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

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

Carbohydrate Polymers 78 (2009) 549–556 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/ca...

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Carbohydrate Polymers 78 (2009) 549–556

Contents lists available at ScienceDirect

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

Emulsifying behaviour and rheological properties of the extracellular polysaccharide 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 b

REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 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 2009 Received in revised form 18 May 2009 Accepted 20 May 2009 Available online 31 May 2009 Keywords: Pseudomonas oleovorans Glycerol byproduct Extracellular polysaccharide (EPS) Rheology Bioemulsifier Emulsification index

a b s t r a c t The functional properties of a novel extracellular polysaccharide (EPS) produced by Pseudomonas oleovorans grown on glycerol byproduct, generated by the biodiesel industry, were investigated. The EPS is a high 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 has pseudoplastic fluid behaviour in aqueous media. The apparent viscosity was stable for the pH range 2.9–7.1 and NaCl concentrations up to 1.0 M. Though the apparent viscosity decreased at high temperatures, at alkaline conditions and at NaCl concentrations of 2.0 M, pseudoplastic fluid behaviour was retained. The EPS was capable of stabilizing water emulsions with several hydrophobic compounds, including hydrocarbons, vegetable and mineral oils. It retained its emulsifying activity during exposure to wide temperature (30–50 °C) and pH (2–12) variations, as well as to the presence of NaCl at concentrations as high as 2.0 M. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction A huge variety of polymers, such as polysaccharides, are naturally produced by microorganisms. Their application in numerous industrial areas, is mainly due to their rheological properties that allow the formation of viscous solutions at low concentrations (0.05–1.0%) and stability over wide temperature, pH and ionic strength ranges (Kumar & Mody, 2009, chap. 10). Many microbial polymers can also find useful applications as bioemulsifiers due to their ability to stabilize emulsions between water and hydrophobic compounds. Examples of bioemulsifiers include: RAG-1 emulsan and biodispersan, produced by Acinetobacter calcoaceticus strains (Rosenberg, Rubinovitz, Gottlieb, Rosenhak, & Ron, 1988; Sar & Rosenberg, 1983), and alasan, produced by Acinetobacter radioresistens (Navon-Venezia et al., 1995). Other biopolymers with potential applications as bioemulsifiers include extracellular polysaccharides 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 chemical counterparts, because they are biodegradable, less toxic and have activity under a wider variety of conditions (Banat, Makkar, & Cameotra, 2000). Due to their wide diversity in composition and structure, bioemulsifiers are characterized by improved func* Corresponding author. Tel.: +351 21 2948357; fax: +351 21 2948385. E-mail address: [email protected] (M.A.M. Reis). 0144-8617/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2009.05.016

tionality and stability, which broadens the spectrum of potential applications, which include: the oil and petroleum industries, water and soil bioremediation, metal treatment and processing, detergents and laundry supplies, agriculture, textile manufacturing, 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 the lack of experience in their applications. Operating costs may be reduced by using low-cost substrates, which account for 10–30% of the overall production cost. Biopolymer recovery from the cultivation broth also has a considerable impact on the final product cost (Makkar & Cameotra, 2002). Hence, bioemulsifiers that can be recovered by simple and inexpensive techniques will probably have a more competitive price. Pseudomonas oleovorans has been reported to produce extracellular biosurfactants capable of forming stable oil in water emulsions when grown in two-liquid phase cultures, such as n-decane (Schmid, Kollmer, & Witholt, 1998). In previous work, we have reported on the characteristics of a new extracellular polysaccharide (EPS) produced by P. oleovorans from pure glycerol (Freitas et al., 2009). The EPS is a high molecular weight heteropolysaccharide (1.0–5.0  106), composed by neutral sugars and acyl groups substituents. Preliminary tests have shown that this new biopolymer forms viscous aqueous solutions with pseudoplastic fluid behaviour and has an emulsifying activity against n-hexadecane comparable to other bioemulsifiers. Glycerol byproduct from the biodiesel industry has proven to be a suitable carbon substrate for EPS production by P. oleovorans (unpublished data), in spite

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of containing some impurities. The use of glycerol byproduct as a feedstock is however a very important cost reduction factor. Although the EPS chemical composition and molecular weight were 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 stabilizing capacity of the EPS produced by P. oleovorans grown on glycerol byproduct from the biodiesel industry. The emulsifying behaviour and the rheological properties were investigated for several EPS concentrations, as well as under different conditions of temperature, pH and ionic strength.

