Bulgarian Antarctic Research: Life Sciences

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BULGARIAN ANTARCTIC RESEARCH VOLUME 4

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BULGARIAN ANTARCTIC RESEARCH Life Sciences Vol. 4

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Bulgarian Antarctic Institute

Bulgarian Academy of Sciences

BULGARIAN ANTARCTIC RESEARCH Life Sciences Volume 4

Edited by

Acad. V. Golemansky & Dr. N. Chipev

Sofia - Moscow 2004

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Bulgarian Academy of Sciences

© PENSOFT Publishers ISBN 954-642-219-3 (Vol. 4) First published 2004 All rights reserved

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form by any means, electronic, mechanical, photo copying, recording or otherwise, without the prior written permission of the copyright owner.

Pensoft Publishers, Geo Milev str., No. 13a, 1111 Sofia, Bulgaria E-mail: [email protected], www.pensoft.net

Printed in Bulgaria, December 2004

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Contents SOIL CHARACTERISTICS AND MICROBIAL ABUNDANCE OF MOSS AND COASTAL HABITATS OF LIVINGSTON ISLAND – ANTARCTICA A. Kenarova, V. Bogoev .................................................................................................................. 1

LIPID COMPOSITION AND β-GLUCOSIDASE PRODUCTION FROM CRYPTOCOCCUS VISHNIACII AL4 K. Pavlova, M. Zlatanov, G. Angelova, I. Savova ....................................................................... 9

KERATINASE PRODUCTION OF THERMOPHILIC ACTINOMYCETES SPECIES FROM ANTRACTICA A. Gushterova, M. Noustorova, K. Pavlova ............................................................................. 23

ISOLATION AND TAXONOMIC STUDY OF ANTARCTIC YEASTS FROM LIVINGSTON ISLAND FOR EXOPOLYSACCHARIDE-PRODUCING K. Pavlova, A. Gushterova, I. Savova, M. Nustorova .............................................................. 27

BIOCHEMICAL CHARACTERISTIC OF ANTARCTIC YEASTS K. Pavlova, M. Zlatanov, L. Koleva, I. Pishtiyski ...................................................................... 35

EFFECT OF TEMPERATURE AND SODIUM CHLORIDE ON THE BIOMASS AND FATTY ACIDS COMPOSITION OF ANTARCTIC YEAST STRAIN SPOROBOLOMYCES ROSEUS AL8 L. Koleva, K. Pavlova, M. Zlatanov ............................................................................................ 47

ALKALOIDS FROM THE ANTARCTIC STRAIN MICROBISPORA AERATA SUBSP. NOV. IMBAS-11A. ISOLATION, SEPARATION AND PHYSICO-CHEMICAL PROPERTIES V. Ivanova, U. Graefe, R. Schlegel, A. Gusterova, K. Aleksieva, M. Kolarova, R. Tzvetkova ........................................................................................................... 55

ARRHENIA RETIRUGA (BULL.: FR.) REDHEAD VAR. ANTARCTICA HORAC – ONE AGARICAL FUNGUS FROM LIVINGSTON ISLAND, SOUTH SHETLANDS (THE ANTARCTIC) M. Gyosheva and R. Metcheva ..................................................................................................... 65

DISTRIBUTION OF FRESHWATER ALGAE ON LIVINGSTON ISLAND, SOUTH SHETLANDS ISLANDS, ANTARCTICA .II. (CYANOPROKARYOTA) D. Temniskova-Topalova, R. Zidarova ....................................................................................... 69

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CONTENTS

ADDITIONAL DATA AND SUMMARIZED CHECK-LIST ON THE RHIZOPODS (RHIZOPODA: AMOEBIDA & TESTACEA) FROM LIVINGSTON ISLAND, SOUTH SHETLANDS, THE ANTARCTIC V. Golemansky, M. Todorov ........................................................................................................ 83

REVIEW ON THE FREE-LIVING COPEPODS (CRUSTACEA) FROM THE REGION OF THE BULGARIAN ANTARCTIC BASE, LIVINGSTON ISLAND I. Pandourski, A. Apostolov ........................................................................................................ 95

HEAVY METALS AND TOXIC ELEMENTS CONTENT IN GENTOO PENGUINS (PYGOSCELIS PAPUA) FEATHERS DURING MOULT R. Metcheva, L. Yurukova ........................................................................................................... 101

BLOOD CHEMISTRY STUDIES AND DIFFERENTIAL COUNTING OF LEUCOCYTES IN GENTOO PENGUINS (PYGOSCELIS PAPUA) IN RELATION OF MOULTING AND NON-MOULTING STAGES E. Trakijska, K. Stojanova, R. Metcheva ................................................................................... 107

GENTOO PENGUIN COLONY ESTIMATES USING DIGITAL PHOTOGRAPHY R. Metcheva, P. Zehtindjev, Y. Yankov ...................................................................................... 115

COMPARATIVE STUDIES ON THE CONTENTS OF CHEMICAL ELEMENTS IN SOIL COVER FROM LIVINGSTON ISLAND, ANTARCTICA M. Sokolovska, J. Bech ................................................................................................................ 123

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© PENSOFT Publishers AND MICROBIAL ABUNDANCE OF MOSS AND COASTAL BulgarianHAntarctic ABITATS ...Research SOIL CHARACTERISTICS 1 Sofia – Moscow Life Sciences, vol. 4: 1-8, 2004

Soil characteristics and microbial abundance of moss and coastal habitats of Livingston Island – Antarctica A. KENAROVA, V. BOGOEV Department of Ecology and Nature Protection, Faculty of Biology – St. Kliment Ohridsky University of Sofia, 8 Dragan Tzankov St., 1164 Sofia, Bulgaria

ABSTRACT Soil samples from three different types of habitats (moss, moss- ornithogenic and coastal- ornithogenic) were studied according to their nutrient and microbiological characteristics. The soils tested are relatively well stocked with organic matter but poor in inorganic nitrogen and phosphorus. The concentration of organic matter is an individual characteristic of the samples but not of the soil groups. The stock of nitrogen and phosphorus is the chemical characteristic of the soil groups which differ them each other significantly. The bioligical differences of soil groups are given by their microbiological characteristics, especially the fungal and bacterial biomass as well as the abundance of saprotrophic bacteria. The abundance of oligotrophic bacteria cannot be assumed as a biological characteristic of soil groups because of the statistically unsignificant difference in the inter group variance of this parameter. KEY WORDS Antarctica, Livingston island, soil habitats, soil microorganisms.

INTRODUCTION The extreme conditions of Antarctica determine the specific character of soil microbiota, inhabiting the continental, coastal or island part of the Continent. The seasonal changes of the climate influence much more the biota of the island and coastal part than the inner continental one where the soil annualy is freezed and ice covered. *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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A. KENAROVA, V. BOGOEV

It is the point of freeze- thaw transitions rather than the temperature itself which is critical for the onset of microbial activity. Such transitions lead not only to existence of free water in the environment but also to release of organic substances by the microorganisms, cryptogamic flora and invertebrates (Collins et al. 1975; Block 1984; Smith 1984). The turn between winter inactive condition to one of summer microbial activity can be fulfilled within the limits of a day (Wynn-Williams 1980). At the coastal and island part of Antarctica two basic types of habitats have been formed in respect of the higher concentrations of organic matter in soil – communities of mosses in the damper parts and ornithogenic coastal soils. The rate of organic decomposition is a factor for the existence of the moss habitat itself (Wynn-Williams 1980, 1988). The balance between growth and decomposing of moss determines whether or not on a certain place a peat field or moss covering will be formed (Davis 1986). In the peat-moss communities there are a great number of heterotrophic and autotrophic microorganisms (Christie 1987; Davis 1981; Burn 1984; Lister et al. 1987). The basic source of nitrogen, phosphates and nutrients on the cold oligotrophic soils is the guano secreted by the penguins. Published data show that despite the great number of bacteria, less than 8% of the populations are metabolically active even during summer (Ramsy 1983; Gushterova 1999). The bacteria from the alimentary tract of penguins probably cannot adapt to low temperatures of the environment and/or are inhibited by different antibiotic substances like the acrylic acid secreted from some sea phytoplankton, consumed by Euphansia superba, which serves as food for the penguins (Sieburth 1969). The subject of this study is to determine the differences between abiotic and biotic characteristics of the moss, moss-ornithogenic and ornithogenic soils from the Livingston Island. MATERIALS AND METHODS Soil samples from Livingston Island were taken in January 2002 from the Bulgarian Antarctic expedition. They were dried in room temperature, then sifted and kept in sterile paper bags at 4°C. The samples were divided into three groups according to the type of habitats they were taken from – moss (1), moss- ornitogenic (2) and coastal- ornitogenic (3). The pH was measured by pH-meter in water extract – 1g soil:25ml distillated water. The amount of organic matter was determined after Tyurin’s method based on oxidation of organic matter by potassium dichromate (Kaurichev 1980). The concentration of phosphoates and inorganic nitrogen (ammonium and nitrate) were determined in a way recommended in “Methods of soil analyses..” (Baker and Norman 1982; Keeney and Nelson 1982).

