Production of emulsifier by a strain of Production of emulsifier by a strain of Production of emulsifier by a strain of Production of emulsifier by a strain of Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa (

. The intention of this work was to evaluate the growth of Pseudomonas aeruginosa (C1 LBPVMA-UFAL) on lubricant oil and verify the production of rhamnolipid biosurfactant. Tests in solid medium containing lubricant oil supplemented with different sources of nitrogen were used to evaluate the growth of P. aeruginosa . The growth medium used for production of rhamnolipid was (g L -1 ): yeast extract, 0.1; NaCl, 1.0; MgSO 4 , 0.2; MnSO 4 .H 2 O, 0.02; 0.5% lubricant oil (w v -1 ). The biosurfactant concentration was detected 24h after the inoculation, during the exponential phase, with the maximum value obtained up to 120h, although no drastic alteration of pH had been verified. The emulsifier activity was also first detected after 24h of incubation, with maximum production after 120h. The characterization of the produced biosurfactant was performed through thin layer chromatography, which showed the presence of two spots with R f values equal to 0.71 and 0.5, revealed by reagents specific to rhamnolipids. These results suggested that two types of rhamnolipidic biosurfactant are produced by the strain of P. aeruginosa in limited conditions of nutrients, able to use lubricant oil as main carbon source. This bacterium, isolated from agro-industrial effluent, showed potential to bioremediation assays of contamination with petrol and his derivates.

Production of emulsifier by a strain of Production of emulsifier by a strain of Production of emulsifier by a strain of Production of emulsifier by a strain of Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa (C1 LBPVMA (C1 LBPVMA (C1 LBPVMA (C1 LBPVMA----UFAL) using lubricant oil as main carbon source UFAL) using lubricant oil as main carbon source UFAL) using lubricant oil as main carbon source UFAL) using lubricant oil as main carbon source

Introduction Introduction Introduction Introduction
Petrol is a complex mixture of many compounds such as alkanes, aromatics, resins and asphaltenes, which could potentially be eliminated by microbial degradation (BARATHI; VASUDEVAN, 2001).In general, microorganisms may require some physiological adaptation to mineralize or grow on substrates with low solubility in water.Through evolution, bacteria have adapted themselves to feeding on water-immiscible materials by manufacturing and using a surface active product that helps them in the aqueous phase.So, they Acta Scientiarum.Technology Maringá, v. 32, n. 1, p. 33-36, 2010 adsorb, emulsify and disperse or solubilize the water immiscible material (HEALY et al., 1996).The use of synthetic surfactants in treating hydrocarbon systems contaminating soil improves the restoration of these areas, increasing the surface for microbial attack.However, they may promote accumulation in the ecosystem causing serious environmental damage (CUNHA;LEITE, 2000).Biosurfactants in many cases have proved to be more effective than chemical surfactants, and have added benefit to be biodegradable (BANAT, 1995).The rate of hydrocarbon biodegradation is dependant of the biosurfactant effect, in two ways: by increasing solubilization and dispersion of the hydrocarbon, and by changing the affinity between microbial cells and hydrocarbons, by inducing increases in cell surface hydrophobicity (ZHANG;MILLER, 1995).
The best studied class of glycolipidic biosurfactants is the rhamnolipidc one, produced by several species of Pseudomonas.P. aeruginosa can produce them from substrates including alkanes, glycerol, olive oil and mannitol (LANG; WULLBRANDT, 1999).The great metabolic diversity of the Pseudomonas genus makes possible the use of these microorganisms for environmental treatment.The objective of this work was to evaluate the growth of different species of Pseudomonas on lubricant oil and verify the production of rhamnolipid biosurfactant.

