 4), respectively. At low deformation, the linear viscoelastic response of PS-EDIV culture broths is dominated by the elastic component up to a strain of circa 200%, whereupon the system begins to yield and respond nonlinearly with the viscous component dominating (G > G ) as shown in Figs. 3 and 4. The storage modulus averaged approximately 55.5 Pa and loss modulus 8 Pa, which indicates the more gel-like character of the biomass–polymer-solution. The limiting value  L , that indicates the range of the linearviscoelastic interval, was quantified with the amplitude sweep as well. As pointed out directly in Figs. 3 and 4 (dashed line), a deformation of about 21% can be fairly taken as the limiting value  L of the LVE interval. 3.4. Frequency sweep The mechanical spectra of PS-EDIV culture broth are shown in Fig. 5. Solid-like behavior of PS-EDIV broth is observed at 20 ◦ C as

Fig. 3. Storage modulus G () and loss modulus G () of the PS-EDIV-containing culture broth with cBTM = 11.1 g/L, cPS-EDIV = 13.1 g/L at a final shear stress of 50 Pa and different frequencies.

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Fig. 4. Storage modulus G () and loss modulus G () of the PS-EDIV-containing culture broth with cBTM = 11.1 g/L, cPS-EDIV = 13.1 g/L at a frequency of 1.6 Hz (ω = 10 s−1 ) and different shear stress values at the end of each measurement.

shown in Fig. 5. The common characteristic feature of the curves is that G (storage modulus) predominates over G (loss modulus, data not shown) through the accessible frequency range studied. It means that the polysaccharide containing culture broth behaves more elastic than viscous. However, notice that G remains almost linear (with an averaged value of 33.4 Pa) over three decades, showing only a slight frequency dependence. While G was nearly independent of the frequency, kept an average value of 3.6 Pa (data not shown). Similar behavior was observed for microbial polysaccharide (deacetylated Na-rhamsan) [14]; mixed polysaccharide systems [20]; bacterial polysaccharides formed by fermentation [21] and mixtures of agar and ␬-carrageenan [22]. 3.5. Thixotropic loop The thixotropic behavior of PS-EDIV broth is illustrated in Fig. 6 for two different PS-EDIV culture broth concentrations (cBTM = 1.4 g/L, cPS-EDIV = 2 g/L, and cBTM = 1.7 g/L, cPS-EDIV = 2.5 g/L), with a clear shear-thinning behavior. The shear stress values in the increasing ramp were always greater than those in the decreasing ramp suggesting an incomplete structure recovery leading to a thixotropic behavior. No hysteresis that is shear- or time-dependent effect was visible for the lowest concentration, cBTM = 1.4 g/L, cPS-EDIV = 2 g/L (A), while for the higher concentration, cBTM = 1.7 g/L, cPS-EDIV = 2.5 g/L (B), an overshoot together with a hysteresis loop were seen (see insert in Fig. 6). Notices, that this overshoot could be better appreciate in a lin–lin scale. This is evident result of the nonlinear viscoelastic behavior. Hysteresis loops can depend on the ramp duration, selected shear rates, and inertial characteristics of the rheometric system [23]; therefore, start-up tests and stepwise sequences at different values of shear rate were

Fig. 6. Thixotropic loops in a log-scale representation at different PS-EDIV concentrations: (A) cBTM = 1.4 g/L, cPS-EDIV = 2 g/L, and (B) cBTM = 1.7 g/L, cPS-EDIV = 2.5 g/L. Solid lines represent the best fit of the data by using the Herschel–Bulkley model. Insert shows a lin–lin representation for (B). Table 2 Fitting parameters for the Herschel–Bulkley model and experiments (Fig. 6). Estimations were made with increasing and decreasing shear stress, (A) cBTM = 1.4 g/L, cPS-EDIV = 2 g/L; and (B) cBTM = 1.7 g/L, cPS-EDIV = 2.5 g/L.  0 is a function of cPS-EDIV and depends of the shear history from the sample. Moreover, while n is almost constant in all the cPS-EDIV studied, K showed a clear dependence of cPS-EDIV (data not showed). 0

