Solution properties of an exopolysaccharide from a Pseudomonas strain obtained using glycerol as sole carbon source

Carbohydrate Polymers 78 (2009) 526–532

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Solution properties of an exopolysaccharide from a Pseudomonas strain obtained using glycerol as sole carbon source Loic Hilliou a,d,*, Filomena Freitas b, Rui Oliveira b, Maria A.M. Reis b, David Lespineux c, Christian Grandfils c, Vítor D. Alves d a

I3N – Institute for Nanostructures, Nanomodelling and Nanofabrication, Department of Polymer Engineering, University of Minho, Campus de Azurem, 4800-058 Guimarães, Portugal REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal c Interfacultary Research Centre of Biomaterials (CEIB), University of Liège, B-4000 Liège, Belgium d REQUIMTE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 3 April 2009 Received in revised form 15 May 2009 Accepted 18 May 2009 Available online 23 May 2009 Keywords: Exopolysaccharide (EPS) Viscosity Glycerol Polyelectrolyte Light scattering

a b s t r a c t We report the solution properties of a new exopolysaccharide (EPS) obtained from a Pseudomonas strain fed with glycerol as the sole source of carbon. This high molecular mass (3  106 g mol1) biopolymer is essentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolyte character. The Smidsrod parameter B computed from the ionic strength dependence of the intrinsic viscosity indicates that the EPS backbone is rather flexible. In salt free aqueous solutions, the zero shear viscosity scaling with concentration follows a typical polyelectrolyte behavior in bad solvent, whereas at high ionic strength the rheological response is reminiscent from neutral polymers. Light scattering data indicate that the EPS adopts a globular conformation as a result of hydrophobic interactions. EPS solutions are stable within 4 days as particle sizing does not indicate EPS aggregation. Both globular conformation and stability against precipitation from solution are attributed to the low charge density of the polyelectrolyte. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Microbial polysaccharides, such as xanthan, gellan, pullulan and bacterial alginate, may represent alternatives to polysaccharides obtained from plants (Guar gum, Arabic gum or pectins), algae (alginate, carrageenan or agar) and crustacean (chitin) (Stephen, 1995). In fact, microbial production is a much more controlled process, originating a product with tuned chemistry and properties and with a constant availability over time. This constitutes an advantage over the natural biopolymers isolated from plants and algae, whose availability and physical–chemical properties are dependent on external factors, such as climate conditions and the season of the year (Sutherland, 2001). The most used carbon sources for EPS production have been sugars, namely glucose and sucrose, applied for instance in the production of xanthan gum (García-Ochoa, Santos, Casas, & Gómez, 2000) and bacterial alginate (Peña, Trujillo-Roldán, & Galindo, 2000). However, the high cost of these carbon sources has a direct impact on production costs, which limits the market potential of

* Corresponding author. Address: I3N – Institute for Nanostructures, Nanomodelling and Nanofabrication, Department of Polymer Engineering, University of Minho, Campus de Azurem, 4800-058 Guimarães, Portugal. Tel.: +351 253 510 320; fax: + 351 253 510 339. E-mail address: [email protected] (L. Hilliou). 0144-8617/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2009.05.011

these biopolymers. In order to decrease the production costs, it is important to look for less expensive carbon sources, like industrial wastes or industrial by-products (Kumar, Mody, & Jha, 2007). Sugar molasses and potato starch wastes are examples of low cost carbon sources already used for the production of microbial polysaccharides such as exopolysaccharide based on cellulose (Paterson-Beedle, Kennedy, Melo, Lloyd, & Medeiros, 2000) and pullulan (Barnett, Smith, Scanlon, & Israilides, 1999). More recently, glycerol, a by-product of many industrial processes, mainly from biodiesel production, has been generated in large quantities far beyond current consumption in traditional applications. Interesting applications for glycerol are still lacking. We reported recently the production of a new microbial polysaccharide by a Pseudomonas strain using glycerol as the sole carbon source (Freitas et al., 2009; Reis et al., 2008). The biopolymer is a high molecular weight extracellular heteropolysaccharide composed of neutral sugars (galactose, mannose, glucose and rhamnose) and acyl groups (pyruvil, succynil and acetyl). This exopolysaccharide (EPS) is amorphous, as inferred by thermal analysis and solid-state NMR. It possesses flocculating and emulsifying properties, along with film-forming capacity, making it a good alternative to other natural and microbial polysaccharides. A systematic study of the EPS aqueous solutions properties is of major importance in order to screen potential application of this product to industrial activities such as water treatment, food,

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pharmaceutical, cosmetic, mining, paper and oil recovery. Preliminary rheological studies showed that the crude EPS has viscosity enhancing properties similar to Guar gum. Here, we explore in greater detail the solution properties of a purified EPS sample, in order to rationalize the good viscoelastic properties. EPS in salt free solutions as well as in NaCl solutions with various ionic strengths are studied at concentrations ranging from the dilute regime to the concentrated regime, using light scattering techniques and rheometry.

