Carbohydrate Polymers 78 (2009) 549–556

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Carbohydrate Polymers

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Emulsifying behaviour and rheological properties of the extracellularpolysaccharide produced by Pseudomonas oleovorans grown on glycerol byproduct

Filomena Freitas a, Vitor D. Alves b, Mónica Carvalheira a, Nuno Costa a, Rui Oliveira a, Maria A.M. Reis a,*

a REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugalb REQUIMTE-Department of Chemical Engineering, Universidade do 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 31 March 2009Received in revised form 18 May 2009Accepted 20 May 2009Available online 31 May 2009

Keywords:Pseudomonas oleovoransGlycerol byproductExtracellular polysaccharide (EPS)RheologyBioemulsifierEmulsification index

0144-8617/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.carbpol.2009.05.016

* Corresponding author. Tel.: +351 21 2948357; faxE-mail address: amr@dq.fct.unl.pt (M.A.M. Reis).

a b s t r a c t

The functional properties of a novel extracellular polysaccharide (EPS) produced by Pseudomonas oleovo-rans grown on glycerol byproduct, generated by the biodiesel industry, were investigated. The EPS is ahigh molecular weight (4.6 � 106) heteropolysaccharide, composed by neutral sugars (galactose, 68%;mannose, 17%; glucose, 13%; rhamnose, 2%; fucose, 4%) and acyl groups (3.04%). This biopolymer haspseudoplastic fluid behaviour in aqueous media. The apparent viscosity was stable for the pH range2.9–7.1 and NaCl concentrations up to 1.0 M. Though the apparent viscosity decreased at high tempera-tures, at alkaline conditions and at NaCl concentrations of 2.0 M, pseudoplastic fluid behaviour wasretained. The EPS was capable of stabilizing water emulsions with several hydrophobic compounds,including hydrocarbons, vegetable and mineral oils. It retained its emulsifying activity during exposureto wide temperature (30–50 �C) and pH (2–12) variations, as well as to the presence of NaCl at concen-trations as high as 2.0 M.

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1. Introduction

A huge variety of polymers, such as polysaccharides, are natu-rally produced by microorganisms. Their application in numerousindustrial areas, is mainly due to their rheological properties thatallow the formation of viscous solutions at low concentrations(0.05–1.0%) and stability over wide temperature, pH and ionicstrength ranges (Kumar & Mody, 2009, chap. 10). Many microbialpolymers can also find useful applications as bioemulsifiers dueto their ability to stabilize emulsions between water and hydro-phobic compounds. Examples of bioemulsifiers include: RAG-1emulsan and biodispersan, produced by Acinetobacter calcoaceticusstrains (Rosenberg, Rubinovitz, Gottlieb, Rosenhak, & Ron, 1988;Sar & Rosenberg, 1983), and alasan, produced by Acinetobacterradioresistens (Navon-Venezia et al., 1995). Other biopolymers withpotential applications as bioemulsifiers include extracellular poly-saccharides produced by Pseudomonas tralucida (Appaiah &Karanth, 1991), Pseudoalteromonas sp. (Gutierrez, Schimmield,Haidon, Black, & Green, 2008), Zoogloea sp. (Lim, Kim, Kim, Yoo,& Kong, 2007) and Enterobacter cloaceae (Iyer, Mody, & Jha, 2006).

The use of bioemulsifiers is advantageous comparing to chemi-cal counterparts, because they are biodegradable, less toxic andhave activity under a wider variety of conditions (Banat, Makkar,& Cameotra, 2000). Due to their wide diversity in compositionand structure, bioemulsifiers are characterized by improved func-

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tionality and stability, which broadens the spectrum of potentialapplications, which include: the oil and petroleum industries,water and soil bioremediation, metal treatment and processing,detergents and laundry supplies, agriculture, textile manufactur-ing, pulp and paper processing, paints, cosmetics, pharmaceuticals,personal care products and food processing (Rosenberg & Ron,1999). Presently, their use is limited by production costs and thelack of experience in their applications. Operating costs may be re-duced by using low-cost substrates, which account for 10–30% ofthe overall production cost. Biopolymer recovery from the cultiva-tion broth also has a considerable impact on the final product cost(Makkar & Cameotra, 2002). Hence, bioemulsifiers that can berecovered by simple and inexpensive techniques will probablyhave a more competitive price.

