Article

DOi: 10.2478/V10133-010-0084-5

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bIoMass productIon bY antarctIc YEast straIns: an InvEstIgatIon on tHE LIpId coMposItIon

M. Zlatanov1, K. Pavlova2, G. Antova1, M. Angelova-Romova1, K. Georgieva2, S. Rousenova-Videva2 1 University of Plovdiv, Department of chemical technology, Plovdiv, Bulgaria 2 Institute of Microbiology, Bulgarian Academy of Sciences, Laboratory of Applied Microbiology, Sofia, Bulgaria correspondence to: M. Zlatanov e-mail: magzlat@uni-plovdiv.bg

Psychrophilic yeast strains Rhodotorula glutinis AL107, Sporobolomyces roseus AL108, Cryptococcus albidus AL55, Cryptococcus laurentii AL56 and Cryptococcus laurentii AL58 isolated from soil sample taken from the region of the Bulgarien base on Livingston Island, Antarctica, were studied. The biomass production was followed after cultivation of the yeasts in a medium with pH 5.3 at 15C for 120 h. The biomass concentration by psychrophilic yeast strains was: R. glutinis AL107-6.05 g/l, S. roseus AL108-5.78 g/l, Cr. albidus AL55, Cr. laurentii AL56 and Cr. laurentii AL58-6.52 g/l, 6.84 g/l and 6.24 g/l, respectively. The extracted and separated lipids from the samples were supplied to analysis and the compositions of fatty acids, phospholipids, sterols as well as tocopherols were determined. Unsaturated fatty acids, mainly oleic (58.6-63.5%) and of saturated palmitic (18.2-24.5%), predominated in triacylglycerols. Sterols (0.1-0.3%) were valued in the dry yeast biomass. The content of phospholipids, mainly phosphatidylcholine, phosphatidylinositole and phosphatidylethanolamine was found to be in the range of 0.2-1.6%. The quantity of tocopherols was 0-26.3 mg/kg. All of tocopherol classes were established. Biotechnol. & Biotechnol. eq. 2010, 24(4), 2096-2101 Keywords: psychrophilic yeast strains, biosynthesis of the yeasts, lipids, fatty acids, phospholipids, sterols, tocopherols the existence of yeast in extreme Antarctic conditions is related to the content of lipids which have a structural and protective role in the cell. Membrane lipid composition has been considered to account for the lower and upper temperatures of growth of the psychrophilic yeasts. Phospholipids especially phosphatidylcholine have important significance for building of cell membrane structures. lipid characterization of yeasts and preparation of lipids as biologically active products were studied by a large number of researchers (1, 2, 6, 8, 11, 13, 14, 18, 19, 20, 22, 27). lipids synthesized by microorganisms have been mainly used in pharmaceutical industry, for technical purposes and more rarely as fodder. Microbial lipids have been known as a source of special oils and fats with high industrial potential for application and evaluated as an alternative source of animal and plant oils (10, 11). they have the potential to be used for interesterification and as a substitute for certain dietary lipids. this paper discusses the lipid content and composition of the biologically active lipid constituents as fatty acids, phospholipids, sterols and tocopherols in the selected Antarctic yeast strains.

abstract

Introduction

Search for novel and biotechnologically unexplored microorganisms motivates the vast interest of scientists in the last two-three decades to the habitants of the extreme niches. now-a-day, it is clear that some extremophiles have novel metabolic pathways and so might serve as a source of exopolysaccharides and enzymes with novel properties and uses (12, 15). Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and Polar Regions. the ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently extreme conditions. One such defiance is lower membrane permeability of cells. the decrease of temperatures has an adverse effect on the physical properties and functions of membranes, normally leading to a reduction in membrane fluidity. Several studies have clearly indicated that the ability to modulate membrane fluidity by regulating the synthesis of fatty acids is very crucial for low temperature adaptation (9, 24). lower growth temperatures produce a higher content of unsaturated fatty acids compared to the saturated fatty acids. this phenomenon applies to bacteria or yeasts (6, 9, 21, 24) and in several species of psychrophilic yeasts so that the unsaturated fatty acids would constitute 50.0-90.0% of the total fatty acid composition as in species of Mrakia, Candida, Torulopsis, Leucosporidium and Cryptococcus (25, 26). 2096