Material and methods
Microorganism and screening of nitrogen source Microorganism and screening of nitrogen source Microorganism and screening of nitrogen source Microorganism and screening of nitrogen source The bacteria used in this work were isolated from agro-industrial effluent ('S.A. Usina Coruripe Açúcar e Álcool'), in the 'Laboratório de Bioquímica do Parasitismo Vegetal e Microbiologia Ambiental -LBPVMA', at the 'Instituto de Química e Biotecnologia' -'Universidade Federal de Alagoas/Brazil -UFAL'.Later, this isolate was identified by morphological and biochemical analysis as Pseudomonas aeruginosa.
The influence of nitrogen source was studied in the medium containing 0.5% lubricant oil (w v -1 ), 0.1% NaCl, 0.1% of one of the three different nitrogen sources [yeast extract, NaNO 3 or (NH 4 ) 2 SO 4 ] and 15.0 g L -1 agar.The inoculum volume was estimated to obtain approximately 1 x 10 3 cells mL -1 .The plates were incubated at 30ºC, in the dark, for 72h.The number of viable cells was measured in parcels of the liquid medium (cells mL -1 ), using a Newbauer chamber and light microscope (640 X).

Growth conditions Growth conditions Growth conditions Growth conditions
The growth medium used for production of rhamnolipid was (g L -1 ): yeast extract, 0.1; NaCl, 1.0; MgSO 4 , 0.2; MnSO 4 .H 2 O, 0.02.The fermentation process was carried out in 150 mL Erlenmeyer flasks, containing 50 mL of medium, in triplicate.After that, 0.5 % of lubricant oil (w v -1 ) was added.The final pH in the medium was adjusted to 7.0, using 0.1 N KOH.The inoculum volume was estimated to obtain approximately 1 x 10 5 cells mL -1 .Submerged microbial cultures were incubated in the dark, at 30ºC, 200 rpm, for 120h.The growth was monitored each 24h, by cell counting as well as optical density at 600 nm.

Analytical methods Analytical methods Analytical methods Analytical methods
Rhamnolipid concentration was determined in triplicate by using orcinol assay (CHANDRASEKARAN; BEMILLER, 1980).To 0.1 mL of each sample from the cells culture supernatant, 0.9 mL of a solution was added containing 0.19% orcinol (in 53 % H 2 SO 4 ), and the mixture was heated at 80ºC for 30 min.Then, the samples were cooled for 15 min.at room temperature and the A 421 nm was measured.The rhamnolipid concentration was evaluated based on a standard curve prepared with different concentrations of L-rhamnose (equivalent mg rhamnose mL -1 ).
The assay of emulsification activity was determined by the method of Cirigliano and Carman (1984) modified.To 2 mL of each sample from the cells culture supernatant, it was added 1 mL of cyclohexane, and then mixed using vortex for 2 min.The resulting uniform emulsion was allowed to sit for 10 min.After that, its absorbance was measured at 540 nm.The blank contained 2 mL of the filtered sterilized medium.