Pseudomonas oleovorans has been reported to produce extracel-lular biosurfactants capable of forming stable oil in water emul-sions when grown in two-liquid phase cultures, such as n-decane(Schmid, Kollmer, & Witholt, 1998). In previous work, we havereported on the characteristics of a new extracellular polysaccha-ride (EPS) produced by P. oleovorans from pure glycerol (Freitaset al., 2009). The EPS is a high molecular weight heteropolysaccha-ride (1.0–5.0 � 106), composed by neutral sugars and acyl groupssubstituents. Preliminary tests have shown that this new biopoly-mer forms viscous aqueous solutions with pseudoplastic fluidbehaviour and has an emulsifying activity against n-hexadecanecomparable to other bioemulsifiers. Glycerol byproduct from thebiodiesel industry has proven to be a suitable carbon substratefor EPS production by P. oleovorans (unpublished data), in spite

550 F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556

of containing some impurities. The use of glycerol byproduct as afeedstock is however a very important cost reduction factor.Although the EPS chemical composition and molecular weightwere not significantly influenced by the use of glycerol byproduct,the impact upon polymer properties must be evaluated.

In this work, we report on the emulsion forming and stabilizingcapacity of the EPS produced by P. oleovorans grown on glycerolbyproduct from the biodiesel industry. The emulsifying behaviourand the rheological properties were investigated for several EPSconcentrations, as well as under different conditions of tempera-ture, pH and ionic strength.

2. Materials and methods

2.1. Biopolymer production and extraction

Pseudomonas oleovorans NRRL B-14682 was grown on a slightlymodified Medium E* (Freitas et al., 2009), supplemented with glyc-erol byproduct to give a concentration between 25 and 50 g L�1.Glycerol byproduct, supplied by Fábrica Torrejana de Biocombustí-veis SA, Portugal, had a glycerol content of 89% and residual con-tents of methanol (0.04%), organic material (0.4%), ashes (6.8%)and water (3.5%). EPS production was performed in a 10 L bioreac-tor (BioStat B-plus, Sartorius), as described by Freitas et al. (2009).The bioreactor was operated in a batch mode during the first 24 hof cultivation and, then, in a fed-batch mode, by supplying the bio-reactor with cultivation Medium E*, with a glycerol concentrationof 200 g L�1, at a constant rate of 20 mL h�1.

Throughout the cultivation, culture broth samples were recov-ered from the bioreactor and centrifuged (18,000g, 15 min) for cellseparation. The cell-free supernatant was stored at �20 �C for thedetermination of glycerol and ammonium concentrations. Celldry weight (CDW), glycerol and ammonium concentrations, as wellas the measurement of the culture broth viscosity, were performedas described by Freitas et al. (2009).

For extracting the EPS, the culture broth was recovered from thebioreactor, diluted with deionised water for viscosity reductionand centrifuged (20,000g, 1 h), for cell separation. The volume ofwater added to dilute the culture broth varied from 1:1, for lowviscosity samples, to 1:7, for highly viscous samples. The EPS inthe cell-free supernatant was precipitated by the addition of ace-tone (3:1). The precipitated EPS was washed with acetone, dis-solved in deionised water and freeze dried.

2.2. Physicochemical characterization

For the determination of the sugar composition, the EPS (2–3 mg) was dissolved in 5 mL deionised water and hydrolyzed withtrifluoroacetic acid (TFA) (0.1 mL TFA 99%), at 120 �C, for 2 h. Thehydrolysate was used for the quantification of the constituentmonosaccharides by liquid chromatography (HPLC) using a Carb-oPac PA10 column (Dionex), equipped with an amperometricdetector. The analysis was performed at 30 �C, with sodiumhydroxide (NaOH 4 mM) as eluent, at a flow rate of 0.9 mL min�1.The acid hydrolysate of the EPS was also used for the identificationand quantification of acyl groups, as described by Freitas et al.(2009).

The total inorganic content was determined as described by Fre-itas et al. (2009). For determination of the protein content, 5.5 mLsamples of aqueous EPS solutions (4.5 g L�1) were mixed to 1 mL20% NaOH and placed at 100 �C for 5 min. After cooling on ice,170 lL of CuSO4�5H2O (25% v/v) were added. After mixing, thesamples were centrifuged (3500g, 5 min) and the optical densitywas measured at 560 nm (Spectrophotometer Helios Alpha,

Thermo Spectronic, UK). Albumin (Merck) solutions (0.5–3.0 g L�1) were used as protein standards.

Average molecular weights (Mw) and the polydispersity index(Mn/Mw) were obtained by size exclusion chromatography, as de-scribed by Freitas et al. (2009).

2.3. Rheology

The effect of EPS concentration, pH, temperature and ionicstrength on the EPS solutions viscosity was studied using a con-trolled stress rheometer (ARG2, TA Instruments Inc., New Castle,DE, USA), equipped with a cone and plate geometry (diameter4 cm, angle 2 degrees). During the experiments, the shearinggeometry was covered with paraffin oil in order to prevent waterloss. Flow curves were determined using a steady state flow rampin the range of shear rate from 1 s�1 to 700 s�1. The shear rate wasmeasured point by point with consecutive 60 s steps of constantshear rate. The viscosity was recorded for each point to obtainthe flow curves.