2. Materials and methods 2.1. Biopolymer production and extraction Pseudomonas oleovorans NRRL B-14682 was grown on a slightly modified Medium E* (Freitas et al., 2009), supplemented with glycerol byproduct to give a concentration between 25 and 50 g L1. Glycerol byproduct, supplied by Fábrica Torrejana de Biocombustíveis SA, Portugal, had a glycerol content of 89% and residual contents of methanol (0.04%), organic material (0.4%), ashes (6.8%) and water (3.5%). EPS production was performed in a 10 L bioreactor (BioStat B-plus, Sartorius), as described by Freitas et al. (2009). The bioreactor was operated in a batch mode during the first 24 h of cultivation and, then, in a fed-batch mode, by supplying the bioreactor with cultivation Medium E*, with a glycerol concentration of 200 g L1, at a constant rate of 20 mL h1. Throughout the cultivation, culture broth samples were recovered from the bioreactor and centrifuged (18,000g, 15 min) for cell separation. The cell-free supernatant was stored at 20 °C for the determination of glycerol and ammonium concentrations. Cell dry weight (CDW), glycerol and ammonium concentrations, as well as the measurement of the culture broth viscosity, were performed as described by Freitas et al. (2009). For extracting the EPS, the culture broth was recovered from the bioreactor, diluted with deionised water for viscosity reduction and centrifuged (20,000g, 1 h), for cell separation. The volume of water added to dilute the culture broth varied from 1:1, for low viscosity samples, to 1:7, for highly viscous samples. The EPS in the cell-free supernatant was precipitated by the addition of acetone (3:1). The precipitated EPS was washed with acetone, dissolved 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 with trifluoroacetic acid (TFA) (0.1 mL TFA 99%), at 120 °C, for 2 h. The hydrolysate was used for the 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 acid hydrolysate of the EPS was also used for the identification and quantification of acyl groups, as described by Freitas et al. (2009). The total inorganic content was determined as described by Freitas et al. (2009). For determination of the protein content, 5.5 mL samples of aqueous EPS solutions (4.5 g L1) were mixed to 1 mL 20% NaOH and placed at 100 °C for 5 min. After cooling on ice, 170 lL of CuSO45H2O (25% v/v) were added. After mixing, the samples were centrifuged (3500g, 5 min) and the optical density was measured at 560 nm (Spectrophotometer Helios Alpha,

Thermo Spectronic, UK). Albumin (Merck) solutions (0.5– 3.0 g L1) were used as protein standards. Average molecular weights (Mw) and the polydispersity index (Mn/Mw) were obtained by size exclusion chromatography, as described by Freitas et al. (2009). 2.3. Rheology The effect of EPS concentration, pH, temperature and ionic strength on the EPS solutions viscosity was studied using a controlled stress rheometer (ARG2, TA Instruments Inc., New Castle, DE, USA), equipped with a cone and plate geometry (diameter 4 cm, angle 2 degrees). During the experiments, the shearing geometry was covered with paraffin oil in order to prevent water loss. Flow curves were determined using a steady state flow ramp in the range of shear rate from 1 s1 to 700 s1. The shear rate was measured point by point with consecutive 60 s steps of constant shear rate. The viscosity was recorded for each point to obtain the flow curves. 2.4. Emulsifying activity The standard assay for emulsifying activity was based on the method described by Cooper and Goldenberg (1987), using n-hexadecane 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 mm diameter  180 mm) and stirred in the vortex at 2400 rpm for 2 min. After 24 h, the emulsification index (E24) was determined as follows: E24 ¼ he =hT  100, where he (mm) is the height of the emulsion 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-hexadecane was evaluated for different EPS concentrations, temperatures, pH and ionic strength. The emulsifying assay was also applied to other hydrophobic compounds (hydrocarbons and oils), using the same ratio of 0.6% EPS solution:hydrophobic compound (2:3 v/v). The tested compounds included oils (corn oil, sunflower oil, olive oil, paraffin oil and cedar wood oil, purchased at the local supermarket; oleic acid, Panreac; and silicone oil, Wacker) and hydrocarbons (benzene, Merck; chloroform, Sigma; cyclohexane, Panreac; n-decane, Merck; dimethylether, Riedel de Haen; n-hexane, Riedel de Haen; isooctane, Merck; toluene, Sigma; xylene, Merck). All tests were performed at room temperature. The capacity of the EPS to stabilize emulsions after being subjected to high temperatures and to an autoclaving process was also assessed. For this test, EPS aqueous solutions (0.6%) were placed at 80, 100 and 120 °C for 1 h, or autoclaved (120 °C, 1 bar, 20 min). After cooling to room temperature, the emulsions were prepared with n-hexadecane (EPS solution:hexadecane ratio of 2:3) and left for 24 h to determine E24.

