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 strainobtained 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, Portugalb REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugalc Interfacultary Research Centre of Biomaterials (CEIB), University of Liège, B-4000 Liège, Belgiumd REQUIMTE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal

a r t i c l e i n f o

Article history:Received 3 April 2009Received in revised form 15 May 2009Accepted 18 May 2009Available online 23 May 2009

Keywords:Exopolysaccharide (EPS)ViscosityGlycerolPolyelectrolyteLight scattering

0144-8617/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.carbpol.2009.05.011

* Corresponding author. Address: I3N – Institute foelling and Nanofabrication, Department of PolymeMinho, Campus de Azurem, 4800-058 Guimarães, Portfax: + 351 253 510 339.

E-mail address: [email protected] (L. Hilliou).

a b s t r a c t

We report the solution properties of a new exopolysaccharide (EPS) obtained from a Pseudomonas strainfed with glycerol as the sole source of carbon. This high molecular mass (3 � 106 g mol�1) biopolymer isessentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolytecharacter. The Smidsrod parameter B computed from the ionic strength dependence of the intrinsic vis-cosity indicates that the EPS backbone is rather flexible. In salt free aqueous solutions, the zero shearviscosity scaling with concentration follows a typical polyelectrolyte behavior in bad solvent, whereasat high ionic strength the rheological response is reminiscent from neutral polymers. Light scattering dataindicate that the EPS adopts a globular conformation as a result of hydrophobic interactions. EPS solu-tions are stable within 4 days as particle sizing does not indicate EPS aggregation. Both globular confor-mation and stability against precipitation from solution are attributed to the low charge density of thepolyelectrolyte.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Microbial polysaccharides, such as xanthan, gellan, pullulan andbacterial alginate, may represent alternatives to polysaccharidesobtained 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 pro-cess, originating a product with tuned chemistry and propertiesand with a constant availability over time. This constitutes anadvantage over the natural biopolymers isolated from plants andalgae, whose availability and physical–chemical properties aredependent on external factors, such as climate conditions andthe season of the year (Sutherland, 2001).

The most used carbon sources for EPS production have beensugars, namely glucose and sucrose, applied for instance in the pro-duction 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 directimpact on production costs, which limits the market potential of

ll rights reserved.

r Nanostructures, Nanomod-r Engineering, University ofugal. Tel.: +351 253 510 320;

these biopolymers. In order to decrease the production costs, it isimportant to look for less expensive carbon sources, like industrialwastes or industrial by-products (Kumar, Mody, & Jha, 2007). Sugarmolasses and potato starch wastes are examples of low cost carbonsources already used for the production of microbial polysaccha-rides such as exopolysaccharide based on cellulose (Paterson-Bee-dle, Kennedy, Melo, Lloyd, & Medeiros, 2000) and pullulan (Barnett,Smith, Scanlon, & Israilides, 1999).

More recently, glycerol, a by-product of many industrial pro-cesses, mainly from biodiesel production, has been generated inlarge quantities far beyond current consumption in traditionalapplications. Interesting applications for glycerol are still lacking.We reported recently the production of a new microbial polysac-charide by a Pseudomonas strain using glycerol as the sole carbonsource (Freitas et al., 2009; Reis et al., 2008). The biopolymer is ahigh molecular weight extracellular heteropolysaccharide com-posed of neutral sugars (galactose, mannose, glucose and rham-nose) and acyl groups (pyruvil, succynil and acetyl). Thisexopolysaccharide (EPS) is amorphous, as inferred by thermal anal-ysis and solid-state NMR. It possesses flocculating and emulsifyingproperties, along with film-forming capacity, making it a goodalternative to other natural and microbial polysaccharides.

A systematic study of the EPS aqueous solutions properties is ofmajor importance in order to screen potential application of thisproduct to industrial activities such as water treatment, food,

L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532 527

pharmaceutical, cosmetic, mining, paper and oil recovery. Preli-minary rheological studies showed that the crude EPS has viscosityenhancing properties similar to Guar gum. Here, we explore ingreater detail the solution properties of a purified EPS sample, inorder to rationalize the good viscoelastic properties. EPS in salt freesolutions as well as in NaCl solutions with various ionic strengthsare studied at concentrations ranging from the dilute regime to theconcentrated regime, using light scattering techniques andrheometry.