2.4. Emulsifying activity

The standard assay for emulsifying activity was based on themethod described by Cooper and Goldenberg (1987), using n-hexa-decane as the test substance. Briefly, 6 mL of n-hexadecane (Sigma)were added to 4 mL of EPS aqueous solution in a test tube (20 mmdiameter � 180 mm) and stirred in the vortex at 2400 rpm for2 min. After 24 h, the emulsification index (E24) was determinedas follows: E24 ¼ he=hT � 100, where he (mm) is the height of theemulsion layer and hT (mm) is the overall height of the mixture.All tests were performed in duplicate.

The emulsion forming and stability capacity against n-hexadec-ane was evaluated for different EPS concentrations, temperatures,pH and ionic strength. The emulsifying assay was also applied toother hydrophobic compounds (hydrocarbons and oils), using thesame ratio of 0.6% EPS solution:hydrophobic compound (2:3 v/v).The tested compounds included oils (corn oil, sunflower oil, oliveoil, paraffin oil and cedar wood oil, purchased at the local super-market; oleic acid, Panreac; and silicone oil, Wacker) and hydrocar-bons (benzene, Merck; chloroform, Sigma; cyclohexane, Panreac;n-decane, Merck; dimethylether, Riedel de Haen; n-hexane, Riedelde Haen; isooctane, Merck; toluene, Sigma; xylene, Merck). Alltests were performed at room temperature.

The capacity of the EPS to stabilize emulsions after being sub-jected to high temperatures and to an autoclaving process was alsoassessed. For this test, EPS aqueous solutions (0.6%) were placed at80, 100 and 120 �C for 1 h, or autoclaved (120 �C, 1 bar, 20 min).After cooling to room temperature, the emulsions were preparedwith n-hexadecane (EPS solution:hexadecane ratio of 2:3) and leftfor 24 h to determine E24.

3. Results and discussion

3.1. Biopolymer production

EPS production and growth of P. oleovorans using glycerolbyproduct as substrate are presented in Fig. 1A. Bacterial cellgrowth was suppressed within one day of cultivation by imposingnitrogen limiting conditions (<0.1 g NHþ4 L�1). A maximum biomassconcentration of 7.32 g L�1 was reached within 2 days of cultiva-tion. During the stationary growth phase, ammonium concentra-tion was kept at a residual value (below the detection limit) andglycerol concentration was maintained relatively constant (5.0–10.0 g L�1), even though the feeding solution (containing

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Fig. 1. (A) Time course of the cultivation of Pseudomonas oleovorans on glycerol byproduct (glycerol, h; ammonium, 4; CDW, ; EPS, �). (B) Shear rate dependent apparentviscosity built up of the culture broth over time, during the cultivation of P. oleovorans with glycerol byproduct.

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Fig. 2. Sugar composition of the EPS produced by Pseudomonas oleovorans(galactose, s; mannose, h; glucose, 4; fucose, ; glucose, }) and variation of itsmolecular weight over cultivation time ( ).

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200 g L�1 glycerol and 0.9 g NHþ4 L�1) was fed to the bioreactor at aconstant flow rate (20 mL h�1).

Increased EPS production was initiated within one day of culti-vation, when the culture entered the stationary growth phase(Fig. 1A), indicating that the synthesis of extracellular polysaccha-ride by P. oleovorans has non-growth associated kinetics. After5 days of cultivation, the EPS attained a concentration of15.0 g L�1. Considering the time window of effective EPS produc-tion (between days 1.0 and 5.3), the productivity and the net yieldon glycerol were 0.13 g L�1 h�1 and 0.15 g g�1, respectively. Inother experiments performed with P. oleovorans grown on glycerol,both the final EPS concentration and its productivity were im-proved. Typically, the cultivation for EPS production takes between4 and 7 days, with a final production of 7.0–18.0 g L�1 of EPS andmaximum productivities of 0.1–0.2 g L�1 h�1 (data not shown).

Concomitant with EPS production, there was a drastic change inthe culture broth viscosity (Fig. 1B). The culture broth developednon-Newtonian characteristics, acting as a pseudoplastic fluid,where the measured viscosity decreased with increasing shearrate. The apparent viscosity of the culture broth measured at lowshear rates showed an increase of almost three orders of magni-tude (from 4.7 � 10�3 to 2.0 Pa s) (Fig. 1B). This viscosity built upis a common feature observed in many microbial cultivations forthe production of extracellular polysaccharides (Kumar & Mody,2009) and it usually determines the termination of the run dueto loss of bulk homogeneity of the culture broth.