3. Results and discussion 3.1. Biopolymer production EPS production and growth of P. oleovorans using glycerol byproduct as substrate are presented in Fig. 1A. Bacterial cell growth was suppressed within one day of cultivation by imposing 1 ). A maximum biomass nitrogen limiting conditions (<0.1 g NHþ 4 L 1 concentration of 7.32 g L was reached within 2 days of cultivation. During the stationary growth phase, ammonium concentration was kept at a residual value (below the detection limit) and glycerol concentration was maintained relatively constant (5.0– 10.0 g L1), even though the feeding solution (containing

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A

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Viscosity (Pa.s)

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day 5

100 day 4

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10-2

day 1

0

0

1

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3 4 Time (day)

5

6

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10-3 100

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102 103 Shear Rate (s–1)

Fig. 1. (A) Time course of the cultivation of Pseudomonas oleovorans on glycerol byproduct (glycerol, h; ammonium, 4; CDW, viscosity built up of the culture broth over time, during the cultivation of P. oleovorans with glycerol byproduct.

3.2. Chemical composition and molecular weight The EPS produced by P. oleovorans from glycerol byproduct was recovered from the bioreactor at the end of the cultivation (5 days), but culture broth samples were recovered from the bioreactor at different cultivation times, being the EPS extracted and characterized in terms of its composition and molecular weight (Fig. 2). The glycosyl composition analysis revealed that it was a heteropolysaccharide composed by neutral sugars. Galactose was the most abundant monosaccharide constituent throughout the cultivation, representing 68% of the polymer’s carbohydrate content at the end of the run. Mannose and glucose were also present in significant amounts, accounting for 17% and 13%, respectively, of the carbohydrate content of the EPS. Rhamnose and fucose were present in minor amounts (2% and 4%, respectively). The EPS produced by P. oleovorans from pure glycerol had a similar composition, with galactose being the main sugar constituent (70%) (Freitas et al., 2009). Mannose represented 23% of the carbohydrate content and rhamnose was a minor component (4%). On the other hand, the EPS obtained from pure glycerol had lower glucose content

6

; EPS, ). (B) Shear rate dependent apparent

5.0x106

( (A) )

5

4.0x106

4 3.0x106

Mw

Sugar (g l-1 )

1 200 g L1 glycerol and 0.9 g NHþ ) was fed to the bioreactor at a 4 L 1 constant flow rate (20 mL h ). Increased EPS production was initiated within one day of cultivation, when the culture entered the stationary growth phase (Fig. 1A), indicating that the synthesis of extracellular polysaccharide by P. oleovorans has non-growth associated kinetics. After 5 days of cultivation, the EPS attained a concentration of 15.0 g L1. Considering the time window of effective EPS production (between days 1.0 and 5.3), the productivity and the net yield on glycerol were 0.13 g L1 h1 and 0.15 g g1, respectively. In other experiments performed with P. oleovorans grown on glycerol, both the final EPS concentration and its productivity were improved. Typically, the cultivation for EPS production takes between 4 and 7 days, with a final production of 7.0–18.0 g L1 of EPS and maximum productivities of 0.1–0.2 g L1 h1 (data not shown). Concomitant with EPS production, there was a drastic change in the culture broth viscosity (Fig. 1B). The culture broth developed non-Newtonian characteristics, acting as a pseudoplastic fluid, where the measured viscosity decreased with increasing shear rate. The apparent viscosity of the culture broth measured at low shear rates showed an increase of almost three orders of magnitude (from 4.7  103 to 2.0 Pa s) (Fig. 1B). This viscosity built up is a common feature observed in many microbial cultivations for the production of extracellular polysaccharides (Kumar & Mody, 2009) and it usually determines the termination of the run due to loss of bulk homogeneity of the culture broth.