2. Materials and methods

2.1. Polysaccharide production and recovery

The bioprocess used to obtain the exopolysaccharide has beenreported in detail elsewhere (Freitas et al., 2009). The culture wasgrown on Medium E* (Brandl, Gross, Lenz, & Fuller, 1988), supple-mented with 25 g/l glycerol (Fluka, 86%) as carbon source and3.3 g/l (NH4)2HPO4 as nitrogen source. EPS production was carriedout using Pseudomonas oleovorans NRRL B-14682 and was per-formed in a 10 l bioreactor (BioStat B-plus, Sartorius) operated infed-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 airper volume of reactor per minute). Glycerol and ammonium concen-tration were determined as described by Freitas et al. (2009).

Culture broth samples were diluted with deionised water forviscosity reduction and centrifuged at 48,384g for 1 h. The cell-freesupernatant was subjected to protein thermal denaturation at80 �C during 4 h, followed by their separation by centrifugation(48,384g, 1 h). The polymer was then precipitated from the cell-free supernatant by the addition of cold ethanol 96 vol% (3:1)and separated by centrifugation (27,216g, 15 min). The pellet waswashed with ethanol 96 vol%, redissolved in deionised water, rep-recipitated two times and freeze dried.

2.2. Chemical characterization

The polymer samples were analyzed in terms of sugar composi-tion, acyl groups, inorganic and protein content. For the analysis ofthe sugar composition, 2–3 mg of the extracted EPS were dissolvedin 5 ml deionized water and hydrolyzed with trifluoroacetic acid(TFA) (0.1 ml TFA 99%), at 120 �C, for 2 h. The hydrolysate was usedfor the identification and quantification of the constituent mono-saccharides by liquid chromatography (HPLC) using a CarboPacPA10 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 min�1. The hydro-lysate was also used for the identification and quantification of acylgroups present in the EPS. The analysis was performed by HPLCwith an Aminex HPX-87H column (BioRad), coupled to an ultravi-olet (UV) detector, using sulphuric acid (H2SO4 0.01 N) as eluent, ata flow rate of 0.6 ml min�1 and a temperature of 50 �C. The detec-tion was performed at 210 nm.

For the determination of the EPS protein content, 5.5 ml sam-ples 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 wasmixed with 170 ll of CuSO4 � 5H2O (25% v/v) and centrifuged at3500g for 5 min. The optical density was measured at 560 nm(Spectrophotometer Helios Alpha, Thermo Spectronic, UK). Albu-min (Merck) solutions (0.5–3.0 g/l) were used as protein standards.

The total inorganic content of the extracted EPS was determinedby subjecting the EPS to pyrolysis at a temperature of 550 �C for48 h. The EPS was further analyzed by Inductively Coupled Plasma– Atomic Emission Spectroscopy, to quantify its content in sodium,calcium, potassium, magnesium and iron.

2.3. Biopolymer solutions

The EPS, isolated as described in Section 2.1 after 7 days opera-tion of the bioreactor (see below), was added to hot NaCl solutionsor deionised water and stirred at 80 �C for 1 h. Solutions were thenfurther stirred overnight at room temperature, to ensure completedissolution of the EPS, and then characterized without delay. EPSconcentration ranged from 0.0005 to 0.04 wt% for light scatteringexperiments, and from 0.01 to 1.6 wt% for rheological analysis.Ionic strength of solutions ranged from roughly 10�4 for deionisedwater (Dou & Colby, 2006) to 0.5 M.

2.4. Rheological measurements

EPS solutions prepared as described above were directly loadedin 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 withparaffin oil in order to prevent water loss. Such pre-heating stepwas performed in order to erase any thermal and mechanical his-tory induced by the preparation of EPS solutions, and which mightaffect the rheological response of solutions at 25 �C. Solutions werethen cooled (�5 �C/min) down to 25 �C and time was left for sam-ples to equilibrate as demonstrated by the record of a time inde-pendent dynamic loss modulus G0 0 measured at 1 Hz with a 0.1oscillatory shear strain amplitude. The mechanical spectrum ofthe solution was then recorded at 25 �C by performing a frequencysweep obtained with oscillatory strain amplitudes ranging from0.01 to 0.15. The oscillatory torque response recorded on-lineshowed a sinusoidal wave form for all reported frequencies, thusensuring a linear relationship between the applied sinusoidalstrain and the measured stress. The solutions flow curves werethen obtained from steady stress sweep tests (shear rate measuredover the last 10 s of a step shear stress with 60 s duration, and stea-dy state defined within a 2% tolerance for shear rate variation be-tween two consecutive step shear stresses) performed between 0.1and 100 Pa. Inspection of samples, right after performing the stea-dy stress sweep tests, indicated that no flow instability (inertial,elastic or edge fracture leading to emulsion of the paraffin oil) tookplace within the range of applied stresses. As a result, a smoothshear thinning behavior was observed for all flow curves (see Figs.3 and 5), which confirms that no secondary flow developed duringthe flow tests. For dilute EPS solutions showing viscosities belowthe sensitivity of the stress rheometer (such sensitivity limit wasreached for concentration approaching 0.1 g/dl), a Cannon–Fenskecapillary viscometer (COMECTA S.A., Barcelona, Spain) immersedin a temperature bath at 25 �C was used.