3.2. Chemical composition and molecular weight

The EPS produced by P. oleovorans from glycerol byproduct wasrecovered from the bioreactor at the end of the cultivation (5 days),but culture broth samples were recovered from the bioreactor atdifferent cultivation times, being the EPS extracted and character-ized in terms of its composition and molecular weight (Fig. 2). Theglycosyl composition analysis revealed that it was a heteropolysac-charide composed by neutral sugars. Galactose was the most abun-dant monosaccharide constituent throughout the cultivation,representing 68% of the polymer’s carbohydrate content at theend of the run. Mannose and glucose were also present in signifi-cant amounts, accounting for 17% and 13%, respectively, of the car-bohydrate content of the EPS. Rhamnose and fucose were presentin minor amounts (2% and 4%, respectively). The EPS produced byP. oleovorans from pure glycerol had a similar composition, withgalactose being the main sugar constituent (70%) (Freitas et al.,2009). Mannose represented 23% of the carbohydrate contentand rhamnose was a minor component (4%). On the other hand,the EPS obtained from pure glycerol had lower glucose content

and fucose was not detected. These differences may be related tothe use of glycerol byproduct that contains some impurities notpresent in pure glycerol and may have influenced the EPS sugarcomposition.

The polymer also had acyl groups substituents that accountedfor 3.04% of its dry mass. The presence of acyl group substituentsis common in microbial EPS and they greatly influence polymer’sproperties, namely, solubility and rheology (Rinaudo, 2001). Themain acyl groups detected were pyruvil (2.51%) and succinyl(0.54%), as well as other unidentified organic acids, which werepresent only in residual amounts. The presence of pyruvil and suc-cinyl confer the EPS an anionic character (Freitas et al., 2009). TheEPS obtained from pure glycerol had similar pyruvil content(3.35%) and higher succinyl content (1.04%), but it additionallycontained acetyl (0.38%) (Freitas et al., 2009) that was not detectedin the EPS produced from glycerol byproduct.

The extracted polymer also contained other non-sugar constit-uents that were probably remnants of the culture broth, namely,proteins and inorganic residues. Proteins accounted for 10% ofthe polymer’s mass. The polymer characterized in previous workhad a lower protein content (5%) because the cell-free supernatantwas subjected to protein removal prior to EPS precipitation (Freitaset al., 2009), thus yielding a polymer with a higher degree of purity.In the present work, the extraction procedure was made simpleraiming at generating a final product with a more competitive price.For most of the emulsifiers applications (e.g. oil and petroleum

552 F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556

industries, water and soil bioremediation, metal treatment andprocessing, detergents and laundry supplies, textile manufactur-ing, paints), there is no need for a highly purified bioemulsifier.

As shown by its pyrolysis at 550 �C, the EPS had a total inor-ganic residues content of 23.6%, being the main cations detectedby ICP analysis: sodium (10.77%), potassium (4.70%), calcium(0.10%) and magnesium (0.08%). Cobalt, copper, iron, manganeseand zinc were also detected in trace amounts (<0.01%). No sulphurwas detected, as confirmed by the elemental analysis. On theother hand, the EPS contained 4.34% of phosphorus, as detectedby ICP. Part of these metal cations were probably present in theextracted polymer as counter-ions for the anionic acyl groups orrepresent salts that co-precipitated with the EPS during the recov-ery procedure. The EPS produced from pure glycerol had lowerinorganic residues content (11.6%) (Freitas et al., 2009). This resultwas probably due to the fact that glycerol byproduct had an ashcontent of approximately 6.8%, from which 3.3% were attributedto sodium (detected by ICP analysis), originating from the NaOHused during the biodiesel production process. Moreover, as statedabove, the EPS described in the present study was recovered fromthe cell-free supernatant, without any additional purificationtreatment, in contrast to the previously described polymer whichwas further purified with a thermal treatment and successiveprecipitations.

The polymer’s average molecular weight showed a gradual in-crease from 2.4 � 106 (day 1) to 4.6 � 106 (day 5) (Fig. 2). The ex-tracted polymers were rather homogeneous, as shown by thepolydispersity index that ranged between 2.02 and 2.33. Similarvalues were obtained for the EPS produced from pure glycerol,namely, average molecular weight increasing from 1.0 � 106 to5.0 � 106 during the 4 days cultivation run (Freitas et al., 2009).The EPS produced from pure glycerol had lower polydispersity in-dexes (1.43–2.15) indicating that the extracted polymers weremore homogeneous.

