1. Production of biosurfactants from yeasts cultivated on glycerol A. Giannopoulos, A. Makri and G. Aggelis Unit of Microbiology, Division of Genetics; Cell & Development Biology, Dpt of Biology; University of Patras; Greece Introduction Surfactants are organic molecules that usually consist of a hydrophobic and a hydrophilic part. The hydrophilic part makes surfactants soluble in water, while its hydrophobic part makes them tend to concentrate at interfaces. Depending on the nature of the hydrophilic group, four types of surfactants can be distinguished: anionic, cationic, zwitterionic, and non-ionic surfactants. The major classes of biosurfactants include glycolipids, phospholipids, fatty acids, lipopeptides/lipoproteins, and biopolymeric surfactants. The hydrophobic part of the molecule consist of long-chain fatty acids, hydroxy fatty acids or α-alcyl-β-hydroxy fatty acids. The hydrophilic portion can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol [1]. Surfactants are used in several fields, such as in industrial cleaning, agriculture, health, food, paper, construction and metal industries, textiles, cosmetics, pharmaceutical, petroleum and petrochemical industries, including applications in environmental bioremediation. They are capable of a broad range of functional properties that include emulsification activity, wetting, foaming,, viscosity reduction, phase separation and solubilization ability [2]. The production of biosurfactants by microorganisms grown on glycerol has been studied before. The yeast strain Pseudozyma antarctica produces mannosylerythritol lipids, while the bacterial strain Pseudomonas aeruginosa produces rhamnolipids during cultivation on glycerol. Many more microorganisms have also been studied during the past few years for their ability to produce such molecules [3]. In this work we present the kinetics of six yeasts strains on glycerol and their ability to produce surfactants. Cryptococcus curvatus was found to produce such molecules in significant amount. Further analysis of the crude product was performed, to determine its structure and consistence. Materials and Methods Microorganisms: Cryptococcus curvatus (NRRL Y-1511), Candida tropicalis (NRRL Y-12968), Pichia ciferrii (NRRL Y-1031), Candida guilliermondi (NRRL Y-2075), Candida diddensiae (NRRL Y-7589), Williopsis saturnus (NRRL Y-17396). Culture conditions: Microorganisms were cultivated in batch cultures at T=28 °C, with glycerol being the only carbon and energy substrate. Lipid extraction: According to Folch protocol [4]. GC analysis: Fatty acid analysis was performed after trans-methylation according to the AFNOR method [5], in an Agilent Technologies 7890 A device equipped with a HP-88 (J&W scientific) column (60 m x 0.25 mm). Conditions: carrier gas helium, flow rate 1 ml/min, oven T=200 °C, injector T=250 °C, detector (FID) T=280 °C. HPLC analysis: Glycerol was determined in filtered (through 0.2 μ m pore size bacteriological filter, Whatman) aliquots of the culture by an HPLC apparatus (Ultimate 3000, Dionex, Germering, Germany) equipped with an HPX-87H column and a R.I. detector. Conditions: eluant H 2 SO 4 0.004 N, flow rate 0.9 ml/min, T =55 ο C. Emulsification activity: Determination of biosurfactants’ emulsification activity was performed according to Cirigliano & Carman [6]. Broth samples were collected after centrifugation of the cultures at 15.000 rpm for 15 min, at T=4 °C. The samples were filtrated (through 0,45 μ m pore size bacteriological filters, Whatman). 2 ml of the filtrate were added to 2 ml of sodium acetate buffer 0,1 M (pH=3) and 1 ml of olive oil. The mixture was shaken on vortex for 30 sec. The resulting emulsion was then incubated for 30 min. after which its absorbance was measured at 540 nm. The blank used contained 2 ml of sterile medium containing glycerol. Isolation of biosurfactant: Broth from cultures of C. curvatus was collected after centrifugation (15.000 rpm for 15 min, at T=4 °C), filtered (through 0,45 μ m pore size bacteriological filters, Whatman), preserved at 4 οC overnight along with equal quantity (1:1 w/w) of HCl 6 N and then treated with chloroform to isolate the surfactant molecules. The