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The amount of fungal and bacterial biomass was calculated after microscopic measurement of fungal mycelium length (light microscopy) and bacterial number counting (epiflourescent microscopy); from the average values of measured parameters the amount of fungal and bacterial biomass was calculated (Zviagincev and Zaiceva 1979; Zviagincev at al. 1978). Abundance of heterotrophic and oligotrophic bacteria was counted on selective media- heterotrophs on a tenfold diluted nutrient agar and oligotrophs on a salt agar amended with 10% soil extract. After inoculation the petri dishes were cultivated at 11-12°C for 7-10 days. Statistical analyses were processed according to the inner- and inter soil groups’ variations of microbiological and chemical characteristics (Moroney 1969). RESULTS AND DISCUSSION The soils from the three habitats (moss, moss-ornithogenic and coastal-ornithogenic) are poor in inorganic nitrogen and phosphorus and comparatively well provided with organic matter (Tabl.1.). Similar data concerning the chemistry of soils from Livingston Island were also published by Nustorova et al. (2002). The reason for the low quantities of inorganic nitrogen and phosphorus in the soil samples, compared to the existing organic matter, is most likely the low microbial activity, inhibited from the unfavourable conditions of the environment. Of the three soils groups investigated, the best supplied with biogenic elements are moss-ornithogenic soils and these conditions of the specific environment favor the better metabolic activity of microorganisms. This question is the subject of a thorough discussion by many authors (Tearle 1987; Wynn-Williams 1980, 1982; Pugh et al. 1982; Davis 1986; Christie 1987). The consideration of statistical analysis find out the existence of difference between inter- and inner soil group variability of nitrogen, phosphorus and pH values (Table 2). Although their common characteristic of low level of the inorganic nitrogen and phosphorus reserves, soils from the different habitats have group identity in respect of the average levels of these parameters. The existing inner-group variation Table 1. Physic- chemical characteristics of the soil groups. Soil group of habitats

pH

organic matter %

NO3-N mg kg-1

NH4-N mg kg-1

HPO4 -P mg kg-1

moss moss-ornitogenic coastal-ornitogenic

6.27 6.56 6.92

5.22 12.16 9.08

2.4 6.4 1.7

16 48 18

20 32 24

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Table 2. Analysis of variance and significance of the difference between inner- and inter soil groups variability. Parameter organic matter pH nitrogen HPO42- -P bacterial biomass fungal biomass oligotrophs saprotrophs

Source of variation

Sums of squares

Degrees of freedom

Variance estimate

F

inter-group inner-group inter-group inner-group inter-group inner-group inter-group inner-group inter-group inner-group inter-group inner-group inter-group inner-group inter-group inner-group

221 628.22 377.5 19.74 19.49 0.00874 19.64 0.046 0.182 0.051 5134.5 2675.67 88.39 772.38 17.02 15.47

2 16 2 16 2 16 2 16 2 16 2 16 2 16 2 16

110.5 39.26 188.79 1.23 9.74 0.00054 9.84 0.0029 0.091 0.0032 2567.25 167.23 44.19 48.27 8.51 0.967

2.81 153.48 18 037 3 393 28.44 15.35 0.92 8.8

Snedecor’s F(1% level of variance ratio) =5.8

in the values of nitrogen, phosphorus and pH is many times lower than the intergroup one. The level that the quantity of organic matter varies in the groups of soil patterns is greater than in the every one inner-group, but the difference is not statistically significant. The quantity of organic matter may be assumed as characteristic of the particular soil sample but not for the entire habitat. For example, the inner-group variability in the organic matter within the second group soils is so great that the soil samples may be divided into subgroups: 2A – average value of the organic content is 2.1% and corresponding to that subgroups 2B – 13% and 2C – 21.4%. Microbiological analyses of soil samples determine the amount of bacterial and fungal biomass as well as the abundance of different groups of microorganisms, which form a heterothrophic block. The results of bacterial and fungal biomass determination by groups of soil samples are represented on Fig.1. The amount of fungal biomass (42 mg g-1 soil) is by as much 2.5 orders of magnitude higher than the bacterial biomass (0.96 mg g-1 soil). A similar proportion is normal about slightly acidic soils but here it is reinforced because of the unfavourable condition of the environment. In regions with a cold climate, slow cycles of sub-

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SOIL CHARACTERISTICS AND MICROBIAL ABUNDANCE OF MOSS AND COASTAL HABITATS ... fungal biomassxE-2 mgg-1soil bacterial biomassxE-4 mgg-1 soil

50 45 Microbial biomass

5

40 35 30 25 20 15 10 5 0 1

2

3

Soil groups

Fig. 1. Fungal and bacterial biomass of the three different soil groups.

stances are observed and there the soil fungi have a major importance for the mineralisation of death organic matter. The amount of fungal biomass varies between different groups of soil samples as it is best represented in soils influenced by animal excrements. The amount of bacterial biomass in the first and the third groups of soils is approximately equal and about 2 times lower in comparison with the second group. The differences of fungal and bacterial biomass between the soils of the habitats studied is clearly defined and the inter-group variability is statistically significant. In that case it may be accepted that in the specific conditions of the soil environments the abundance of microorganisms should be taken as their biological characteristic. The difference between inter- and inner-variability in fungal biomass is smaller than the one in the bacterial biomass. The fungal biomass is distributed more homogeneously in soils of different habitats and it may be interpreted as a smaller dependence by factors of the environment. Inner-group variability of fungal and bacterial biomasses is greatest in ornithogenic soils and decreases for the other two soil groups, maybe due to the nature of animal excrements and the degree of their decomposition. Very often, if we are interested in the way a certain process runs, we take into consideration not only the biomass and the number of the microorganisms but also the number of the representatives from specific eco-physiological groups (like het-

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Number of heterotrophs

erotrophic bacteria). The total number of heterotrophs (saprotrophs and oligotrophs) in the different groups of soil samples is shown in Fig. 2. Oligotrophic bacteria in the soils of Antarctica predominate in their number (for the three soil samples it is 5,82×105 cfu g-1 on the average) and they exceed around six times the number of the saprotrophs (0,99×105 cfu g-1). The inner-group variability in the number of oligotrophs is commensurate with the inter-group one and the difference between them is not significant. The intergroup variability of saprotrophs is greater than the inner- group one and the difference between them is statistical significant and it can assumed as microbial characteristics of soil groups. What is interesting is the proportion between the two main groups of heterotrophic microorganisms in the specific groups of soil samples. The abundance of heterotrophs is smallest in the first group of samples (mossnhabitats) while there the number of saprotrophs and oligotrophs is in rough figures equal. Considering the soil groups of moss-ornitogenic and coastal-ornitogenic, the abundance of saprotrophs and oligotrophs is higher in comparison to the first group around 2.5 and 14-16 times, respectively. The soils from different habitats may be distinguished for the stored biogenic elements with comparatively equal but authentically important differences between their values. The concentration of organic matter in soils cannot be accepted as a habitat characteristic, but only as a specification of a certain soil sample. The microbial characteristics of the examined habitats give us knowledge of their biological difoligotrophsxE+5 cfug-1soil saprotrophsxE+5 cfug-1soil

10 9 8 7 6 5 4 3 2 1 0 1

2

3

Soil groups

Fig. 2. Abundance of oligotrophs and saprotrophs in the soil groups.