3.3. Rheological properties

The analysis of the rheological properties of the EPS in aqueousmedia showed evidence of pseudoplastic fluid behaviour, as theviscosity was influenced by shear rate (Fig. 3A). This behaviour isexpected for solutions of polysaccharides, resulting from theirpolymeric structure and high molecular weight (Rao, Suresh, &Suraishkumar, 2003). In addition, the viscosity is recovered forlow shear rates, after the imposition of high shear rate values(Fig. 3A). The flow behaviour of the culture broth at the end of

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Fig. 3. (A) Dependence of the apparent viscosity on the shear rate of an 1.5% EPS solutionshear rate from 700 s�1 to 1 s�1 ( ); and of the culture broth at the end of the 5 days cultsolutions as a function of polymer concentration at a shear rate of 5 s�1. The experimen

the cultivation, when the EPS concentration was 15.0 g L�1, wasidentical to the flow behaviour of a 1.5% aqueous solution preparedwith the freeze dried polymer (Fig. 3A). This result confirms thatthe viscosity built up observed during the cultivation was con-ferred by the presence of the EPS in the culture broth. Moreover,the fact that the EPS retained the same flow behaviour shows thatthe extraction process did not alter the polymer’s viscosityproperties.

The viscosity of EPS solutions at different concentrations (0.2–1.5%) was investigated (Fig. 3B). As expected, the apparent viscos-ity increased with increasing concentration. As the polymer con-centration becomes higher, the individual molecules start tooverlap, inducing the formation of intermolecular junctions and,hence, limiting polymer chain arrangement and stretching. Conse-quently, there is an increase of the solution’s viscosity (Bae, Oh,Lee, Yoo, & Lee, 2008).

Industrial processes frequently involve exposure to extremes oftemperature, pH, pressure and ionic strength. Hence, it is impor-tant to evaluate how the EPS rheological properties are influencedby those conditions, as well as its reversibility. The EPS in aqueoussolution was exposed to temperature (15–120 �C) and pH (2.9–11.8) ranges, and to autoclaving (simultaneous high pressure andtemperature conditions), as well as to NaCl concentrations up to2.0 M.

The effect of temperature on the flow behaviour of EPS aqueoussolutions was investigated by measuring the apparent viscosity atdifferent temperatures (Fig. 5A). It is evident that the EPS is sensi-tive to the temperature, as shown by the viscosity reduction ob-served as the temperature was increased. Nevertheless, thepseudoplastic fluid behaviour was retained even for the highesttemperature tested (data not shown). Moreover, low viscositymay be an advantageous characteristic for several industrial appli-cations, such as oil tankers cleaning and the recovery of solidwastes (Martinez-Checa, Toledo, Vilches, Quesada, & Calvo,2002). Viscosity reduction with increasing temperature between10 and 90 �C was also observed for other bacterial polysaccharides,such as xanthan gum (García-Ochoa et al., 2000) and bacterial algi-nate (Chen, Chen, Chang, & Su, 1985).

An additional study to investigate the effect of temperature onthe viscosity of the EPS was conducted by heating a 0.8% EPS solu-tion from 17 to 80 �C and cooling it back to 17 �C (Fig. 4A) at a con-stant shear rate of 5 s�1. The results show that the viscosity isreduced with increasing temperatures, but it regains its originalviscosity upon cooling, indicating that the EPS is not degraded byheating up to 80 �C. Instead, as the temperature is raised, the inter-

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F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556 553

action between the molecules is decreased and the polymer struc-ture is loosened, thus decreasing the viscosity of the solution. Thefact that the EPS regains its original viscosity after being exposed tohigh temperatures envisages its use in applications were the prod-ucts are processed at high temperatures (e.g. pasteurization) butthe final product is used at room temperature. The rheologicalproperties of the final product thus remain stable, regardless ofbeing stored in the refrigerator, at room temperature of heated.This behaviour was also observed for xanthan gum, for which theviscosity is fully reversible between 10 and 80 �C (García-Ochoaet al., 2000), and for bacterial alginate for temperatures up to75 �C (Chen et al., 1985).

The effect of extreme temperatures on the EPS flow curves wasevaluated by subjecting 0.8% aqueous solutions to high tempera-tures (100 and 120 �C, for 1 h), and to an autoclaving process(120 �C, 1 bar, for 20 min) (Fig. 4B). The EPS did not regain its ori-ginal apparent viscosity after being exposed to temperatures equalor higher than 100 �C. In fact, about half of the viscosity was lostwhen the polymer was heated to 100 �C, being even further re-duced after the treatment at 120 �C. Nevertheless, though havinga lower viscosity, the solutions continued to exhibit pseudoplasticfluid behaviour. There was no significant difference between heat-ing to 120 �C and combining this temperature with a pressure of1 bar. Bacterial alginate shows a similar behaviour: when heated

above 75 �C the original viscosity of the solution is not restoredupon cooling, while for higher temperatures (121 �C) the viscosityis practically destroyed (Chen et al., 1985). These results suggestthat the high temperatures tested caused either a partial EPS deg-radation or an alteration of its tertiary structure with the polymerchains adopting a different intermolecular arrangement. This issueis to be addressed in future studies.

