Identification and characterization of bioemulsifier

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Bioresource Technology 101 (2010) 5186–5193

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Identification and characterization of bioemulsifier-producing yeasts isolated from effluents of a dairy industry A.S. Monteiro a,*, M.R.Q. Bonfim a, V.S. Domingues a, A. Corrêa Jr. a, E.P. Siqueira b, C.L. Zani b, V.L. Santos a a b

Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, 31270-901, Belo Horizonte, MG, Brazil Laboratório de Química de Produtos Naturais, Centro de Pesquisas René Rachou Fundação Oswaldo Cruz, 30190-002, Belo Horizonte, MG, Brazil

a r t i c l e

i n f o

Article history: Received 2 October 2009 Received in revised form 7 February 2010 Accepted 8 February 2010 Available online 15 March 2010 Keywords: Bioemulsifier Yeasts Dairy industry

a b s t r a c t New bioemulsifier-producing yeasts were isolated from the biological wastewater treatment plant of a dairy industry. Of the 31 bioemulsifier-producing strains, 12 showed emulsifying activity after 2 months of incubation, with E24 values ranging from 7% to 78%. However, only Trichosporon loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 exhibited high emulsion-stabilizing capacity, with E24 values of 78%, 67%, and 66%, respectively. These isolates were shown to induce a strong emulsion stabilizing activity rather than the reduction of the interfacial tension. These strains exhibited similar growth rates in the exponential growth phase, with a clear acceleration after 24 h and stabilization of the activity after 144 h. Emulsification and stability properties of the bioemulsifiers were compared to those of commercial surfactants after the addition of NaCl and exposure to temperature of 100 °C. The compounds produced by the isolates appeared to be lipid–polysaccharide complexes. Gas chromatograph analysis of the lipidic fraction of the bioemulsifiers from CLV20, CLOA40, and CLOA70 shows the prevalence of (9Z,12Z)-octadeca-9,12-dienoic acid, in concentrations of 42.8%, 25.9%, and 49.8%, respectively. The carbohydrate composition, as determined by GC–MS of their alditol acetate derivatives, showed a predominance of mannose, galactose, xylose and arabinose. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Biosurfactants are amphiphilic compounds produced by a variety of microorganisms that contain hydrophobic and hydrophilic moieties. These compounds are able to accumulate between fluid phases, thus reducing surface and interfacial tension at the fluid surface and interface (Desai and Banat, 1997). They are of two types, low-molecular weight biosurfactants which are generally glycolipids and lipopeptides and high-molecular weight biosurfactants, which are generally lipopolysaccharides, lipoproteins or a combination of both (Healy et al., 1996; Rosenberg and Ron, 1999; Christofi and Ivshina, 2002). These high-molecular weight compounds are associated with production of stable emulsions, but the lowering of the surface tension or interfacial tension is not a usual trait of them and they are frequently called as bioemulsifiers (Bognolo, 1999). Microbial biosurfactants have recently been studied intensively because of their useful functional properties, such as emulsification, phase separation, wetting, foaming, solubilization, corrosion inhibition, viscosity reduction, and inhibition of biofilm formation.

* Corresponding author. Fax: +55 31 34092733. E-mail address: [email protected] (A.S. Monteiro). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.02.041

Biosurfactants have marked advantages over chemically synthesized surface-active compounds and can replace conventional surfactants in many areas including agriculture, industrial cleaning, the food and beverage, textile, cosmetics, and pharmaceutical industries (Dehghan-Noude et al., 2005). Their advantages are numerous: they have many potential applications due to their novel structural characteristics and physical properties; they are produced from renewable substrates; they can be readily modified by genetic engineering, biologically or biochemically; and they are biodegradable (Mulligan and Gibbs, 1993) In addition, they present low toxicity and remain stable at extremes of pH, salinity, and temperature (Banat et al., 2000). Despite such advantages, these molecules have not been commercialized due to their low yields and high costs of production. While high production costs can be tolerated for biosurfactants/bioemulsifiers used in low-volume, highlypriced products such as those used in cosmetics and medicinal products, high production costs are incompatible with applications that require high volumes of low-priced surfactants, such as enhanced oil recovery (EOR) (Kuyukina et al., 2001). The foci for reduction of biosurfactant/bioemulsifier production costs are the microorganisms (selected, adapted, or engineered for high yields), the process (selected, designed, and engineered for low capital and operating costs), the microbial growth substrate

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or process feedstock (selected for their low cost), and the process by-products (minimized or managed as saleable products rather than treated and discarded as wastes) (Mulligan and Gibbs, 1993). Some bacteria are able to synthesize biosurfactants, but do so in relatively low concentrations (Morita et al., 2007) as higher concentrations would damage their cell membranes. Some yeasts, on the other hand, can produce biosurfactants in higher concentrations (Cooper and Paddock, 1984). However, the literature describes only a few strains that produce biosurfactants/bioemulsifiers, namely Candida bombicola, C. antarctica, Pseudozyma aphidis, C. lipolytica and Yarrowia lipolytica (Cavalero and Cooper, 2003; Kim et al., 1999; Morita et al., 2007; Sarubbo et al., 2007; Amaral et al., 2006). The majority of the yeasts described so far produce lowmolecular weight glycolipid biosurfactants or high-molecular weight polymeric compounds. In this study, we used an enrichment culture method to isolate yeasts from the activated sludge wastewater treatment plant of a dairy industry; these yeasts are capable of producing bioemulsifiers when cultivated in vegetal oil medium. We describe here the physiological characteristics of the strains, their identification by molecular methods, and the production and the preliminary characterization of the bioemulsifiers produced.