Materials and Methods

Yeast strains The five yeast strains Rhodotorula glutinis Al107, Sporobolomyces roseus Al108, Cryptococcus albidus Al55, Cryptococcus laurentii Al56 and Cryptococcus laurentii Al58 included in this study were isolated from soil from the region of the Bulgarian base on livingston island, Antarctica. the strains were maintained on malt slant agar and stored at 4c. Biotechnol. & Biotechnol. eq. 24/2010/4

culture media and fermentation conditions the basal medium containing (g/l): sucrose- 40.0, (nh4)2So42.5, Kh2Po4- 1.0, MgSo4.7h2o- 0.5, naci- 0.1, cacl2.2h2o0.01 and yeast extract- 1.0. the initial ph was adjusted to ph 5.3 and the medium was sterilized for 30 min at 112c. the cultivation proceeded in shaken 500-ml Erlenmeyer flasks containing 100 ml medium inoculated with 10% (V/V), 48 h inoculum, for 120 h at 15c and 220 rpm. analytical methods All solvents and reagents were analytical grade and were used without additional purification. Reference phospholipids and fatty acid methyl esters were purchased from Fluka (chemie Gmbh, Switzerland). Reference tocopherol isomers and individual sterols were purchased from Merck (Darmstadt, Germany). tlc (thin-layer chromatography) plates were prepared in the laboratory using Silica gel G 60 (Merck, Darmstadt, Germany). biomass production Biomass was separated by centrifugation at 6000 x g, washed twice with distilled water and lyophilised. Dry weight of the biomass prepared by Antarctic yeast strains was determined after drying at 105c until constant weight (13). Isolation of glyceride oil and determination of oil content Biomasse (50 g sample) was air-dried and the oil was extracted with n-hexane in Soxhlet for 8 h. the solvent was partly removed in rotary vacuum evaporator, the residue was transferred in pre-weight glass vessels and the rest of the solvent was removed under stream of nitrogen to a constant weight to determine the oil content (23). Fatty acids the total fatty acid composition as well as the fatty acid composition of phospholipids was determined by gas chromatography (Gc) after transmethylation of the respective sample with 2% h2So4 in ch3oh at 50oc according to christie (10). Fatty acid methyl esters (FAME) were purified by Silica gel tlc on 20 cm x 20 cm plates covered with 0.2 mm Silica gel 60 G layer (Merck, Darmstadt, Germany) with mobile phase n-hexane:acetone 100:8 l. Gc was performed on a HP 5890 (hewlett Packard Gmbh, Austria) gas chromatograph equipped with a 30 m x 0.25 mm (i.D.) capillary innoWax column (cross-linked PeG, hewlett Packard Gmbh, Austria) and a flame ionization detector (FID). The column temperature was programmed from 165oc to 240oc at 4oc/min and held at this temperature for 10 min; injector and detector temperatures were 250oC. Nitrogen was the carrier gas at a flow rate 0.8 ml/min; split was 100:1. Identification was performed by comparison of retention times with those of a standard mixture of fatty acids subjected to Gc under identical experimental conditions (3). phospholipids Another part (10 g) of air-dried biomass was subjected to Folch extraction according to christie (10). lipids from Polar strains Biotechnol. & Biotechnol. eq. 24/2010/4

were isolated from the total lipids by column chromatography (10). Briefly, the sample (100 mg) was applied on a 40 cm x 2 cm glass column packed with Silica gel Unisil 100-200 mesh (Clarkson Chemicals Co., USA) and eluted in sequence with chloroform (for neutral lipids, sterols and sterol esters), acetone (sterol glycosides) and with methanol to isolate phospholipids. the phospholipid classes were isolated by two-dimensional thin-layer chromatography (7) on 20 cm x 20 cm glass plates with 0.2 mm Silica gel 60 G layer (Merck) impregnated with 1% aqueous (nh4)2So4. In the first direction the plate was developed with chloroform:methanol:ammonia, 65:25:5 l and in the second- with chloroform:acetone:methanol:acet ic acid:water, 50:20:10:10:5 l. the individual phospholipids were detected and identified by spraying with specific reagents according to christie (10). Dragendorff test (detection of choline-containing phospholipids), ninhydrin spray (for phospholipids with free amino groups), and Shiffs reagent (for inositol containing phospholipids) were employed. Additional identification was performed by comparing the respective Rf values with those of authentic commercial standards subjected to Silica gel tlc under identical experimental conditions. the quantification was carried out spectrophotometrically against a standard curve by measuring the phosphorous content at 700 nm after scrapping the respective phospholipid spot and mineralization of the substance with a mixture of perchloric acid and sulphuric acid 1:1 l. sterols the oil was hydrolized with ethanolic Koh (10); sterols were extracted with light petroleum ether and purified by TLC under the above conditions prior the Gc analysis. Sterol content was determined on HP 5890 gas chromatograph (hewlett Packard Gmbh, Austria) equipped with 25 m x 0.25 mm hP5 capillary column (Agilent technologies, Santa clara cA, USA) and flame ionization detector (4). Temperature gradient was set from 90oc (held for 2 min) to 290oc at 15oc/min then to 310oc at 4oc/min and held at this temperature for 10 min. the injector temperature was 300oc and the detector temperature was 320oC. Nitrogen was the carrier gas at a flow rate 0.8 ml/ min; split 100:1. tocopherols tocopherols were determined directly in the oil by high performance liquid chromatography (hPlc) on a MerckHitachi (Merck, Darmstadt, Germany) instrument equipped with 250 mm x 4 mm nucleosil Si 50-5 column (Merck, Darmstadt, Germany) and fluorescent detector Merck-Hitachi F 1000. the operating conditions were as follows: mobile phase of n-hexane:dioxan 96:4 l, flow rate 1 ml/min, excitation 295 nm, emission 330 nm. 20 kl 1% solution of oil were injected (5, 16). Tocopherols were identified by comparing the retention times with those of authentic individual tocopherols. the tocopherol content was calculated on the base of tocopherol peak areas in the sample vs. tocopherol peak area of standard a-tocopherol solution. 2097