2.5. Light scattering

EPS solutions were passed through 0.45 lm polysulfone filtersprior to analysis. Multi Angle Laser Light Scattering (MALLS) datawere obtained by injecting the filtered EPS solutions at a flow rateof 19 ml/h with a syringe pump (Vial Medical, Program II) in the K5flow cell of a MALS detector (Dawn, Wyatt Technology Corp., SantaBarbara, 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). TheMALLS and RI data were recorded with ASTRA software (Version4.73.04, Wyatt Technology Corp., Santa Barbara, CA). DynamicLight 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 variationsof light scattering intensity were analysed at 25 �C at an angle of90� using the BI9000 correlator (Brookhaven Instruments Corp.,Holtsville, NY). The mean light scattering intensities were analysed

528 L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532

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

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

EPS solutions with a concentration of 0.04 wt%, prepared at var-ious ionic strengths, were analysed by Size Exclusion Chromatogra-phy 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 onthe SEC-MALLS system. The latter combines a HPLC pump (HewlettPackard quaternary 1050), an autoinjector (Hitachi-Merck,Lachrom L7200, model 1405–040), and a set of two analyticalSEC linear columns (PL aquagel-OH mixed 8 lm, 30 � 7.5 mm)protected by a guard column. UV (Beckman UV model 266 fixedat 254 nm) signals were recorded together with MALS and RI sig-nals (the same MALLS and RI detector as described above wereused) in order to follow the purity and molecular mass distributionof the polysaccharide. The SEC columns were equilibrated for 24 hbefore running the analysis at a flow rate of 0.7 ml/min at roomtemperature.

0.00 0.02 0.04 0.06 0.08 0.100

4

8

12

16

20

24

28

η spe /

c (

dl/g

)

c (wt %)

Fig. 1. Huggins extrapolation to intrinsic viscosity for the EPS in aqueous solutionswith 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.

3. Results and discussion

3.1. EPS production and chemical characterization

The cultivation run lasted for 7 days, but bacterial cell growthwas ended after 3 days of operation by imposing nitrogen limit-ing conditions (concentration below 0.1 g NH4

+/l). Nevertheless,a residual ammonium concentration was kept in the bioreactorby the addition of mineral medium. The bioreactor was fed withmineral medium, supplemented with a glycerol concentration of200 g/l. EPS synthesis was initiated during the exponential phaseof bacterial growth, but the maximum production rate wasobserved after the culture has entered the stationary phase. Theviscosity of the culture broth showed a dramatic increase withcultivation time (Freitas et al., 2009). As a result, appropriate mix-ing, aeration or control of bioreactor parameters could no longerbe performed and the cultivation was eventually terminated atday 7. At that stage, the final EPS concentration achieved was13.3 g/l, corresponding to a maximum productivity of 2.8 g/l dayand an EPS yield of 0.19 g/g on a glycerol basis. The productivityachieved from glycerol is in the range of values referred for xan-than gum (3.1�12.2 g l�1 d�1) (García-Ochoa et al., 2000) andbacterial alginate (0.43–1.53 g l�1 d�1) (Peña et al., 2000), pro-duced under optimized conditions using glucose and sucrose ascarbon sources.

Sugar analysis indicated that the overall sugar content in thepurified EPS was 79.2 wt%, galactose being the most abundantmonosaccharide, accounting for 70 wt% of the total carbohydratecontent, followed by mannose (23 wt%), glucose (4 wt%) and rham-nose (3 wt%). The total inorganic content was 11 wt%, where themain 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 repre-sented 4.8 wt% of the overall mass and the protein content was5.0 wt%. The EPS recovered after 7 days of cultivation is thereforea galactose-rich polysaccharide with charges (due to the pyruviland succinyl groups) on the polymer backbone, and cations, actingeither as counter-ions or taking part of some precipitated salts dur-ing the recovery and purification processes.