2. Methods 2.1. Isolation and selection of bioemulsifier-producing yeasts Samples (250 mL each) were collected in duplicate in sterile 500mL glass flasks from three points of the liquid surface layers of the aerated lagoon, the stabilization lagoon, and the aeration tank of the wastewater treatment plant of a dairy industry in Minas Gerais State, Brazil. The samples collected were preserved in ice and transported for microorganism isolation. Twenty milliliters of each sample were inoculated into a 180 mL mineral medium (MM) containing 3.4 g K2HPO4, 4.3 g KH2PO4, 0.3 g MgCl22H2O, 1 g (NH4)2SO4, 0.5 g yeast extract and 0.2 g chloramphenicol per liter. These flasks were then supplemented with sunflower oil (20 g/L) as the sole carbon source, and incubated at 25 °C on a rotary shaker at 200 rpm for 96 h. After incubation, 1 mL samples were removed from culture and diluted up to 109 in sterile saline solution (0.85% NaCl). Aliquots of 0.1 mL of each dilution were plated onto potato dextrose agar medium (PDA; Biobras, Brazil) supplemented with chloramphenicol (0.2 g/L), and incubated at 25 °C for 120 h. Individual colonies on the plates, representative of each of the morphotypes, were purified by streaking at least three times on PDA and evaluated for bioemulsifier production. The strains isolated were maintained in GYMP broth (w/v-2% glucose, 0.5% yeast extract, 1% malt extract, 0.2% NaH2PO4) with 20% (v/v) glycerol added at 80 °C. The bioemulsifier production assays were carried out in Erlenmeyer flasks containing 50 mL of MM with 20 g/L of sunflower oil added and incubated in an orbital shaker (200 rpm) at 28 °C for 144 h. After incubation, the yeast cells were separated from the growth medium by centrifugation at 5000 rpm for 20 min at 4 °C. The growth of the strains was estimated by optical density (OD) measurements at 600 nm. To avoid the influence of residual oil present in the medium in this analysis, the OD measurements were made from pellet resuspended in saline solution (0.85%). As necessary, suspensions were diluted in saline solution to obtain OD values inside the linearity range of the spectrophotometric method. The supernatant was filtrated through a 0.45-lm Millipore membrane and refrigerated at 4 °C until detection of emulsifying activity (E24) by the method of Cameron et al. (1988). In these assays, 4-mL aliquots of the cell-free filtrate were mixed with 6 mL of toluene in a test tube and vortexed vigorously for 2 min. After 24 h, the proportion of toluene emulsified was

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compared with the total volume of toluene added. The emulsification index (E24) was calculated by the formula: height of the emulsion layer/total height  100. To verify the stability of the emulsions, the emulsification index was also evaluated 2 months after the emulsions were prepared as described above. 2.2. Identification of yeasts Conventional identification of the yeasts was done following the taxonomic keys of Kurtzman and Fell (1998), and their identities confirmed by comparing the D1/D2 variable domains of the large-subunit rDNA sequence and of 5.8 rRNA-ITS region with other sequences retrieved from the GenBank. For genomic DNA extraction, cells of a pure culture from an YM agar (24–48 h old) were removed, resuspended in 2 mL saline solution, and centrifuged at 13,000 rpm for 5 min. The supernatant was discarded; the pellet was resuspended in 400 lL extraction buffer (2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris–HCl, pH 8.0, 10 mM EDTA) (Lãs Heras-Vazquez et al., 2003) and then transferred to a 1.5-mL conical tube for further cell disruption for at least 20 s using approximately 80 mg of acidwashed glass beads (Sigma, 150–212 l) in combination with a strong vortex. The suspension containing disrupted cells was incubated for 10 min at 65 °C in order to complete the cell rupture and was the genomic DNA extracted by adding equal volumes of phenol–chloroform–isoamylalcohol (25:24:1), followed by isopropanol precipitation. Subsequently, the pellet was washed with 70% ethanol and dissolved in 20–50 lL TE. 2.3. PCR protocol The 5.8S-ITS region was amplified using PCR with primers ITS1 (50 -TCCGTAGGTGAACCTGCGC-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) as described by White et al. (1990). The D1/D2 variable domains of the large-subunit rDNA were amplified with PCR using primers NL1 (50 -GCATATCAATAAGCGGAGGAAAAG-30 ) and NL4 (50 -GGTCCGTGTTTCAAGACGG-30 ), according to the methods of Lachance et al. (1999). The amplified DNA was concentrated, cleaned (Kit Wizard Plus SV Minipreps DNA Purification System; Promega, USA), and sequenced in a MegaBACE™ 1000 automated sequencing system (Amersham Biosciences, USA). The sequence data were aligned using the Electropherogram Quality Analysis program (http:// asparagin.cenargen.embrapa.br). The yeasts were identified by searching databases using the BLAST sequence analysis tool (http://.ncbi.nlm.nih.gov/BLAST/). 2.4. Isolation, purification, and characterization of the bioemulsifier Surface-active compounds were recovered from the cell-free culture broths of Trichosporon loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 grown in 100 mL liquid MM supplemented with sunflower oil (20 g/L) at 28 °C on a reciprocal rotary shaker (200 rpm for 144 h), by extracting with ethyl acetate or precipitating with ethanol. Further, the surface tension of the cell-free culture broths was determined by the Ring method (Haba et al., 2000) using a KRUSS tensiometer (K10T Hamburgo). These cell-free culture broths (50 mL) were filtered through a 0.45-lm Millipore membrane and extracted twice with 10 mL of ethyl acetate, and the solvent was evaporated at 25 °C. In the ethanol precipitation method, the cell-free culture broths were mixed with four volumes of cold ethanol (1:4 v/v) and maintained at 4 °C for 48 h. The precipitate formed was collected by centrifugation at 5000 rpm for 20 min and treated with chloroform–methanol (2:1, v/v) in order to remove residual oils, and dried at 60 °C to constant weight. The resulting white powder was dissolved in deionized