results and discussion

biomass production Pigment-forming R. glutinis Al107 and S. roseus Al108 were selected as the strains with the highest content of total lipids in the biomass and Cr. albidus Al55, Cr. laurentii Al56 and Cr. laurentii Al58 as -carotene source (12). The biomass production was: R. glutinis Al107- 6.05 g/l, S. roseus Al1085.78 g/l, Cr. albidus Al55, Cr. laurentii Al56 and Cr. laurentii Al58- 6.52 g/l, 6.84 g/l and 6.24 g/l, respectively. general characteristics of the oil Main characteristics of yeasts and lipids were determined such as: lipid content, content of total phospholipids, sterols and tocopherols. the results are shown in table 1. the level of lipids in the investigated biomass synthesized by S. roseus Al108, cultivated at 15oc was highest- 7.4%. the content of lipids in the yeasts of Cryptococcus (2.4-5.6%) was significantly higher than that established earlier in other strains from the same species, when it was 1.0-1.9% (27). the quantities of sterols were in the same range (0.1-0.3%). the content of tocopherols varied from 9.1 to 26.3 mg/kg, except for S. roseus Al108, where only traces were established. the phospholipid percentage was also higher in comparison with the data of previous investigations (27). Fatty acid composition of triacylglycerols Fatty acid composition of triacylglycerols in the microbial lipids determined by capillary gas chromatography is presented in table 2. in biomass of R. glutinis Al107, Cr. albidus Al55, Cr. laurentii Al56 and Cr. laurentii Al58 twelwe fatty acids were detected while almitoleic (16:1), erucic acid (c22:1) were not established in triacylglycerols of S. roseus Al108 and linolenic acid (18:3) in Cr. albidus Al55. Fatty acid analysis of biomass synthesized from the five strains revealed oleic acid (18:1) to be the predominant fatty acid (58.6-63.5%). the values of the polyunsaturated fatty acids were linoleic- 3.6% and linolenic- 2.2% for strain R. glutinis Al107, respectively 4.7% and 2.3% for strain S. roseus Al108. these levels were higher than the content of polyunsaturated fatty acids in biomasses of Cryptococcus. the obtained data about unsaturated acids (66.1-70.5%) corresponds to the results reported by earlier investigations (13, 26, 27). the quantity of palmitic acid, the main saturated acid, was 18.2-24.5% and stearic acid- 3.55.2%. the other acids were presented in negligible quantities. Unsaturated acids predominated in all investigation samples of triacylglycerols. phospholipid composition Data about phospholipid composition are presented in table 3. The main classes of phospholipids were identified in all samples, but the quantitative composition was different. in the phospholipid fraction of R. glutinis Al107 phosphatidylethanolamine (49.2%) predominated, followed by phosphatidylcholine and phosphatidylinositol. in the 2098