3.2. Effect of ionic strength on the chain size and conformation indilute solutions

Data obtained for EPS solutions with the Cannon–Fenske capil-lary viscometer are presented in Fig. 1. Intrinsic viscosities [g] werecomputed from data fitting to the Huggins equation

g0 � gS

cgS¼

gspe

c¼ ½g� þ kH½g�2c ð1Þ

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

½g� ¼ ½g�1 þ Bð½g�0:1Þ1:3M

�12 ð2Þ

where M is the ionic strength, [g]1 is the intrinsic viscosity at infi-nite ionic strength and [g]0.1 the intrinsic viscosity measured at0.1 M NaCl. Parameter B is a constant which is related to the stiff-ness of the polymer. In this framework, the slope S obtained fromthe Smirod representation displayed in Fig. 2, is related to parame-ter B as

B ¼ S

ð½g�0:1Þ1:3 ð3Þ

From the data in Fig. 2 we find B = 0.1 ± 0.003, which indicates thatthe present EPS chain is not rigid under such salt conditions (0.1 MNaCl). Rather, it shows a flexible conformation approaching the onecommonly found for various extracellular polysaccharides and nat-ural carbohydrate polymers (Ding, LaBelle, Yang, & Montgomery,2003; Ren, Ellis, Sutherland, & Ross-Murphy, 2003).

Results from SEC-MALLS analysis give additional inputs on theEPS conformation in 0.1 M NaCl. 60% of the EPS was recovered afterSEC-MALLS analysis, whereas a mobile phase with lower ionicstrength essentially leads to dramatic EPS adsorption on the SECcolumn. As such, a fair quantitative estimation of both molecular

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

NaCl (M) [g] (dl g�1) kH Mw (106 g mol�1) Rg (nm) A2 (10�3 ml g�2) Rh (nm)

0.01 14 ± 0.4 1.05 ± 0.100.02 10.6 ± 0.7 1.23 ± 0.15 3.0 ± 0.5 99.3 ± 7.0 �2.5 ± 1.2 127.0 ± 17.80.05 7 ± 1 2.3 ± 0.7 2.7 ± 0.5 98.4 ± 7.1 �2.9 ± 2.0 131.2 ± 28.20.1 6.2 ± 0.3 2.58 ± 0.25 3.4 ± 0.5 103.8 ± 6.3 �1.5 ± 1.0 128.1 ± 23.90.25 3.2 ± 0.5 96.1 ± 6.6 �2.7 ± 0.7 128.2 ± 16.60.5 4.9 ± 0.4 4.5 ± 0.5 – – –

L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532 529

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

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

case, additional characterization is needed to check whetherbranching occurs in the EPS backbone.

Alternatively, large values of the Huggins coefficient can becaused by EPS aggregation (interchain interactions). Indeed, thechemical analysis of the EPS indicates that this hydrophobic poly-electrolyte is composed by less than 5 wt% of charged groups. TheEPS is thus weakly charged and we may question the occurrence ofcounterion condensation leading to precipitation of globular con-formers (Dobrynin, Colby, & Rubinstein, 1995). Therefore, DLSwas performed at a concentration of 0.08 wt% in solution with var-ious ionic strengths, and over a 4 days period. The hydrodynamicradii Rh of EPS extracted from the diffusion coefficients measuredduring the first day are reported in Table 1. Data indicate that with-in the experimental error, Rh remains constant over a period of 4days (results not shown). In addition, no precipitation wasobserved over the same period. Therefore, EPS aggregation can beruled out. Rh is quantitatively in harmony with the radius of gyra-tion Rg determined by the extrapolation (with a second order poly-nomial) at zero scattering angle in the Zimm plots. Rg values fordifferent ionic strengths are reported in Table 1, along with the cor-responding molecular mass Mw obtained by extrapolation to zeroconcentration in the Zimm analysis. The ratio Rg/Rh is roughly0.8, and indicates a compact spherical conformation (Aseyev,Klenin, Tenhu, Grillo, & Geissler, 2001; Yang & Zhang, 2009) forall ionic strengths. This result is in agreement with the chain con-formation determined from the SEC-MALLS analysis. We note thatall conformational parameters obtained from light scattering areroughly insensitive to the ionic strength (see Rg and Rh in Table1). This is in contrast to data obtained with capillary viscosimetry.We note here that light scattering data are treated with theassumption of spherical scatterers (Rh is obtained through the dif-fusion constant and the Einstein–Stoke relation), or of point-wisescatterer (Rg is obtained with a Zimm analysis where the particlescattering factor is assumed to be quadratic with the scatteringangle). Therefore, additional scattering experiments will be neededto check whether the EPS conformation is more extended (worm-like chain) at lower ionic strengths where larger values for [g] arefound.