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water (5 mg/mL) and preserved at 4 °C until used in the assays to detect emulsifying and surfactant activities, according to Morita et al. (2007). In the assays, aliquots (50 lL each) of the extracts (5 mg/mL) were added to the surface of polystyrene plates (90  15 mm). After 10 min, the droplets diameters (mm) were measured. All determinations were done in triplicate. The extracts with emulsifying and surfactant activities were characterized for their protein, lipid, and sugar content. Protein was measured according to the Bradford method (1976) using bovine serum albumin as the standard. Carbohydrates were determined by the phenol–sulphuric acid method using D-glucose as the standard (Dubois et al., 1956) and the lipid concentration was determined according to the modified method of Piretti et al. (1988). In the assays, samples of 500 mg were stored in 100 mL sealed flasks containing 10 mL 5 M NaOH in 95% aqueous methanol. After incubation at 100 °C for 5 h, each reaction was neutralized with 1 mL HCl (37%) and incubated in a rotary shaker (200 rpm) at 25 °C for 10 min. After adding 20 mL ethyl acetate the flask was incubated under agitation for additional 15 min. The organic phase was separated and the solvent eliminated at 50 °C for 24 h. The dried lipid pellet was weighted and the lipid concentration of the extract expressed in milligrams per 100 mg of extract. For purification, the crude water-soluble bioemulsifier was then applied to a Sephacryl S-200 column (1.6  57 cm, Pharmacia K 16/ 70 column) coupled to an FPLC system (Pharmacia). The column was pre-equilibrated with deionized water and eluted with 0.1 M degassed PBS buffer. Fractions (2 mL each) were collected, with the flow rate maintained at 1.0 min/mL and monitored by absorbance at 280 nm. Total sugars (by phenol–sulphuric acid method), total protein (by Bradford method) and emulsification activity (E24) were determined. The fractions with higher emulsifying activity and carbohydrates were pooled and lyophilized in a freeze dryer (Labconco. Corporation, Kansas City, MO) and were characterized for their lipid and carbohydrate composition. In order to determine fatty acid composition of the lipid moiety of the bioemulsifier, the lipids extracted according to the method of Piretti et al. (1988) were methylated with the boron fluoride–methanol reagent (14%) in the proportion of 1 mL reagent per 10 mg of lipid. The resulting sample was stored in 2-mL microtubes and incubated at 95 °C in a water bath for 15 min. Fatty acid methyl esters (FAMEs) were extracted three times with n-hexane and analysed in a Varian model 3380 gas chromatograph using He as carrier gas. A flame ionization detector (FID) and a CP-Sil 88 capillary column (50 m  0.25 mm ID) were used. Column temperature was initially set at 170 °C for 1 min and programmed to rise linearly to 250 °C at a rate of 4 °C/min; the thermal program was halted isothermally at this temperature. The injector and detector block temperatures were 250 °C. A bacterial fatty acid–methyl ester mixture (TM 37, FAME, Mix 47885; Supelco, USA) was used as the reference standard. To verify the presence of free fatty acids, these procedures were carried out on samples of crude and pure bioemulsifiers, not hydrolyzed with 5 M NaOH. The carbohydrate composition of the bioemulsifier was determined by gas chromatography and mass spectrometry. A lyophilized sample of bioemulsifier (1 mg) was hydrolyzed in a sealed tube with 150 lL of 2 M trifluoroacetic acid (CF3COOH) at 120 °C for 4 h. After evaporation the residue was washed twice with methanol; the sample was then reduced with 1 M aqueous sodium borohydride (NaBH4, 100 lL) and acetylated with a mixture of potassium acetate (100 lg) and acetic anhydride (100 lL) at 100 °C for 2 h. The excess reagent was removed by evaporation and the sample washed several times with ethanol. The alditol acetates were extracted with ethyl acetate and water (1:1, v:v) and analysed by GC–MS (SHIMADZU, model QP 5050 A) equipped with a PTE-5-Supelco (30 m  0.25 mm ID, 0.25 lm film) column using He as carrier gas. Column temperature was programmed to in-

crease from 100 °C (1 min) to 200 °C at a rate of 4 °C/min, followed by 20 °C/min to 300 °C, and the column was maintained at this temperature for 5 min. These procedures were carried out for crude and pure bioemulsifier samples, without hydrolysis, to verify the presence of free carbohydrates in these samples. Sugars were identified by comparing the relative retention times of sample peaks with standards. The following sugar standards used for the identification were purchased from Supelco USA: glucose, mannose, galactose, ramnose, fucose, ribose, arabinose, and xylose. The results were recorded and processed using Class 3.02 software (Shimadzu) and expressed as relative peak areas for each sugar. 2.5. Kinetics of microbial growth and production of bioemulsifier The inocula of T. loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 were prepared from cells grown on Sabouraud Agar (Difco, USA) for 48 h at 25 °C, washing in saline solution (0.85% NaCl, w/v) and resuspending in flasks containing 50 mL MM. Optical densities (OD) at 600 nm were measured, and the absorbance values were used to determine the inoculum’s volume to be transferred to assay flasks to obtain a cell density of 0.02 OD using the following formula: (350 mL  0.02)/OD600 nm of the cell suspension. The cell concentrations were confirmed by colony-forming units counts, by inoculating 100 lL volumes in duplicate on PDA agar, and were obtained values corresponding to 3.32 log10 cfu/mL. The growth and bioemulsifier production assays were performed in 1-L Erlenmeyer flasks containing 350 mL of MM supplemented with 20 g/L of sunflower oil and incubated in an orbital shaker (180 rpm) at 28 °C for 144 h. Growth, bioemulsifier production, and emulsifying activity (E24) were estimated after 4, 6, 8, 16, and then at 24 h intervals until the total duration of incubation reached 144 h. The cell populations were monitored by yeast colony counts of appropriate decimal dilutions on Sabouraud agar. Later, plates containing 30–300 yeast colonies after incubation at 25 °C for 48 h were used to calculate the average number of colony-forming units (cfu/mL). For recovery of bioemulsifier, cells were separated from the culture broth by centrifugation at 5000 rpm for 20 min at 4 °C. The supernatant was filtered through a 0.45-lm Millipore membrane, and four volumes of cold ethanol were added. The white precipitate formed was collected by centrifugation at 5000 rpm for 20 min and treated with chloroform–methanol (2:1, v/v), in order to remove residual oils, and dried at 60 °C to constant weight. The values obtained were used to estimate production (g/L) and volumetric production rate (g/L/h) at each time-point evaluated. 2.6. Stability studies The effect of the addition of NaCl (30% w/v) on emulsion stability was investigated. After addition of the salt to a 1% w/v solution of bioemulsifier crude extract in deionized water, the emulsifying activity was assessed by the emulsification index method (E24) as described earlier. The effect of the temperature on the bioemulsifier activity was evaluated by keeping the extract at 100 °C in a water bath for 60 min, and then cooling it to room temperature before the emulsification assays. Synthetic surfactants Tween 80 and Triton X-100 (Sigma Chemical Co.) at 0.2% (w/v) were used as controls. 2.7. Statistical analysis The experimental data are presented in terms of arithmetic means of at least three replicates. All statistical calculations were made using Sigma-Stat 3.5 statistical software (Jandel Scientific, San Rafael, CA). Analyses of variance (ANOVA) and Duncan’s means comparison test with a significance level of 0.05 were applied. Pearson’s correlation coefficient was used to test for a correlation