S. roseus Al108, Cr. laurentii Al56, Cr. laurentii Al58 the main component was phosphatidylcholine (37.5%, 43.5% and 55.7%, respectively) and in the Cr. albidus Al55phosphatidylinositol (55.3%). these compositions were different from those of other Antarctic yeasts, where high quantities of lysophosphatidylcholine (more then 10.0%) were established (27). Fatty acid composition of the main phospholipid classes- phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine was determined (table 4). Monounsaturated oleic acid predominated, but its content varied in a significant range. The highest quantity of oleic acid was established in the phosphatidylcholine fraction (22.0-63.0%) and the lowest was in the phosphatidylinositol (1.3-28.4%). in the phosphatidylethanolamine fraction the content of oleic acid was too low: for Cr. laurentii Al55 and Cr. laurentii Al56- 11.3% and 2.3 %, respectively. in the phosphatidylethanolamine of Cr. albidus Al55 very high content of palmitoleic acid (18.5%) was valued. A significant quantity of unsaturated fatty acids, mainly linoleic, was found in phosphatidylinositol of Cr. laurentii Al56 (18.3%) and phosphatidylethanolamine of Cr. laurentii Al58 (30.2%). linolenic acid was mainly established in the phosphatidylinositol fraction (4.6-15.9%). Palmitic acid (8.6-37.0%) predominated among the saturated acids. in the phosphatidylinositol fraction of all yeasts a significantly high percentage of unusual saturated fatty acids, such as margarinic (9.1-28.0%) and arahinic acid (9.1-32.3%) was established. these contents were about 10 times higher than the ones in other lipids. the percentage of wide-spread saturated palmitic acid was lower than the common occurrence. Behenic acid (13.8%) was fixed in phosphatidylcholine of Cr. laurentii Al58. We may conclude that fatty acid composition of phospholipids varies in large limits depending on the species of yeasts. in all cases when fatty acid composition of triacylglycerols in phospholipids was compared it was observed that a higher content of saturated fatty acids (c16:0, c17:0, c18:0, c20:0) was identified at the expense of the lower content of oleic and linoleic acids. these differences between fatty acid composition of triacylglycerols and phospholipids may be due to different phases of the biosynthesis of those compounds and the stages of biosynthesis and accumulation of fatty acids. According to Munchi and all (17) phospholipids are synthesized first and then triacylglycerols are accumulated. The first stage is also characterized by a high concentration of saturated fatty acids, especially palmitic, stearic, margarinic, behenic which are accumulated in phospholipids. in the second stage the unsaturated fatty acids (oleic and linoleic) are synthesized and they are accumulated in triacylglycerols. tocopherol composition individual qualitative and quantitative tocopherol composition of the oils established by hPlc is presented in table 5. Biotechnol. & Biotechnol. eq. 24/2010/4

General composition of Antarctic yeasts and their lipids compounds in the yeast biomass Lipids, % wt phospholipids, % wt in the biomass in the lipids sterols, % wt in the biomass in the lipids tocopherols, mg/kg in the biomass in the lipids Yeast strains

tabLE 1

R. glutinis aL107 S. roseus aL108 Cr. albidus aL55 Cr. laurentii aL56 Cr. laurentii aL58 4.3 0.2 4.6 0.3 7.7 9.1 213.4 7.4 1.2 14.8 0.1 0.4 tr. tr. 5.6 1.6 28.6 0.2 3.1 25.2 588.1 5.2 0.8 15.4 0.3 5.9 26.3 609.1 2.4 1.5 62.5 0.1 1.5 11.2 268.4 tabLE 2

Fatty acid composition of triacylglycerols Fatty acids* 12:0 14:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 saturated / unsaturated Yeast strains, % wt R. glutinis aL107 S. roseus aL108 1.0 1.1 21.4 1.4 1.7 4.9 58.6 3.6 2.2 2.2 1.6 0.3 33.9 66.1 0.4 0.5 18.2 1.3 4.0 63.5 4.7 2.3 3.8 1.3 29.5 70.5 Cr. albidus aL55 0.7 3.5 22.6 0.4 0.4 4.0 62.7 2.1 0.6 1.8 1.2 33.6 66.4

Cr. laurentii aL56 Cr. laurentii aL58 0.1 1.4 24.5 0.3 0.2 5.2 62.3 1.2 1.3 1.2 1.3 1.0 33.9 66.1 0.2 0.3 21.1 0.3 0.2 3.5 62.4 2.8 2.7 3.6 1.5 1.4 30.4 69.6

*12:0- Lauric acid; 14:0- Myristic acid; 16:0- Palmitic acid; 16:1- Palmitoleic acid; 17:0- Margarinic acid; 18:0- Stearic acid; 18:1- Oleic acid; 18:2- linoleic acid; 18:3- Linolenic acid; 20:0- Arahinic acid; 22:0- Behenic acid; 22:1- erucic acid