3.2. Rheology of salt free solutions

Fig. 3 shows the flow curves of EPS in deionised water for con-centrations ranging from 0.2 to 1 wt%. Curves are characterized bya Newtonian plateau at lower shear rate followed by a shear thin-ning behavior. Data in Fig. 3 were fitted with a Cross equation

g ¼ g0

1þ ðs _cÞmð4Þ

in order to obtain a quantitative estimate of the zero shear viscosityg0 and relaxation time s. The concentration dependence of the spe-cific viscosity computed from g0 and the solvent viscosity isdisplayed in Fig. 4. Data reported for concentrations below0.1 wt% were obtained with the Cannon–Fenske capillary. Videoimaging of the solutions meniscus in the capillary showed thatthe wall shear rates ranged between 1 and 100 s�1. Inspection of

100 101 102 103

10-2

10-1

100

η (P

a.s)

γ (s-1)

Fig. 3. Shear rate dependence of the steady state viscosity of EPS solutions indeionised 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 thedata.

100 101 102 103

10-2

10-1

100

η (P

a.s)

γ (s-1)

Fig. 5. Shear rate dependence of the steady shear viscosity of EPS solutions in 0.1 MNaCl. 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)) tothe data.

530 L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532

Fig. 3 indicates that these shear rates belong to the Newtonianregime of dilute EPS solutions. The extension of Eq. (1) to higherorders in the concentration is often used to extract useful empiricalstructure-properties relationships from the viscous properties ofvarious polymer solutions (Clasen & Kulicke, 2001). However, aswe are dealing with a charged EPS, we naturally focus our attentionto the theory of polyelectrolyte solutions (Dobrynin & Rubinstein,2005) to analyze the data. The theory predicts power law relation-ships between the concentration and the solution dynamic proper-ties such as the zero shear viscosity and the longest relaxation time.As such, experimental data are to be compared to the predictedexponents of power laws (indicated in Fig. 4 by lines and numbers).Fig. 4 reveals 3 concentration regimes defined by different scaling ofthe specific viscosity with the EPS concentration. At low concentra-tion, a power law with exponent ½ is observed which conforms tothe scaling expected for the semi dilute and non entangled regimeof either hydrophobic polyelectrolytes or polyelectrolytes in saltfree solutions (Dobrynin et al., 1995). A crude estimate for the over-

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

lap concentration c* which defines the onset of the semi dilute nonentangled regime is given by the concentration at which gspe � 1 (Dicola et al., 2004; Larson, 1999). Data in Fig. 4 suggest thatc* � 0.01 wt%. Taking into account the fact that the molecular massMw of the EPS does not depend on the ionic strength, we can com-pute the size Rg of the equivalent EPS coil in salt free conditionsusing the definition of the overlap concentration c* and