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between growth, E24, and bioemulsifier production. Values of P < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Isolation and identification of bioemulsifier-producing yeasts A total of 132 yeasts were isolated from samples from the wastewater treatment plant of a dairy industry in Minas Gerais State – Brazil. Thirty-one isolates were isolated from samples from the aerated lagoon (CLA), 29 from the stabilization lagoon, and 72 from the aeration tank. Thereafter, we studied the growth of the isolated strains in a mineral medium supplemented with sunflower oil. Fifty of these yeasts showed growth in this medium. As the second step, we demonstrated the secretion of bioemulsifiers by the strains that showed vigorous growth in sunflower oil mineral medium by measuring the emulsifying activity (E24) of their broth. The profile of the emulsifying activity obtained from the culture medium of strains that produced emulsifying substances is presented in Table 1. The values of the emulsifying activity of these yeasts range from 14% to 78% after 24 h. Among these 31 yeasts, only CLV20, CLOA40, and CLOA70 showed stable bioemulsifier activity after 60 days. Morphological and biochemical properties and sequence analysis of rDNA of the 31 strains of yeast selected revealed that 16 strains belong to the genus Trichosporon, 8 to the genus Geotrichum, 6 to the genus Galactomyces, and 1 to the genus Issatchenkia (Table 2). 3.2. Determination of surface tension in cell-free culture broths The strains T. loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70, which showed higher emulsifying activity Table 1 Growth, emulsifying activity, and stability of the emulsions of cell-free mineral medium. Isolates

Growth (OD660 nm)

EA (E24)a

EA (2 months)b

CLV7 CLV9 CLV10 CLV11 CLV14 CLV15 CLV17 CLV20 CLV24 CLV26 CLA1 CLA2 CLA7 CLA8 CLA10 CLA11 CLA30 CLA40 CLOA7 CLOA8 CLOA10 CLOA16 CLOA18 CLOA19 CLOA20 CLOA22 CLOA23 CLOA27 CLOA40 CLOA70 CLOA 71

12.5 ± 1.2 13.2 ± 0.8 14.2 ± .5 15.3 ± 0.66 14.3 ± 0.57 12.5 ± 0.44 15.6 ± 1.4 13.7 ± 1.1 14.8 ± 0.4 16.4 ± 0.47 17.3 ± 0.36 13.7 ± 0.38 13.4 ± 0.84 14.7 ± 0.37 15.4 ± 0.56 15.3 ± 0.76 19.5 ± 0.22 13.6 ± 0.34 18.2 ± 0.46 14.3 ± 1.31 17.3 ± 0.64 13.8 ± 0.53 12.4 ± 0.27 13.7 ± 1.4 17.6 ± 0.92 13.7 ± 0.64 12.3 ± 0.44 14.6 ± 0.60 17.6 ± 1.2 15.8 ± 0.43 16.3 ± 0.30

14.14 ± 0.79 36.27 ± 0.85 61.57 ± 0.33 63.73 ± 0.85 42.65 ± 1.47 36.27 ± 0.20 38.73 ± 0.85 75.60 ± 0.85 43.14 ± 0.85 44.61 ± 0.85 36.27 ± 0.65 22.44 ± 0.85 29.90 ± 0.74 58.05 ± 1.22 66.35 ± 1.25 56.74 ± 0.85 44.61 ± 0.85 45.10 ± 0.85 71.08 ± 0.00 18.03 ± 0.85 58.82 ± 0.85 59.31 ± 0.85 45.10 ± 0,82 44.61 ± 0,85 44.12 ± 0,85 37.25 ± 0,85 37.75 ± 0,85 18.14 ± 0.85 77.78 ± 0.82 66.67 ± 0.85 59.31 ± 0.85

7.32 ± 0.06 14.71 ± 0.00 16.25 ± 0.72 35.29 ± 0.85 – – 22.06 ± 1.70 61.29 ± 0.24 35.29 ± 0.85 – – – – – – 29.54 ± 0,49 – – – – – – 35.78 ± 0.82 31.86 ± 0.00 – – – – 77.45 ± 0.82 65.80 ± 0.00 –

Data are means ± standard deviations of three independent experiments. Emulsifying activity (EA) evaluated after 24 h (a) or 2 months (b).

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and emulsion stability after 60 days, were evaluated for their ability to secrete substances capable of reducing the surface tension of the growth medium. After 144 h of growth, surface tension of cellfree culture broths observed was 36, 41, and 52 mN/m for CLV20, CLOA40, and CLOA70, respectively. These values represent a reduction of 48%, 40%, and 25%, respectively, in the surface tension of the growth medium when compared to the initial values. These values fall within the range of values reported in the literature for yeasts of genus Candida and Yarrowia (Haba et al., 2000; Amaral et al., 2006). None of the other strains showed significant correlation between reduction of surface tension and emulsion formation. The reduction of surface or interfacial tension and emulsifying activity has often been used as the primary criterion for screening microorganisms for bioemulsifier production. However, the reduction of surface tension and emulsifying activity does not necessarily correlate. Furthermore, some microbial emulsifiers such as the sophorolipids from Torulopsis bombicola have been shown to reduce surface tension and interfacial tension but have not proved to be good emulsifiers (Cooper and Paddock, 1984). Yeasts belonging to genus Candida have been known to be one of the major producers of biosurfactants. The mannosylerythritollipids from Candida sp. SY16 and sophorolipids from C. bombicola 22214 have been reported to reduce the surface tension to below 33 mN/m (Kim et al., 1999; Cooper and Paddock, 1984). However, Haba et al. (2000), studying biosurfactant production by bacteria of genus Pseudomonas and yeasts, found that tensoactive substances produced by C. glabrata and C. lipolytica reduced surface tension to the range 35–45 mN/m. Yeasts from the arthroconidial ascomycetous genus Geotrichum Link: Fries represent the anamorphic state of Dipodascus Lagerheim and Galactomyces Redhead and Malloch (Kurtzman and Robnett, 1988). It is a ubiquitous fungus found in plant tissues, silage (soil, milk, air, and water (Pottier et al., 2007; O’brien et al., 2005). Galactomyces geotrichum (anamorph Geotrichum candidum) is widely used in the dairy industry as secondary/adjunct cultures in many semi-fresh, soft and semi-hard cheeses made of cow’s, goat’s, and ewe’s milk (Pottier et al., 2007). It grows very early during the ripening process and stimulates the development of bacterial flora (Guéguen, 1984). It plays an important role in the catabolism of triglycerides and casein, and also in cheese organoleptic properties and appearance (Boutrou and Guéguen, 2005). Yeast strains belonging to genus Trichosporon are widely distributed and have been isolated from various sources including plant and animal matter, air, soil, industrial wastewaters, ligneous pulp, and activated sludge (Middelhoven et al., 2001; Santos and Linardi, 2001). This genus comprises yeasts with different biochemical pathways. Trichosporon species were reported to utilize benzene, and phenol (Middelhoven et al., 2001; Santos and Linardi, 2001). The described metabolic potential of Trichosporon species has made the application of these yeasts for environmental purposes very promising (Kaszycki et al., 2006). To our knowledge, this is the first report on biosurfactant/bioemulsifier production by yeasts of genera Galactomyces, Geotrichum, and Trichosporon isolated from dairy industry effluents. The majority of yeasts reported in the literature belong to the genera Candida and Pseudozyma (Kim et al., 1999; Morita et al., 2007). Furthermore, future work should focus on the capacity of yeasts to produce bioemulsifiers and biosurfactants from different carbon sources, especially from industrial residues, and optimize the conditions for production using a factorial experimental design. 3.3. Isolation, purification, and characterization of bioemulsifiers Of the two extraction methods used, the method with ethyl acetate did not extract surfactants or emulsifying compounds from cell-free broths of T. loubieri CLV20, Geotrichum sp. CLOA40, and

