ANALYSIS OF FATTY ACIDS IN THE BIOEMULSIFIER FROM YARROWIA LIPOLYTICA EH 59

Alicja Szulc¹, Łukasz Ławniczak¹, Paweł Cyplik², Andrzej Olszanowski^1
1 Institute of Chemical Technology and Engineering, Poznań University of Technology, Poland
² Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland

ABSTRACT

Contradictory opinions exist regarding the fatty acids content of the bioemulsifier obtained from Yarrowia lipolytica yeast. Our research with Y. lipolytica EH 59 strain showed that cells cultivated on glucose, glycerol or soybean oil excreted a bioemulsifier which does not contain fatty acids permanently bound to polysaccharide-protein matrix. Fatty acids profiles of cell membranes and the bioemulsifier posed similar composition, indicating that rather than being a selective excretion, they are impurities from the cells responsible for the detection of fatty acids in the bioemulsifier.

Key words: bioemulsifier, biosurfactant, fatty acids, Yarrowia lipolytica, yeast.

INTRODUCTION

Microbial growth is often associated with the production of extracellular compounds, which may be of potential industrial interest [7,16,17,20,21,22,23,24]. Some of them, including lipopeptides, glycolipids, fatty acids and polymeric substances were found to be surface active [6,9]. Numbers of bacterial species were recognized to produce biosurfactants (i.e., rhamnolipids produced by Pseudomonas æruginosa) [1,2,10,15]. Yeast are also capable of producing surfactants during growth on substrates such as fatty acids, glycerol or simple sugars. Yarrowia lipolytica ATCC 8662 strain has been reported to produce significant amounts of biosurfactant called liposan [4,14]. It was suggested that apart from polysaccharides and proteins, liposan also contains fatty acids [14]. On the other hand, Cirigliano and Carman [4] suggested the opposite. Thus the aim of this study was to obtain liposan using various carbon sources and study its fatty acids content. Additionally, comparison of fatty acids profiles of the biosurfactant and cellular lipid composition was investigated.

MATERIAL AND METHODS

Microorganism and chemicals
Yarrowia lipolytica EH 59 was kindly donated by Umweltvorschungszentrum GmbH, Leipzig-Halle, Germany and was maintained on YM agar plates stored at 4°C. The commercially available chemicals were purchased from Merck Eurolab GmbH (Darmstadt), Fluka, Riedel de Haen (Seelze) and Sigma – Aldrich Laborchemikalien GmbH (Seelze).

Culture conditions
For bioemulsifier production, the yeast were cultivated in 500-ml Erlenmeyer flasks [100 ml 0.6% (w/v) yeast nitrogen base medium (YNB)], supplemented with 5, 10 or 15 gL^-1 of glucose, glycerol or sunflower oil. The flasks were inoculated with approximately 1×10⁸ cells and incubated at 30° C at 120 rpm for 168 hours. The pH was adjusted to 5.5±0.1 every 24 h using 2 N NaOH. Growth was monitored every 24 hours by aseptically collecting samples (4 ml) during the experiment and measuring their optical density at 600 nm. All experiments were performed in triplicates.

Isolation of the bioemulsifier from Y. lipolytica EH 59
Two methods of bioemulsifier separation were applied. The first method was performed according to the procedure described by others [5,8,11,18,19], in which growth the culture was filtered through a filter paper and later through a cellulose membrane. The cell-free medium was transferred to a separatory funnel and extracted with 500 ml of chloroform-methanol solution (2:1). The creamy precipitate formed between the aqueous and the organic phase was collected on cellulose filter and nitrogen stream dried.