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2.3. Biopolymer solutions The EPS, isolated as described in Section 2.1 after 7 days operation of the bioreactor (see below), was added to hot NaCl solutions or deionised water and stirred at 80 °C for 1 h. Solutions were then further stirred overnight at room temperature, to ensure complete dissolution of the EPS, and then characterized without delay. EPS concentration ranged from 0.0005 to 0.04 wt% for light scattering experiments, and from 0.01 to 1.6 wt% for rheological analysis. Ionic strength of solutions ranged from roughly 104 for deionised water (Dou & Colby, 2006) to 0.5 M.

2. Materials and methods 2.4. Rheological measurements 2.1. Polysaccharide production and recovery The bioprocess used to obtain the exopolysaccharide has been reported in detail elsewhere (Freitas et al., 2009). The culture was grown on Medium E* (Brandl, Gross, Lenz, & Fuller, 1988), supplemented with 25 g/l glycerol (Fluka, 86%) as carbon source and 3.3 g/l (NH4)2HPO4 as nitrogen source. EPS production was carried out using Pseudomonas oleovorans NRRL B-14682 and was performed in a 10 l bioreactor (BioStat B-plus, Sartorius) operated in fed-batch mode, with controlled pH (6.75–6.85) and temperature (30 °C), and at a constant air flow rate of 0.125 vvm (volume of air per volume of reactor per minute). Glycerol and ammonium concentration were determined as described by Freitas et al. (2009). Culture broth samples were diluted with deionised water for viscosity reduction and centrifuged at 48,384g for 1 h. The cell-free supernatant was subjected to protein thermal denaturation at 80 °C during 4 h, followed by their separation by centrifugation (48,384g, 1 h). The polymer was then precipitated from the cellfree supernatant by the addition of cold ethanol 96 vol% (3:1) and separated by centrifugation (27,216g, 15 min). The pellet was washed with ethanol 96 vol%, redissolved in deionised water, reprecipitated two times and freeze dried. 2.2. Chemical characterization The polymer samples were analyzed in terms of sugar composition, acyl groups, inorganic and protein content. For the analysis of the sugar composition, 2–3 mg of the extracted EPS were dissolved in 5 ml deionized water and hydrolyzed with trifluoroacetic acid (TFA) (0.1 ml TFA 99%), at 120 °C, for 2 h. The hydrolysate was used for the identification and quantification of the constituent monosaccharides by liquid chromatography (HPLC) using a CarboPac PA10 column (Dionex), equipped with an amperometric detector. The analysis was performed at 30 °C, with sodium hydroxide (NaOH 4 mM) as eluent, at a flow rate of 0.9 ml min1. The hydrolysate was also used for the identification and quantification of acyl groups present in the EPS. The analysis was performed by HPLC with an Aminex HPX-87H column (BioRad), coupled to an ultraviolet (UV) detector, using sulphuric acid (H2SO4 0.01 N) as eluent, at a flow rate of 0.6 ml min1 and a temperature of 50 °C. The detection was performed at 210 nm. For the determination of the EPS protein content, 5.5 ml samples of 4.5 g/l aqueous solutions were mixed to 1 ml 20% NaOH, placed at 100 °C for 5 min and cooled on ice. Each sample was mixed with 170 ll of CuSO4  5H2O (25% v/v) and centrifuged at 3500g for 5 min. The optical density was measured at 560 nm (Spectrophotometer Helios Alpha, Thermo Spectronic, UK). Albumin (Merck) solutions (0.5–3.0 g/l) were used as protein standards. The total inorganic content of the extracted EPS was determined by subjecting the EPS to pyrolysis at a temperature of 550 °C for 48 h. The EPS was further analyzed by Inductively Coupled Plasma – Atomic Emission Spectroscopy, to quantify its content in sodium, calcium, potassium, magnesium and iron.