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ferences that is determined through the quantity of biomass (fungal and bacterial) and the number of saprotrophic bacteria. The difference in the number of oligotrophic bacteria cannot be assumed as a habitat characteristic because of the statistically insignificant difference in the intergroup variance of this parameter. REFERENCES BACKER DALE E, NORMAN H. SHUR.1982. Atomic absorption and flame emission spectrometry. In: Methods of soil analyses: chemical and microbiological properties (Eds: Page, A.L., Miller, R.H., Keeney, D.R.). 2nd ed. Am.Soci. Agronomy, Soil Sci.Society Am., Madison, Wis., 13-27 BLOCK W. (1984): Terrestrial microbiology, invertebrates and ecosystems. In: Antarctic Ecology (Ed: Laws, R.M.). Academic Press, London. vol.1,163-236. BURN A.J.1984. Energy partitioning in the Antarctic collembolan Cryptopygus antarcticus. Ecol. Entomol. 9, 11-21. CHRISTIE P. 1987. Nitrogen in two contrasting Antarctic bryophyte communities. J. Ecol. 75, 73-94. COLLINS N.J., J.H. BACKER, P.J. TILBROOK. 1975. Signy Island, maritime Antarctic. In: Structure and Function of Tundra Ecosystems (Eds.: Rosswall, T. and Heal, O.W.). Ecol. Bull. (Stockholm). 20, 345-374 DAVIS R.C. 1981. Structure and function of two Antarctic terrestrial moss communities. Ecol. Monogr. 5, 125-143. DAVIS R.C. 1986. Environmental factors influencing decomposition rate in two Antarctic moss communities. Polar Biol. 5, 95-104. GUSHTEROVA A.M., M. NUSTOROVA, R. TSVETKOVA, G. SPASOV, V. CHIPEVA. 1999. Investigations of the microflora in penguin’s excrements in the Antarctic. Bul. Antarctic Research, Life Science, vol.2, 1-7. KAURICHEV M.C.(ed.) 1980. Analises of organic matter concentration in soil samples. Handbook of Soil Analyses. Moscow, “Kolos”, 272, (in russian). KEENEY D.R., D.W. NELSON. 1982. Nitrogen- inorganic forms. In: Methods of soil analyses: chemical and microbiological properties (Eds: Page, A.L., Miller, R.H., Keeney, D.R.). 2nd ed. Am.Soci. Agronomy, Soil Sci.Society Am., Madison, Wis., 643-698 LISTER A., M.B. USHER, W. BLOCK. 1987. Description and quantification of field attack rates by predatory mites. An example using an electrophoresis method with a species of Antarctic mite. Oecologia. 72, 185-191. MORONEY M.J (ed.). 1969. The analysis of variation and co- variation. Facts from Figures. Penguin Books Ltd. , London, 371-457 NUSTOROVA M., M. GROZEVA, A. GUSHTEROVA. 2002. Study on soils from the region of Livingston Island (Antarctica). Bul. Antarctic Research, Life Science, vol.3, 21-27.

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PUGH G.J.F., D. ALLSOPP. 1982. Micro-fungi on Signy Island, South Okney Islands, South Atlantic Ocean. Br.Antarct.Surv.Bull. 57, 55-68 RAMSAY A.J. 1983. Bacterial biomass in ornithogenic soils of Antarctica. Polar Biol. 1, 221-225 SIEBURTH J.M.C.N. 1963. Bacterial habitats in the Antarctic environments. In: Symposium of Marine Microbiology (Ed: Oppenheimer, C.H.). Charles C.Thomas, Springfield, 533-548 SMITH R.I.L. 1984. Colonization and recovery by cryptogams following recent volcanic activity on Deception Island, South Shetland Islands. Br.Antarct.Surv.Bull. 62, 25-51 TEARLE P.V. 1987. Cryptogamic carbohydrate release and microbial response during spring freeze-thaw cycles in Antarctic fellfield fines. Soil Biol.Biochem. 19, 381-390. WYNN-WILLIAMS D.D. 1980. Seasonal fluctuations in microbial activity in Antarctic moss peat. Biol.J.Linn.Soc. 14, 11-28 WYNN-WILLIAMS D.D. 1982. Simulation of seasonal changes in microbial activity of maritime Antarctic peat. Soil Biol.Biochem. 14, 1-12. WYNN-WILLIAMS D.D. 1988. Cotton strip decomposition relative to environment factors in the maritime Antarctic. In: Cotton Strip Assay: An Index of Decomposition in Soils (Eds: Harrison, A.F., Latter, P.M. and Walton, D.W.H.). ITE Symp., Institute of Terrestrial Ecology, Granade-over-Sands vol.24, 126-133. ZVIAGINCEV D.G., E.A. DIMITRIEV, P.A. KOGEVIN. 1978. Investigation of soil microorganisms by epifluorescent mycroscopy. Microbiologya. 47(6), 1091-1096, (in russian). ZVIAGINCEV D.G., B.E. ZAICEVA. 1979. Short time changes in fungal and bacterial biomasses in soil samples. Microbiologya. 48(6), 1082-1085, (in russian).

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©LPENSOFT PublishersAND β-GLUCOSIDASE PRODUCTION FROM CRYPTOCOCCUS Bulgarian AntarcticAL Research IPID COMPOSITION VISHNIACII 9 4 Sofia – Moscow Life Sciences, vol. 4: 9-21, 2004

Lipid composition and β-glucosidase production from Cryptococcus vishniacii AL4 K. PAVLOVA 1, M. ZLATANOV2, G. ANGELOVA3, I. SAVOVA4 1

Department of Microbial Biosynthesis and Biotechnologies, 26 Maritza Blvd, 4002 Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria, E-mail: [email protected] 2

3

University of Plovdiv, Dept. of Chemical Technology, 4000 Plovdiv, Bulgaria

Higher Institute of Food and Flavour Industry, Dept. of Microbiology, 4002 Plovdiv, Bulgaria National Bank for Industrial Microorganisms and Cell Cultures,

4

125 Tzarigradsko Shosse Blvd 2, 1113 Sofia, Bulgaria

ABSTRACT A yeast strain isolated from Antarctic lichen samples taken from the region of the Bulgarian base on Livingston Island was identified as Cryptococcus vishniacii AL4, according to morphological, cultural and physiological characteristics. The strain was cultivated in YPD medium with the addition of 0.17M NaCl and 0.85M NaCI at temperatures 4°C and 18°C for determining the effect of these factors on fatty acid composition. The amount of unsaturated fatty acid in the triacylglycerol fraction was 76.1% and 75.9% at temperatures of cultivation 4°C and 18°C. The content of sterols and phospholipids was respectively 36.0 mg and 111.0 mg per kg dry biomass. The major phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidyl- ethanolamine) were 69.5%. The amount of tocopherols was mainly δ-tocopherol. The strain Cr. vishniacii AL4 was also studied for producing the enzyme β-glucosidase. The biosynthesis of the enzyme was examined on different carbone sources – glucose, cellobiose, glucose plus methyl-β-D-glucopyranoside, glucose plus cellobiose, glucose plus salicin under aerobic conditions. The highest levels of exocellular (56.67 U/ml) and endocellular (140.10 U/ml) β-glucosidase activities were obtained in the medium with 1.0% cellobiose at 24°C.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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KEYWORDS Cryptococcus vishniacii AL4, fatty acid, phospholipids, sterol, tocopherols, β-glucosidase