K

n

Up Down

0.751 0.299

0.013 0.017

0.742 0.685

Up Down

9.656 5.003

0.036 0.057

0.729 0.702

A

B

applied. Herschel–Bulkley model (Table 2) is in excellent agreement with result of PS-EDIV culture broth (see solid lines in Fig. 6). 3.6. Cox–Merz rule From dynamic measurements, the variations of the complex dynamic viscosity |*| (given by |*| = (G2 + G2 )1/2 /ω) as a function of angular frequency (in rad/s), can be compared to the flow curves. The frequency sweep was carried out in the LVE interval, where the complex viscosity |*| is equivalent to the shear viscosity  (Cox–Merz superposable) [24]. The frequency sweep resulted in a viscosity of 26.1 Pa s at a shear rate of 1 s−1 (Fig. 7) which correlates well with rotational measurements performed at the same shear rate [18]. In contrast, for xanthan [25] and EPS produced by C. capsulata [26] were reported some deviations to the Cox–Merz rule; which were interpreted in terms of specific interactions between chain segments, occurring in addition to normal topological entanglements [27], or to the making and breaking of non-covalent (hydrogens) bonds [28]. 4. Discussion

Fig. 5. Dependency of the Storage modulus G on the angular frequency ω in the LVE-range at given shear stress [Pa] or rather deformation [%], cBTM = 5.4 g/L, cPS-EDIV = 6.4 g/L.

The designing of a technical process for the microbial production of an exopolysaccharide and, in particular, for the subsequent design of an optimal down stream processing requires an adequate knowledge about the dependency of rheological properties of the culture broth on pH-value, temperature and salt concentration. For this reason, the main rheological characteristics of PS-EDIVcontaining culture broths of S. pituitosa were analyzed. The viscosity of the culture broth was not influenced by the temperature within the measurement range of 20–60 ◦ C. The rela-

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Fig. 7. Cox–Merz representation at predefined shear stress [Pa] or deformation [%], cBTM = 5.4 g/L, cPS-EDIV = 6.4 g/L. Solid line represents the shear viscosity at same conditions.

tively constant viscosity of approximately 4.9 Pa s indicates that no thermal decomposition of the biomass–polymer network occurred which is advantageous for the production process and handling of the culture broth. However, while pH-values ranging from 6.0 to 12.0 did not significantly influence the viscosity of the broth, pHvalues in the range of 4–5.3 tend to increment the viscosity up to 90% (see also Fig. 1). This behavior has to be carefully taken into account for pH-control strategies during cultivation, since zones with increased local viscosities would arise at the place where the acid dosage takes place. Especially regulating pH in the range of 5.3–6.0, this phenomenon could be a very important process issue, because it could lead to pH oscillations or bang–bang operation of the controller caused by bad mixing of the culture broth and asynchronous sensor/actuator fluctuations or delay. Analogous to the temperature, the presence of the cells in the polymer-containing culture broth might theoretically help to stabilize the zone of increased viscosities considering the establishment of a network of the exopolysaccharide with the cells as function of the pH-value, however this hypothesis could not be demonstrated in this work. Furthermore, gelation was not observed in spite of the present polyvalent cations in the biomass–polymer-solution. It can be presumed that gelation is prevented by steric inhibition through the glucose side chains of the polymer [16]. The density of the polymer network is developed only weakly due to steric inhibition by the glucose side chains. The slight stabilizing effect of FeCl3 might be explained by the polyvalent charge of Fe3+ -ions which are thus able to interact with more than one polymer strand, thereby affecting a relatively low-density polymer network [29]. Because the polymer strands are aligned in the direction of rotation after shearing, the estimated viscosities at decreasing shear rates are lower than the viscosities at increasing shear rates. The present salts did not influence the network and, accordingly, the viscosity of the polymer did not increase. The discrepancy between the flow index of the reference and the samples shown in Fig. 2 might be explained by the deacylation mode; the reference was not deacylated, while the samples were deacylated by treating with NaOH and HCl. This probably caused the decline of stability due to the absence of functional acetyl and glyceryl groups which otherwise support the formation of bipolar bonds between the polymer strands. The amplitude sweep experiments (Figs. 3 and 4) revealed the gel character of the sample and were essential for identifying the LVE interval used in further oscillatory measurements. A frequency sweep could only be carried out within the LVE interval, because the laws of Hooke and Newton only apply to this interval.