The study of the pH effect on the apparent viscosity of the EPSwas performed by changing the pH of a 1.2% aqueous solution (pH7.1) by the addition of small drops of HCl 4.0 M or NaOH 5.0 M.The results are presented in Fig. 5B for a shear rate of 5 s�1. Itcan be concluded that the viscosity properties of the EPS werenot influenced by acidic conditions, since it was not significantlyaltered by lowering the pH from 7.1 to 5.3 and 2.9. In contrast,alkaline conditions reduced the viscosity in about 28% and 54%by increasing the pH from 7.1 to 10.1 and 11.8, respectively. Evenso, the solutions still retained a considerable viscosity and thepseudoplasticity was maintained (data not shown). Bacterial algi-nate solutions have a similar behaviour, namely, they are stablefor pH 4–10, but its viscosity decreases above pH 10 (Chenet al., 1985). Although the viscosity of xanthan solutions is notsignificantly affected by changes in pH between 1 and 13 (Gar-cía-Ochoa et al., 2000), it is maximal for neutral pH, decreasingfor lower or higher values (López, Vargas-García, Suarez-Estrella,

554 F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556

& Moreno, 2004). Also, it was observed that xanthan’s composi-tion is altered at pH 9 or higher, and for pH lower than 3 (Lópezet al., 2004).

The intermolecular arrangement of charged polysaccharidesmay be expanded by electrostatic repulsion, or contracted by elec-trostatic attraction between polymer chains (Lai & Yang, 2007). Theelectrostatic force is therefore expected to have an important effecton the conformation of the EPS, and the related physical–chemicalproperties such as viscosity. Therefore, in this study, the flowbehaviour of the EPS in the presence of increasing NaCl concentra-tion was addressed (Fig. 5C). The results show that the apparentviscosity of the EPS solutions was not altered by increasing the io-nic strength up to 1.0 M NaCl. This fact may be related to ions pres-ent already in the polymer sample. However, higher values of ionicstrength (2.0 M NaCl) resulted in a 26% reduction of the solutionviscosity. The raised concentration of counter-ions experiencedwhen the ionic strength got higher, may have made the moleculescontract even further due to charge shielding effect, thus resultingin a lower viscosity.

3.4. Emulsion forming and stabilizing capacity

In previous studies, the EPS produced by P. oleovorans fromglycerol has shown to possess the capacity to stabilize hydrocar-bon/water emulsions, with an emulsifying activity against n-hexa-decane comparable to other bioemulsifiers (Freitas et al., 2009). Inthe present study, the emulsion forming and stabilizing capacityof this new biopolymer was studied in greater detail. Severalhydrophobic substances were tested, including vegetable andmineral oils, and hydrocarbons. Hexadecane was used as the testsubstance to study the emulsion-stabilizing capacity of the EPS asa function of polymer concentration, temperature, pH and ionicstrength.

A criterion for determining the emulsion-stabilizing capacity ofan emulsifier consists on evaluating its ability to maintain at least50% of the original emulsion volume 24 h after its formation(Willumsen & Karlson, 1997). Considering this criterion, the EPSproduced by P. oleovorans has proven to possess emulsion-stabiliz-ing capacity for several hydrocarbons, including n-hexadecane,n-hexane, xylene, chloroform, benzene, toluene, as shown byemulsification indexes higher than 50% (Table 1). The high emulsi-fication indexes observed reflect the stability of the emulsions thusformed. Moreover, those emulsions were stable for several weeks(data not shown). The other hydrocarbons tested, namely, cyclo-hexane, n-decane and diethyl ether, though having lower emulsifi-cation indexes (45%, 35% and 25%, respectively), formed stableemulsions that did not break within several days after their prep-aration. Little emulsion-stabilizing capacity, with emulsions break-ing up after only a few minutes, was observed for most of the oils

Table 1Emulsification index (E24) for the EPS produced by Pseudomonas oleovorans againstseveral hydrophobic compounds. All emulsions were prepared at room temperature,by mixing a 0.8% EPS solution with each of the hydrophobic compound in a vortex(2400 rpm, 2 min); the emulsions were left at room temperature for 24 h todetermined E24.

Hydrocarbons E24 (%) Oils E24 (%)

n-Hexadecane 70 Paraffin oil 30n-Hexane 75 Silicone oil <10Cyclohexane 40 Cedarwood oil 30n-Decane 35 Palm oil <10Xylene 60 Sunflower oil 20Diethylether 25 Corn oil <10Chloroform 70 Olive oil 65Benzene 60 Oleic acid <10Toluene 65

tested, including corn oil, palm oil, silicone oil and oleic acid.Cedarwood oil, paraffin oil and sunflower oil gave rise to water:oilstable emulsions, yet their emulsification indexes were lower than50% (Table 1). The only tested vegetable oil for which the EPS hasshown good emulsion stabilizing capacity was olive oil, with anemulsification index of 65% (Table 1). These results show thatthe EPS emulsion forming and stabilizing capacity is specific forcertain hydrophobic compounds. A similar behaviour has been re-ported for other microbial bioemulsifiers (Das, Mukherjee, & Sen,2009; Kumar, Mody, & Jha, 2007; Martinez-Checa et al., 2002; Mataet al., 2006; Nitschke & Pastore, 2006; Sarubbo, de Luna, &Campos-Takaki, 2006).