INTRODUCTION Yeast biodiversity, which increases with the availability of water and energy, probably predominates on continental Antarctica (Vishniac, 1996). The psychrophilic species Cryptococcus vishniacii was discovered in Antarctica with a maximum growth temperature of 20°C. It occurs widely in the rose desert (“dry valleys”) but is unknown outside Antarctica (Vishniac & Hempling, 1979; Vishniac, 1999). Yeasts are of special interest because of their ability to accumulate high amounts of oil and their relatively high growth rates in a nitrogen limited culture. Their fatty acid profile of the triacylglycerol fraction resembles that of plant oils but oil productivity and composition vary sometimes significantly, according to the strain (Ratledge, 1982). Lipid characterization of the yeasts and preparation of lipids as biologically active products were studied by a large number of researchers (Aggelis et al., 1996; Celligoi et al., 1997; Jacob, 1993; Nojoma et al., 1999; Ratledge & Boulton, 1985; Ratledge, 1988, Saxena et al., 1998). The fatty acids of Cryptococcus antarcticus strains grown at 13°C were 87-93% unsaturated and those of the type of C. vishniacii were 91% unsaturated. Neither species produced linolenic acid under this condition (Vishniac & Kurtzman, 1992). Whole cell fatty acid composition has been used increasingly for the identification of ascomycetous (Augostin & Kock, 1989; Kock et al., 1985) and basidiomycetous yeasts (Smith et al., 1989). Vishniac & Kurtzman (1992) showed habitat temperature was reflected in the fatty acid composition and they used this fact as a factor for distinguishing four biotypes of Cryptococcus. The effect of culture conditions on lipid production has been clarified by several studies of technological interest (Aggelis et al., 1996; Choi et al., 1982; Evans & Ratledge, 1984a; Evans & Ratledge, 1984b). The effect of concentrations of sodium chloride was investigated by Vishniac & Kurtzman (1992) and the results showed that C. antarcticus biotypes were more halotolerant (growth rates estimated to fall to 0 between 1.45 and 1.70M NaCl) than the type strain of C. vishniacii (growth rate estimated to fall to 0 at 1.2M NaCl), although they were less halotolerant than the type strain of Cryptococcus albidus var. albidus (growth rate estimated to fall to 0 at 1.83M NaCl). β-Glucosidase has a great application in the wine and fruit juice industry for hydrolysis of glucosides in general sources. While the glycosides are not volatile and possess no aromatic flavour, their aglycones represent an important potential source

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of fragrant compounds. The microbial β-glucosidase could hydrolyze efficiently bond glucosides and liberate the aromatic compounds without modification (Aggelis et al., 1996; Grossman et al., 1997; Gunata et al., 1990; Shoseyov et al., 1990; Vasserot, 1993). The yeasts belonging to the genera Candida, Pichia, Brettanomyces, Saccharomyces and Zygosaccharomyces possessed the ability to synthesize the enzyme β-glucosidase (Gueguen et al., 1994; Gueguen et al., 1995; Mateo & Di Stefano, 1997; Riccio, 1999; Saha & Bothast, 1996; Vasserot, 1990). Forty-eight yeast strains from the genera Candida, Debaryomyces, Kluyveromyces and Pichia were screened for production of extracellular glucose-tolerant and thermophilic β-glucosidase using p-nitrophenyl-δ-D-glucoside as substrate. Enzymes from 15 yeast strains showed very high glucose tolerance. The β-glucosidases from all these strains hydrolyzed cellobiose (Saha & Bothast, 1996). Candida wickerhamii produced β-glucosidase when they grew on either glucose or cellobiose but the activity was 64-fold higher on cellobiose. An intracellular β-glucosidase was isolated from cellobiose-fermenting yeast Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth with the highest level of production in a medium containing cellobiose as a carbohydrate source. The activity of the enzyme was the highest against aryl-β-1,4-glucosides (Skory et al., 1996). In this paper we present the results of identification of an Antarctic yeast strain, its lipid composition as well as its ability for biosynthesis of β-glucosidase. MATERIALS AND METHODS Different lichen samples were taken by the Bulgarian Antarctic expedition (19992000) from the region of our base on Livingston Island (62o48' S and 61º15' W). They were analyzed for the presence of yeasts. Yeasts were isolated and maintained using previously described methods (Pavlova et al., 2001). A yeast strain identified as Cryptococcus vishniacii AL4 was deposited at the National Bank for Industrial Microorganisms and Cell Cultures, Bulgaria. It was carried out morphology, growth characteristics, sporulation behavior, pseudomycelium formation, assimilation of carbon and nitrogen sources and fermentation sugars using standard methods (Barnett et al., 1990; Kurtzman & Fell, 1998). The colonies were obtained and described on glucose peptone agar (YPD), containing (w/v): 0.3% yeast extract, 2.0% glucose, 1.0% peptone, 2.0% agar. The strain was inoculated in liquid YPD for the determination of cultural characteristics. The assimilation of hydrocarbons was studied using yeast nitrogen base (YNB, Sigma) with the addition of 1.0 % (w/v) hydrocarbon as a sole carbon source and incubation at temperature 25ºC was carried out. Nitrate, nitrite, ethylamine, L-lysine and cadaverine were used as a sole source of nitrogen. The ability of the strains to metabolize certain sugars anaerobically was tested using Durham tubes. Records were taken up to three

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weeks. The diazonium blue B (DBB) test was used for differentiating ascomycetes from basidiomycetes yeast. The yeast strain was tested for lipid content in a medium containing: peptone 10.0 g/l, yeast extract 3.0 g/l, glucose 10.0 g/l, and pH 5.0. Inoculum was prepared by growing cells from malt agar in 10 ml of liquid medium. After 48 h 2.0% (v/v) of the inoculum was added in 200 ml Erlenmeyer flasks containing 20 ml medium. The strain was cultivated on a rotary shaker at 220 min-1 at 18ºC and 24°C for 96 h. Biomass was liophilizated on – 64°C. Different carbon sources were used for biosynthesis of β-glucosidase: glucose (Fluka) 10.0 g/l, D(+)-cellobiose (Fluka Garantie) 10.0 g/l, glucose 10.0 g/l plus cellobiose 3.0 g/l, glucose 10.0 g/l plus methyl-β-D-glucopyranoside (Fluka Biochemika) 3.0 g/l, glucose 10.0 g/l plus salicin (Merk) 3.0 g/l. The culture was centrifuged (4000g for 20 min) and washed twice with distilled water. The cells and culture supernatant were assayed for enzymatic activity. Cells were centrifuged at 4000g for 30 min, washed twice with phosphate buffer pH 7.0, resuspended in this buffer and disintegrated with glass beads in a Braun apparatus (B. Braun Melsungen AG, Melsingen, Germany). The cell suspension was centrifuged at 10 000 × g for 20 min. The lipids were extracted in a Soxhlet apparatus with n-hexane for 8 h. After rotation vacuum distillation of the solvent the extracted oils were weighed. The fatty acid composition of triacylglycerols was identified by capillary GC of their methyl esters. The esterification was carried out by the Metcalfe and Wang technique (1981). Methyl esters were purified by TLC. Determination was accomplished on a Pay – Unicam 304, provided with a flame-ionization detector, 30-m capillary column Innowax (Scotia Pharmaceutical) impregnation at column temperature 165-225°C, at a gradient of 4 K per min, detector temperature 300°C, injector temperature 280°C, carrier gas N2. Lipids were extracted according to Folch (1957). Polar lipids were divided from nonpolar lipids by column chromatography. The phospholipid constituents were separated by two-directional TLC and estimated spectrophotometrically at 700 nm (Beshkov & Ivanova, 1972). The amount of sterols (as ergosterol) was determined spectrophotometrically after separation by TLC on silica-gel 60 G (Merck) according to Ivanov et al. (1972). Tocopherols were analyzed directly in the oils by HPLC with fluorescence detection (Ivanov & Aitzetmuller, 1995). Merck-Hitachi unit fitted with a Nucleosil Si 50-5 250×4 mm column and with a fluorescent detector Merck-Hitachi F 1000 was used: excitation was done at 295 nm, emission measured at 330 nm, mobile phase was nhexane dioxin (94:4), rate of mobile phase was 1 ml/min; the peaks were identified using anthentic individual tocopherols. β-glucosidase activity was assayed by measuring the amount of p-nitrophenol (pNP) liberated from p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate. En-