EPS solutions prepared as described above were directly loaded in the pre-heated (80 °C) cone and plate geometry (diameter 6 cm, angle 0.2 rad) of a stress rheometer (ARG2, TA Instruments Inc., New Castle, DE, USA) and the shearing geometry was covered with paraffin oil in order to prevent water loss. Such pre-heating step was performed in order to erase any thermal and mechanical history induced by the preparation of EPS solutions, and which might affect the rheological response of solutions at 25 °C. Solutions were then cooled (5 °C/min) down to 25 °C and time was left for samples to equilibrate as demonstrated by the record of a time independent dynamic loss modulus G00 measured at 1 Hz with a 0.1 oscillatory shear strain amplitude. The mechanical spectrum of the solution was then recorded at 25 °C by performing a frequency sweep obtained with oscillatory strain amplitudes ranging from 0.01 to 0.15. The oscillatory torque response recorded on-line showed a sinusoidal wave form for all reported frequencies, thus ensuring a linear relationship between the applied sinusoidal strain and the measured stress. The solutions flow curves were then obtained from steady stress sweep tests (shear rate measured over the last 10 s of a step shear stress with 60 s duration, and steady state defined within a 2% tolerance for shear rate variation between two consecutive step shear stresses) performed between 0.1 and 100 Pa. Inspection of samples, right after performing the steady stress sweep tests, indicated that no flow instability (inertial, elastic or edge fracture leading to emulsion of the paraffin oil) took place within the range of applied stresses. As a result, a smooth shear thinning behavior was observed for all flow curves (see Figs. 3 and 5), which confirms that no secondary flow developed during the flow tests. For dilute EPS solutions showing viscosities below the sensitivity of the stress rheometer (such sensitivity limit was reached for concentration approaching 0.1 g/dl), a Cannon–Fenske capillary viscometer (COMECTA S.A., Barcelona, Spain) immersed in a temperature bath at 25 °C was used. 2.5. Light scattering EPS solutions were passed through 0.45 lm polysulfone filters prior to analysis. Multi Angle Laser Light Scattering (MALLS) data were obtained by injecting the filtered EPS solutions at a flow rate of 19 ml/h with a syringe pump (Vial Medical, Program II) in the K5 flow cell of a MALS detector (Dawn, Wyatt Technology Corp., Santa Barbara, CA) irradiated by a Uniphase Argon laser (488 nm; 10 mW) and placed in series with the refractive index (RI) detector (Optilab DSP, Wyatt Technology Corp., Santa Barbara, CA). The MALLS and RI data were recorded with ASTRA software (Version 4.73.04, Wyatt Technology Corp., Santa Barbara, CA). Dynamic Light Scattering (DLS) was also performed on the same solutions, using an optical assembly equipped with a 20 mW HeNe laser (Photocor Instruments, Inc., College Park, MD). The time variations of light scattering intensity were analysed at 25 °C at an angle of 90° using the BI9000 correlator (Brookhaven Instruments Corp., Holtsville, NY). The mean light scattering intensities were analysed

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in triplicate on 1 min sampling. The DLS signals were analysed with the DYNALS software (Version 2.8.3, Alango Ltd., Tirat Carmel, Israel). The refractive index increment (dn/dc) was 0.16, as measured with the RI detector for a set of solutions with various concentrations and ionic strengths.

3.2. Effect of ionic strength on the chain size and conformation in dilute solutions Data obtained for EPS solutions with the Cannon–Fenske capillary viscometer are presented in Fig. 1. Intrinsic viscosities [g] were computed from data fitting to the Huggins equation

2.6. Size-exclusion chromatography combined with MALLS (SEC-MALLS)

g0  gS gspe ¼ ¼ ½g þ kH ½g2 c cgS c

EPS solutions with a concentration of 0.04 wt%, prepared at various ionic strengths, were analysed by Size Exclusion Chromatography coupled with Multiple Angle Laser Light Scattering (SEC-MALLS). Solutions were filtrated on a glass fibre filter (0.3 lm; Pall, Life Sciences, Type A/E) before their injection on the SEC-MALLS system. The latter combines a HPLC pump (Hewlett Packard quaternary 1050), an autoinjector (Hitachi-Merck, Lachrom L7200, model 1405–040), and a set of two analytical SEC linear columns (PL aquagel-OH mixed 8 lm, 30  7.5 mm) protected by a guard column. UV (Beckman UV model 266 fixed at 254 nm) signals were recorded together with MALS and RI signals (the same MALLS and RI detector as described above were used) in order to follow the purity and molecular mass distribution of the polysaccharide. The SEC columns were equilibrated for 24 h before running the analysis at a flow rate of 0.7 ml/min at room temperature.

where g0 is the zero shear viscosity of the solution, gS is the solvent viscosity, gspe is the specific viscosity, kH the Huggins coefficient and c the EPS concentration. Values calculated for [g] and kH using Eq. (1) are gathered in Table 1 for all ionic strength studied. As expected for a polyelectrolyte, the intrinsic viscosity is a decreasing function of the ionic strength, since the chain conformation changes from a stretched conformation to a coil-like or globular one. We can rationalize these data by using the Smirod empirical prediction (Smidsrod & Haug, 1971) for the ionic strength dependence of the intrinsic viscosity

