On-line version ISSN 0717-3458
Electron. J. Biotechnol. vol.9 no.4 Valparaíso July 2006
Production and stability studies of the bioemulsifier obtained from a new strain of Candida glabrata UCP 1002
Leonie Asfora Sarubbo
Juliana Moura de Luna
Galba Maria de
Financial support: The work was financed by
Keywords: biosurfactant, Candida, emulsifier, fermentation, glucose, vegetal oil.
Evaluation of both tenso-active and emulsifying activities indicated that a biosurfactant was produced by the newly isolated and promising strain Candida glabrata isolated from mangrove sediments. The extracellular water-soluble emulsifying agent was isolated and identified as a heteropolymer. The maximum of bioemulsifier production was observed when the strain was grown on soluble and insoluble substrates cotton seed oil plus glucose, reaching values of 10.0 g/l after 144 hrs at 200 rpm. The cell-free culture broth containing the examined agent lowered the surface tension of the medium to 31 mN/m. Stable and compact emulsions with emulsifying activity of 75% of cotton seed oil were detected. The emulsification capacity remained practically unaltered within a wide
Surfactants and emulsifiers
The success of biosurfactant production depends on the development of cheaper processes and the use of low cost raw
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 is to investigate the production of a biosurfactant by Candida glabrata isolated from mangrove sediments and to
Candida glabrata UCP 1002 was isolated from mangrove sediment collected in the City of
Cultures were grown on a mineral medium containing 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. The final pH of the medium was 5.7.
The Candida glabrata was grown in solid medium at
Emulsification activity was measured using the method described by Cooper and Goldenberg (1987) whereby 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 rpm for 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
The efficiency of the biosurfactant as an emulsifying agent was measured on the cell-free broth. Variations between
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
Surface tension and critical micellar concentration (CMC) were determined on cell-free broth obtained by centrifuging the cultures at 10000 x g for 15 min with a Tensiometer model Sigma 70 (KSV Instruments LTD - Finland) using the Du Nouy ring method at room temperature. The CMC was determined by measuring the surface tensions of dilutions of cell-free broth in distilled water up to a constant value of surface tension. Measurements of surface tension from distilled water and from the
The 144-h culture was refrigerated for 24 hrs to solidify the remaining oil and to effect yeast settling. The culture was filtered through Whatman no. 1 filter paper and centrifuged at 10000 x g for 15 min. The cell-free broth was concentrated (500 ml) by freeze drying and extracted three
Protein concentration in the isolated bioemulsifier was determined by the Lowry method (Lowry et al. 1951) using Bovine serum albumin as a
Figure 1 shows the biomass concentration, pH and emulsification index of Candida glabrata cultivation in mineral medium containing 7.5% of cotton seed oil plus 5% of glucose. 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. The profile of emulsification activity production was observed in three independently run fermentations. 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. Conversely, the emulsification of n-hexadecane started after the microorganism entered the stationary growth phase, with maximum activity of 67% after 96 hrs of cultivation.
For the measurements of surface tension of distilled water after different dilutions of the cell-free broth after 144 hrs of cultivation, it was found that the emulsifier agent obtained from glucose plus cotton seed oil could lower the surface tension of water (air-water interface) from 68 mN/m to 31 mN/m (CMC), which shows that it is a good surfactant (Figure 2). This result is similar to others surfactants produced by yeast from carbohydrate and vegetal oil as substrates (Davila et al. 1992; Zhou and Kosaric, 1995; Garcia-Ochoa and Casas, 1999).
The efficiency of the bioemulsifier containing cell-free broth is shown in Figure 3. The results 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, however, for n-hexadecane emulsification, the reduction of cell-free broth volume decreased the emulsification capacity.