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zyme solution 0.4 ml was mixed with 0.4 ml of 5 mmol/l of pNPG in 100-mmol/ l citrate-phosphate buffer (pH 5.0). The reaction mixture was incubated at 30ºC for 30 min and subsequently 1.2 ml of carbonate buffer (0.2 mmol/l, pH 10.2) was added to stop the reaction. The liberated pNP in this mixture was measured at 400 nm by a spectrophotometer Shimadzu, Japan. All assays were performed in duplicate and averaged. One unit (U) of enzyme activity is that which released 1 mmol pnitrophenol min-1. RESULTS AND DISCUSSION Yeast strain was isolated from lichen taken from the region of the Bulgarian Antarctic Base on Livingston Island. The strain was selected and was identified using the criteria of yeast classification proposed by Barnett et al. (1990) and Kurtzman & Fell, (1998). The morphological, cultural and growth characteristics and biochemical properties of the strain is shown in Table 1. Table 1. Morphological characteristic and biochemical properties of the strain AL4 Characteristics Shape of cells Size of cells Sediment Shape of coloniaSurfaceMargin Colour Teliospores Ascospores Pseudomycel True mycel Ring Film Diazonium Blue (DBB) Urease b-glucosidase Amylase Protease Grown in vitamin free medium Starch formation Acetic acid production Grown at 25ºC Grown at 30ºC Grown at 37ºC (+) – positive, (-) – negative, (W) – weak

Strain AL4 ovoidal to elongate 3.4 – 7.1 x 3.8 – 12.7 mm little smooth, dullentire cream + + + + + + -

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The results indicated that the cells were ovoidal to elongate (3.4-7.1) µm × (3.812.7) µm. The strain formed a little sediment. The culture was cream-coloured. The surface of colonia was smooth, dull and the margin was entire. The strain was not able to form pseudomycel, true mycel, ring and film. The strain was DBB positive. It synthesized β-glucosidase, urease and did not synthesize amylase and protease. The strain did not produce acid from sugars. It was able to grow at 4°C to 25°C but did not grow at 30°C and 37°C. The assimilation of carbon compounds is shown in Table 2. The results indicated that the strain grew well on medium with D-glucose, D-xylose, L-arabinose, sucrose, trehalose, cellobiose, salicin, raffinose, melizitoze, xylitol, D-glucoronat and methanol. The utilized nitrogen sources included nitrate and nitrite, lysine, ethylamine. Cadaverin was not utilized as a carbon source. The taxonomic study of the yeast allowed us to classify the strain as Cryptococcus vishniacii AL4 as regards of yeast classification criteria proposed by Barnett, at al., (1990), Kurtzman & Fell, (1998). Table 2. Utilization of carbon sources by yeast strain AL4 Characteristics

AL4

Characterictics

AL4

D - Glucose D- Galactose L- Sorbose D- Glucosamine D- Ribose D- Xylose L- Arabinose D- Arabinose L- Rhamnose Sucrose Maltose Trehalose Me a-D-glucoside Cellobiose Salicin Melibiose Lactose Raffinose Melezitose Inulin Starch Glycerol

+ D, W + + + D + + + + + D D -

Erythritol Ribitol Xylitol L-Arabinitol D- Glucitol Manitol Galactitol Myo- Inositol D - Glucuronate Succinate Citrate Methanol Ethanol D- Gluconate DL-Lactate Fermentation Nitrate Nitrite Ethylamine L- Lysine Cadaverine

+ D + D + D, W + + + -

(+) – positive, (-) - negative, (D) – delayed growth, (W) – weak growth, (VW) – very weak growth

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Lipid composition of Cryptococcus vishniacii AL 4 and effect of temperature cultivation (4°C and 18°C) on fatty acid were studied. Lipids were extracted from the yeast strain when it reached the stationary phase of growth in order to get a stable fatty acid composition. It is known that the fatty acid compositions of yeast lipid vary with the growth phase (Aggelis, 1996; Hanson & Dostalek, 1986; Kaneko, 1976). The effect of temperature and sodium chloride on the fatty acid composition of C. vishniacii AL4 is presented in Table 3. The fatty acid profile of the triacylglycerol fraction of the yeast strain was different under cultivation at 4°C and 18°C. The fatty acid composition of the C. vishniacii AL 4 showed a predominance of C18 unsaturated fatty acids at both growth temperatures. The main saturated fatty acid was palmitic (C16:0) acid. Oleic, palmitic and linoleic acids were the predominant fatty acids in the triacylglycerol fractions of C. vishniacii AL4.- the strain synthesized 17.6% palmitic, 48.2% oleic and 19.1% linoleic acids at 4°C and 19.7% palmitic, 61.3% oleic and 12.1% linoleic acid at 18°C. Oleic, linoleic and palmitic acids as the major constituents of lipids were synthesized by the yeast strains Cryptococcus vishniacii ATCC 36649 (Vishniac & Kurtzman, 1992). Akhtar et al. (1998) as well reported that fatty acid analysis of lipids extracted from strains Apiotrichum curvatum ATCC 105 67, Cryptococcus albidus ATCC 56 297, Lipomyces starkegi ATCC 12659 and Rhodosporidium toruloides ATCC 10788 revealed palmitic, oleic, linoleic and stearic acids as the principal fatty acids in the triacylglycerol fractions. The yeast strain Cryptococcus vishniacii AL4 tested in this study had insignificant differences in quantities of unsaturated fatty acids in the triacylglycerol fraction irrespective of the temperature – 76.1% at 4°C and 75.9% at 18°C, but the concetration of individual fatty acids varied as a function of the Table 3. Effect of incubation temperature and sodium chloride on the fatty acid composition of Cryptococcus vishniacii AL4 Fatty acid Laurinic (C12:0) Myristic (C14:0) Palmitic(C16:0) Palmoleic(C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic(C18:2) Linolenic(C18:3) Saturated Unsaturated

Cryptococcus vishniacii AL4 4°C without NaCl 18°C without NaCl 0.17M NaCl 0.2±0.1 0.1 ± 0.1 17.5 ± 1.2 4.5 ± 1.1 5.9 ± 1.3 48.2 ± 4.2 19.1 ± 1.8 4.3 ± 1.1 33.9 76.1

0.5±0.2 0.2±0.1 19.7±1.7 2.4±30.3 3.6±1.0 61.3±6.3 12.1±1.2 0.1±0.1 20.1 75.9

4.2±0.8 0.2±0.1 19.2±1.3 1.0±0.4 3.6±0.8 257.1±5.7 13.3±2.1 1.0±0.8 27.6 72.4

0.85M NaCl 0.1±0.1 0.1±0.1 20.1±1.8 2.2±0.3 3.1±1.0 57.8±6.3 15.7±2.1 0.2±0.1 24.1 75.9

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temperature and these results were in close agreement with those previously obtained by Watson et al. (1976) The fatty acid composition of C. vishniacii AL4 showed that the addition of different concentration of NaCl (0.17M and 0.85M) had no effect on the quantity of oleic, palmitic and linoleic acids. The experiments with different concentrations of NaCl indicated that the strain C. vishniacii AL4 was slightly halotolerant, which was in agreement with the investigations of Vishniac & Kurtzman (1992) about Cryptococcus vishniacii ATCC 36649 T. General composition of the investigated lipids of C. vishniacii AL4 is presented in Table 4. The sterols were 36.0 mg/kg, phospholipids – 111mg/kg, tocopherols – 0.1 mg/kg in dry biomass. The data in the table showed that phosphatidylcholine, phosphatidylethanolamine, phosphatidic acids and phosphatidylinositol were found to be the major components in the phospholipid fraction. The quantity of tocopherols was mainly δ-tocopherol. Table 4. Lipid composition of Cryptococcus vishniacii AL4 Component Sterols Phospholipids Tocopherols

Cryptococcus vishniacii AL4 General composition in lipid fraction, % (W/V) in dry biomass, mg/kg in lipid fraction, % (W/V) in dry biomass, mg/kg in lipid fraction, % (W/V) in dry biomass, mg/kg

0.2 36.0 2.0 111.0 5.3 0.1

Phospholipids, % Phosphatidylcholine Phosphatidylinositol Phosphatidylethanolamine Phosphatidic acids Lysophosphatidylcholine Lysophosphatidylethanolamine Phosphatidylserin Sphyngomielin Monophosphatidylglycerol Diphosphatidylglycerol

