Rhamnolipids Production by a€ Pseudomonas ...

SPECIAL THEME: GREEN SURFACTANTS – Synthesis, Properties, Performance and Application

y

Roberta B. Lovaglio1 , Vinícius L. da Silva1 , Tulio de Lucca Capelini1 , Marcos N. Eberlin2 , Rudolf Hausmann3 , Marius Henkel4 and Jonas Contiero1

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Rhamnolipids Production by a Pseudomonas eruginosa LBI Mutant: Solutions and Homologs Characterization This paper evaluates the effect of additives (NaCl and ethanol) on the solution properties of rhamnolipids. The properties are the surface activity, aggregate formations and emulsifying activity as well as the synergistic effects of additives and pH variations on the physical properties of rhamnolipids. Additionally, analysis of fatty acids and rhamnolipid homologues produced using different carbon sources was performed by mass spectrometry. The results indicate that this biosurfactant maintain its properties in the presence of additives. NaCl decreases the size and number of aggregates formed in solutions without pH control, while ethanol to rhamnolipid solutions reduces critical micelle concentration and favors aggregation of monomers. The profiles of fatty acids produced by P. aeruginosa LBI 2A1 varied according to the carbon source used, however for rhamnolipids there was no difference. Key words: Rhamnolipids, Pseudomonas aeruginosa LBI mutant, biosurfactant, physical properties, critical micelle concentration (CMC)

Rhamnolipidproduktion mittels Pseudomonas aeruginosa-LBI-Mutante: Charakterisierung der Lösungen und Homologen. In diesem Beitrag wurde der Einfluss der Additive (NaCl und Ethanol) auf die Eigenschaften von Rhamnolipidlösungen bestimmt. Die Eigenschaften sind die Oberflächenaktivität, die Bildung von Aggregaten und die Emulsionsbildung. Des Weiteren wurden bestimmt die synergistischen Effekte der Additive und der Einfluss der pH-Wertänderungen auf die physikalischen Eigenschaften der Rhamnolipide. Eine Analyse der Fettsäuren und der mit verschiedenen Kohlenstoffquellen erzeugten Rhamnolipidhomologen wurde mittels Massenspektrometrie durchgeführt. Die Ergebnisse machen deutlich, dass dieses Biotensid seine Eigenschaften in Gegenwart der Additive behält. Ohne pH-Wert-Kontrolle verringert NaCl die Anzahl und Größe der in Lösung gebildeten Aggregate, während Ethanol in den Rhamnolipidlösungen die kritische Mizellenbildungskonzentration (CMC) reduziert und die Aggregation der Monomere bevorzugt. Die von P. aeruginosa LBI 2A1 erzeugten Fettsäureprofile variierten aufgrund der verwendeten Kohlenstoffquelle, für die Rhamnolipide machte das aber keinen Unterschied. Stichwörter: Rhamnolipid, Pseudomonas aeruginosa-LBI-Mutante, Biotensid, physikalische Eigenschaften, kritische Mizellenbildungskonzentration (CMC) 1

2 3 4

UNESP – Universidade Estadual Paulista, Department of Biochemistry and Microbiology, Institute of Biological Sciences, Av. 24-A, 1515 Bela Vista, CEP 13506-900, Rio Claro, SP, Brazil Thomson Mass Spectrometry Laboratory, Institute of Chemistry, Campinas State University, UNICAMP, Campinas, SP, Brazil Institute of Food Science and Biotechnology (150), Section Bioprocess Engineering (150k), University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany

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1 Introduction

Rhamnolipids produced by P. aeruginosa strains are one of the most studied biosurfactants, and are capable of reducing the surface tension of water from a value of 72 mN/m to values of *30 mN/m [1]. The critical micelle concentrations (CMC) of these compounds typically range from 5 – 200 mg/L, depending on the proportion of different homologues in the mixture [2, 3]. This class of surfactants is capable of maintaining surface activity and emulsifications under extreme conditions of temperature and pH [4, 5]. According to Vinson [6], biosurfactants form microaggregates consisting of micelles, vesicles, and bilayers, and the morphology of these aggregates is affected by surfactant concentration [7], pH [8], ionic strength [9], and co-solutes such as alcohols or metal contaminants [8, 10]. Rhamnolipids have a wide variety of applications and have demonstrated great potential for use in decontamination of water and soil polluted by oil and heavy metals [11]. Furthermore, rhamnolipids can be used as emulsifiers and solubilizers in the food processing, cosmetic, and pharmaceutical industries. Many of the industrial and environmental applications in which biosurfacants are used involve extreme conditions of pH, pressure, and temperature, and the possible presence of additives such as ethanol and NaCl. If rhamnolipids are to replace existing chemical surfactants in such applications, they must be able to maintain their surface activity, emulsification, and aggregation characteristics under the conditions described above. The rhamnolipids produced by P. aeruginosa have been described by Lang and Wagner [12] as a mixture of the following species homologs: RL1 – Rha2C10C10; RL2 – RhaC10C10; RL3 – Rha2C10 and RL4 – RhaC10. According to Abdel-Mawgoud [13] there are *60 homologs produced by Pseudomonas species and other bacterial families. The physical properties of rhamnolipids depend on the composition of the homologues, which in turn is determined by the bacterial strain, culture conditions, and medium composition [14]. Identification of the homologues which comprise a rhamnolipid mixture is of paramount importance for characterizing its physical properties and potential applications. This study was conducted to evaluate the effects of NaCl and ethanol addition on rhamnolipid solutions, as well as the synergistic effects of additives and pH variations on the physical properties of rhamnolipids. Additionally, analysis of fatty acids and rhamnolipid homologues produced using different carbon sources was performed by mass spectrometry.