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Table 2 Yeasts identified by sequence analysis of the 5.8 rRNA-ITS and 26 rRNA D1/D2 regions. Isolates

Species

Identity (%)

GenBank accession numbers

Sequencea

CLV7 CLV9 CLV10 CLV11 CLV14 CLV15 CLV17 CLV20 CLV24 CLV26 CLA1 CLA2 CLA7 CLA8 CLA10 CLA11 CLA30 CLA40 CLOA7 CLOA8 CLOA10 CLOA16 CLOA18 CLOA19 CLOA20 CLOA22 CLOA23 CLOA27 CLOA40 CLOA70 CLOA 71

Trichosporon asahii Geotrichum sp. Geotrichum sp. Trichosporon dulcitum Trichosporon sp. Geotrichum sp. Galactomyces sp. Trichosporon loubieri Trichosporon loubieri Trichosporon montevideense Trichosporon loubieri Trichosporon mycotoxinivorans Trichosporon asahii Geotrichum sp. Galactomyces geotrichum Trichosporon montevideense Trichosporon loubieri Geotrichum sp. Trichosporon montevideense Galactomyces sp. Issatchenkia orientalis Trichosporon asahii Galactomyces geotrichum Trichosporon montevideense Geotrichum sp. Geotrichum sp. Galactomyces geotrichum Galactomyces geotrichum Geotrichum sp. Trichosporon montevideense Trichosporon asahii

99 99 98 99 98 98 97 99 99 99 99 99 99 98 100 100 99 99 99 98 99 99 99 100 100 99 99 100 99 100 99

GU299462 GU299463 GU299464 GU299465 GU299466 GU299467 GU299450 GU299451 GU299452 GU299453 GU299454 GU299455 GU299468 GU299469 GU299470 GU299456 GU299457 GU299472 GU299458 GU299459 GU299473 GU299471 GU299474 GU299460 GU299475 GU299476 GU299477 GU299478 GU299479 GU299461 GU299480

2 2 2 1 2 2 1 1 1 1 1 2 2 1 2 1 1 2 1 2 2 2 1 1 2 2 2 2 2 1 2

a (1) 18S ribosomal rRNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence obtained with primers (TS1/ITS4). (2) 26S ribosomal RNA genes, partial sequence obtained with primers (NL1/NL4).

T. montevideense CLOA70. However, tensoactive compounds were detected in the extracts obtained from precipitation with ethanol as evaluated with the emulsifying activity assay (E24) and the method of collapsing droplets on polystyrene plates. The values of emulsifying activity for T. loubieri CLV20, Geotrichum sp. CLOA40 and T. montevideense CLOA70 were 68%, 65%, and 64%, respectively. The diameter of all droplets was 7 mm. This value represents an increase of 2 mm in the droplet diameter when compared to that of the control sample (deionized water). The undetected biosurfactant activity in the ethyl acetate extracts suggested the absence of low-molecular weight biosurfactants such as glycolipids, sophorolipids, and mannosylerythritollipids (MELs). These compounds are preferentially extracted by non-polar solvents such as ethyl acetate and methyl-tertiary-butyl ether (MTBE) (Kuyukina et al., 2001; Morita et al., 2007; Cavalero and Cooper, 2003). Biosurfactants containing high-molecular weight carbohydrates, lipids, and protein are preferentially extracted from cell-free growth medium by precipitation with acetone, ethanol, or chloroform–methanol (Sarubbo et al., 2007). These data suggest that the compounds produced by yeasts posses similar composition and molecular weights. On average, approximately 7.0, 6.4, and 6.5 g of crude material per liter of cell-free broths of T. loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 could be recovered after precipitation with ethanol. The precipitates with surfactant and emulsifying activities were characterized by their proteins, carbohydrates, and lipids contents. The results showed that the bioemulsifiers were composed only by lipids and carbohydrates, representing 78.7% and 87.5% in CLV20; 88.8% and 21.3% in CLOA40; and 12.5% and 11.2% in CLOA70. The chromatographic fractions from the crude bioemulsifier were evaluated for their total sugar, and emulsifying activity, allowing the identification of a single peak with fractions showing positive results for total carbohydrates and emulsifying activity

(data not shown). Gas chromatography of the lipid fraction of bioemulsifier from CLOA40 showed that (9Z,12Z)-octadeca-9,12-dienoic acid, octadecanoic acid, 9-octadecenoic acid and tridecanoic acid are the major acids accounting for 90% of the total fraction (Table 3). In CLOA70, (9Z,12Z)-octadeca-9,12-dienoic acid, 10-octadecenoic, docosanoic acid, and hexadecanoic acid are the major acids accounting for 85.98% of total acids. In CLV20, (9Z,12Z)-octadeca-9,12-dienoic acid, (9E,11E)-octadeca-9,11-dienoic acid, 9octadecenoic acid and hexadecanoic acid are the major acids accounting for 92% of total fatty acids. The fatty acids detected were supplied only after hydrolysis of bioemulsifier samples. Free fatty acids were not detected in raw bioemulsifier. A similar composition of fatty acids has been described for surfactants produced