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3 2.0x106 2 1.0x106

1 0

0.0 0

1

2

3

4

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6

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

and fucose was not detected. These differences may be related to the use of glycerol byproduct that contains some impurities not present in pure glycerol and may have influenced the EPS sugar composition. The polymer also had acyl groups substituents that accounted for 3.04% of its dry mass. The presence of acyl group substituents is common in microbial EPS and they greatly influence polymer’s properties, namely, solubility and rheology (Rinaudo, 2001). The main acyl groups detected were pyruvil (2.51%) and succinyl (0.54%), as well as other unidentified organic acids, which were present only in residual amounts. The presence of pyruvil and succinyl confer the EPS an anionic character (Freitas et al., 2009). The EPS obtained from pure glycerol had similar pyruvil content (3.35%) and higher succinyl content (1.04%), but it additionally contained acetyl (0.38%) (Freitas et al., 2009) that was not detected in the EPS produced from glycerol byproduct. The extracted polymer also contained other non-sugar constituents that were probably remnants of the culture broth, namely, proteins and inorganic residues. Proteins accounted for 10% of the polymer’s mass. The polymer characterized in previous work had a lower protein content (5%) because the cell-free supernatant was subjected to protein removal prior to EPS precipitation (Freitas et al., 2009), thus yielding a polymer with a higher degree of purity. In the present work, the extraction procedure was made simpler aiming at generating a final product with a more competitive price. For most of the emulsifiers applications (e.g. oil and petroleum

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industries, water and soil bioremediation, metal treatment and processing, detergents and laundry supplies, textile manufacturing, paints), there is no need for a highly purified bioemulsifier. As shown by its pyrolysis at 550 °C, the EPS had a total inorganic residues content of 23.6%, being the main cations detected by ICP analysis: sodium (10.77%), potassium (4.70%), calcium (0.10%) and magnesium (0.08%). Cobalt, copper, iron, manganese and zinc were also detected in trace amounts (<0.01%). No sulphur was detected, as confirmed by the elemental analysis. On the other hand, the EPS contained 4.34% of phosphorus, as detected by ICP. Part of these metal cations were probably present in the extracted polymer as counter-ions for the anionic acyl groups or represent salts that co-precipitated with the EPS during the recovery procedure. The EPS produced from pure glycerol had lower inorganic residues content (11.6%) (Freitas et al., 2009). This result was probably due to the fact that glycerol byproduct had an ash content of approximately 6.8%, from which 3.3% were attributed to sodium (detected by ICP analysis), originating from the NaOH used during the biodiesel production process. Moreover, as stated above, the EPS described in the present study was recovered from the cell-free supernatant, without any additional purification treatment, in contrast to the previously described polymer which was further purified with a thermal treatment and successive precipitations. The polymer’s average molecular weight showed a gradual increase from 2.4  106 (day 1) to 4.6  106 (day 5) (Fig. 2). The extracted polymers were rather homogeneous, as shown by the polydispersity index that ranged between 2.02 and 2.33. Similar values were obtained for the EPS produced from pure glycerol, namely, average molecular weight increasing from 1.0  106 to 5.0  106 during the 4 days cultivation run (Freitas et al., 2009). The EPS produced from pure glycerol had lower polydispersity indexes (1.43–2.15) indicating that the extracted polymers were more homogeneous. 3.3. Rheological properties The analysis of the rheological properties of the EPS in aqueous media showed evidence of pseudoplastic fluid behaviour, as the viscosity was influenced by shear rate (Fig. 3A). This behaviour is expected for solutions of polysaccharides, resulting from their polymeric structure and high molecular weight (Rao, Suresh, & Suraishkumar, 2003). In addition, the viscosity is recovered for low 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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Viscosity (Pa.s)

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the cultivation, when the EPS concentration was 15.0 g L1, was identical to the flow behaviour of a 1.5% aqueous solution prepared with the freeze dried polymer (Fig. 3A). This result confirms that the viscosity built up observed during the cultivation was conferred by the presence of the EPS in the culture broth. Moreover, the fact that the EPS retained the same flow behaviour shows that the extraction process did not alter the polymer’s viscosity properties. The viscosity of EPS solutions at different concentrations (0.2– 1.5%) was investigated (Fig. 3B). As expected, the apparent viscosity increased with increasing concentration. As the polymer concentration becomes higher, the individual molecules start to overlap, inducing the formation of intermolecular junctions and, hence, limiting polymer chain arrangement and stretching. Consequently, there is an increase of the solution’s viscosity (Bae, Oh, Lee, Yoo, & Lee, 2008). Industrial processes frequently involve exposure to extremes of temperature, pH, pressure and ionic strength. Hence, it is important to evaluate how the EPS rheological properties are influenced by those conditions, as well as its reversibility. The EPS in aqueous solution was exposed to temperature (15–120 °C) and pH (2.9– 11.8) ranges, and to autoclaving (simultaneous high pressure and temperature conditions), as well as to NaCl concentrations up to 2.0 M. The effect of temperature on the flow behaviour of EPS aqueous solutions was investigated by measuring the apparent viscosity at different temperatures (Fig. 5A). It is evident that the EPS is sensitive to the temperature, as shown by the viscosity reduction observed as the temperature was increased. Nevertheless, the pseudoplastic fluid behaviour was retained even for the highest temperature tested (data not shown). Moreover, low viscosity may be an advantageous characteristic for several industrial applications, such as oil tankers cleaning and the recovery of solid wastes (Martinez-Checa, Toledo, Vilches, Quesada, & Calvo, 2002). Viscosity reduction with increasing temperature between 10 and 90 °C was also observed for other bacterial polysaccharides, such as xanthan gum (García-Ochoa et al., 2000) and bacterial alginate (Chen, Chen, Chang, & Su, 1985). An additional study to investigate the effect of temperature on the viscosity of the EPS was conducted by heating a 0.8% EPS solution from 17 to 80 °C and cooling it back to 17 °C (Fig. 4A) at a constant shear rate of 5 s1. The results show that the viscosity is reduced with increasing temperatures, but it regains its original viscosity upon cooling, indicating that the EPS is not degraded by heating up to 80 °C. Instead, as the temperature is raised, the inter-