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

2 Materials and Methods

2.2.2.2 Turbidity measurements

2.1

Turbidity of the prepared biosurfactant solutions was measured at 600 nM using a Shimadzu, UV-2550 UV/Vis spectrophotometer (Shimadzu, Kyoto, Japan) at room temperature. All measurments were made in triplicate.

Rhamnolipid production

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P. aeruginosa strain LBI 2A1 was used for the production of rhamnolipids. Culture media and growth conditions used for rhamnolipid production were previously described by Muller [15]. Fermentations, using corn soapstock and sunflower oil as carbon sources were performed in a bioreactor, while rhamnolipids from castor oil was carried out in Erlenmeyer flasks at 37 8C, 200 rpm agitation, for 120 h on a rotary shaker. Ramnolipids isolated from sunflower oil were used to characterize the properties of solutions, and rhamnolipid homologues produced during fermentation of P. aeruginosa with 3 different carbon sources were profiled. 2.2

Rhamnolipid Extraction

Fermented broth was centrifuged at 4 000 rpm for 30 min, and equal volumes of cell free supernatant and n-hexane were thoroughly mixed in a volumetric flask. The mixture was then allowed to settle until the organic and aqueous phases separated. The organic phase was removed and 85 % H3PO4 1 : 100 (v/v) was added to aqueous phase to cause preciptiation of rhamnolipids. The biosurfactants were extracted with ethyl acetate 1 : 1.25 (v/v). The mixture was then shaken for 10 min, allowed to settle, and the upper phase was removed. This extraction process was then repeated using the lower phase. The extracted rhamnolipids were concentrated using a rotary evaporator, and the viscous yellowish product was dissolved in methanol and concentrated again for evaporation of residual solvent at 45 8C. 2.2.1

Rhamnolipid Solution

Solutions of rhamnolipids were prepared in ultrapure water at concentrations of 1.0, 10, 20, 30, 40, 50, 100, 250, 500, and 1 000 mg/L. The influence of pH was studied in the range of pH 4 – 8; and pH was adjusted using HCl (0.1 M) and NaOH (0.1 M), respectively. The effects of additives on rhamnolipid solutions were evaluated by addition of NaCl (0.4 – 0.8 M) and C2H5OH (0.3, 0.5, and 0.7 M). Control solutions were prepared: 1. without additives, and 2. without pH adjustment. These solutions were used for emulsification testing and measurments of surface tension and turbidity. 2.2.2 Emulsifying Activity (E24)

A 2-mL portion of kerosene was added to 2 mL of rhamnolipid solution and the mixture was shaken at high speed for 2 min. All tubes were kept closed to avoid evaporation. After 24 h, the emulsification index was calculated by dividing the measured height of the emulsion layer (EH) by the total height (TH) of the mixture, and multiplying by 100 (Equation 1). E24 ¼ ðEH=THÞ 2.2.2.1

100

Mass spectrometry

Electrospray ionization mass spectra were recorded on a high-resolution Q-Tof (Micromass, UK) mass spectrometer with a quadrupole (Qq) orthogonal time-of-flight configuration. The equipment was calibrated using H3PO4. Rhamnolipid samples were individually diluted with a solution of methanol/water (1 : 1) + 0.1 % ammonium hydroxide to ensure deprotonation of rhamnolipids and injected directly onto the ESI ionization device via a syringe pump at a flow of 10 mL/min. The spectra were acquired in negative mode in a mass range 100 – 1 500 m/z using a capillary voltage of 3.5 kV, and a cone voltage of 35 V; desolvation gas was heated at 100 8C. ESI tandem mass spectra were acquired by mass-selecting the target ion using a quadrupole mass analyzer, followed by 25 eV collision induced dissociation with argon in the collision cell. The rhamnolipid material was dissolved in methanol : water (1 : 1, v/v), filtered (0.22 mm), and introduced into the source at a rate of 15 mL/min–1 with a syringe pump. 3 Results and Discussion 3.1