In the second method, the culture medium was stored at 4°C for 24 h to ensure cell sedimentation. The broth of nitrogen-limited culture on glycerol was separated from cells by centrifugation at 10000×g for 10 min. The resulting cell-free medium was then filtered through a 0.45 µm membrane (Millipore). Approximately 80 ml of the cell-free filtrate was transferred to a separatory funnel and supernatant was acidified to pH 2 with H₂SO₄ and extracted with 50 ml of ethyl acetate. The aqueous phase was extracted again two times with 50 ml of ethyl acetate. A white precipitate formed in the aqueous phase after the first extraction was collected on filter paper (Whatman no. 42) and dried in the nitrogen stream. The organic phase was dried with anhydrous Na₂SO₄ before evaporation in a Büchi R-144 flash evaporator. Both, the crude precipitate and the organic phase, were subjected to fatty acids profile analyses. The precipitate was weighted after drying.

Analysis of fatty acids in the emulsifier
To determine the fatty acids composition of bioemulsifier, 5 mg of each crude precipitate was treated with 2 ml of 2% H₂SO₄ in methanol  at 90°C for 2h. Later, the sample was subjected to extraction with n-hexane. Obtained FAMEs were analysed by a GC-MS (HP6890N-HP5973, Hewlett Packard, USA) on a BPX-5 column (30 m × 0.32 mm, 0.25 mm film thickness, SGE, Germany). Helium was used as a carrier gas at a constant flow of 2 ml min^-1. Injector temperature was set at 280°C. The oven temperature programme was run for 1 min isothermal at 70°C, from 70°C to 150°C (8°C min^-1), from 150°C to 220°C (2°C min^-1) and then for 1 min isothermal at 220°C, from 220°C to 300°C (20°C min^-1). Mass spectra were collect in a full scan mode (m/z 30–400) at a transfer line temperature of 280°C, a source temperature of 230°C. Sum of the peak areas of the FAMEs were used to determine their relative amounts. The fatty acids were precisely identified by co-injection of a reference compounds (Supelco, Bellefonte, USA).

Analysis of fatty acids in the biomass
Biomass from the samples was harvested by centrifugation (7000×g for 10 min), washed two times with potassium phosphate buffer (50 mM, pH 7.0) and stored at -20°C prior to the lipid extraction. Frozen cells were washed with 5 ml of 95% ethanol  and subsequently with 5 ml of n-hexane  in order to remove extracellular fat from the cell surface. Lipids were extracted with a chloroform-methanol-water mixture according to a method described by Bligh and Dyer [3]. Fatty acid methyl esters (FAMEs) were prepared by using extracted lipids and boron trifluoridmethanol (15 min at 80°C) applying the method of Morrison and Smith [12] and extracted with hexane. The methylesters were analysed by fatty acid composition with GC-MS as described earlier.

RESULTS AND DISCUSSION

Bioemulsifier production
The amount of the bioemulsifier excreted to the medium was clearly dependent on the amount of growth substrate. It was determined that 15 gL^-1 was the optimum amount of glucose for bioemulsifier production (Table 1). Similar results were obtained from cells cultivated on glycerol with 1.34 g of bioemulsifier produced by a culture with 15 gL^-1 glycerol. As expected the bioemulsifier production is somehow correlated with the amount of obtained biomass. The maximal biomass yield was 6.5 gL^-1 when 15 gL^-1 of glucose  in the medium.

Table 1. The amount of the bioemulsifier excreted to the medium by Y. lipolytica EH 59

Growth substrate	Glucose			Glycerol [gL-1]
C source [gL-1]	5	10	15	5	10	15
Emulsifier [g/l]	0.12	0.73	1.28	0.22	0.84	1.34
Biomass yield [g/l]	1.9	4.1	6.5	1.2	3.8	5.1

Due to analytical problems we did not attempt to produce and isolate bioemulsifier from yeast grown on sunflower oil.

