BIP - Production and stability studies of the bioemulsifier obtained from a new strain of Candida glabrata UCP 1002

Surfactants and emulsifiers are indispensable components of daily life. They are widely used in the pharmaceutical, cosmetic, petroleum and food industries (Makkar and Cameotra, 1998; Lang, 2002). Most of these compounds are of petroleum origin, which are not easily biodegradable and their manufacturing processes and by-products can be environmentally hazardous. Increased environmental awareness and strict legislation has made environmental compatibility of surfactants an important factor in their applications for various uses (Maier and Soberon-Chavez, 2000). Several different microbial products that exhibit surface-active properties have been identified in the past. These so called biosurfactants are produced by certain bacteria and by a number of yeasts and filamentous fungi. They include low-molecular-weight glycolipids, lipopeptides and high-molecular-weight lipid-containing polymers such as lipoproteins, lipopolysaccharide-protein complexes and polysaccharide-protein-fatty acid complexes (Ron and Rosenberg, 2001). Considering the biosurfactants are readly biodegradable and can be produced in large amounts by microorganisms and thus are not dependent on petroleum-derived products, they might be able to replace, in some instances, the traditional synthetic surfactants (Banat et al. 2000). The literature shows that a wide range of carbon sources, including agricultural renewable resources, like sugars and oils, are suitable carbon sources for production of ecologically safe biosurfactants with good properties. (Gallert and Winter, 2002). Among yeasts, Candida species have been widely employed for insoluble substrates fermentation and have been reported to produce surface active agents (Sarubbo et al. 1999; Sarubbo et al. 2001). The objective of this work was to investigate the production of a biosurfactant by Candida glabrata UCP 1002 (isolated from mangrove sediment collected in the City of Rio Formoso, Pernambuco State, Brazil (Gomes et al. 2000). The production of the emulsifier was carried out in Erlenmeyer’s flasks containing 100 ml of the production medium (0.1% NH₄NO₃,0.02% KH₂PO₄, 0.02% MgSO₄.7H₂O, 0.3% yeast extract and 7.5% cotton seed oil plus 5.0% glucose as substrates) and shaking at 200 rpm for 144 hrs at 27ºC.

An emulsion is formed when one liquid phase is dispersed as microscope droplets in another liquid continuous phase, stabilizing the emulsion. The emulsification activity is assayed by the ability of the surfactant to generate turbidity due to suspended hydrocarbons in an aqueous system. The emulsification index, a rapid and simple method used to describe the presence of an emulsion, as described by Cooper and Goldenberg (1987). The simple methodology consisted of 6 ml of n-hexadecane or cotton seed oil was added to 4 ml of the culture broth free of cells in a graduated screwcap test tube and vortexed at 3000 rpmfor 2 min. The emulsion stability was determined after 24 hrs, and the emulsification index was calculated by dividing the measured height of the emulsion layer by the mixture's total height and multiplying by 100.

Considering the potential of application of biosurfactants in the environment or in industries, it is important to study their behaviour under extreme conditions. Stability studies were done using the cell-free broth obtained centrifuging the cultures at 10000 x g for 15 min. 4 ml of the culture broth free of cells were heated at 80ºC, and cooled to room temperature, after which the emulsification activity was measured. The emulsification capacity of culture broth free of cells was also determined after exposure at lower temperature (0-4ºC). To study the pH stability of the cell-free broth, the pH of the cell-free broth was adjusted to different pH values (2 to 12) and the emulsification activity was measured. The culture liquid pH was adjusted with 1 M NaOH. The effect of NaCl concentrations (2 to 10%) on the emulsification capacity of the culture broth free of cells was also determined.

Surface tension at the air/water and oil/water interfaces can easily be measured with a tensiometer equipment. When a surfactant is added to air/water or oil/water systems at increasing concentrations, a reduction of surface tension is observed up to a critical level, above which amphyphilic molecules associate readily to form supramolecular structures like micelles, bilayers, and vesicles. This value is known as the critical micelle concentration (CMC). CMC is defined by the solubility of a surfactant within an aqueous phase. Surface tension and CMC were determined on cell-free broth with a Tensiometer model Sigma 70 (KSV Instruments LTD - Finland).