Effect of NaCl on rhamnolipid solutions

The surface tension of ultrapure water decreased with increasing concentrations of rhamnolipids. As shown in Fig. 1, there was an initial transition point at 30 mg/L, above these concentrations decrease of measures was lower, the second transition point was at 100 mg/L, where the measures tended to stabilization. A profile of surface tension versus rhamnolipid concentration has been previously presented in the literature. Pornsunthorntawee [4] found two transition zones, one at 40 mg/L, which was associated with formation of monorhamnolipid micelles, and another at 200 mg/L, which was regarded as the critical micelle concentration (CMC) of the biosurfactant used in the study. Cohen and Exerowa [16] reported the presence of three transition points, which were related to the early formation of aggregates, formation of mono-rhamnolipid micelles, and the CMC of the rhamnolipid mixture.

ðEquation1Þ

Surface Activity Measurements

Surface tension was determined by the Du-No y ring method with a Kr ss K6 Tensiometer (Kr ss, Hamburg, Germany). Water ST was measured at the start of each set of experiments to calibrate the tensiometer. Experiments were performed at (25 € 1) 8C, and all measurements were made in triplicate.

398

2.3

Figure 1 Effect of NaCl (0.4 and 0.8 M) on the surface tension (mN m–1) of rhamnolipid solutions with concentrations ranging from 1 to 1000 mg L–1

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

The presence of homologues in different proportions and with varying degrees of hydrophobicity is due to variations in the number of transition areas, leading to micelle formation at different rhamnolipid concentrations [16, 17]. In the current study, there was a gradual reduction of surface tension at rhamnolipid concentrations ranging from 1 to 20 mg/L. This resulted from the increased number of monomers that were arranged on the surface of the solution, producing changes in surface activity. At rhamnolipid concentrations between 20 and 30 mg/L, there was a high concentration of free monomers organized at the air/liquid interface, producing the greatest slope in the surface tension curve. At concentrations > 30 mg/L, the decrease in slope was reduced due to formation of aggreagates which decrease the availability of free monomers. The CMC of the rhamnolipid mixture was *100 mg/L, and there was a tendancy for meansurements of surface tension to stabilize above this concentration. Addition of NaCl produced no change in the surface activity of rhamnolipid solutions prepared at concentrations of 1 and 10 mg/L. The transition zones observed for the control solutions remained the same at each NaCl concentration evaluated (Fig. 1). The effect of NaCl addition decreased with increasing concentrations of rhamnolipids. While surface tension initially appeared to be lower in rhamnolipid solutions containing NaCl, addition of NaCl did not affect the surface tension of rhamnolipid solutions > 200 mg/L concentration. Helvacı [18] reported that addition of NaCl to rhamnolipid solutions caused a decrease in surface tension due to the interaction of electrolytes with surfactant carboxyl groups. This interaction caused a reduction in repulsion between rhamnolipid monomers and a subsequent packaging effect which increased surface activity. The minimum surface tension of rhamnolipid solutions containing NaCl was not altered, indicating that the biosurfactant produced by P. aeruginosa LBI 2A1 cultured in sunflower oil is tolerant to increased ionic strength, and maintains its surface activity. This characteristic makes it suitable for use in bioremediation of sea water in coastal regions. The relationship between turbidity and formation of rhamnolipid micelles and aggregates has been previously descibed [4, 7]. Therefore, the turbidity of a control solution was measured to assess the increase in the numbers and sizes of rhamnolipid aggregates resulting from increased concentration (Fig. 2). At low rhamnolipid concentrations, there was a small increase in turbidity measurements which intesified after reaching a concentration of 100 mg/L. This indicated an in-