3.1. EPS production and chemical characterization The cultivation run lasted for 7 days, but bacterial cell growth was ended after 3 days of operation by imposing nitrogen limiting conditions (concentration below 0.1 g NH4+/l). Nevertheless, a residual ammonium concentration was kept in the bioreactor by the addition of mineral medium. The bioreactor was fed with mineral medium, supplemented with a glycerol concentration of 200 g/l. EPS synthesis was initiated during the exponential phase of bacterial growth, but the maximum production rate was observed after the culture has entered the stationary phase. The viscosity of the culture broth showed a dramatic increase with cultivation time (Freitas et al., 2009). As a result, appropriate mixing, aeration or control of bioreactor parameters could no longer be performed and the cultivation was eventually terminated at day 7. At that stage, the final EPS concentration achieved was 13.3 g/l, corresponding to a maximum productivity of 2.8 g/l day and an EPS yield of 0.19 g/g on a glycerol basis. The productivity achieved from glycerol is in the range of values referred for xanthan gum (3.112.2 g l1 d1) (García-Ochoa et al., 2000) and bacterial alginate (0.43–1.53 g l1 d1) (Peña et al., 2000), produced under optimized conditions using glucose and sucrose as carbon sources. Sugar analysis indicated that the overall sugar content in the purified EPS was 79.2 wt%, galactose being the most abundant monosaccharide, accounting for 70 wt% of the total carbohydrate content, followed by mannose (23 wt%), glucose (4 wt%) and rhamnose (3 wt%). The total inorganic content was 11 wt%, where the main cations detected were sodium (5.2 wt%) and potassium (1.0 wt%), with minor amounts of calcium (0.1 wt%), zinc (0.06 wt%) and magnesium (0.05 wt%). The acyl groups represented 4.8 wt% of the overall mass and the protein content was 5.0 wt%. The EPS recovered after 7 days of cultivation is therefore a galactose-rich polysaccharide with charges (due to the pyruvil and succinyl groups) on the polymer backbone, and cations, acting either as counter-ions or taking part of some precipitated salts during the recovery and purification processes.

1

½g ¼ ½g1 þ Bð½g0:1 Þ1:3 M 2

ð2Þ

where M is the ionic strength, [g]1 is the intrinsic viscosity at infinite ionic strength and [g]0.1 the intrinsic viscosity measured at 0.1 M NaCl. Parameter B is a constant which is related to the stiffness of the polymer. In this framework, the slope S obtained from the Smirod representation displayed in Fig. 2, is related to parameter B as

B¼

S

ð3Þ

ð½g0:1 Þ1:3

From the data in Fig. 2 we find B = 0.1 ± 0.003, which indicates that the present EPS chain is not rigid under such salt conditions (0.1 M NaCl). Rather, it shows a flexible conformation approaching the one commonly found for various extracellular polysaccharides and natural carbohydrate polymers (Ding, LaBelle, Yang, & Montgomery, 2003; Ren, Ellis, Sutherland, & Ross-Murphy, 2003). Results from SEC-MALLS analysis give additional inputs on the EPS conformation in 0.1 M NaCl. 60% of the EPS was recovered after SEC-MALLS analysis, whereas a mobile phase with lower ionic strength essentially leads to dramatic EPS adsorption on the SEC column. As such, a fair quantitative estimation of both molecular

28 24

η spe / c (dl/g)

3. Results and discussion

ð1Þ

20 16 12 8 4 0 0.00

0.02

0.04

0.06

0.08

0.10

c (wt %) Fig. 1. Huggins extrapolation to intrinsic viscosity for the EPS in aqueous solutions with different ionic strength adjusted by the addition of NaCl. ( ): 0.01 M; (}): 0.025 M; (D): 0.05 M; (s): 0.1 M; (h): 0.5 M. Solid lines are fits of Eq. (1) to the data.