The emulsion-stabilizing capacity of the EPS as a function ofpolymer concentration was tested for three of the hydrocarbonsthat gave the highest E24 in the previous study, namely, n-hexadec-ane, xylene and n-hexane (Table 1). For both n-hexadecane and xy-lene, the Willumsen and Karlson (1997) criterion for the emulsion-stabilizing capacity of a given compound is fulfilled for an EPS con-centration of 0.6% (Fig. 6A and B). E24 was further improved forhigher EPS concentrations, reaching indexes of 100% and 80% fora polymer concentration of 1.0%, with n-hexadecane and xylene,respectively. On the other hand, an EPS concentration of 0.6%was found to be optimal to stabilize emulsions with n-hexane, asthere was no further improvement in E24 for higher polymer con-centrations (Fig. 6C). E24 has been reported to be proportional tothe surfactant concentration till the CMC (critical micellar concen-tration) value is reached (Cooper & Goldenberg, 1987). CMC isprobably reached at a lower concentration for n-hexane than forn-hexadecane and xylene, thus explaining the differences observedfor the hydrocarbons.

The emulsifying activity of the EPS produced by P. oleovorans isnoteworthy, especially in comparison with the activity of chemicalsurfactants. The chemical surfactants Tween 20, Tween 80 and Tri-ton X-100 have emulsifying indexes of 43%, 40% and 38%, respec-tively, for emulsions with xylene (Martinez-Checa et al., 2002),while the EPS had a E24 of 60%. The EPS emulsifying activity is alsobetter than some plant gums, namely, Karaya gum, Tragacanthgum and Arabic gum, that were reported to form emulsions withxylene with E24 of 69%, 67% and 33%, respectively, at considerablyhigher gum concentrations of 3.5% (Ashtaputre & Shah, 1995). Xan-than gum, a bacterial polysaccharide, exhibits a similar E24 (61%)for a polymer concentration of 0.35% (Iyer et al., 2006).

In many industrial processes, emulsifiers/surfactants are ex-posed to extremes of temperature, pressure, pH and/or ionicstrength. The emulsion forming and stabilizing capacity of theEPS was tested under different conditions of temperature (20–120 �C), pressure, pH (2–12) and NaCl concentration (0–2.0 M).The capacity to form emulsions, as well as the emulsion’s stability,under different conditions depends, not only on the emulsifierused, but also on the hydrophobic compound employed.

Emulsions prepared with n-hexadecane and 0.8% EPS aqueoussolutions were subjected to different temperatures in the range20–60 �C (Fig. 7A). The emulsions formed were thermostable fortemperatures up to 50 �C. However, higher temperatures (60 �C)caused a reduction of the emulsification index, but the emulsionwas maintained. During heating to 100 and 120 �C, as well as auto-claving, the emulsions were completely broken up (data notshown). The emulsions with n-hexadecane were also submittedto freezing/thawing procedures. The emulsions were stable duringfreezing but they were broken up during the thawing process.These results show that the emulsions with n-hexadecane cannotwithstand the extreme temperature conditions tested. Neverthe-less, the same tests need to be performed with other hydrophobiccompounds using the temperature ranges used for specific applica-tions to fully assess the capacity of the EPS to stabilize emulsions atextreme conditions.

0

10

20

30

40

50

60

70

80

90

100

0.0 0.2 0.4 0.6 0.8 1.0EPS concentration (%)

0.0 0.2 0.4 0.6 0.8 1.0

A B

EPS concentration (%)0.0 0.2 0.4 0.6 0.8 1.0 1.2

EPS concentration (%)

C

Em

ulsi

fica

tion

inde

x (%

)

Fig. 6. Emulsification index as a function of EPS concentration for n-hexadecane (A), xylene (B) and n-hexane (C). The tests were performed at room temperature (�20 �C) andneutral pH, with EPS solutions prepared in deionised water.

0

10

20

30

40

50

60

70

80

20 30 40 50 60

Temperature (ºC)

Em

ulsi

fica

tion

inde

x (%

)

0 2 4 6 8 10 12

pH

0.0 0.4 0.8 1.2 1.6 2.0

NaCl (M)

A CB

Fig. 7. Influence of temperature (A), pH (B) and NaCl concentration (C) on the emulsification index of emulsions formed between 0.6% EPS aqueous solutions and n-hexadecane.