Analysis of fatty acids in biomass
Y. lipolytica EH 59 exhibited storage lipid accumulation when glucose was used as a substrate. No significant changes in fatty acids profiles were observed during microbial growth, regardless of the substrate used. Analyses of fatty acids extracted from biomass after 48 h of cultivation (15 gL^-1 glucose) showed that the C18:1 was the dominant fatty acid (Table 2). The C16:0, C18:0 and C18:2 were detected at the same level of about 15%. The shift from glucose to glycerol affected the lipid composition. Both the amount and the degree of unsaturation of accumulated fatty acids increased compared to the yeast cultivated on glucose (data not shown). The most significant differences were observed for palmitoleic (C16:1) and oleic (C18:1) fatty acids (Table 2). As a result, the unsaturated to saturated fatty acids ratio was higher for the yeast cultivated on glycerol. The cells cultivated on sunflower oil exhibited similar fatty acid profile to those cultivated on glucose. Accumulation of a single-cell oil (yield 20%, data not shown) was observed for yeast cultivated on all carbon substrates (glycerol, glucose and sunflower oil). Recently, other Y. lipolytica strain has been reported to produce significant amounts of lipids, rich in both non-saturated acids when grown on fatty substrates [13].

Table 2. Fatty acids profile of Y. lipolytica EH 59 cell lipids cultivated on various carbon sources

Growth substrate	Fatty acids [%]*
	16:0	16:1	18:0	18:1	18:2	others
glucose	16.5	4.0	16.8	30.7	13.3	18.6
glycerol	14.1	12.2	3.6	50.3	16.8	2.8
sunflower oil	20.2	5.8	15.1	36.7	13.0	9.4

Analysis of fatty acids in the bioemulsifier
No significant differences were found between the crude precipitate obtained using the two described methods (data not shown), as well as between fatty acids in the crude precipitate and the organic phase (Fig. 1). This can be attributed to the fact that fatty acids were not bound with the organic matrix of the precipitate (this was additionally confirmed by chemical analyses). These results correspond well to those obtained by Cirigliano and Carman [4]. In addition to this, the fatty acids profile of the cells cultivated on glucose (Table 1) was identical to the profile of the bioemulsifier (Fig. 1). This supports the statement that traces of fatty acid found in liposan by Pareilleux et al. [14] were probably an artefact from cells impurities. Similar results were obtained for cells cultivated on glycerol, with slightly higher amount of alpha-linolenic acid (C18:3) and lower of oleic acid (C18:1) (data not shown). The composition of liposan was confirmed to consist of carbohydrates (83%) and  protein (17%), which corresponds well to the composition described by Cirigliano et al. [4].

Fig. 1. Fatty acids profile from the organic phase and the bioemulsifier (crude precipitate), produced by Y. lipolytica EH 59 cultivated on glucose

CONCLUSIONS

1. The bioemulsifier produced by Y. lipolytica EH 59 does not contain fatty acids permanently bound to polysaccharide-protein matrix.

2. It was possible to obtain the bioemulsifier from cells of Y. lipolytica EH 59 grown on "water soluble" carbon sources like glucose or glycerol.

3. Composition of intracellular fatty acids depends on the growth substrate nature.

ACKNOWLEDGEMENTS

We are thankful to L. Chrzanowski and M. Owsianiak for fruitful discussions and comments on the manuscript. The valuable contribution from our colleagues, ensuring proper handling with yeast and pleasant atmosphere while working in the university laboratories is greatly appreciated. Financial support from PUT BW 32/003/09.

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Accepted for print: 6.11.2009

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Alicja Szulc
Institute of Chemical Technology and Engineering,
Poznań University of Technology, Poland
M. Skłodowskiej-Curie 2, 60-965 Poznań, Poland
Phone: +48-61-665-37-16
email: alicja_szulc@wp.pl

Łukasz Ławniczak
Institute of Chemical Technology and Engineering,
Poznań University of Technology, Poland
M. Skłodowskiej-Curie 2, 60-965 Poznań, Poland

Paweł Cyplik
Department of Biotechnology and Food Microbiology,
Poznań University of Life Sciences, Poland
Wojska Polskiego 48, 60-627 Poznań, Poland

Andrzej Olszanowski
Institute of Chemical Technology and Engineering,
Poznań University of Technology, Poland
M. Skłodowskiej-Curie 2, 60-965 Poznań, Poland

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