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Fig. 3. (A) Dependence of the apparent viscosity on the shear rate of an 1.5% EPS solution with increasing the shear rate from 1 s1 to 700 s1 ( ) followed by decreasing the shear rate from 700 s1 to 1 s1 ( ); and of the culture broth at the end of the 5 days cultivation run with increasing the shear rate (w). (B) Apparent viscosity of EPS aqueous solutions as a function of polymer concentration at a shear rate of 5 s1. The experiments were carried out at 25 °C in both cases.

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Fig. 4. (A) Apparent viscosity of a 0.8% EPS solution during heating (j), followed by cooling ( ) (the solution was heated from 17 to 80 °C with a heating rate of 3 °C/min, kept at 80 °C for 30 s and cooled back to 17 °C with a cooling rate of 3 °C/min). The measurements were made at a shear rate of 5 s1. (B) Flow curves for 0.8% EPS solutions exposed to different thermal treatments: starting solution ( ), solution subjected to 100 °C for 1 h ( ), solution subjected to 120 °C for 1 h ( ) and autoclaved solution ( ) (all measurements were made after cooling the solutions to 25 °C).

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Fig. 5. Influence of temperature (A), pH (B) and NaCl concentration (C) on the apparent viscosity of a 1.2% EPS solution (measured at a shear rate of 5 s1 and at a temperature of 25 °C).

action between the molecules is decreased and the polymer structure is loosened, thus decreasing the viscosity of the solution. The fact that the EPS regains its original viscosity after being exposed to high temperatures envisages its use in applications were the products are processed at high temperatures (e.g. pasteurization) but the final product is used at room temperature. The rheological properties of the final product thus remain stable, regardless of being stored in the refrigerator, at room temperature of heated. This behaviour was also observed for xanthan gum, for which the viscosity is fully reversible between 10 and 80 °C (García-Ochoa et al., 2000), and for bacterial alginate for temperatures up to 75 °C (Chen et al., 1985). The effect of extreme temperatures on the EPS flow curves was evaluated by subjecting 0.8% aqueous solutions to high temperatures (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 original apparent viscosity after being exposed to temperatures equal or higher than 100 °C. In fact, about half of the viscosity was lost when the polymer was heated to 100 °C, being even further reduced after the treatment at 120 °C. Nevertheless, though having a lower viscosity, the solutions continued to exhibit pseudoplastic fluid behaviour. There was no significant difference between heating to 120 °C and combining this temperature with a pressure of 1 bar. Bacterial alginate shows a similar behaviour: when heated

above 75 °C the original viscosity of the solution is not restored upon cooling, while for higher temperatures (121 °C) the viscosity is practically destroyed (Chen et al., 1985). These results suggest that the high temperatures tested caused either a partial EPS degradation or an alteration of its tertiary structure with the polymer chains adopting a different intermolecular arrangement. This issue is to be addressed in future studies. The study of the pH effect on the apparent viscosity of the EPS was performed by changing the pH of a 1.2% aqueous solution (pH 7.1) 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 s1. It can be concluded that the viscosity properties of the EPS were not influenced by acidic conditions, since it was not significantly altered 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. Even so, the solutions still retained a considerable viscosity and the pseudoplasticity was maintained (data not shown). Bacterial alginate solutions have a similar behaviour, namely, they are stable for pH 4–10, but its viscosity decreases above pH 10 (Chen et al., 1985). Although the viscosity of xanthan solutions is not significantly affected by changes in pH between 1 and 13 (García-Ochoa et al., 2000), it is maximal for neutral pH, decreasing for lower or higher values (López, Vargas-García, Suarez-Estrella,