isolation was followed by determination of the dry weight of the surfactant after lyophilization, GC analysis for determination of its fatty acid composition, HPLC analysis to determine the sugar moiety. Figure 1: Kinetics diagram of Cryptococcus curvatus cultivated on glycerol (30 g/l) as sole carbon source. Biomass (X, g/l), cellular lipids (L, g/l), glycerol (Glol, g/l), emulsification activity of biosurfactant (U/l x 10 -1 ; U/g DW x 10 -1 ; 1U=1 unit shift in absorbance at 540 nm) Table 1: Indicative evidence from the kinetics of C. guilliermondi, C. tropicalis, C. diddensiae, P. ciferrii and W. saturnus cultivated in media of glycerol (30 g/l) as sole carbon source (n.d.: not determined; O.D. values under 0.05 are counted as 0.00 Units of emulsification activity, meaning there is no biosurfactant produced). Yeastst (h)Biomass (g/l) Percentage of lipids (L/X %) Glol (g/l)Emulsification activity U/g DW x 10 -1 U/l x 10 -1 C. guilliermondi 435.03 3.66 15.45 0.00 735.99 5.71 10.07 0.00 1196.04 6.29 8.27 0.00 C. tropicalis 260.95 n.d. 28.27 0.00 681.09 4.5927.97 0.00 971.30 4.7827,73 0.00 C. diddensiae 273.89 n.d. 20.11 0.00 496.08 0.5616.28 0.00 937.08 0.73 8.76 0.00 P. ciferrii 324.03 3.2821.9510.0840.60 564.80 4.4217.70 8.2339.50 885.6511.9611.30 5.1829.00 W. saturnus 527.86 3.08 7.88 0.00 779.39 3.53 1.88 0.00 959.55 3.79 0.42 0.00 Intracellular lipids Yeastt (h)C16:0C16:1C18:0C18:1C18:2C18:3 (Δ9,12,15) Others Cryptococcus curvatus 4325.65 0.8712.9043.3613.042.79 1.39 4825.95 0.7812.3642.7312.202.50 3.04 6525.83 0.7411.0143.9313.002.66 2.82 7225.82 0.7912.1045.8311.772.22 1.48 9624.26 0.5513.9046.2411.792.29 0.43 11225.95 0.8211.8447.0411.052.38 0.92 12026.08 0.8511.5847.3711.242.06 0.82 Table 2: Fatty acid composition of intracellular lipids accumulated in C. curvatus, compared to the fatty acid composition of the surfactant molecules it produces. Surfac tant Yeastt (h)C16:0C16:1C18:0C18:1C18:2C18:3 (Δ9,12,15) Others Cryptococcus curvatus 14431.25-24.2731.6612.82-- References [1] Mulligan et al. 2001. Engineering Geology 60 (2001) 371-380. [2] Ciapina et al. 2006. Applied Biochemistry and Biotechnology 880 Vol. 129-132, 2006. [3] Desai and Banat. 1997. Microbiology and Molecular Biology Reviews Mar. 1997, p. 47–64 Vol. 61, No. 1 [4] Folch et al. 1957. J Biol Chem 199:833–841. [5] AFNOR (1984) Association Française pour Normalisation. Paris, p 95 [6] Cirigliano and Carman.1984. Applied and Environmental Microbiology, Oct. 1984, p.747-750 Acknowledgments Financial support was provided by REVOIL SA as a scholarship, in the field of renewable energy sources Results and discussion Growth of a variety of yeasts was studied on nitrogen-limited media, containing glycerol (30 g/l) as sole carbon and energy source. All strains studied, except for C. tropicalis, showed significant growth (biomass produced up to 10 g/l). C. curvatus and P. ciferrii consumed almost half of the initial quantity of glycerol and accumulated a significant amount of intracellular lipids (up to 12%), comparing to the other yeasts studied. Furthermore, while P. ciferrii and C. curvatus demonstrate emulsification activity of about 100 U/g DW, the other yeast strains show no activity, when grown on glycerol as sole carbon source. Fatty acid composition of intracellular lipids of these yeasts and of the biosurfactant produced by C. curvatus was determined by GC analysis. Palmitic (C16:0) and oleic acids (C18:1) are the major fatty acids accumulated in the cells of C. curvatus, followed by linoleic acid (C18:2). The biosurfactant presented a differentiated fatty acid composition, meaning that it is newly synthesized and not intracellular lipids excreted in the growth environment. The HPLC analysis of the biosurfactant produced by C. curvatus presented a sugar component which could not be identified by comparing its RT with those of common sugars. In conclusion, C. curvatus was found to produce a quantity of 200 mg/l of biosurfactant agents with significant emulsification activity. This product could be of great potential interest in various areas of industry and in bioremediation processes. Figure 2: Isolation of biosurfactant produced by Cryptococcus curvatus.