Screening of nitrogen Screening of nitrogen Screening of nitrogen Screening of nitrogen source source source source
The influences of the nitrogen source (yeast extract, ammonium and nitrate) on the growth of P. aeruginosa isolated was studied using the medium containing 0.5% lubricant oil expressed in log of the number of cells mL -1 , after 72h of incubation, in the dark, at 30ºC.The maximum concentrations of cells in media supplemented with yeast extract, NaNO 3 or (NH 4 ) 2 SO 4 were respectively 5.3 x 10 7 , 5 x 10 6 and 3.8 x 10 6 mL -1 (  (1984), which demonstrated the influences of nitrogen source (yeast extract) on biosurfactant production from P. aeruginosa DSM2659.According to these authors, in the absence of yeast extract, the biomass concentration of P. aeruginosa decreases, and a moderate accumulation of glucose occurs during the biosurfactant production.The results of both turbidity and viable cell counting indicate the production of biosurfactant by P. aeruginosa C1 using lubricant oil as the main carbon source.The initial concentration of biosurfactant was 29.62 eq mg rhamnose L -1 , reaching the maximum value around of 36 eq mg rhamnose L -1 at 120h after the inoculation.The results of cellular growth and biosurfactant production are shown in Figure 1.In comparison with data obtained by Déziel et al. (1996), who investigated the capacity of biosurfactant production by P. aeruginosa 19SJ on salt medium supplemented with 2% of naphthalene or 2% mannitol, separately, the isolate of P. aeruginosa studied here was more efficient in the production of biosurfactant, since the 19SJ strain had its maximal productivity around 1.6 and 0.4 eq mg rhamnose L -1 using mannitol and naphthalene, respectively.Zhang et al. (2005) suggested that the hydrocarbon biodegradation probably occurs with increase of cell surface hydrophobicity after extraction of lipopolysaccharides from the cellular envelope by rhamnolipids, which subsequently stimulates uptake via direct contact between cells and hydrocarbon droplets.This observation is supported by data of Ozdemir and Malayoglu (2004), which showed the role of R1 and R2 rhamnolipids produced by P. aeruginosa in the transport and assimilation of hydrocarbon.Abalos et al. (2004) verified that the consortium between P. aeruginosa and other microorganisms increased the biodegradation of crude oil, with subsequent rhamnolipid production.
Property emulsifier of biosurfactant produced in lubricant Property emulsifier of biosurfactant produced in lubricant Property emulsifier of biosurfactant produced in lubricant Property emulsifier of biosurfactant produced in lubricant oil medium oil medium oil medium oil medium The emulsification activity was first detected after 24h the incubation, with maximum production after 120h (Figure 1).Surface active agents assist degradation of hydrocarbon pollutants by facilitating desorption from the soil, and/or by dispersing small droplets that are more easily attacked by microorganisms.In previous work, Zhang and Miller (1995) demonstrated surfactant effects on hydrocarbon biodegradation, and it depends on the structure of the surfactant, on the physical state and amount of alkane dispersion and emulsification, and on the degrading isolate of microorganism.Although these factors are interdependent, each one can be considered separately in order to help interpret the system as whole.

Characterization of the biosurfactant obtained Characterization of the biosurfactant obtained Characterization of the biosurfactant obtained Characterization of the biosurfactant obtained
The biosurfactant extracted from the concentrated culture supernatant was analyzed by TLC, and visualized with specific reagents, producing spots with different Rf values (retention factor).The spots with Rf 0.71 and Rf 0.5 showed positive reactions for glycids (Molish reagent) and lipids (rhodamine B reagent), but negative reactions for amino groups (ninhydrin and UV), and can be seen in Table 2.These results suggested that P. aeruginosa C1 (LBPVMA-UFAL) produces two types of rhamnolipids in lubricant oil medium.In general, some authors reported monorhamnolipids as the predominant component of rhamnolipid surfactant mixture, whereas many reports described the dirhamnolipids as the main component present.In contrast, the predominance of mono or dirhamnolipid forms and the relationship with surfactant production characteristics are not well understood, but some attempts have been made (NITSCHKE et al., 2005).

Conclusion Conclusion Conclusion Conclusion
These results suggest a correlation between growth of the isolate of Pseudomonas aeruginosa obtained from agro-industrial effluent (C1 LBPVMA-UFAL) in oil lubrificant medium and the production of biosurfactant with emulsifier activity.Because of its origin, this strain of P. aeruginosa has also potential application in consortium of microorganisms to remediation of environments polluted by hydrocarbon oil derivatives.

Table 1 )
. The choice of the nitrogen Acta Scientiarum.Technology Maringá, v. 32, n. 1, p. 33-36, 2010 sources was based on the results here obtained and on the observations reported by Guerra-Santos et al.

Table 1 .
Screening of different nitrogen sources for growth of

Table 2 .
Thin layer chromatography parameters of the different forms of rhamnolipids found in the growth medium of Pseudomonas aeruginosa C1 (LBPVMA-UFAL), after 120h of incubation (in the dark, at 30ºC, 200 rpm).Bromocresol green: reagent used for detection of free carboxylic acid; d Rhodamine 6G: reagent used for detection of lipids; e Iodine vapours: reagent used for detection of organic compounds; f Molish: reagent used for detection of glycids. c