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& Moreno, 2004). Also, it was observed that xanthan’s composition is altered at pH 9 or higher, and for pH lower than 3 (López et al., 2004). The intermolecular arrangement of charged polysaccharides may be expanded by electrostatic repulsion, or contracted by electrostatic attraction between polymer chains (Lai & Yang, 2007). The electrostatic force is therefore expected to have an important effect on the conformation of the EPS, and the related physical–chemical properties such as viscosity. Therefore, in this study, the flow behaviour of the EPS in the presence of increasing NaCl concentration was addressed (Fig. 5C). The results show that the apparent viscosity of the EPS solutions was not altered by increasing the ionic strength up to 1.0 M NaCl. This fact may be related to ions present already in the polymer sample. However, higher values of ionic strength (2.0 M NaCl) resulted in a 26% reduction of the solution viscosity. The raised concentration of counter-ions experienced when the ionic strength got higher, may have made the molecules contract even further due to charge shielding effect, thus resulting in a lower viscosity. 3.4. Emulsion forming and stabilizing capacity In previous studies, the EPS produced by P. oleovorans from glycerol has shown to possess the capacity to stabilize hydrocarbon/water emulsions, with an emulsifying activity against n-hexadecane comparable to other bioemulsifiers (Freitas et al., 2009). In the present study, the emulsion forming and stabilizing capacity of this new biopolymer was studied in greater detail. Several hydrophobic substances were tested, including vegetable and mineral oils, and hydrocarbons. Hexadecane was used as the test substance to study the emulsion-stabilizing capacity of the EPS as a function of polymer concentration, temperature, pH and ionic strength. A criterion for determining the emulsion-stabilizing capacity of an emulsifier consists on evaluating its ability to maintain at least 50% of the original emulsion volume 24 h after its formation (Willumsen & Karlson, 1997). Considering this criterion, the EPS produced by P. oleovorans has proven to possess emulsion-stabilizing capacity for several hydrocarbons, including n-hexadecane, n-hexane, xylene, chloroform, benzene, toluene, as shown by emulsification indexes higher than 50% (Table 1). The high emulsification indexes observed reflect the stability of the emulsions thus formed. Moreover, those emulsions were stable for several weeks (data not shown). The other hydrocarbons tested, namely, cyclohexane, n-decane and diethyl ether, though having lower emulsification indexes (45%, 35% and 25%, respectively), formed stable emulsions that did not break within several days after their preparation. Little emulsion-stabilizing capacity, with emulsions breaking up after only a few minutes, was observed for most of the oils

Table 1 Emulsification index (E24) for the EPS produced by Pseudomonas oleovorans against several 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 to determined E24. Hydrocarbons

E24 (%)

Oils

E24 (%)

n-Hexadecane n-Hexane Cyclohexane n-Decane Xylene Diethylether Chloroform Benzene Toluene

70 75 40 35 60 25 70 60 65

Paraffin oil Silicone oil Cedarwood oil Palm oil Sunflower oil Corn oil Olive oil Oleic acid