crease in the number and size of aggregates precisely at the CMC of this biosurfactant. A rhamnolipid solution with a concentration of 1.0 g/L demonstrated a maximum absorbance of 0.65, and the slope of the turbidity line indicated stabalization of the trubidity values. When compared to a control solution, addition of NaCl to rhamnolipid solutions did not produce a decrease in turbidity measurements. Increasing NaCl concentrations from 0.4 to 0.8 M further reduced the numbers and sizes of surfactant aggregates (Fig. 2). Emulsification tests performed with rhamnolipid solutions containing salt and kerosene as hydrophobic agents showed no emulsions between phases after 24 h. However, these solutions maintained their ability to reduce surface tension in the presence of NaCl. Also, as previously mentioned by Lovaglio [5], addition of a small concentration of NaCl (10–3 M) was beneficial for emulsification of benzene, kerosene, and soybean oil. Measurements of turbidity and surface tension were made using rhamnolipid solutions (1.0 g/L) in the pH range of 4.0 – 8.0 (Fig. 3). Changes in pH did not cause significant changes in the surface tension of solutions, as the greatest difference between measurements at various pH values was 0.3 mN/m. However, changes in pH did cause changes in the aggregation behavior of rhamnolipids, as reflected by changes in turbidity. When compared to a solution without pH adjustment, the absorbance of the solutions at pH 4, 7, and 8 diminished; however, a significant increase in turbidity was observed for solutions at pH 5 and 6, which coincided with a small decrease in surface tension in this pH range. Ishigami [19] reported that the pKa for rhamnolipids is 5.6. Therefore, rhamnolipid solutions with pH values between 5 and 6 should contain both protonated and non-protonated monomers, which should facilitate interaction between monomers due to a lack of electrostatic repulsion, and favor formation of rhamnolipid aggregatges. We next evaluated the effect of pH on the turbidity of rhamnolipid solutions containing 0.4 and 0.8 M NaCl. Control solutions consisted of (1) a solution without NaCl and pH adjusted solution in a range from 5.0 to 8.0 and (2) an NaCl solution without pH adjustment (Fig. 4). The rhamnolipid solutions in which pH was adjusted showed increased tubidity due to formation of rhamnolipid aggregates throughout the range of pH values tested. When compared to the control solution (without pH adjustment, but containing NaCl), addition of NaCl resulted in an increase in the numbers and sizes of aggregates formed in solutions at pH 6, 7, and 8. In the pH range of 5 – 6, increasing salt concentrations caused a slight reduction in the OD of the solution. At a

Figure 2 Effect of NaCl (0.4 and 0.8 M) on turbidity of rhamnolipid solutions with concentrations ranging from 1 to 1000 mg L–1

Figure 3 Turbidity and surface tension measurements of rhamnolipid solutions (1.0 g L–1) with pH ranging from 4.0 to 9.0

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

The surface tension curves of rhamnolipid solutions containing varying amounts of ethanol were very similar and the CMC values of these solutions were reduced when compared to the control solution. Additionally, measurements of surface tension became stabilized at rhamnolipid concentratons < 100 mg/L (Fig. 5). Products used for cosmetic and cleaning purposes normally contain alcohol, and rhamnolipids can be incorporated into these products as emulsifying agents. It is therefore important to determine the biosurfactant activity of rhamnolipids in the presence of alcohol. Pornsunthorntawee [4] reported that addition of increasing amounts of ethanol caused an increase in the CMC of rhamnolipids produced by P. aeruginosa SP4. The analyzed soultions were prepared in phosphate buffer with pH 7.4. Under such conditions, the rhamnolipid molecules are deprotonated and hence more susceptible to reaction with ad-

ditives used in the current study. In the present study, measurements of surface tension were obtained using rhamnolipid solutions prepared with ultra pure water, and the pH values after addition of ethanol and NaCl were 4.30 € 0.04 and 4.0 € 0.5, respectively. Therefore, in these solutions, the rhamnolipids were protonated and less reactive. Addition of increasing amounts of ethanol caused a rise in turbidity measurements of rhamnolipid solutions, indicating that alcohol promotes aggregation of surfactant monomers (Fig. 6). This result corroborates to those obtained for surface tension measurements, which showed decreases in the CMC of rhamnolipids produced by P. aeruginosa LBI 2A1. In solutions with a controlled pH, increases in ethanol concentration did not produce a significant difference between turbidity measurements at each pH value. However, a decrease in hydrogen ion concentration was accompanied by a decrease in absorption (OD) at all conditions tested (Fig. 7a). These results agree with those of Pornsunthorntawee [4], which showed a reduction in aggregates following addition of ethanol at pH 7.4. While the results presented above (Fig. 6) indicate increased aggregation behavior with addition of alcohol, the pH of the solutions was 4.30 € 0.04, demonstrating the influence of hydrogen ions on the solution properties of rhamnolipids. Changes in pH can influence the structures formed by rhamnolipid monomers at concentrations above the CMC. Changes in the morphology of rhamnolipid aggregates from lamellar structures to vesicles and micelles with increasing pH have been reported to occur between pH 5.8 and 8.0 [8]. Rhamnolipid solutions containing ethanol but without pH adjustment were evaluated for their ability to emulsify kerosene. After 24 h, there was no emulsion between the hydrophilic and hydrophobic phases. We therefore adjusted the pH of the solutions in an attempt to increase emulsification activity (Fig. 7b). We next quantified the emulsification activity (E24) of rhamnolipid solutions containing ethanol throughout an entire range of pH values and ethanol concentrations. Solutions containing 0.7 M ethanol demonstrated higher rates of emulsification; however, there was no significant difference in emulsion formation at the different pH values tested. Addition of ethanol to rhamnolipid solutions at pH 7.4 increased the efficiency of Sudan III dye encapsulation. Ethanol may act as a co-surfactant and interact with rhamnolipid molecules through hydrogen bonding, leading to formation of rhamnolipid-ethanol aggregates, which can increase the solubility of nonpolar dyes such as Sudan III [4].