F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556 555

On the other hand, even though emulsions generally need to beas stable as possible, there are several products/applications wherethe opposite effect is desired, so that the emulsion can be control-lably destabilized when required (Chabrand, Kim, Zhang, Glatz, &Jung, 2008). Emulsion breakdown and subsequent separation tooil and water (demulsification) is important in several industries,such as petroleum, nuclear, environmental or oil extraction tech-nologies, where it can be performed by enzymatic, physical and/or chemical treatments.

An additional test was performed to test the capacity of the EPSto form and stabilize emulsions at high temperatures that con-sisted in exposing aqueous solutions of the biopolymer to temper-atures of 80 and 100 �C, freezing/thawing and autoclaving, prior tothe preparation of the emulsions with n-hexadecane. The EPS re-tained its capacity to form stable emulsions after being frozen/thawed, as well as after being heated to 80 and 100 �C. Freezing/thawing or heating the EPS solution to 80 �C did not reduce theemulsion forming capacity against n-hexadecane, being the E24

identical to the solution not submitted to those procedures. Bythe contrary, heating to 100 �C reduced E24 for n-hexadecane to25%, while heating to 120 �C or autoclaving led to loss of emulsify-ing capacity. This result is in agreement with the reduction of theviscosity observed when the EPS solutions were subjected to thesame treatment (see Section 3.3). Most likely, the chemical compo-sition of the EPS or its tertiary structure were affected by the treat-ments at high temperature and/or pressure, which caused a changeof its properties.

The emulsions formed with n-hexadecane were relatively stablefor the pH range tested (2–12) with negligible variation observed

as the pH was changed (Fig. 7B), while the presence of NaCl causedan increase of E24 from 48.3% to 62.3%, as the NaCl concentrationwas increased up to 2.0 M (Fig. 7C).

Acidic conditions seem to decrease the emulsifying activity ofsome bacterial biopolymers, such as the biopolymers producedby Arthrobacter sp RAG-1 (Rosenberg, Zuckerberg, Rubinovitz, &Gutnick, 1979) and Pseudoalteromonas sp. strain TG12 (Gutierrezet al., 2008), as well as commercial polysaccharides (e.g. xanthangum and Arabic gum) (Gutierrez et al., 2008). By the contrary,the rhamnolipid-type biosurfactant produced by Pseudomonas flu-orescens formed emulsions whose stability increased with pH from2 to 12 (Abouseoud, Maachi, Amrane, Boudergua, & Nabi, 2008).The differences observed among the various biopolymers used asemulsion stabilizers are most likely attributable to their diversechemical composition and structure.

On the other hand, the emulsion stability under different pH isalso highly dependent on the compound that is to be emulsified.For example, the exopolysaccharide produced by Enterobacter clo-aceae has shown to be able to emulsify hexane, being the emul-sions stable for the pH range 2–10. By the contrary, E. cloaceaegroundnut oil emulsions stability was decreased as the pH was in-creased from 2 to 10 (Iyer et al., 2006).

P. fluorescens biosurfactant formed emulsions whose stabilityshowed little changes by the presence of NaCl in concentrationsup to 4.0 M (Abouseoud et al., 2008). Nevertheless, as was ob-served for pH, the emulsion stability is also dependent on the com-pound used. The emulsions formed by the exopolysaccharideproduced by E. cloaceae with hexane were stable in the presenceof NaCl in the range of 0.1–1.0 M, but groundnut oil emulsions sta-

556 F. Freitas et al. / Carbohydrate Polymers 78 (2009) 549–556

bility was improved by increasing the NaCl concentration (Iyeret al., 2006).

4. Conclusions

Results obtained with this study show that the EPS produced byP. oleovorans can be used as an emulsion forming and stabilizingagent between aqueous solutions and hydrophobic compounds.The stability of the emulsions under diverse conditions, such astemperature, pressure, pH and ionic strength, coupled with itsgood viscosity, makes the P. oleovorans polysaccharide as a poten-tial emulsifier to be used in many food and pharmaceutical formu-lations. In particular, the stability of the emulsions and of the EPSsolutions viscosity at acidic pH values may promote its applicationin food products containing citric acid and ascorbic acid, wheregelation is not required. Moreover, the high and stable emulsifyingactivity at various pH in the presence of salt, renders this productsuitable for its application in enhanced oil recovery. The ability tostabilize water/olive oil emulsions, together with its good viscosity,suggest the application of the EPS in food products (e.g. mayon-naise), as well as in cleaning agents in the food industry.

Acknowledgements

This project was financially supported by 73100, Lda., under theproject ‘‘Production of biopolymers from glycerol”, 2005/2008. Theauthors acknowledge Prof. Ana Ramos from CQFB/REQUIMTE, FCT-UNL, for the molecular weight determination. Vítor D. Alvesacknowledges Fundação para a Ciência e a Tecnologia, Pos-doc fel-lowship SFRH/BPD/26178/2005.

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