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Table 1 Intrinsic viscosity [g], Huggins coefficient kH, Molecular mass Mw, radius of gyration Rg, second virial coefficient A2, and hydrodynamic radius Rh obtained from EPS dilute solutions at various ionic strengths (NaCl). NaCl (M)

[g] (dl g1)

kH

0.01 0.02 0.05 0.1 0.25 0.5

14 ± 0.4 10.6 ± 0.7 7±1 6.2 ± 0.3

1.05 ± 0.10 1.23 ± 0.15 2.3 ± 0.7 2.58 ± 0.25

4.9 ± 0.4

4.5 ± 0.5

Mw (106 g mol1)

Rg (nm)

A2 (103 ml g2)

Rh (nm)

3.0 ± 0.5 2.7 ± 0.5 3.4 ± 0.5 3.2 ± 0.5 –

99.3 ± 7.0 98.4 ± 7.1 103.8 ± 6.3 96.1 ± 6.6 –

2.5 ± 1.2 2.9 ± 2.0 1.5 ± 1.0 2.7 ± 0.7 –

127.0 ± 17.8 131.2 ± 28.2 128.1 ± 23.9 128.2 ± 16.6

weight and radius of gyration is not amenable. Nevertheless, a qualitative picture of the EPS conformation under such salt conditions is provided by the Flory plot displayed in the inset of Fig. 2. The scaling of the radius of gyration Rg with the molecular weight Mw shows a power law behavior with an exponent of roughly 0.24, and therefore suggests a rather compact and globular conformation (the exponent for a compact coil in a theta solvent is 0.33 (Yang & Zhang, 2009)). We may therefore, suspect that such a globular conformation stems from bad solvent condition for the biopolymer. MALLS performed at different ionic strengths actually supports the picture of a hydrophobic polyelectrolyte for the EPS. For all experimental conditions studied, negative slopes for lines at constant scattering angles in the Zimm plots indicate that the second virial coefficient A2 is negative (see corresponding values in Table 1). This result suggests that water is not a good solvent for the EPS, and as such a globular, string or necklace conformation could be expected for this hydrophobic polyelectrolyte (Dobrynin & Rubinstein, 1999), depending on the charge density of the polyelectrolyte. Negative values for A2 are also in harmony with the large Huggins coefficients reported in Table 1. The large Huggins coefficients suggest the presence of large intra or interchain interactions (Cheng et al., 2002). The presence of acyl groups on galactose rich backbones is known to induce such interactions (Rinaudo, 2004). Huggins coefficients show an increase with the ionic strength, which correlates with a decrease in the intrinsic viscosity. A similar qualitative trend for the ionic strength dependence of both kH and [g] was recently reported (Rotureau, Dellacherie, & Durand, 2005) for a series of chemically modified dextrans and was related to increased hydrophobic interactions achieved through branching of short hydrocarbon chains. In the present

case, additional characterization is needed to check whether branching occurs in the EPS backbone. Alternatively, large values of the Huggins coefficient can be caused by EPS aggregation (interchain interactions). Indeed, the chemical analysis of the EPS indicates that this hydrophobic polyelectrolyte is composed by less than 5 wt% of charged groups. The EPS is thus weakly charged and we may question the occurrence of counterion condensation leading to precipitation of globular conformers (Dobrynin, Colby, & Rubinstein, 1995). Therefore, DLS was performed at a concentration of 0.08 wt% in solution with various ionic strengths, and over a 4 days period. The hydrodynamic radii Rh of EPS extracted from the diffusion coefficients measured during the first day are reported in Table 1. Data indicate that within the experimental error, Rh remains constant over a period of 4 days (results not shown). In addition, no precipitation was observed over the same period. Therefore, EPS aggregation can be ruled out. Rh is quantitatively in harmony with the radius of gyration Rg determined by the extrapolation (with a second order polynomial) at zero scattering angle in the Zimm plots. Rg values for different ionic strengths are reported in Table 1, along with the corresponding molecular mass Mw obtained by extrapolation to zero concentration in the Zimm analysis. The ratio Rg/Rh is roughly 0.8, and indicates a compact spherical conformation (Aseyev, Klenin, Tenhu, Grillo, & Geissler, 2001; Yang & Zhang, 2009) for all ionic strengths. This result is in agreement with the chain conformation determined from the SEC-MALLS analysis. We note that all conformational parameters obtained from light scattering are roughly insensitive to the ionic strength (see Rg and Rh in Table 1). This is in contrast to data obtained with capillary viscosimetry. We note here that light scattering data are treated with the assumption of spherical scatterers (Rh is obtained through the diffusion constant and the Einstein–Stoke relation), or of point-wise scatterer (Rg is obtained with a Zimm analysis where the particle scattering factor is assumed to be quadratic with the scattering angle). Therefore, additional scattering experiments will be needed to check whether the EPS conformation is more extended (wormlike chain) at lower ionic strengths where larger values for [g] are found. 3.2. Rheology of salt free solutions Fig. 3 shows the flow curves of EPS in deionised water for concentrations ranging from 0.2 to 1 wt%. Curves are characterized by a Newtonian plateau at lower shear rate followed by a shear thinning behavior. Data in Fig. 3 were fitted with a Cross equation

g¼

Fig. 2. Ionic strength dependence of the intrinsic viscosity of the EPS. The line is a fit of Eq. (2) to the data, from which the stiffness parameter B is obtained. Inset: Flory representation of EPS in 0.1 M NaCl obtained from SEC-MALS analysis. The line is a linear fit to the data giving the slope indicated in the inset.