Figure 5 Effect of ethanol on surface tension (mN m–1) of rhamnolipid solutions with concentrations ranging from 1 to 1000 mg L–1

Figure 6 Effect of ethanol on turbidity of rhamnolipid solutions with concentrations of 1–1000 mg L–1

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Figure 4 Effect of pH on turbidity of rhamnolipid solutions (1 g L–1) containing NaCl (0.1, 0.4, and 0.8 M) as an additive

neutral pH, addition of two different concentrations of salt resulted in similar aggregation behaviors. In basic pH there is an inversion of the relations mentioned for pH 5 and 6, since increasing salt concentrations caused a rise in turbidity measurements of solutions. Increasing concentrations of OH– favor deprotonation of rhamnolipids, which facilitates binding of Na+ ions, decreases electrostatic repulsion, and favors formation of aggregates at higher NaCl concentrations. Emulsification tests were conducted using rhamnolipid solutions containing salt and with pH adjustment. After 24 h, no emulsion was detected between the phases. 3.2

Effect of ethanol on rhamnolipid solutions

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(a) Figure 7

(b) Effect of pH on turbidity (a) and emulsifying activity (b) of rhamnolipid solutions (1 g L–1) containing ethanol (0.3, 0.3, and 0.3 M) as an additive

Our results showed that addition of ethanol to rhamnolipid solutions did not cause changes in surface activity, and favored an increase in the number and size of aggregates formed. Also, emulsification activity was increased in the presence of alcohol (0.3, 0.5, and 0.7 M), when the pH of the solutions was adjusted to 5, 6, 7 or 8. Our results also demonstrated that rhamnolipids can be successfully used as biosurfactants in industrial processes which require the presence of additives and involve varia-

tions in pH. As observed in experiments using alcohol as an additive, control of pH can exert a positive effect on maintaining the emulsifying activity of rhamnolipids. 3.3

Characterization of rhamnolipid homologs

ESI(-) – MS analysis was used the examine the structures of rhamnolipids produced by P. aeruginosa LBI 2A1 cultured with different carbon sources. Figure 8 shows the fragmen-

Figure 8 ESI(-) – MS from rhammnolipids produced by P. aeruginosa LBI 2A1 cultured with different carbon sources (a) sunflower oil (b) corn soapstock (c) castor oil

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

tation ions from mass spectra obtained from rhamnolipids produced from sunflower oil (Fig. 8a – m/z 213, 239, 279, 281, 325, 475, 503, 649, and 677); corn soapstock (Fig. 8b bm/z 131, 213, 239, 279, 281, 299, 329, 475, 503, 649, and 677) or castor oil (Fig. 8c m/z 131, 213, 239, 241, 257, 297, 409, 411, 427, 437, 453, 481, 503, and 649). These results show that different compositions and ratios of homologues were produced by P. aeruginosa cultured with each different carbon source. According to Catharino [20], ions m/z 279 and m/z 281 correspond to linoleic and oleic acid, respectively, which are fatty acids present at high concentrations in both corn soapstock and sunflower oil [21]. Therefore, it appears that these fatty acids are not produced by P. aeruginosa strain LBI 2A1. The free and associated b-hydroxy acids produced by P. aeruginosa LBI 2A1 cultured using three different carbon sources are shown in Table 1. P. aeruginosa cultured using different carbon sources produced lipids containing different compositions and ratios of fatty acids. The profiles of b-hydroxy acids produced from sunflower oil and corn soapstock were similar to those previously described in the literature [22, 23]. According to D ziel [24], these fatty acids are not derived from degradation of rhamnolipids, but rather are rhamnolipid intermediates. Hydroxy-fatty acids have surfactant activity, and are capable of reducing the surface tension of water below the levels achieved Elemental Composition Structure/Unsaturations b-hydroxy acid