Table 3 Fatty acid composition of bioemulsifiers isolated from yeasts CLV20, CLOA40, and CLOA70. Fatty acids (%)

Decanoic acid Undecanoic acid Hexadecanoic acid Octadecanoic acid 10-Octadecenoic acid (9Z,12Z)-Octadeca-9,12-dienoic acid (9E,11E)-Octadeca-9,11-dienoic acid 9-Octadecenoic acid 2-Pentenoic acid Docosanoic acid Tridecanoic acid 11-(2-Hidroxylpentil)undecanoic acid Tetradocosanoic acid 2-Methoxydecanoic acid Areas of peaks not identified

Strains CLV20

CLOA40

CLOA70

0.63 0 7.09 0.29 0.60 42.82 34.23 8.06 1.80 1.82 1.44 0 0 0 1.22

0 4.35 0 19.70 0 25.87 0 26.39 0 0.31 8.12 0.98 0 1.27 13.01

0 0 5.09 4.13 22.19 49.79 0 0 0 8.91 0 0 3.7 1.8 4.39

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by Y. lipolytica IMUFRJ 50682 (Amaral et al., 2006) and Penicillium sp. (Luna-Velasco et al., 2007). Carbohydrates in the bioemulsifier was determined by GC–MS of alditol acetate derivatives. In the bioemulsifier from CLOA40, most sugars were identified as mannose (41%), glucose (8%), and galactose (44%). The predominant sugars in the bioemulsifier from CLOA70 were xylose (55%), arabinose (28%), ribose (9%), and mannose (4%). In the bioemulsifier from CLV20, most sugars were identified as galactose (44%), mannose (41%), and glucose (8%). This composition is similar to that described for a surfactant produced by Y. lipolytica IMUFRJ 50682, which showed a predominance of mannose (Amaral et al., 2006). Biosurfactants of the hydrophilic group composed of a single type of monosaccharide have also been described, such as that produced by T. bombicola, which presents only sophorose in its constitution (Cooper and Paddock, 1984). The biomolecular composition of these polymers may have influenced their emulsifying activity. Several studies have suggested that both the total amount and distribution of fatty acids and carbohydrates in an emulsifier play an important role in its emulsifying activities (Kim et al., 1997a,b, 2000). The fraction of 3-hydroxymyristic acid residues in lipopeptide lichenicin A produced by Bacillus licheniformis was reported to be one of the most important factors influencing its surfactant activity (Kim et al., 2000). Kim et al. (1997a,b) reported that the 3-hydroxydodecanoic acid content of emulsan played a role in its emulsifying activity. In other study, Kim et al. (1997a,b) evaluated the emulsifying activity of various emulsan samples with the different degrees of branching of the carbohydrate backbone obtained from Acinetobacter calcoaceticus under different culture conditions and the authors observed that this activity had a linear correlation to the branching degrees of the carbohydrate backbone. 3.4. Kinetics of microbial growth and bioemulsifier production The growth, emulsifying activity, bioemulsifier production, and the volumetric production rates of T. loubieri CLV20, Geotricum sp. CLOA40, and T. montevideense CLOA70 in mineral medium supplemented with sunflower oil (20 g/L) are shown in Fig. 1(A–C). For all strains, emulsifier/biosurfactant production follows an exponential growth phase. Bioemulsifiers production continues even when microbial growth ceases. About 50.5%, 54.6%, and 51.6% of bioemulsifiers in the CLV20, CLOA40, and CLOA70 strains, respectively, were obtained in the exponential growth phase in the first 72 h. These results indicate that production of bioemulsifier is partially growth-associated, as the peaks of bioemulsifier production and biomass did not coincide. Kinetics of partially or totally growth-associated production have been reported Pseudomonas aeruginosa LBI (Benincasa et al., 2002), and Bacillus subtilis LB5a (Nitschke and Pastore, 2006). Where production is associated with growth, a direct correlation between the use of substrate and the production of biosurfactant is observed (Desai and Desai, 1993). In this study, a linear correlation between cellular concentration (cfu/mL) of yeasts CLV20 (r = 0.628), CLOA40 (r = 0.774) and CLOA70 (r = 0.676) and bioemulsifier production in the logarithmic phase was observed (P < 0.05). This behavior could be explained by the fact that the emulsifier is being produced to improve the carbon source assimilation. The lower values of correlation coefficients can be explained by the delay in the biosurfactant/ bioemulsifier production in relation to the biomass production. The amounts of bioemulsifier produced (7.0 g/L for CLV20, 6.5 g/L for CLOA40, and 6.3 g/L CLOA70) are within the range described for yeasts from vegetal oils. Sarubbo et al. (2007) reported that 8 g/L of protein–lipid–polysaccharide complex bioemulsifier was produced by C. lipolytica when canola oil (10%) was used as carbon source. A total of 14.9 g/L of sophorolipid was produced by Pichia anomola grown in 8% soybean oil (Thaniyavarn et al.,

Fig. 1. Time course of cell growth and bioemulsifier production by T. loubieri CLV20 (A), Geotrichum sp. CLOA40 (B), and T. montevideense CLOA70 (C) grown in a mineral medium with sunflower oil (20 g/L) and incubated in an orbital shaker at 180 rpm at 28 °C. Log cfu/mL (-d-), Emulsifying index (E24) of the culture medium (-s-), production of bioemulsifier (g/L) (-D-) and volumetric production rate (g/L/h) (-?-). The standard deviation from the mean values based on three replicate experiments of E24 was lower than 5.7% and for those of growth data was lower than 0.98%.