Rg ¼ 3Mw4pqNAc�

� �13

where NA is the Avogadro number and q the solvent

density (de Gennes, 1979). We obtain Rg = 228 nm usingMw = 3.0 � 106 and c* = 0.01 wt%. Rg is roughly twice the value ob-tained with static light scattering for the lowest ionic strength stud-ied (see Table 1), and as such is indicative of a rather extendedconformation in salt free solutions. A second power law behaviorwith exponent 3/2 is observed for concentration in excess ofce = 0.05 wt%. This behavior corresponds to the scaling predictedfor semi dilute and entangled polyelectrolytes in salt free solutions(Dobrynin et al., 1995). We note that the salt free EPS solution ispoorly entangled as the viscosity at ce is only 6 times larger thanthe viscosity of the solvent. Such a behavior was also reported formodel hydrophobic polyelectrolytes in salt free solutions exhibitinga 10 times smaller molecular mass (Di cola et al., 2004) than thepresent EPS. The ratio ce/c* gives the width of the semi dilute nonentangled regime, and is expected to be as large as 1000 (Dobryninet al., 1995). The ratio for the present EPS is rather small when com-pared to those measured with model hydrophobic polyelectrolytes(Di cola et al., 2004; Dou & Colby, 2006), and as such validates thepicture of a weakly charged EPS (Dou & Colby, 2006). Departurefrom the 3/2 scaling at highest concentrations marks the onset ofa third concentration regime: for concentrations larger than c**, asteeper dependence of the specific viscosity with the concentrationis observed and as such defines the onset of the concentrated re-gime. This high concentration behavior contrasts with the concen-tration scaling, gspe � c0, predicted for the so-called beadcontrolled regime of semi dilute entangled polyelectrolytes in badsolvent (Dobrynin & Rubinstein, 1999). However, rheologicalevidence for this concentration regime is still lacking (Di colaet al., 2004) as it requires highly charged polyelectrolytes exhibitingmany entangled beads on a bead-necklace conformation (Dobrynin& Rubinstein, 1999, 2005). The inset in Fig. 4 presents the mechan-ical spectrum measured at the highest concentration studied. Thehigh frequency part of the spectrum shows the onset of a crossover

L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532 531

between G0 and G0 0. No elastic plateau is observed for the storageshear modulus G0, and as such no clear evidence for entanglementsbetween EPS chains is obtained. Therefore, we still miss a clearexplanation for the steep increase of free salt EPS solution viscosityat higher concentrations, as the interplay between screened electro-static interactions, polyelectrolyte conformation and possible repta-tion 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. Theshear rate dependence of the steady state viscosity data exhibits aNewtonian behaviour at the lowest concentrations, whereas ashear thinning is observed for more concentrated solutions. Datafitting with Eq. (4) yields the zero shear viscosity and allows thecorresponding plot of gspe as a function of EPS concentration inFig. 6. The concentration dependence of gspe shows essentiallytwo regimes characterized by different power laws. At low concen-tration, we identify the overlap concentration c* � 0.05 wt% (con-centration corresponding to gspe = 1) which is larger than theoverlap concentration measured in salt free solutions. This overlapconcentration can be compared to the value c* = 0.1 wt% computedfrom the value of [g] measured at 0.1 M NaCl (see Table 1), andusing the relationship c* � 0.77/[g], devised for closely packedspherical and impenetrable coils (Graessley, 1980). The departurebetween these two estimates is fairly acceptable if we take intoaccount the general approximation (Larson, 2005) underlying therelationship between c* and [g]. Under the assumption of coil con-formation, the overlap concentration at which gspe = 1 corresponds

to a radius of gyration Rg ¼ 3Mw4pqNAc�

� �1=3¼ 140 nm, which is roughly

in agreement with the EPS conformation obtained from MALLS(Rg = 103.8 nm). The closer agreement between light scatteringand rheology as compared with results obtained with salt freesolutions, stems from the EPS globular conformation at higherionic strength, which as such validates the use of the above rela-tionship between Rg and c*. A concentration scaling showing apower law behaviour with exponent 5/4 is first observed, whereasdeparture from this scaling occurs for concentration beyondce = 0.4 wt% where a second concentration regime characterized

Fig. 6. Concentration dependence of the specific viscosity of EPS solutions in 0.1 MNaCl. 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 semidilute entangled regimes, respectively. Lines in the insets indicate slopes 1 and 2 forthe terminal behavior of G0 0 and G0 , respectively.

by a power law scaling with exponent 15/4 is evidenced. Bothexponents correspond to the concentration scaling predicted forthe semi dilute and semi dilute entangled regimes, respectively,of neutral polymers in good solvent (de Gennes, 1979). Entangle-ments also show up as an elastic plateau in the high frequencyregime of the mechanical spectrum recorded with the highestEPS concentration (lower inset in Fig. 6). As such, the viscousenhancing character of the present EPS in concentrated (1 wt%)solutions with high ionic strengths originates from entanglementsbetween EPS chains.