with rhamnolipids. D ziel [24] also presented additional evidence for the surfactant properties of hydroxy-fatty acids. Mutant strains of Pseudomonas rhlB– capable of producing hydroxylated fatty acids, but not rhamnolipids, demonstated a swimming type of motility which was dependent on surfactant agents, while rhlA– mutants showed a lack of motility. P. aeruginosa cultured using castor oil as a carbon source produced high amounts of hydroxylated fatty acids. The ion m/z 257 was observed only in such samples, with a relative abundance of 84 %, and the fragmentation pattern corresponded to dihydroxy C14:1 (Fig. 9a). Ion m/z 437 indicated the presence of a b-hydroxy acid identified as C14:1 C12:1 (Fig. 9b). According to the compilation of existing rhamnolipid counterparts [13, 22, 25], there are previous no records of a fatty acid with such a structure. The production of di-and tri-hydroxy acids by Pseudomonas aeruginosa from ricinoleic acid, the main component of castor oil, has been previously described by Kim [26] and Kuo [27]. These investigators reported the production of 10,12-dihydroxy-8-octadecenoic acid and 7,10,12-trihydroxy-8-octadecenoic, respectively. The addition of hydroxyl groups to fatty acids increases their viscosity and reactivity, allowing their use in production of resins, lubricants, cosmetics, plastics, and other synthetic materials. Furthermore, several hydroxylated fatty acids have shown antimicrobial activity against fungi and bacteria [28 – 30].

Nomenclature/ [M-H]– Fragmentations m/z

Relative Abundance (%) S.O

C.S

C.O

C12H22O3/C12 : 1

b-hydroxydodecenoil [213] 55,59 e 113

7

32

100

C14H24O3/C14 : 2

b-hydroxy tetra-di-decenoil [239] 59

40

71

13

C14H26O3 /C14 : 1

b-hydroxy tetradecenoil [241] 59, 127, 205, 223

14

36

86

C14H26O4 /C14 : 1-OH

b-di-hydroxy tetradecenoil [257] 59, 81, 83, 197

0

0

84

C16H26O5 C10 : 2C6

b-hydroxy-di-decenoil-b-hydroxihexanoate

8,5

22

30

C18H34O3 C18 : 1- OH

ou ácido ricinolêico [297] 113, 183, 279

C16H28O5

b-hydroxy-octenoil-b-hydroxioctanoate

0

67

0

C 8 : 1C 8

[299] 59, 141, 155, 253, 281

C18H32O5

b-hydroxy-octanoil-b-hydroxidecenoate

8

13

0

C8 C10 : 1

[327] 159, 213, 281 5,5

14,5

0

5

6

0

0

0

12,7

0

0

14

0

0

41

0

0

43

0

0

29

C18H34O5

b-hydroxy-octanoil-b-hydroxidecanoate

C10C8/C8C10

[329] 59, 159, 187, 283, 311

C20H38O5

b-hydroxy-decanoil-b-hydroxidecanoate

C10C10

[357] 159, 187, 215, 239, 279, 311

C24H42O5

b-hydroxy-dodecenoil-b-hydroxidodecenoate

C12: 1C12 : 1

[409] 151, 195, 213

C24H44O5

b-hydroxy-dodecanoil-b-hydroxidodecenoate

C12: 1C12/C12C12: 1

[411] 113, 197, 213, 215

C26H46O5

b-hydroxi-dodecenoil-b-hydroxitetradecenoato

C12: 1C14 : 1

[437] 213, 223, 241

C26H46O6 C12 : 1C14 : 1/ 14 : 1C12 : 1

b-hydroxi-dodecenoil-b-dihydroxitetradecenoato

C28H50O6

b-hydroxy-tetradecenoil-b-dihydroxitetradecenoate

C14 : 1C14 : 1/C14 : 1C14 : 1

[481] 213, 223, 239, 241, 257, 285

[453] 195, 213, 239, 257

Table 1 Chemical composition and relative abundance of fatty acids produced by Pseudomonas aeruginosa LBI 2A1 (S.O – sunflower oil; C.S – corn soapstock; C.O – castor oil)

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

(a)

(b)

A total of 9 different rhamnolipid homologues were produced by P. aeruginosa LBI 2A1 under the conditions used in this study, and these homologues included both mono-and dirhamnolipids. Except for RhaC8C10 (m/z 475), which was not produced when castor oil was used as a carbon source, numerous other homologues were produced in various proportions by cells grown with all other carbon sources, and ions m/z 503 (RhaC10C10) and 649 (RhaRhaC10C10) were prevalent in all samples (Table 2). Nitschke [31] reported the production of 10 different rhamnolipid homologs by P. aeruginosa LBI when using soybean soapstock as a carbon source. The variations seen in homologue production are primarly due to the bacterial strain, culture medium, culture conditions, and culture age [9]. The physico-chemical properties of rhamnolipid mixtures can be influenced by the types and content of individual homologues. For example, mono-rhamnolipids have a higher critical micelle concentration and will bind to metals more strongly than cationic dirhamnolipids [32]. The profiles of rhamnolipids produced by P. aeruginosa LBI 2A1 cultured with different carbon sources were very similar; however, this was not true for the hydroxylated fatty acids. When castor oil was used as a carbon source, there was a high abundance of ions corresponding to fatty acids with alkyl chains ‡ 10 carbon atoms, and the use of castor oil may have favored synthesis of such fatty acids. Mata-