30 <10 30 <10 20 <10 65 <10

tested, including corn oil, palm oil, silicone oil and oleic acid. Cedarwood oil, paraffin oil and sunflower oil gave rise to water:oil stable emulsions, yet their emulsification indexes were lower than 50% (Table 1). The only tested vegetable oil for which the EPS has shown good emulsion stabilizing capacity was olive oil, with an emulsification index of 65% (Table 1). These results show that the EPS emulsion forming and stabilizing capacity is specific for certain hydrophobic compounds. A similar behaviour has been reported for other microbial bioemulsifiers (Das, Mukherjee, & Sen, 2009; Kumar, Mody, & Jha, 2007; Martinez-Checa et al., 2002; Mata et al., 2006; Nitschke & Pastore, 2006; Sarubbo, de Luna, & Campos-Takaki, 2006). The emulsion-stabilizing capacity of the EPS as a function of polymer concentration was tested for three of the hydrocarbons that gave the highest E24 in the previous study, namely, n-hexadecane, xylene and n-hexane (Table 1). For both n-hexadecane and xylene, the Willumsen and Karlson (1997) criterion for the emulsionstabilizing capacity of a given compound is fulfilled for an EPS concentration of 0.6% (Fig. 6A and B). E24 was further improved for higher EPS concentrations, reaching indexes of 100% and 80% for a 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, as there was no further improvement in E24 for higher polymer concentrations (Fig. 6C). E24 has been reported to be proportional to the surfactant concentration till the CMC (critical micellar concentration) value is reached (Cooper & Goldenberg, 1987). CMC is probably reached at a lower concentration for n-hexane than for n-hexadecane and xylene, thus explaining the differences observed for the hydrocarbons. The emulsifying activity of the EPS produced by P. oleovorans is noteworthy, especially in comparison with the activity of chemical surfactants. The chemical surfactants Tween 20, Tween 80 and Triton X-100 have emulsifying indexes of 43%, 40% and 38%, respectively, for emulsions with xylene (Martinez-Checa et al., 2002), while the EPS had a E24 of 60%. The EPS emulsifying activity is also better than some plant gums, namely, Karaya gum, Tragacanth gum and Arabic gum, that were reported to form emulsions with xylene with E24 of 69%, 67% and 33%, respectively, at considerably higher gum concentrations of 3.5% (Ashtaputre & Shah, 1995). Xanthan 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 exposed to extremes of temperature, pressure, pH and/or ionic strength. The emulsion forming and stabilizing capacity of the EPS 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 emulsifier used, but also on the hydrophobic compound employed. Emulsions prepared with n-hexadecane and 0.8% EPS aqueous solutions were subjected to different temperatures in the range 20–60 °C (Fig. 7A). The emulsions formed were thermostable for temperatures up to 50 °C. However, higher temperatures (60 °C) caused a reduction of the emulsification index, but the emulsion was maintained. During heating to 100 and 120 °C, as well as autoclaving, the emulsions were completely broken up (data not shown). The emulsions with n-hexadecane were also submitted to freezing/thawing procedures. The emulsions were stable during freezing but they were broken up during the thawing process. These results show that the emulsions with n-hexadecane cannot withstand the extreme temperature conditions tested. Nevertheless, the same tests need to be performed with other hydrophobic compounds using the temperature ranges used for specific applications to fully assess the capacity of the EPS to stabilize emulsions at extreme conditions.

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100

A

90

B

C

Emulsification index (%)

80 70 60 50 40 30 20 10 0 0.0

0.2

0.4 0.6 0.8 1.0 EPS concentration (%)

0.0

0.2

0.4 0.6 0.8 1.0 EPS concentration (%)

0.0

0.2

0.4 0.6 0.8 1.0 EPS concentration (%)

1.2

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) and neutral pH, with EPS solutions prepared in deionised water.

80

A

B

C

Emulsification index (%)

70 60 50 40 30 20 10 0 20

30

40

50

60

0

2

4

Temperature (ºC)

6

8 pH

10

12

0.0

0.4

0.8

1.2

1.6

2.0

NaCl (M)

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 nhexadecane.

On the other hand, even though emulsions generally need to be as stable as possible, there are several products/applications where the opposite effect is desired, so that the emulsion can be controllably destabilized when required (Chabrand, Kim, Zhang, Glatz, & Jung, 2008). Emulsion breakdown and subsequent separation to oil and water (demulsification) is important in several industries, such as petroleum, nuclear, environmental or oil extraction technologies, where it can be performed by enzymatic, physical and/ or chemical treatments. An additional test was performed to test the capacity of the EPS to form and stabilize emulsions at high temperatures that consisted in exposing aqueous solutions of the biopolymer to temperatures of 80 and 100 °C, freezing/thawing and autoclaving, prior to the preparation of the emulsions with n-hexadecane. The EPS retained 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 the emulsion forming capacity against n-hexadecane, being the E24 identical to the solution not submitted to those procedures. By the contrary, heating to 100 °C reduced E24 for n-hexadecane to 25%, while heating to 120 °C or autoclaving led to loss of emulsifying capacity. This result is in agreement with the reduction of the viscosity observed when the EPS solutions were subjected to the same treatment (see Section 3.3). Most likely, the chemical composition of the EPS or its tertiary structure were affected by the treatments at high temperature and/or pressure, which caused a change of its properties. The emulsions formed with n-hexadecane were relatively stable for the pH range tested (2–12) with negligible variation observed