3.4. Flow master curves and aggregation

Data displayed in Figs. 3 and 5 could be superimposed by scal-ing each flow curve horizontally with the relaxation time s andvertically with the respective zero shear viscosity g0 computedwith Eq. (4) for each concentration. The resulting master curvesare presented in Fig. 7. Similar master curves were obtained withvarious polysaccharides (Morris, Cutler, Ross-Murphy, Rees, &Price, 1981) including strongly interacting cellulose (Burchard,2001), with various EPSs (Goh, Hemar, & Singh, 2005; Gorret, Re-nard, Famelart, Maubois, & Doublier, 2003), and also with polyiso-butylene solutions in a mixture of polybutene oil and dekalin for allconcentration regimes (Nogueiro & Maia, 2003). The concentrationdependence of Cross parameters m and s is depicted in the inset ofFig. 7 for salt free EPS solutions and EPS solutions prepared in 0.1 MNaCl. Comparison of both sets of data indicates that a similar con-centration scaling is observed for these non linear parameters. Wetherefore, suspect that electrostatic interactions do not affect qual-itatively the flow behavior of semi dilute entangled hydrophobicpolyelectrolytes in the high shear rate limit. Exponent m is virtu-ally not depending on the concentration, reaching a value of 0.7and 0.8 for the salt free solutions and the solutions in 0.1 M NaCl,respectively. These values are reminiscent from the almost univer-sal exponent m = 0.76 found for many polysaccharides (Morris,1990), including those presenting strong interactions such ashydrogen bonding between polymer chains, and also compare wellwith the asymptotic exponent m = 0.82 predicted for well entan-gled flexible polymers in good solvent (Graessley, 1974). Thedependence s � c3 (suggested by the solid lines indicating a slopeof 3 in the inset of Fig. 7) is similar to that referred for weakly

Fig. 7. Master curves for EPS flow curves in deionised water (lines) and in 0.1 MNaCl (open squares). Inset: concentration dependence of parameters m (solidsymbols) and s (open symbols) of Cross equation in deionised water (square) and in0.1 M NaCl (triangles). Note that the concentrations correspond to the semi diluteentangled regime for both solvents.

532 L. Hilliou et al. / Carbohydrate Polymers 78 (2009) 526–532

charged model polyelectrolytes in the semi dilute entangled con-centration regime (Dou & Colby, 2006). A power law behaviours � c2.75 is predicted for entangled polymer melts (de Gennes,1979), whereas exponents ranging from 2.75 to 2.85 were mea-sured for semi dilute polymer solutions in a theta solvent (Adam& Delsanti, 1985). Exponents well in excess of 3 are usually re-ported for polysaccharides exhibiting strong interchain association(Burchard, 2001). Therefore, we confirm that the rheologicalbehaviour displayed in Fig. 7 corresponds to semi dilute entangledpolymer solutions in bad solvent, presenting no strong interactionbetween chains. As such, aggregates are likely not to be formedwithin the range of concentration studied.

4. Conclusions

A microbial polysaccharide has been produced by a Pseudomo-nas strain using glycerol as the sole carbon source. The purifiedEPS is a high molecular mass polysaccharide, and is essentiallymade of galactose monomers with pyruvate and succinate groupsimparting a polyelectrolyte character. The picture emerging fromintrinsic viscosity and light scattering data is that the EPS is a flex-ible (within the limitation of the physical meaning of parameter B(Ding et al., 2003; Ren et al., 2003)) hydrophobic polyelectrolytewhich adopts a compact globular conformation in the presenceof salt and which does not show any tendency to aggregation inthe dilute regime, at least within a period of 4 days. Complemen-tary scattering techniques are however, needed to elucidate theEPS conformation in solution, as large Huggins coefficients aremeasured with capillary viscosimetry and suggest a significantbranching on the EPS backbone. The EPS shows good viscousenhancing properties (a 1 wt% solution is roughly a 1000 timesmore viscous than water) and a strong shear thinning. This rheo-logical behavior originates from entanglements between EPSchains in solutions with high ionic strength. In salt free solutions,the concentration scaling of the EPS zero shear viscosity shows atypical weakly charged hydrophobic polyelectrolyte behavior.However, the high viscosity measured in the concentrated regimestill needs to be rationalized, even if the non linear rheology sug-gests the disentanglement of EPS chains.

Acknowledgements

The authors acknowledge the Portuguese company 73100 forthe financial support, under the project ‘‘Production of biopoly-mers from glycerol”, 2005/2008. Vítor D. Alves acknowledges Fun-dação para a Ciência e a Tecnologia, Pos-doc fellowship SFRH/BPD/26178/2005.

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