g0 1 þ ðsc_ Þm

ð4Þ

in order to obtain a quantitative estimate of the zero shear viscosity g0 and relaxation time s. The concentration dependence of the specific viscosity computed from g0 and the solvent viscosity is displayed in Fig. 4. Data reported for concentrations below 0.1 wt% were obtained with the Cannon–Fenske capillary. Video imaging of the solutions meniscus in the capillary showed that the wall shear rates ranged between 1 and 100 s1. Inspection of

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0

10

0

10

10

η (Pa.s)

η (Pa.s)

10

-1

10

-1

10

-2

-2 0

10

10

1

2

10

10

3

-1

γ (s )

10

0

10

1

10

2

10

3

-1

γ (s )

Fig. 3. Shear rate dependence of the steady state viscosity of EPS solutions in deionised water. Solution concentrations from bottom to top are 0.17, 0.27, 0.33, 0.42, 0.54, 0.7 and 1.0 wt%. Lines represent fits of the Cross model (Eq. (4)) to the data.

Fig. 5. Shear rate dependence of the steady shear viscosity of EPS solutions in 0.1 M NaCl. Solution concentrations from bottom to top are 0.06, 0.18, 0.26, 0.37, 0.5, 0.6, 0.75, 0.9, 1.1, 1.28 and 1.6 wt%. Lines represent fits of the Cross model (Eq. (4)) to the data.

Fig. 3 indicates that these shear rates belong to the Newtonian regime of dilute EPS solutions. The extension of Eq. (1) to higher orders in the concentration is often used to extract useful empirical structure-properties relationships from the viscous properties of various polymer solutions (Clasen & Kulicke, 2001). However, as we are dealing with a charged EPS, we naturally focus our attention to the theory of polyelectrolyte solutions (Dobrynin & Rubinstein, 2005) to analyze the data. The theory predicts power law relationships between the concentration and the solution dynamic properties such as the zero shear viscosity and the longest relaxation time. As such, experimental data are to be compared to the predicted exponents of power laws (indicated in Fig. 4 by lines and numbers). Fig. 4 reveals 3 concentration regimes defined by different scaling of the specific viscosity with the EPS concentration. At low concentration, a power law with exponent ½ is observed which conforms to the scaling expected for the semi dilute and non entangled regime of either hydrophobic polyelectrolytes or polyelectrolytes in salt free solutions (Dobrynin et al., 1995). A crude estimate for the over-

lap concentration c* which defines the onset of the semi dilute non entangled regime is given by the concentration at which gspe  1 (Di cola et al., 2004; Larson, 1999). Data in Fig. 4 suggest that c*  0.01 wt%. Taking into account the fact that the molecular mass Mw of the EPS does not depend on the ionic strength, we can compute the size Rg of the equivalent EPS coil in salt free conditions using the definition of the overlap concentration c* and  13 w Rg ¼ 4p3M where NA is the Avogadro number and q the solvent qNA c

Fig. 4. Concentration dependence of the specific viscosity of EPS solutions in deionised water. The inset is the mechanical spectrum ((h): G0 ; (j): G0 0 ) recorded at a concentration of 1 wt%, with solid lines indicating slopes 1 and 2 for the terminal behavior of G0 0 and G0 , respectively.

density (de Gennes, 1979). We obtain Rg = 228 nm using Mw = 3.0  106 and c* = 0.01 wt%. Rg is roughly twice the value obtained with static light scattering for the lowest ionic strength studied (see Table 1), and as such is indicative of a rather extended conformation in salt free solutions. A second power law behavior with exponent 3/2 is observed for concentration in excess of ce = 0.05 wt%. This behavior corresponds to the scaling predicted for semi dilute and entangled polyelectrolytes in salt free solutions (Dobrynin et al., 1995). We note that the salt free EPS solution is poorly entangled as the viscosity at ce is only 6 times larger than the viscosity of the solvent. Such a behavior was also reported for model hydrophobic polyelectrolytes in salt free solutions exhibiting a 10 times smaller molecular mass (Di cola et al., 2004) than the present EPS. The ratio ce/c* gives the width of the semi dilute non entangled regime, and is expected to be as large as 1000 (Dobrynin et al., 1995). The ratio for the present EPS is rather small when compared to those measured with model hydrophobic polyelectrolytes (Di cola et al., 2004; Dou & Colby, 2006), and as such validates the picture of a weakly charged EPS (Dou & Colby, 2006). Departure from the 3/2 scaling at highest concentrations marks the onset of a third concentration regime: for concentrations larger than c**, a steeper dependence of the specific viscosity with the concentration is observed and as such defines the onset of the concentrated regime. This high concentration behavior contrasts with the concentration scaling, gspe  c0, predicted for the so-called bead controlled regime of semi dilute entangled polyelectrolytes in bad solvent (Dobrynin & Rubinstein, 1999). However, rheological evidence for this concentration regime is still lacking (Di cola et al., 2004) as it requires highly charged polyelectrolytes exhibiting many entangled beads on a bead-necklace conformation (Dobrynin & Rubinstein, 1999, 2005). The inset in Fig. 4 presents the mechanical spectrum measured at the highest concentration studied. The high frequency part of the spectrum shows the onset of a crossover