2008), and 0.56 g/L of bioemulsifier (fatty acids) was produced by C. ingens CB-21 grown in 2% corn oil (Amézcua-Vega et al., 2007). The volumetric production rates of the bioemulsifier increased in the exponential phase (first 72 h of incubation), reaching values

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of around 0.125, 0.140, and 0.121 g/L/h for CLV20, CLOA40, and CLOA70, respectively. After this period, a decline in production was observed (Fig. 1A–C). These strains present the highest emulsifying activities (E24) after 96 h of cultivation, with similar values (76%), which coincides with the period of the maximum bioemulsifier production. In the logarithmic growth phase (72 h), emulsifying activity was linearly correlated with growth; the values of linear correlation coefficient (r) observed were 0.868 for CLV20, 0.958 for CLOA40, and 0.923 for CLOA70 (P < 0.05). The E24 values were also correlated with bioemulsifier production, with values of linear correlation coefficient of 0.902 for CLV20, 0.889 for CLOA40, and 0.885 for CLOA70 (P < 0.05). The emulsifying agents produced by yeasts are synthesized in the presence of a wide range of hydrocarbons and other oils such as vegetable oils (Morita et al., 2007; Kim et al., 1999). With the intention of reducing the cost of production, waste frying oils, cheap agricultural renewable resource, and wastes containing these substrates have been proposed as promising carbon sources for biosurfactant/bioemulsifier production by microorganisms. We are now working on the optimization of culture media based on vegetable oil and glycerol-containing biodiesel wastes to increase the productivity and on new applications of biosurfactants/bioemulsifiers produced by selected yeasts. Similar to bacteria, yeasts produce a number of bioemulsifier with high emulsifying capacity. These molecules from yeasts are particularly promising because several of the yeasts are of food grade (Rosenberg and Ron, 1999). 3.5. Stability studies The effect of NaCl (30%w/v) on the stability of bioemulsifier from CLV20, CLOA40, and CLOA70 is shown in Fig. 2. The addition of NaCl was ineffective in influencing the activity of bioemulsifiers. Exposing bioemulsifiers from strains CLOA70 and CLV20 to a temperature of 100 °C for 60 min had no effect on the activity of the bioemulsifier (Fig. 3). This highlights the usefulness of the biosurfactants/bioemulsifiers in the food, pharmaceutical, and cosmetics industries where heating to achieve sterility is of paramount importance. For the strain CLOA40, a fall in the emulsification index (from 74.8% to 40.4%) was detected after exposure of the bioemulsifiers to a temperature of 100 °C. Emulsification activities and emulsion stability of two synthetic surfactants (Tween 80 and Triton X-100) were compared with those of the bioemulsifiers from strains of yeasts under study using toluene as a substrate. The values of emulsifying activity of the bioemulsifiers obtained from strains CLOA70 and CLV20 did not vary

Fig. 3. Comparison of emulsifying activity of bioemulsifiers from T. loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 with chemical surfactants Triton X-100 and Tween 80, after exposure at 100 °C for 60 min (- -). Control (-j-).

statically with the values of Tween 80 and Triton X-100. But in stability assays, the synthetic surfactants showed no emulsifying activity in the presence of NaCl (30%) (Fig. 2). Some authors have shown that the emulsifying activity of biosurfactants can be equal to or greater than that of synthetic surfactants. Anand et al. (2009) reported that the emulsifying activity of biosurfactant produced by Pseudoxanthomonas sp. PNK-04 using toluene as hydrophobic substrate was 60% compared with 35% for Triton X-100 and 50% for Tween 80. Maneerat and Phetrong (2007) compared the emulsifying activity of biosurfactants and that of Tween 80 after exposure to 100 °C and observed similar values. However, the emulsifying activity of biosurfactants was inhibited by the addition of 12% NaCl. Generally in this study, the emulsion stability of the bioemulsifiers from yeasts was found to be superior to that of the two synthetic surfactants, suggesting that these tensoactives compounds could be used as emulsifying and emulsion-stabilizing agents for numerous industrial applications. 4. Conclusion In this work, we identified new yeasts strains of genus Trichosporon, Geotrichum, and Galactomyces that showed greater emulsifying activities after growth in mineral medium supplemented with sunflower oil. These strains present high potential for applications in a variety of industrial sectors, such as food and cosmetics for formulation of emulsions, and in areas such as removal of oils in tanks and ducts, especially by their emulsifying activity. These bioemulsifiers have high stability compared with Tween 80 and Triton X-100 with respect to presence of electrolytes. They clearly demonstrate their potential for commercial applications that involve extreme environmental conditions. Acknowledgements The authors are thankful for the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), and Comissão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References

Fig. 2. Comparison of emulsifying activity of bioemulsifiers from T. loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 with chemical surfactants, Triton 100X and Tween 80 after addition of NaCl (30%w/v) (- -). Control (-j-).

Amaral, P.F.F., Silva, J.M., Lehocky, M., Barros-Timmons, A.M.V., Coelho, M.A.Z., Marrucho, I.M., Coutinho, J.A.P., 2006. Production and characterization of a bioemulsifier from Yarrowia lipolytica. Process Biochemistry 41, 1894–1898.