as the pH was changed (Fig. 7B), while the presence of NaCl caused an increase of E24 from 48.3% to 62.3%, as the NaCl concentration was increased up to 2.0 M (Fig. 7C). Acidic conditions seem to decrease the emulsifying activity of some bacterial biopolymers, such as the biopolymers produced by Arthrobacter sp RAG-1 (Rosenberg, Zuckerberg, Rubinovitz, & Gutnick, 1979) and Pseudoalteromonas sp. strain TG12 (Gutierrez et al., 2008), as well as commercial polysaccharides (e.g. xanthan gum and Arabic gum) (Gutierrez et al., 2008). By the contrary, the rhamnolipid-type biosurfactant produced by Pseudomonas fluorescens formed emulsions whose stability increased with pH from 2 to 12 (Abouseoud, Maachi, Amrane, Boudergua, & Nabi, 2008). The differences observed among the various biopolymers used as emulsion stabilizers are most likely attributable to their diverse chemical composition and structure. On the other hand, the emulsion stability under different pH is also highly dependent on the compound that is to be emulsified. For example, the exopolysaccharide produced by Enterobacter cloaceae has shown to be able to emulsify hexane, being the emulsions stable for the pH range 2–10. By the contrary, E. cloaceae groundnut oil emulsions stability was decreased as the pH was increased from 2 to 10 (Iyer et al., 2006). P. fluorescens biosurfactant formed emulsions whose stability showed little changes by the presence of NaCl in concentrations up to 4.0 M (Abouseoud et al., 2008). Nevertheless, as was observed for pH, the emulsion stability is also dependent on the compound used. The emulsions formed by the exopolysaccharide produced by E. cloaceae with hexane were stable in the presence of NaCl in the range of 0.1–1.0 M, but groundnut oil emulsions sta-

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bility was improved by increasing the NaCl concentration (Iyer et al., 2006). 4. Conclusions Results obtained with this study show that the EPS produced by P. oleovorans can be used as an emulsion forming and stabilizing agent between aqueous solutions and hydrophobic compounds. The stability of the emulsions under diverse conditions, such as temperature, pressure, pH and ionic strength, coupled with its good viscosity, makes the P. oleovorans polysaccharide as a potential emulsifier to be used in many food and pharmaceutical formulations. In particular, the stability of the emulsions and of the EPS solutions viscosity at acidic pH values may promote its application in food products containing citric acid and ascorbic acid, where gelation is not required. Moreover, the high and stable emulsifying activity at various pH in the presence of salt, renders this product suitable for its application in enhanced oil recovery. The ability to stabilize water/olive oil emulsions, together with its good viscosity, suggest the application of the EPS in food products (e.g. mayonnaise), as well as in cleaning agents in the food industry. Acknowledgements This project was financially supported by 73100, Lda., under the project ‘‘Production of biopolymers from glycerol”, 2005/2008. The authors acknowledge Prof. Ana Ramos from CQFB/REQUIMTE, FCTUNL, for the molecular weight determination. Vítor D. Alves acknowledges Fundação para a Ciência e a Tecnologia, Pos-doc fellowship SFRH/BPD/26178/2005. References Abouseoud, M., Maachi, R., Amrane, A., Boudergua, S., & Nabi, A. (2008). Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens. Desalination, 223, 143–151. Appaiah, K. A. A., & Karanth, N. G. K. (1991). Insecticide specific emulsifier production by hexachlorocyclohexane utilizing Pseudomonas tralucida Ptm+ strain. Biotechnology Letters, 13, 371–374. Ashtaputre, A. A., & Shah, A. K. (1995). Emulsifying property of a viscous exopolysaccharide from Sphingomonas paucimobilis. World Journal of Microbiology Biotechnology, 11, 219–222. Bae, I. Y., Oh, I.-K., Lee, S., Yoo, S.-H., & Lee, H. G. (2008). Rheological characterization of levan polysaccharides from Microbacterium laevaniformans. International Journal of Biological Macromolecules, 42, 10–13. Banat, I. M., Makkar, R. S., & Cameotra, S. S. (2000). Potential commercial applications of microbial surfactants. Applied Microbiology and Biotechnology, 53, 495–508. Chabrand, R. M., Kim, H.-J., Zhang, C., Glatz, C. E., & Jung, S. (2008). Destabilization of the emulsion formed during aqueous extraction of soybean oil. Journal of the American Oil Chemical Society, 85, 383–390. Chen, W.-P., Chen, J.-Y., Chang, S.-C., & Su, C.-L. (1985). Bacterial alginate produced by a mutant of Azotobacter vinelandii. Applied and Environmental Microbiology, 49, 543–546. Cooper, D. G., & Goldenberg, B. G. (1987). Surface active agents of two Bacillus species. Applied and Environmental Microbiology, 53, 224–229. Das, P., Mukherjee, S., & Sen, R. (2009). Substrate dependent production of extracellular biosurfactant by a marine bacterium. Bioresource Technology, 100, 1015–1019.

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