The isolation of the emulsifier was performed by solvent extraction. The cell-free broth was concentrated (500 ml) by freeze drying and extracted three times with chloroform (1:1, by vol.). Protein concentration in the isolated bioemulsifier was determined by the Lowry method (Lowry et al. 1951). Carbohydrates were determined by the phenol-sulfuric acid method (Hanson and Phillips, 1981). The lipid composition of the crude bioemulsifier was determined according to Manocha et al. (1980).

Results and Discussion

The kinetic behaviour is important to describe the profile of the bioemulsifier production related to growth. The Maximum biomass concentration was achieved after 72 hrs. After 48 hrs of growth, a diauxic behaviour was observed, probably due to the consumption of other substrate used in the fermentation. During the exponential growth phase, culture medium pH gradually decreased from 5.7 to 2.6, after which it remained around 3.0. Emulsification of cotton seed oil increased with increasing biomass formation, reaching its optimum nearly at about 24 hrs, and after 48 hrs of growth it showed with constant values around 75% until the end of cultivation.

For the measurements of surface tension, it was found that the emulsifier agent obtained from glucose plus cotton seed oil could lower the surface tension of water to 31 mN/m (CMC), which showed that it is a good surfactant.

The efficiency of the bioemulsifier containing cell-free broth showed that the biosurfactant form Candida glabrata was efficient in emulsificating the cotton seed oil once no significant variation in the emulsification index was observed for this substrate. The effect of added NaCl concentrations showed a reduction of approximately 20% of emulsification activity with the addition of up to 10% (w/v) sodium chloride for both substrates (cotton seed oil and hexadecane), showing a relative tolerance over these salt concentrations. The effect of thermal treatment on the emulsifier activity of Candida glabrata culture showed that no appreciable changes in emulsification capacity occurred, if the cell-free broth was heated, once only 10% of activity was lost at 80ºC. The lost of emulsifier activity could be explained by the denaturation of proteinaceous compounds of the bioemulsifier during heating. There was no significant change at lower temperature (4ºC). The pH of the cell-free broth was varied from 2 to 12 to test the effect of pH on emulsification capacity. No appreciable effect on activity was observed along the pH range, although it was observed an increase at pH 12, especially for cotton seed oil emulsification. Extremes of pH could possibly transform less surface-active species into more active emulsifiers by increased ionization.

The examined agent was isolated from the culture filtrate of Candida glabrata. The precipitate collected in the aqueous phase recovered 100% of the emulsification activity of n-hexadecane that was present in the culture filtrate, while the emulsification activity of the cotton seed oil increased 25%. The average yield of precipitate in the aqueous phase was approximately 10.0 g/l. The emulsification activity of both the isolated biosurfactant and the cell-free remained stable for a reasonable period (more than 4 weeks) under low temperature and upon sterilization.

Bioshyntesis of biosurfactants from a variety of bacteria and yeasts has been reported (Davila et al. 1992; Zhou and Kosaric, 1995; Daniel et al. 1999; Lang and Wulbrandt, 1999), most commonly involving rhamno-lipids, trehalose and sophorose-lipids. These usually contain various hydroxy fatty acids and carbohydrates and are characterized by unique surfactant properties (Ron and Rosenberg, 2001). Preliminary analysis of the bioemulsifier produced by the new strain Candida glabrata indicated that it was a heteropolymer, which consisted of 45% protein, 20% lipid and 10% carbohydrate. Liposan, an extracellular emulsifier synthesized by Candida lipolytica was composed of 93% carbohydrate and 7% protein (Cirigliano and Carman, 1984). Other polymeric emulsifiers containing proteins, carbohydrates and lipids were also produced by Candida lipolytica when grown in babassu vegetal oil (Sarubbo et al. 1999) or glucose (Sarubbo et al. 2001) as the sole carbon sources. The results obtained in this work showed that this new strain of Candida glabrata represents a valuable source of new compounds with surface-active properties, with potential of application in different industries.

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