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Figure 9 ESI(-) – MS/MS from ions m/z 257 (a) and m/z 437 (b) from rhamnolipids produced by P. aeruginosa LBI 2A1, using castor oil as the carbon source

Sandoval [9] observed that variations in carbon source did not change the composition of rhamnolipids produced by P. aeruginosa UG2, which corroborates these results. Moreover, D ziel [33] described qualitative and quantitative changes in rhamnolipid homologues synthesized by P. aeruginosa 57RP cultured in the presence of mannitol or naphthalene. The two major ions resulting from m/z 503 fragmentation (Ramnosil-b-hidroxydecanoil-b-hydroxydecanoate) were m/z 333 and 169, which originate from the rupture of the ester bond between the alkyl chains, resulting in fragments RhaC10 (m/z 333) and C10 (m/z 169). The same rupture occurred for RhaRhaC10C10 (m/z 649). The greater abundance of ions related to counterparts RhaC10C10 and RhaRhaC10C10 indicates ramnosyl transferase (RhlB) preference for substrates with ten carbons, and this is reinforced by the low abundance of ions corresponding to b-hydroxydecanoyl-b-hydroxydecanoate fatty acids (Table 1). According to L pine [22], the enzyme rhamnosyl transferase (RhlB) has a preference for long-chain, saturated substrates. Fragmentation of ions corresponding to rhamnolipids containing alkyl chains with different carbon numbers allowed us to determine the position of fatty acids in the rhamnolipid structure. The fragments generated after rupture of the ester bonds between the fatty acids in these

403

Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

Elemental Composition Rhamnolipids

C24H44O9

Structure/ Unsaturations RhaC8C10(a)

[M-H]–m/z

Relative abundance (%) S.O

475

(a)2,9

8

RhaC10C8(b) RhaC10C10

503

C28H50O9

RhaC10C12: 1/RhaC12: 1C10

529

C28H52O9

RhaC10C12(a)

531

C32H58O13

100 7,8 (a)3,7

9

(b)

Tenside Surfactants Detergents downloaded from www.hanser-elibrary.com by Hanser Verlag (Office) on October 7, 2014 For personal use only.

C34H60O13

RhaRhaC8C10

621

3

RhaRhaC10C10 RhaRhaC10C12 : 1

(c)

RhaRhaC10C12(e) RhaRhaC12C10

0

100

100

8,8

12

10

16

2,4

5

3,6

91

81

8

8,5

14

18,8

1,8 649 675

73 7

RhaRhaC12: 1C10(d) C34H62O13

9,7

(b)1,7

RhaRhaC10C8 C32H58O13

C.O

(b)2,6

C26H48O9

RhaC12C10

C.S

(c)6,7 (d)0,5

677

12,7

(f)

(e)8 (f)2,5

Table 2 Chemical composition and relative abundance of rhamnolipid homologues produced by Pseudomonas aeruginosa LBI 2A1 (S.O – sunflower oil; C.S – corn soapstock; C.O – castor oil)

rhamnolipids were: m/z 475 (RhaC8C10/RhaC10C8) – m/z 305 (RhaC8), 333 (RhaC10): m/z 531 (RhaC10C12/RhaC12C10) – m/z 333 (RhaC10), 361 (RhaC12): m/z 621 (RhaRhaC8C10/ RhaRhaC10C8) – m/z 451 (RhaRhaC8), 479 (RhaRhaC10): m/z 675 (RhaRhaC10C12 1/RhaRhaC12: 1C10) – m/z 479 (RhaRhaC10), 505 (RhaRhaC12: 1): m/z 677 (RhaRhaC10C12/ RhaRhaC12C10) – m/z 479 (RhaRhaC10), and 507 (RhaRhaC12). In all cases analyzed, there was a tendency for rhamnolipids with shorter alkyl chains to be associated with rhamnose. This tendancy was even greater when an unsaturated fatty acid (RhaRhaC10C12:1) was present, with the ion corresponding to RhaC10 showing a relative abundance of 96 %. D ziel [34] observed similar results, and reported a higher abundance of homologues with short-chain fatty acids adjacent to the sugar moiety. The results obtained with fermentation using different carbon sources and subsequent analysis by mass spectrometry suggest that in the presence of a hydrophobic carbon source, synthesis of the lipid portion of rhamnolipids may not involve a de novo process. This is because use of a hydroxylated fatty acid (ricinoleic acid) as a carbon source resulted in production of di-hydroxylated fatty acids. According to Zhu and Rock [35], the stereochemistry of b-hydroxy fatty acids in rhamnolipids corresponds to the stereochemisty of intermediates produced during fatty acid biosynthesis, and not the intermediates of b-oxidation. This suggests that the lipid portions of rhamnolipids are synthesized by the classical route of fatty acid synthesis, using two carbon units (FASII). According to Schweizer and Hoang [36], the FASII enzymatic machinery of P. aeruginosa is the same as that described for E. coli. However, our results suggest the presence of other enzymes that may be responsible for diversion of fatty acids away from b-oxidation and towards synthesis of rhamnolipids. As previously described, when castor oil was used as a cabon source, the highest relative abundance of MS ions was related to fragmentation of hydroxy acids. In this case, rhamnose production might have been the limiting factor for rhamnolipid biosynthesis, since, from oxidation of fatty