47.2 6.9 15.4 8.1 4.3 5.5 5.1 5.5 1.1 0.9 Tocopherols, %

α - tocopherol β - tocopherol δ - tocopherol

trace trace 99.9

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The strain Cr. vishniacii AL4 was examined for β-glucosidase biosynthesis in liquid media with different carbon sources. The effect of a variety of carbon sources on the growth and β-glucosidase production by this strain under aerobic conditions is represented in Table 5. Cellobiose (1%) induced the maximum production of exocellular β-glucosidase (56.67U/ml) and endocellular β-glucosidase (140.10U/ml). The medium containing glucose (1.0%) and methyl- δ-D-glucopiranoside (0.3%) was suitable for exocellular β-glucosidase (54.25U/ml) production. Our results confirmed the reports of other authors indicating that cellobiose was the best carbohydrate source [Skory, 1996). The ability of C. vishniacii AL4 to metabolize cellobiose correlates to its ability to synthesize β-glucosidase. When glucose, glucose plus cellobiose and glucose plus salicin were used as substrates, the β-glucosidase production was less effective. Glucose repressed the biosynthesis of the enzyme by C. vishniacii AL4. Many studies have shown that glucose represses β-glucosidase synthesis by catabolic repression (Gueguen et al., 1994; Gueguen et al., 1995; Leclerc et al., 1984; McQuillan &Halvorsen., 1963; Vasserot et al., 1991). The time course of growth and β-glucosidase biosynthesis by C. vishniacii AL4 was studied on medium with 0.5% cellobiose under cultivation at 18°C and 24°C (Figure 1 and Figure 2). The maximum exocellular and endocellular β-glucosidase was achieved in the late stationary phase (72 h) at a temperature of incubation 24°C (Figure 2). This indicated that the isolated strain from Antarctica was adapted to higher temperature for β-glucosidase biosynthesis. In conclusion, the selected yeast strain C. vishniacii AL4 showed a significant potential to produce the enzyme β-glucosidase. This approach opens up new possibilites in studying the biosynthsis, properties and applications of the enzyme in hydrolysing of cellulose- containing materials. Table 5. Synthesis of β-glucosidase on different carbon sources under aerobic conditions Carbonsource biomass (g/l) Glucose 1.0% Glucose 1.0% and Cellobiose 0.3% Glucose 1.0% and Methyl-glucopyranoside 0.3% Glucose 1.0% and Salicin 0.3% Cellobiose 1.0%

Cryptococcus vishniaciis AL4 exo-b-glucosidase(U/ml) endo-b-glucosidase(U/ml)

4,88 6.59

9.16 7.18

35.00 70.83

6.89

54.25

59.16

4.75

9.00

69.81

4.94

56.67

140.10

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6

120

5

100

4

80

3

60

2

40

1

20

0

0

30

Growth (g/L)

Exo-β-glucosidase (U/ml)

40

20

10

Endo-β-glucosidase (U/ml)

K. PAVLOVA , M. ZLATANOV, G. ANGELOVA, I. SAVOVA

18

0

0

growth exo-β−glucosidase endo-β-glucosidase

24

48

72

96

Time (h)

50

30

20

Growth (g/L)

Exo-β-glucosidase (U/ml)

40

7

140

6

120

5

100

4

80

3

60

2

40

1

20

0

0

10

0

growth exo-β-glucosidase endo-β-glucosidase

0

24

48

72

Endo-β-glucosidase (U/ml)

Fig. 1. Time course of growth and β-glucosidase biosynthesis by Cryptococcus vishniacii AL4 on medium with 0.5 % cellobiose at 18° C.

96

Time (h)

Fig. 2. Time course of growth and β-glicosidase biosynthesis by Cryptococcus vishniacii AL4 on medium with 0.5 % cellobiose at 24° C.

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REFERENCES AGGELIS G., D. STATHAS, N. TAVONLARIS, M. KOMATIS. 1996. Composition of lipids by some strains of Candida species. Production of single cell oil in a chemostat culture. Folia Microbiol 41: 299–302. AKHTAR P., L.I. GRAY, A. ASGHAR. 1998. Chemical characterization and ste-reospecific analysis of lipids synthesized by certain yeast strains. J Food Lipids 5: 299-311. AUGOSTIN O.P.H., J.F. KOCK. 1989. Differantiation of yeast species and strains within a species by cellular fatty acid analysis. 1. Application of an adopted technique to differentiate between strains of Saccharomyces cerevisiae. J Microbiol Meth 10: 9–23. BARNETT J., R.W. PAYNE, D. YARROW. 1990. Yeasts: Characteristics and Identification. Second Ed. Cambridge: Cambridge University Press. ISBN 0-521 35056-5. BESHKOV M., L. IVANOVA. 1972. Determination of phospholipids in lipid mixtures. Sci Works HIFFI, Plovdiv, Bulgaria 20: 231–234. CELLIGOI M.C., D.F. ANGELIA, J.B. BUZATO. 1997. Application of sugar-cane molasses in production of lipids by yeast. Agr Biol Technol 40: 693-698. CHOI S.Y., D.D.Y. RYN, I.S. RNEE. 1982. Production of microbial lipid: Effects of growth rate and oxygen on lipid synthesis and fatty acid composition of Rhodotorula gracilis. Biotechnol Bioeng 14: 1165-1172. EVANS C.T., C. RATLEDGE. 1984a. Effect of nitrogen source on lipid accumu-lation in oleaginous yeasts. J Gen Microbiol 130: 693-1704. EVANS C.T., C. RATLEDGE. 1984b. Influence of nitrogen metabolism on lipid accumulation by Rhodotorula foruloides. J Gen Microbiol 130: 3251–3264. FOLCH M., M. KEESO, G. STANLLY. 1957. A simple method for the isolation and purification of total lipids from animal tissues.J.Bio.Chem 226: 497-498. GROSSMAN M., A. RAPP, W. RIETH. 1997. Enzymatische freistrung gebundener aromastoffe in wein. Dtsch Lebensm Rundsch 1: 7-12. GUEGUEN Y., P. CHERMARDIN, A. ARNAUD, P. GALZY. 1994. Purification and characterization of the endocellular β-glucosidase of a new strain of Candida entomophila isolated from fermenting agave (Agave sp.) juice. Biotechnology and Applied Biochemistry 20: 185-198. GUEGUEN Y., P. CHERMARDIN, A. ARNAUD, P. GALZY. 1995. Comparative study of extracellular and intracellular β-glucosidase of a new strain Zygosaccharomyces bailii isolated from fermenting agave juice. J. Applied Bacteriology 78: 270-280. GUNATA Y.Z., C. BAYONOVE, C. TAPIRO, R.E. CORDONNIER. 1990. Hydo-lysis of grape monoterpenyl-β-D-glucosides by various β-D-glucosidases. J. Agr.Food Chem. 38: 1232-1236. HANSON L., M. DOSTALEK. 1986. Influence of cultivation conditions on lipid production by Cryptococcus albidus. Appl Microbiol Biotechnol 24: 12-18.