Phospholipids composition of Antarctic yeast lipids phospholipids phosphatidylcholine phosphatidylinositol phosphatidylethanolamine phosphatidic acids Lysophosphatidylcholine Lysophosphatidylethanolamine Monophosphatidylglycerine diphosphatidylglycerine sphyngomieline phosphatidylserine

tabLE 3

Yeast strains, % wt R. glutinis aL107 S. roseus aL108 Cr. albidus aL55 Cr. laurentii aL56 Cr. laurentii aL58 19.8 18.5 49.2 12.5 37.5 20.2 17.7 5.3 1.5 3.5 2.3 1.3 2.4 8.3 26.5 55.3 7.3 1.2 1.1 2.1 0.7 5.8 43.5 7.6 20.7 2.7 2.9 6.9 0.4 5.9 9.4 55.7 13.4 19.3 0.8 4.7 0.8 0.1 5.2 2099

Biotechnol. & Biotechnol. eq. 24/2010/4

Fatty acid composition of phospholipids in lipids Yeast strains, % wt R. glutinis aL107 S. roseus aL108 Cr. albidus aL55 Cr. laurentii aL56

tabLE 4

Fatty acids 12:0 14:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 saturated / unsaturated

*pc- Phosphatidylcholine; pEa- Phosphatidyletanolamine; pI- Phosphatidylinositol

PC* PEA* PI* PC PEA PI PC PEA PI PC PEA PI PC PEA PI 14.4 1.0 3.4 0.7 10.7 2.6 5.0 3.0 2.7 4.2 2.0 1.0 1.8 7.1 5.8 9.5 2.7 1.5 19.3 20.8 8.6 31.2 26.0 12.8 37.0 29.1 14.8 16.7 32.2 12.9 29.5 10.9 13.8 1.2 5.2 1.7 3.2 1.1 18.5 1.7 1.5 1.0 0.7 1.0 1.7 3.8 25.7 0.6 4.2 28.0 4.6 12.0 27.2 1.5 9.1 4.5 1.8 25.5 6.3 10.4 4.2 9.4 9.0 7.6 6.2 4.4 9.8 6.0 3.6 6.9 10.7 3.8 6.8 53.5 50.6 8.7 27.1 44.7 2.3 39.1 11.3 1.3 63.0 2.3 28.4 22.0 35.8 7.5 10.7 1.8 2.4 7.8 1.2 2.2 2.1 2.7 5.4 18.3 1.0 30.2 1.7 1.8 2.3 15.5 0.7 2.6 14.3 1.8 6.2 15.9 0.8 15.0 4.6 2.4 0.8 13.6 1.3 3.3 32.3 0.5 4.5 30.1 3.9 13.8 30.0 1.5 36.2 9.1 5.1 1.8 29.1 4.2 1.8 1.6 0.6 1.3 1.8 0.3 13.8 0.5 0.5 0.3 0.3 0.2 0.1 32.8 40.1 73.4 62.7 48.3 81.2 55.9 61.3 82.8 28.8 80.9 47.5 73.8 32.2 77.2 67.2 59.9 26.6 37.3 51.7 18.8 44.1 38.7 17.2 71.2 19.1 52.5 26.2 67.8 22.8 tabLE 5

Cr. laurentii aL58

tocopherol composition of Antarctic yeast lipids tocopherols (t) and tocotrienols (t-3) -T -T-3 b-t -T -T Yeast strains, % wt R. glutinis aL107 S. roseus aL108 8.4 37.7 26.8 27.1 tr. Cr. albidus aL55 85.9 1.6 4.9 3.5 4.1

Cr. laurentii aL56 Cr. laurentii aL58 79.7 3.1 7.2 4.9 5.1 5.0 13.2 33.0 25.2 23.6

All classes of tocopherols (a-, b-, g- and -) were identified in relatively close percentages in the yeast lipids of R. glutinis Al107 and Cr. laurentii Al58. a-tocopherol predominated in Cr. albidus Al55 and Cr. laurentii Al56 (85.9% and 79.7%, respectively). in Cr. albidus Al55, Cr. laurentii Al56 and Cr. laurentii Al58 except tocopherols also - tocotrienol was found (1.6-13.2%).

acknowledgements

The investigations were carried out by the financial support of University of Plovdiv Paisii hilendarski, the Grant X-25/2009.

rEFErEncEs

conclusions

on the basis of the obtained experimental data we may conclude that when the composition of culture media and fermentation conditions vary yeast strains with desired content of microbial lipids can be obtain. the lipids of Cr. albidus Al55 and Cr. laurentii Al58 were very rich in phospholipids (28.6% and 62.5 %, respectively); R. glutinis Al107 and Cr. laurentii Al56- in steros (7.7% and 5.9%, respectively). high level of tocopherols was provided by Cr. albidus Al55- 588.1 mg/kg and Cr. laurentii Al56- 609.1 mg/kg. 2100

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