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between G0 and G00 . No elastic plateau is observed for the storage shear modulus G0 , and as such no clear evidence for entanglements between EPS chains is obtained. Therefore, we still miss a clear explanation for the steep increase of free salt EPS solution viscosity at higher concentrations, as the interplay between screened electrostatic interactions, polyelectrolyte conformation and possible reptation dynamics still needs a theoretical treatment (Dobrynin & Rubinstein, 2005). 3.3. Solution properties at high ionic strength (0.1 M NaCl) Fig. 5 shows the flow curves of EPS solutions at 0.1 M NaCl. The shear rate dependence of the steady state viscosity data exhibits a Newtonian behaviour at the lowest concentrations, whereas a shear thinning is observed for more concentrated solutions. Data fitting with Eq. (4) yields the zero shear viscosity and allows the corresponding plot of gspe as a function of EPS concentration in Fig. 6. The concentration dependence of gspe shows essentially two regimes characterized by different power laws. At low concentration, we identify the overlap concentration c*  0.05 wt% (concentration corresponding to gspe = 1) which is larger than the overlap concentration measured in salt free solutions. This overlap concentration can be compared to the value c* = 0.1 wt% computed from the value of [g] measured at 0.1 M NaCl (see Table 1), and using the relationship c*  0.77/[g], devised for closely packed spherical and impenetrable coils (Graessley, 1980). The departure between these two estimates is fairly acceptable if we take into account the general approximation (Larson, 2005) underlying the relationship between c* and [g]. Under the assumption of coil conformation, the overlap concentration at which gspe = 1 corresponds  1=3 w to a radius of gyration Rg ¼ 4p3M ¼ 140 nm, which is roughly qNA c

531

by a power law scaling with exponent 15/4 is evidenced. Both exponents correspond to the concentration scaling predicted for the semi dilute and semi dilute entangled regimes, respectively, of neutral polymers in good solvent (de Gennes, 1979). Entanglements also show up as an elastic plateau in the high frequency regime of the mechanical spectrum recorded with the highest EPS concentration (lower inset in Fig. 6). As such, the viscous enhancing character of the present EPS in concentrated (1 wt%) solutions with high ionic strengths originates from entanglements between EPS chains. 3.4. Flow master curves and aggregation

in agreement with the EPS conformation obtained from MALLS (Rg = 103.8 nm). The closer agreement between light scattering and rheology as compared with results obtained with salt free solutions, stems from the EPS globular conformation at higher ionic strength, which as such validates the use of the above relationship between Rg and c*. A concentration scaling showing a power law behaviour with exponent 5/4 is first observed, whereas departure from this scaling occurs for concentration beyond ce = 0.4 wt% where a second concentration regime characterized

Data displayed in Figs. 3 and 5 could be superimposed by scaling each flow curve horizontally with the relaxation time s and vertically with the respective zero shear viscosity g0 computed with Eq. (4) for each concentration. The resulting master curves are presented in Fig. 7. Similar master curves were obtained with various polysaccharides (Morris, Cutler, Ross-Murphy, Rees, & Price, 1981) including strongly interacting cellulose (Burchard, 2001), with various EPSs (Goh, Hemar, & Singh, 2005; Gorret, Renard, Famelart, Maubois, & Doublier, 2003), and also with polyisobutylene solutions in a mixture of polybutene oil and dekalin for all concentration regimes (Nogueiro & Maia, 2003). The concentration dependence of Cross parameters m and s is depicted in the inset of Fig. 7 for salt free EPS solutions and EPS solutions prepared in 0.1 M NaCl. Comparison of both sets of data indicates that a similar concentration scaling is observed for these non linear parameters. We therefore, suspect that electrostatic interactions do not affect qualitatively the flow behavior of semi dilute entangled hydrophobic polyelectrolytes in the high shear rate limit. Exponent m is virtually not depending on the concentration, reaching a value of 0.7 and 0.8 for the salt free solutions and the solutions in 0.1 M NaCl, respectively. These values are reminiscent from the almost universal exponent m = 0.76 found for many polysaccharides (Morris, 1990), including those presenting strong interactions such as hydrogen bonding between polymer chains, and also compare well with the asymptotic exponent m = 0.82 predicted for well entangled flexible polymers in good solvent (Graessley, 1974). The dependence s  c3 (suggested by the solid lines indicating a slope of 3 in the inset of Fig. 7) is similar to that referred for weakly