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IVANOV S., K. AITZETMULLER. 1995. Unter suchungen uber die Tocopherol and Tocoteienol zu sammensetzung der Samenole einiger Vertreter der Familie Apiaceae. Fat Sci Technol 97: 24-29. IVANOV S., P. BITCHEVA, B. KONOVA. 1972. Des phytosterols dans les huiles vegatales et les concentres steroliques. Rev Fr Corps Gras 19: 177-180. JACOB, Z. 1993. Yeast lipid biotechnology. Appl Microbiol 39: 185-212. KANEKO H., M. HOSOHARA, M. TANAKA, T. ITOH. 1976. Lipid composition of 30 species of yeast. Lipids 11: 837-844. KOCK J.L.F., P.M. ZATEGAN, P.J. BOTES, B.C. VILJOEN. 1985. Developing a rapid statistical identification process for different yeast species.J. Microbiol. Meth. 4: 3-4. KURTMAN C.P., J.W. FELL. 1998. The Yeasts: a Taxonomic Study. 4th ed. Else-vier Scientific Publisher, Amsterdam (Netherlands) ISBN 0-444-81312-8. LECLERS M., P. GONDE, A. ARNAUD, R. RATOMAHEMINA, P. GALZY, M. NICOLAS. 1984. The enzyme system in a strain of Candida wickerhamii Meyer and Yarrow participating in the hydrolysis of cellodextrins.Journal of General and Applied Microbiology 30: 509-521. MATEO J.J., R.D. DI STEFANO. 1997. Description of the β-glucosidase activity of a wine yeasts. Food Microbiology 14: 583-591. METCALFE L., C. WANG. 1981. Rapid preparation of fatty acid methyl esters using organic base catalyzed transesterification.J.Chromatogr.Sci.19:530-534. McQUILLAN A.M., H.O. HALVORSEN. 1963. Physiolological changes occurr-ing in yeast undergoing glucose repression.J. Bacteriology 84: 31-36. NOJOMA Y., A. KIBAYASHI, A. MATHSUZAKI, T. HATANO, S. FUKUAI. 1999. Isolation and caracterization of fracylglycerol secreting mutant strain from yeast Saccharomyces cerevisiae. J Gen Microbiol 45: 1-6. PAVLOVA K., D. GRIGOROVA, Ts. HRISTOZOVA, A. ANGELOV. 2001. Yeast strains from the Livingston Island, Antarctica.Folia Microbiologica 46: 397-401. RADLEDGE C., C.A. BOUTLON. 1985. Fats and Oils. Pp. 984-1003 in C.L. Cooneg A.E. Humphrey (Eds). Pergamon Press Oxford. RADLEDGE C. 1982. Microbiol fats and oil: assessment potential. Progr Industr Microbiol. 16: 119–206. RADLEDGE C. 1988. Yeast for lipid production. Biochem Soc Trans 16: 1088-1091. RICCIO P., R. ROSSANO, M. VINELLA, P. DOMIZIO, F. ZITO, F. SANSEVRINO, A. D,ELIA, V. ROSI. 1999. Extraction and immobilization in one step of two β-glucosidase released from a yeast strain of Debaryomyces hansenii. Enzyme and Microbial Technology 24: 123-129. SAHA B.C., R.J. BOTHAST. 1996. Glucose tolerant and thermophilic β-gluco-sidase from yeast. Biotechnology Letters 18: 155-158. SAXENA V., C.D. SHARMA, S.D. BHAGAT, V.S. SAINI, N.D.K. ADHIKARI. 1998. Lipid and fatty acid biosynthesis by Rhodotorula minuta JAOCS 75: 501-505.

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SHOSEYOV O., B.A. BRAVDO, D. SIEGEL, A. GOLDMAN, S. GOHEN, L. SHO-SEYOV, K. IKAN. 1990. Immobilizes endo-β-glucosidase enriches flavor of wine and passion fruit juice.J.Agric. Food Chem. 38: 1387-1390. SKORY C.D., S.N. FREER, R.J. BOTHAST. 1996. Properties of an intracellular β-glucosidase purified from the cellobiose fermenting yeast Candida wickerhamii. Appl. Microbiol. Biotech. 46: 353-359. SMITH E.J., P.J. WESTHUIZEN, J.L.F. KOCK, P.M. LATEGAN. 1987. A yeast identification method: the influence of cultural age on the cellular longchain fatty acid composition of three selected basidiomycetous yeast. Syst. Appl. Microbiol. 10: 38-41. VASSEROT Y., A. ARNAUD, P. GALZY. 1993. Evidence for marc monoterpenol glucosides hydrolysis by free or immobilized yeast β-glucosidase. Bioresource Technol. 43: 269-271. VASSEROT Y., P. CHEMARDIN, A. ARNAUD, P. GALZY. 1990. Evidence for the β-glucosidase activity and cellobiose fermentation by various Kloeckera strains. Acta Biotechnologica 5: 451-457. VASSEROT Y., P. CHEMARDIN, A. ARNAUD, P. GALZY. 1991. Purification and properties of a new strain of Candida molischiana able to work at low pH values: possible use in the liberation of bound terpenols. J Basic Microbiology 31: 301 –312. VISHNIAC H.S., C.P. KURTZMAN. 1992. Cryptococcus antarcticus sp. Nov. and Cryptococcus albidosimilis sp. Nov., Basidioblastomycetes from Antarctic soils. International Journal of Systematic Bacteriology 4: 547-553. VISHNIAC H.S., H.W.P. HEMPLING. 1979. Evidence of an indigenous microbio-ta yeast in the dry valley of Antarctica.J.Gen.Microbiology 112: 301-314. VISHNIAC H.S. 1996. Biodiversity of yeast and filamentous microfungi in ter-restrial Antarcic ecosystems. Biodiversity and Conservation 5:1365– 1378. VISHNIAC H.S. 1999. Psychrophilis yeasts pp 315-321 in J Secback (Ed): Enigmatic microorganisms and life in etreme environments. Kluwer Academic Publishers, Dordrecht (Netherlands). WATSON K., H. ARTHU, W.A. SHIPTON. 1976. Leucosporidiu yeasts: obligate psychrophiles which alter membrane – lipid and cytochrome composition with temperature.J. Gen. Microbiol. 97: 11-18.

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© PENSOFT Publishers Research PRODUCTION OF THERMOPHILIC ACTINOMYCETES SPECIESBulgarian FROM AAntarctic NTRACTICA KERATINASE 23 Sofia – Moscow Life Sciences, vol. 4: 23-26, 2004

Keratinase Production of Thermophilic Actinomycetes Species from Antractica A. GUSHTEROVA1, M. NOUSTOROVA2, K. PAVLOVA3 1. Institute of Microbiology – BAS. Acad. G. Bonchev Str.,Bl 26, Sofia 1113 Bulgaria 2. University of Forestry Dep of Ecology. Kl. Ochridski Blvd 10. Sofia – 1756, Bulgaria 3. Laboratory of Applied Microbiology – BAS, 26 Maritza Blvd 4002, Plovdiv, Bulgaria

ABSTRACT Investigation of the C-source concentration (starch on the keratinase production by 2 termophilic actinomycetes strains was carried out. The strains were isolated from Antractic soil. The strains were isolated from Antractic soil. They were checked for proteolytic, caseinolytic and keratinase activity (the data show a positive effect of starch added for all enzymes checked). The distribution of thermophylic actinomycetes in Antrectic soil was found to be poor. Nine thermophylic actinomycetes species has been isolated from 60 soil samples. According to our opinion these strains could be very interesting as a source of biologically active compounds. KEY WORDS Thermophilic actinomycetes, keratinase, Antractic soils.

INTRODUCTION Keratinaceous materials (feather, hair, wool, nails, etc.) represent an undersirable polluting factor of the environment. Since they are practically undigestable by the common proteolytic enzymes they find limited applications in practice. It was found that the structural protein keratin can be degraded by some enzymes (keratinases) produced by species of saprophytic and parasitic fungi (Safranek, Goos, 1982; Lee et al., 1987; Sisentop, Boehm, 1995), some Bacillus strains (Atalo, Gashe, 1993; Cheng et al., 1995) and a few actinomycetes (Muchopadhyay, Chandra, 1990; Boeckle et al., 1995 ). *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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It is shown that thermophilic actinomycetes can thrive using different types of keratin substances as a sole source of nitrogen (Ignatova et al., 1999). Thermophilic species from Antractica produced an antibiotic complex (Ivanova et al. 2001; Ivanova et al., 2002). Some authors (Gross, 1968, Agre, 1986; Pacnicar, Agate, 1986) have established that termophylic actinomycetes are characterized by a quick growing rate at relatively high temperature. Thus these extreme forms of microorganisms have a growing importance for cleaning contaminated soils. Biodegradation by microorganisms possessing keratinolytic activity represents an alternative method for improving the nutritional value of keratin wastes. In this study two thermophylic actinomycetes strains (1a and 2a) isolated from Antractic soils were tested for their ability to utilize wool under different conditions. MATERIAL AND METHODS The objects of our study were 60 samples of soil from Livingston Island, Antractica. The samples were taken from the soil surface with and without plants from a depth of 0 – 15 cm. Microbiological analysis was performed by the dilution method on PKA media: (g) peptone – 5; corn steep liquor -5; NaCh – 5; starch – 10; tap water to 1l as described by Kosmachev (1954). One of the main criteria for the isolated of thermofilic actinomycetes was their growth at temperature 55°C. Altogether 9 thermophilic actinomicetes were isolated and were checked for keratinolytic activity. The isolated strains were cultivated at 55°C in a mineral medium (5g/l NaCh; 5g/l CaCO3, 3,5g/l K2HPO4, 3H2O pH 7, 2) containing sheep wool (6 g/l) as a carbon source. The cultivation was for 5 days, at pH 7.2, t°=55°C. The inoculum age was 18h. Protein and sulphhydryl groups concentrations were estimated using the Bradford method (1976) and the Ellman’s reagent (1959), respectively. The keratinolytic activity was determined by a modified method of Cheng (1995). The absorbance increase at 280 nm was converted into keratinase units (1 KU=0.100 absorbance increase for one hour), while the specific activity was expressed as keratinase units 1 mg/h protein (Mukhopadhyay, Chandra, 1990). The caseinolytic activity was determined according to the method of Johnson et al. (1969). RESULTS AND DISCUSSION The screenings of 9 thermophilic actinomycete strains show that two of them can utilize keratinaceous materials as a sole source of nitrogen. They were tested on min-