A.S. Monteiro et al. / Bioresource Technology 101 (2010) 5186–5193 Amézcua-Vega, C.A., Varaldo, P.H.M., García, F., Leal, E.R., Vázques, R.R., 2007. Effect of culture conditions on fatty acids composition of a biosurfactant produced by Candida ingens and changes of surface tension of culture media. Bioresource Technology 98, 237–240. Anand, S.N., Vijaykumar, M.H., Karegoudar, T.B., 2009. Characterization of biosurfactant produced by Pseudoxanthomonas sp. PNK-04 and its application in bioremediation. International Biodeterioration and Biodegradation 63, 73– 79. Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Applied Microbiology and Biotechnology 53, 495–508. Benincasa, M., Contiero, J., Manresa, M.A., Moraes, I.O., 2002. Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the sole carbon source. Journal of Food Engineering 54, 283–288. Bognolo, G., 1999. Biosurfactants as emulsifying agents for hydrocarbons. Colloids and Surfaces A 152, 41–52. Boutrou, R., Guéguen, M., 2005. Interests in Geotrichum candidum for cheese technology. International Journal Food Microbiology 102, 1–20. Bradford, M.A., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–258. Cameron, D., Cooper, D.G., Neufeld, R.J., 1988. The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier. Applied and Environmental Microbiology 54, 1420–1425. Cavalero, D.A., Cooper, D.G., 2003. The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214. Journal Biotechnology 10, 31–41. Christofi, N., Ivshina, I.B., 2002. Microbial surfactants and their use in fields studies of soil remediation. Journal of Applied Microbiology 93, 915–929. Cooper, D.G., Paddock, D.A., 1984. Production of a biosurfactant from Torulopsis bombicola. Applied and Environmental Microbiology 47, 173–176. Dehghan-Noude, G., Housaindokt, M., Bazzaz, B.S., 2005. Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633. Journal Microbiology 43, 272–276. Desai, J.D., Banat, I.M., 1997. Microbial production of surfactants and their commercial potential. Microbiology Molecular Biology Review 61, 47–64. Desai, J., Desai, A., 1993. Production of biosurfactants. In: Kosaric, N. (Ed.), Biosurfactants, first ed. Production Properties Applications, vol. 10. Marcel Dekker, New York, NY, pp. 65–97. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substance. Analytical Chemistry 28, 350–356. Guéguen, M., 1984. Contribution to knowledge of Geotrichum candidum and in particular to its variation. Consequences for dairy technology. Ph.D. Thesis, Université de Caen Basse-Normandie, Caen, France pp. 487. Haba, E., Espuny, M.J., Busquets, M., Manresa, A., 2000. Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils. Journal of Applied Microbiology 88, 379–387. Healy, M.G., Devine, C.M., Murphy, R., 1996. Microbial production of biosurfactants. Resources, Conservation and Recycling 18, 41–57. Kaszycki, P., Czechowska, K., Petryszak, P., Mie˛dzobrodzki, J., Pawlik, B., Kołoczek, H., 2006. Methylotrophic extremophilic yeast Trichosporon sp.; a soil-derived isolate with potential applications in environmental biotechnology. Acta Biochimica Polonica 53, 463–473. Kim, P., Oh, D.-K., Kim, S.-Y., Kim, J.-H., 1997a. Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1. Biotechnology Letters 19, 457–459. Kim, S.-Y., Oh, D.-K., Kim, J.-H., 1997b. Biological modification of hydrophobic group in Acinetobacter calcoaceticus RAG-1 emulsan. Journal of Fermentation and Bioengineering 84, 162–164. Kim, H.S., Yoon, B.D., Choung, D.H., Oh., H.M., Katsuragi, T., Tani, Y., 1999. Characterization of a biosurfactant, mannosylerythritiol lipid produced from Candida sp. SY16. Applied Microbiology Biotechnology 52, 713–721.

5193

Kim, P., Oh, D.-K., Lee, J.-K., Kim, S.-Y., Kim, J.-H., 2000. Biological modification of the fatty acid group in an emulsan by supplementing fatty acids under conditions inhibiting fatty acid biosynthesis. Journal of Bioscience and Bioengineering 90, 308–312. Kurtzman, C.P., Fell, J.W. 1998. The Yeast, a Taxonomic Study, fourth ed. Elsevier, Amsterdam, Lausanne, New York, Oxford, Shannon, Singapore, Tokyo, p. 420. Kurtzman, C.P., Robnett, C.J., 1988. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 73, 331–371. Kuyukina, M.S., Ivshina, I.B., Philp, J.C., Christofi, N., Dunbar, S.A., Ritchkova, M.I.., 2001. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction. Journal Microbiology Methods 46, 149–156. Lachance, M.A., Bowles, J.M., Starmer, W.T., Barker, J.S.F., 1999. Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Canadian Journal Microbiology 45, 172–177. Lãs Heras-Vazquez, F.J., Mingorance-Cazorla, L., Clemente-Jimenes, J.M.., RodriguezVico, F., 2003. Identification of yeast species from Orange fruit and juice by RFLP and sequence analyses of the 5.8S rRNA gene and the two internal transcribe spacers. FEMS Yeast Research 3, 3–9. Luna-Velasco, M.A., Esparza-Garcia, F., Canizares-Villanueva, R.O., RodriguezVasquez, R., 2007. Production and properties of a bioemulsifier synthesized by phenanthrene-degrading Penicillium sp. Process Biochemistry 42, 310–314. Maneerat, S., Phetrong, K., 2007. Isolation of biosurfactant-producing marine bacteria and characteristics of selected biosurfactant. Songklanakarin Journal of Science and Technology 29, 781–791. Middelhoven, W.J., Scorzetti, G., Fell, J.W., 2001. Trichosporon porosum comb. nov., an anamorphic basidiomycetous yeast inhabiting soil, related to the loubieri/ laibachii group of species that assimilate hemicelluloses and phenolic compounds. FEMS Yeast Research 1, 15–22. Morita, T., Masaaki, K., Fukuoka, T., Imura, T., Kitamoto, D., 2007. Physiological differences in the formation of the glycolipid biosurfactants, mannosylerythritol lipids, between Pseudozyma antarctica and Pseudozyma aphidis. Applied Microbiology Biotechnology 74, 307–315. Mulligan, C.N., Gibbs, B.F., 1993. Factors influencing the economics of biosurfactants. In: Kosaric, N. (Ed.), Biosurfactants, first ed. Production Properties Applications., vol. 10. Marcel Dekker, New York, NY, pp. 329–371. Nitschke, M., Pastore, G.M., 2006. Production and properties of a surfactant obtained from Bacillus subtilis using cassava wastewater. Bioresource Technology 97, 336–341. O’brien, M., O’kiely, P., Forristal, P.D., Fuller, H.T., 2005. Fungi isolated from contaminated baled grass silage on farms in the Irish Midlands. FEMS Microbiology Letters 247, 131–135. Piretti, M.V., Pagliuca, G., Vasina, M., 1988. Transmethylation of neutral and polar lipids with NaBH4 in the presence of NaOH. Chemistry Physics of Lipids 47, 149–153. Pottier, I., Gente, S., Vernoux, J.P., Guéguen, M., 2007. Safety assessment of dairy microorganisms: Geotrichum candidum. International Journal of Food Microbiology 26, 327–332. Rosenberg, E., Ron, E.Z., 1999. High- and low-molecular-mass microbial surfactants. Applied Microbiology Biotechnology 52, 154–162. Santos, V.L., Linardi, V.R., 2001. Phenol degradation by yeast isolated from industrial effluents. Journal General Applied Microbiology 47, 213–221. Sarubbo, L.A., Farias, C.B.B., Campos-Takaki, G.M., 2007. Co-utilization of canola oil and glucose on the production of a surfactant by Candida lipolytica. Current Microbiology 54, 68–73. Thaniyavarn, J., Chianguthai, T., Sangvnich, P., Roongawng, N., Washio, K., Morikawa, M., Thaniyavarn, T., 2008. Production of sophorolipid biosurfactant by Pichia anomala. Bioscience Biotechnology and Biochemistry 72, 2061–2068. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. A Guide to Methods and Applications. Academic Press Inc, San Diego, pp. 315–332.