404

acids provided as carbon source there is deviation of intermediates for hydroxy acids synthesis, reducing acetyl-CoA formation, that would be used for rhamnose production. 4 Conclusions

Addition of NaCl to rhamnolipid solutions does not alter their surface activity, but does decrease the size and number of aggregates formed in solutions without pH control. However, NaCl solutions in which the the pH is controlled at 6, 7 or 8 showed increased aggregation behavior. Addition of ethanol to rhamnolipid solutions reduces critical micelle concentration and favors aggregation of monomers. Such solutions show emulsification activity only under conditions of controlled pH. The profiles of fatty acids produced by P. aeruginosa LBI 2A1 varied according to the carbon source used. The fatty acid profile produced when using castor oil as a carbon source indicated that in the presence of a hydrophobic source, synthesis of the lipid portion of rhamnolipids may occur via b-oxidation, and not de novo synthesis. Acknowledgements

This work was supported by Fundażo de Amparo a Pesquisa do Estado de S¼o Paulo (FAPESP – Process 2007/03983-4). References 1. Parra, J. L., Guinea, J., Manresa, M. A., Robert, M., Mercadé, M. E., Comelles, F. and Bosh, M. P.: Chemical characterization and physicochemical behavior of biosurfactants. J. Am. Oil Soc. 66 (1989) 141 – 145. DOI:10.1007/BF02661805 2. Abalos, A., Pinazo, A., Infante, M. R., Casals, M., García, F. and Manresa, A.: Physicochemical and antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa AT10 from soybean oil refinery wastes. Langmuir, Washington 17 (2001) 1367 – 1371. DOI:10.1021/la0011735 3. Benincasa, M., Abalos, A., Oliveira, I. and Manresa, A.: Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock. Anton. Leeuw. Int. J. G. 85 (2004) 1 – 8. DOI:10.1023/B:ANTO.0000020148.45523.41 4. Pornsunthorntawee A. O., Chavadej, S. and Rujiravanit, R.: Solution properties and vesicle formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4. Colloid Surf. B-Biointerfaces 72 (2009) 1 – 6. DOI:10.1016/j.colsurfb.2009.03.006

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Roberta B. Lovaglio et al.: Rhamnolipids production by a pseudomonas aeruginosa LBI mutant

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Correspondence address Prof. Dr. Jonas Contiero UNESP – Universidade Estadual Paulista Department of Biochemistry and Microbiology Institute of Biological Sciences, Av. 24-A 1515 Bela Vista, CEP 13506-900 Rio Claro, SP Brazil Tel.: +5519 3526 4101 Fax: +5519 35 26 4176 E-Mail: [email protected]

The authors of this paper Roberta B. Lovaglio is a post doc at São Paulo State University. She works in the Industrial Microbiology Laboratory and is involved in the field of Biosurfactants Production. Vinícius L. da Silva is a Ph.D. student at São Paulo State University. He works in the Industrial Microbiology Laboratory and is involved in the field of Biosurfactants Production. Tulio de Lucca Capelini is master on applied microbiology and works with rhamnolipids production. Marcos N. Eberlin is a titular professor at Universidade Estadual de Campinas, and supervisor of ThoMSon Laboratory of Mass Spectrometry. Rudolf Hausmann works with biosurfactants production at Institute of Food Science and Biotechnology, Section Bioprocess Engineering, University of Hohenheim, Stuttgart and Karlsruher Institut für Technologie (KIT). Marius Henkel is Ph.D. student at Karlshure Institute of Technology. He works with rhamnolipids production. Jonas Contiero teaches Biochemistry and industrial Microbiology in the department of Biochemistry and Microbiology at Unesp-Univ. Estadual Paulista and heads a research team LMI (Industrial Microbiology Lab.), involved in the field of Metabolites Production by Microorganisms.

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