Fig. 6. Concentration dependence of the specific viscosity of EPS solutions in 0.1 M NaCl. The upper and lower insets are the mechanical spectra ((h): G0 ; (j): G0 0 ) recorded at the reported concentrations which belong to the semi dilute and semi dilute entangled regimes, respectively. Lines in the insets indicate slopes 1 and 2 for the terminal behavior of G0 0 and G0 , respectively.

Fig. 7. Master curves for EPS flow curves in deionised water (lines) and in 0.1 M NaCl (open squares). Inset: concentration dependence of parameters m (solid symbols) and s (open symbols) of Cross equation in deionised water (square) and in 0.1 M NaCl (triangles). Note that the concentrations correspond to the semi dilute entangled regime for both solvents.

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charged model polyelectrolytes in the semi dilute entangled concentration regime (Dou & Colby, 2006). A power law behaviour s  c2.75 is predicted for entangled polymer melts (de Gennes, 1979), whereas exponents ranging from 2.75 to 2.85 were measured for semi dilute polymer solutions in a theta solvent (Adam & Delsanti, 1985). Exponents well in excess of 3 are usually reported for polysaccharides exhibiting strong interchain association (Burchard, 2001). Therefore, we confirm that the rheological behaviour displayed in Fig. 7 corresponds to semi dilute entangled polymer solutions in bad solvent, presenting no strong interaction between chains. As such, aggregates are likely not to be formed within the range of concentration studied. 4. Conclusions A microbial polysaccharide has been produced by a Pseudomonas strain using glycerol as the sole carbon source. The purified EPS is a high molecular mass polysaccharide, and is essentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolyte character. The picture emerging from intrinsic viscosity and light scattering data is that the EPS is a flexible (within the limitation of the physical meaning of parameter B (Ding et al., 2003; Ren et al., 2003)) hydrophobic polyelectrolyte which adopts a compact globular conformation in the presence of salt and which does not show any tendency to aggregation in the dilute regime, at least within a period of 4 days. Complementary scattering techniques are however, needed to elucidate the EPS conformation in solution, as large Huggins coefficients are measured with capillary viscosimetry and suggest a significant branching on the EPS backbone. The EPS shows good viscous enhancing properties (a 1 wt% solution is roughly a 1000 times more viscous than water) and a strong shear thinning. This rheological behavior originates from entanglements between EPS chains in solutions with high ionic strength. In salt free solutions, the concentration scaling of the EPS zero shear viscosity shows a typical weakly charged hydrophobic polyelectrolyte behavior. However, the high viscosity measured in the concentrated regime still needs to be rationalized, even if the non linear rheology suggests the disentanglement of EPS chains. Acknowledgements The authors acknowledge the Portuguese company 73100 for the financial support, under the project ‘‘Production of biopolymers from glycerol”, 2005/2008. Vítor D. Alves acknowledges Fundação para a Ciência e a Tecnologia, Pos-doc fellowship SFRH/BPD/ 26178/2005. References Adam, M., & Delsanti, M. (1985). Dynamical behavior of semidilute polymer solutions in a Theta solvent: Quasi-elastic light scattering experiments. Macromolecules, 18, 1760–1770. Aseyev, V. O., Klenin, S. I., Tenhu, H., Grillo, I., & Geissler, E. (2001). Neutron scattering studies of the structure of a polyelectrolyte globule in a wateracetone mixture. Macromolecules, 34, 3706–3709. Barnett, C., Smith, A., Scanlon, B., & Israilides, C. J. (1999). Pullulan production by Aureobasidium pullulans growing on hydrolysed potato starch waste. Carbohydrate Polymers, 38, 203–209. Brandl, H., Gross, R. A., Lenz, R. W., & Fuller, R. C. (1988). Pseudomonas oleovorans as a source of poly(b-hydroxyalkanoates) for potential applications as biodegradable polyesters. Applied and Environmental Microbiology, 54, 1977–1982. Burchard, W. (2001). Structure formation by polysaccharides in concentrated solution. Biomacromolecules, 2, 342–353.

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