The effect of added NaCl concentrations on n-hexadecane and cotton seed oil emulsification capacity of the cell-free broth is summarized in Figure
The effect of thermal treatment on the emulsifier activity of Candada 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
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.Bioemulsifier production by yeast Candida utilis varied from 0.26 to 0.93 g/l and depended on process conditions (Shepherd et al. 1995), while the extracellular emulsifying agent from Curvularia lunata yielded 2.6 g/l (Paraszkiewicz et al. 2002). 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 Wullbrandt, 1999), most commonly involving rhamno-lipids, trehalose and sophorose-lipids. These usually contain various hydroxy fatty acids and carbohydrates and
The results obtained in this work
CIRIGLIANO, M.C. and CARMAN, G.M. Isolation of a bioemulsifier from Candida lipolytica. Applied and Environmental Microbiology, October 1984, vol. 48, no. 4, p. 747-750. [ Links ]
COOPER, David G. and GOLDENBERG, Beena G. Surface-active agents from two Bacillus species. Applied and Environmental Microbiology, February 1987, vol. 53, no. 2, p. 224-229. [ Links ]
DANIEL, H-J.; OTTO, R.T.; BINDER, M.;
DAVILA, Anne-Marie; MARCHAL, Rémy and VANDECASTEELE, Jean-Paul. Kinetics and balance of fermentation free from product inhibition: sophorose lipid production by Candida bombicola. Applied Microbiology and Biotechnology, October 1992, vol. 38, no. 1, p. 6-11. [ Links ][CrossRef]
DESAI, J.D. and
GARCIA-OCHOA, F. and CASAS, J.A. Unstructured kinetic model for sophorolipid production by Candida bombicola. Enzyme and Microbial Technology, October 1999, vol. 25, no. 7, p. 613-621. [ Links ][CrossRef]
GOMES, P.F.; NASCIMENTO, A.E.; OKADA, K.;
HANSON, R.S. and PHILLIPS, J.A. Chemical Composition. In: GHERHARDT, P. ed. Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, DC, 1981, p. 328-364. [ Links ]
LANG, S. and WULLBRANDT, D. Rhamnose lipids-biosynthesis, microbial production and application potential. Applied Microbiology and Biotechnology, January 1999, vol. 51, no. 1, p. 22-32. [ Links ][CrossRef]
LOWRY, Oliver H.; ROSEBROUGH, Nira J.; FARR, A. Lewis and RANDALL, Rose J. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, November 1951, vol. 193, no. 1, p. 265-275. [ Links ]
MAIER, R.M. and SOBERON-CHAVEZ, G. Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Applied Microbiology and Biotechnology, November 2000, vol. 54, no. 5, p. 625-633. [ Links ][CrossRef]
MAKKAR, R.S. and CAMEOTRA, S.S. Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtillis. Journal of Industrial Microbiology and Biotechnology, January 1998, vol. 20, no. 1, p. 48-52. [ Links ][CrossRef]
MAKKAR, R.S. and CAMEOTRA, S.S. An update on the use of unconventional substrates for biosurfactant production and their new applications. Applied Microbiology and Biotechnology, March 2002, vol. 58, no. 4, p. 428-434. [ Links ][CrossRef]
MANOCHA, M.S.; SAN-BLAS, G. and CENTENO, S. Lipid composition of Paracoccidioides brasilienses: possible correlation with virulence of different strains. Journal of General Microbiology, March 1980, vol. 117, no. 1, p. 147-154. [ Links ]
O'DONNELL, K.L. Zygomycetes in culture. Palfrey Contributions in Botany.
PARASZKIEWICZ, Katarzyna; KANWAL, Anita and DLUGONSKI, Jerzy. Emulsifier production by steroid transforming filamentous fungus Curvularia lunata. Growth and product characterization. Journal of Biotechnology, January 2002, vol. 92, no. 3, p. 287-294. [ Links ][CrossRef]
SARUBBO, Leonie A.; MARÇAL, Maria Do Carmo; NEVES, Maria Luisa C.; SILVA, Maria Da Paz C.; PORTO, Ana Lúcia F. and CAMPOS-TAKAKI, Galba Maria. Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica. Applied Biochemistry and Biotechnology, July 2001, vol. 95, no. 1, p. 59-68. [ Links ]
SHEPHERD, Rachel; ROCKEY, John; SHUTHERLAND, Ian W. and ROLLER, Sibel. Novel bioemulsifiers from microorganisms for use in foods. Journal of Biotechnology, June 1995, vol. 40, no. 3, p. 207-217. [ Links ][CrossRef]
ZHOU, Q.-H. and KOSARIC, N. Utilization of Canola Oil and lactose to produce biosurfactant with Candida bombicola. Journal of the American Oil and Chemists' Society, 1995, vol. 72, p. 67-71. [ Links ]
Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.