The presence of S. pituitosa cells in the PS-EDIV broth results in an interesting rheological characteristic of the culture broth, since the storage modulus remains nearly constant for the investigated frequency range in frequency sweep experiments (Fig. 5). This is a typical rheological behavior known from cross-linked polymer solutions. But this is misleading, because the PS-EDIV is a linear and not a cross-linked polymer. It is assumed, that this property is due to the linking of the exopolysaccharide to the surface of the cells, as it is the case of other sphingans [30]. That leads to the high viscosity of 26.1 Pa s measured at a shear rate of 1 s−1 . PS-EDIV solutions exhibit shear-thinning behavior, which is a normal behavior for pseudoplastics such as paints, emulsions and dispersions [31]. In the analysis of rheological model applicable to the PS-EDIV culture broth, Herschel–Bulkley equation was in excellent agreement with thixotropy of culture broth (see Fig. 6). Thixotropy indicates continuous breakdown or rearrangement of structure with shearing time [32]. Moreover, the Cox–Merz superposition observed in this PS-EDIV culture broth is generally exhibited for random coil polymers, or structured media (see Fig. 7). Because of its high intrinsic viscosity, the PS-EDIV can be used as a gelling agent, thickener, suspending or binding agent. In terms of performance this natural polysaccharide appears at least as efficient as the usual industrial polysaccharides. These results indicate that the cultivation of S. pituitosa and subsequent purification of PS-EDIV might not encounter extraordinary technical difficulties. With this information, a large scale production of PS-EDIV will be a straightforward process, making economical planning predictable. Acknowledgments The authors gratefully acknowledge the financial support of the (German) Federal Ministry of Education and Research (BMBF) and the Mexican Council for Science and Technology (CONACyT) within the framework of the international cooperation project (MEX 06/015 & J110.531/2006). References [1] Kelco Division of Merck & Co., K9A40: Gellan Gum for Microbial Applications, WAK-Chemie Medical GmbH, 1984. [2] B. Manna, A. Gambhir, P. Ghosh, Production and rheological characteristics of the microbial polysaccharide gellan, Lett. Appl. Microbiol. 23 (1996) 141–145. [3] F. Paul, A. Morin, P. Monsan, Microbial polysaccharides with actual potential industrial applications, Biotechnol. Adv. 4 (1986) 245–259. [4] G.R. Sanderson, Gellan gum, in: P. Harris (Ed.), Food Gels, Elsevier Applied Science, London, New York, 1990, pp. 201–232. [5] R.M. Banik, B. Kanari, S.N. Upadhyay, Exopolysaccharide of the gellan family: prospects and potential, World J. Microbiol. Biotechnol. 16 (2000) 407– 414. [6] C. Mazuel, Pharmaceutical and/or cosmetic composition for local use containing rhamsan gum, US Patent 4,996,197 (1988). [7] M. Amro, Untersuchungen zum Einsatz von Bakterien zur Erhöhung des Entölungsgrades in Eröllagerstätten, Doctoral Dissertation, Technische Universität Clausthal, 1994. [8] A.K. Podolsak, C. Tiu, T. Saeki, H. Usui, Rheological properties and some applications for rhamsan and xanthan gum solutions, Polym. Int. 40 (1996) 155–167. [9] G. Pusch, T. Lötsch, T. Müller, Investigations of the oil displacing efficiency of the suitable polymer products in porous media, aspects of recovery mechanisms during polymer flooding, DGMK Deutsche Wissenschaftliche Gesellschaft für Eröl, Erdgas und Kohle e.V., DGMK-Report, 1987, pp. 295–296. [10] G. Rehage, H. Block, A.K. Wehrhahn, Physico-chemical investigations on polymer-flood media, DGMK Deutsche Wissenschaftliche Gesellschaft für Eröl, Erdgas und Kohle e.V., DGMK-Report, 1987, p. 295. [11] R. Moorhouse, Structure/property relationships of a family of microbial ploysaccharides, in: M. Yalpani (Ed.), Industrial Polysaccharides: Genetic Engineering, Structure/Property Relations and Applications, Bd. 3, Elsevier Science Publishers, Amsterdam, 1987, pp. 187–205. [12] T. Mezger, Das Rheologie-Handbuch: Für Anwender von Rotations- und Oszillations-Rheometern, Curt R. Vincentz Verlag, Hannover, 2000. [13] E. Dreveton, F. Monot, D. Ballerini, J. Lecourtier, L. Choplin, Effect of mixing and mass transfer conditions on gellan production by Auromonas elodea, J. Ferment. Bioeng. 77 (1994) 642–649.

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