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eral liquid medium containing increasing quantities of starch as a carbon source. The initial pH of the medium was adjusted to 7.2. The final pH value was found to be higher (8.5 for both strains) in the control (medium without starch) after 5 days. The final cultivation (table. 1.) pH of the medium strain 1a was over 8 for all different starch concentrations . The pH-value decreases in the media containing 9 or 11 g/l starch for strain 2a. Protein and cystein (thyol groups) concentration was found to decrease together with the increasing of starch concentration for strain 2a. The fluctuation of these parameters for strain 1a was measured. The data obtained with 3 g/l starch concentration in the medium show the highest value of this activity for strain 1a. For the same strain the values of SH and proteins were higher. A simultaneous cleavage of the disulfide bonds during microbial growth has been described for Str. fradiae (Kunert, 1988), Str pactum ( Boeckle Galunsky 1995). Both strains showed relatively high keratinase activity. With the exception of the first three cultivation variants using (1-5 g/l) of starch concentration for strain 2a. The keratinase production of the strain 1a also depends on the starch concentration. This strain showed the highest enzyme activity at the starch concentration of 5 g/l. Such a maximum was obtained at a starch concentration of 9 g/l medium for strain 2a. Our results show that thermophylic actinomycetes are present in the Antractic soils which was somehow unexpected. Some of these strains (1a and 2a) showed proteolytic, caseinolytic and keratinase activity. Table 1. Activity of Termophylic Actinomycete Strains 1a and 2a Strain

Concentration of starch, g/l

Final pH of the medium

Cystein content mmol/ml

Protein content mg/ml

Caseinoly ticactivity CTA/ml

2a

0 1 3 5 7 9 11

8.5 8.28 8.32 8.25 8.42 6.93 5.97

5.24 4.6 3.78 3.85 3.15 2.32 1.96

0.17 0.16 0.15 0.12 0.1 0.097 0.062

1.16 0.3 0 0.88 0.024 1.39 0.38

1a

0 1 3 5 7 9

8.52 8.23 8.26 8.32 8.14 8.11

3.01 2.32 3.01 2.91 2.22 2.22

0.073 0.071 0.095 0.079 0.074 0.068

1.07 0.49 0 0.049 0.41

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These strains could be very interesting as a source of biologically active compounds. ACKNOWLEDGEMENTS The investigation has been accomplished through a Grant B.A. 801/1998 of the Bulgarian National Fund for which is highly appreciated Scientific Reserch. We would also like to express our thanks to Dr. E.Metcheva for the samples that she collected form the Livingston Island. REFERENCES AGRE N. C. 1986. Systematics of Thermophilic Actinomycetes, Puschino Acad. Sci. USSR. ATALO K., B. A. GASHE. 1993. Biotechnol. Lett., 15, 1151-1156. BOECKLE B., B. GALUNSKY, R. MUELLER. 1995. Appl. Environ. Microbiol., 61, 37053710. BRADFORD M. M. 1976. Anal. Biochem., 72, 248-254. CHENG S.-W., H.-M. HU, SH.-WH. SHEN, H. TAKAGI, A. ASANO, Y.-CH. TSAI. 1995. Biosci. Biotech. Biochem., 59, 2239-2243. ELLMAN G. L. 1959. Arch. Biochem. Biophys ., 82, 70-77. GROSS T. J. 1968. Apll. Bacteriol., 31, 36-53. IVANOVA V., M. ORIOL, M-J. MONTES, A. GARCIA, J. GUINEA. 2001. Secondary Metabolites from a Streptomyces Strain isolated from Livingston, Island, Antractica. Zeitscheift fur Naturforschung, 56c: 1-5. IVANOVA V., K. ALEKSIEVA, M. KOLAROVA, V. CHIPEVA, R. SCHEGEL, B. SCHEGEL, U GRAEFE. 2002. Neuropogonines A, B and C, new depsidon type metabolites from Neuropogon sp., an Antractic licheu . Die Pharmazie, 57; 73-73. IVANOVA V., L. YOCHEVA, R. SCHLEGEL, U. GRAEFE, M. KOLAROVA, K. ALEKSIEVA, M. NAIDENOVA. 2002. Antibiotic complex from streptomycetes flavovirens 67, isolated from Livingston Island. Antractica. Bulg. Antract. Reasarch, Life Sciences, 3:35-42. JOHNSON A. J., D. L. KLINE, N. ALKJAERSIG. 1969. Thromb. Diath. Haemorh., 21, 259. KOSMACHEVA. E. 1954. Thermophilic actinomycetes and their antagonistic properties. Ph D Thesis, Inst. Microbial., Moskow. KUNERT J., Z. STRANSKY. 1988. Arch. Microb., 150, 600-601 LEE K. H., K. K. PARK, S. H. PARK, J. B. LEE. YONSEI. 1987. Med J., 28, 131-138. MUKHOPADHYAY R. P., A. L. CHANDRA. 1990. Ind. J. Exp. Biol., 28, 575-577 PAKNIKAR B., D. AGATE. 1986. Current Science, 55, No 18, 927-928 SAFRANEK W. W., R. D. GOOS. 1982. Can. J. Microbiol, 28, 137-140 SISENTOP U., K. H. BOEHM. 1995. Mycoses, 38, 205-209.

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© PENSOFT Bulgarian Antarctic AND TAXONOMIC STUDY OF ANTARCTIC YEASTS FROM LIVINGSTON ISLAND ...Research ISOLATIONPublishers 27 Sofia – Moscow Life Sciences, vol. 4: 27-34, 2004

Isolation and Taxonomic Study of Antarctic Yeasts from Livingston Island for Exopolysaccharide-producing K. PAVLOVA1, A. GUSHTEROVA2, I. SAVOVA3, M. NUSTOROVA4 Department of Microbial Biosynthesis and Biotechnologies, 26 Maritza Blvd., 4002 Plovdiv,

1

Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria, E-mail: [email protected] Institute of Microbiology, Bulgarian Academy of Sciences,

2

26 Acad. G. Bonchev St., 1113 Sofia, Bulgaria National Bank for Industrial Microorganisms and Cell Cultures,

3

125 Tzarigradsko Shosse Blvd., Block 2, 1113 Sofia 4

Department of Ecology, University of Forestry, 10 Kliment Ohridski Blvd., 1156 Sofia, Bulgaria

ABSTRACT Moss, lichen and soil samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeast. Five cultures were obtained and identified as Pseudozyma antarctica AL114, Debariomyces hansenii AL118, Sporobolomyces roseus AL101, Rhodotorula glutinis AL107, Sporobolomyces roseus AL108, according to their morphology, reproductive behaviour, growth at different temperatures, salt concentrations, nutritional characteristics, and various biochemical tests. The strains were examined for biosynthesis of exopolysaccharides on different carbon sources under aerobic conditions. They produced exopolysaccharides (3.90-4.98 g/l) on a medium containing 4% of sucrose at 22°C for 120h and pH 1.88-2.08 of the biosynthetic process. KEY WORDS Yeast, Antarctica, taxonomy, exopolysaccharides.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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K. PAVLOVA, A. GUSHTEROVA, I. SAVOVA, M. NUSTOROVA

INTRODUCTION Various microbiological characteristics of the dry valleys of coastal Antarctica and the continental interior have been published (Atlas et al., 1978; Goto et al., 1969; di Menna1960, 1966a, b). Different strains of Cryptococcus, Candida, Rhodotorula, Sporobolomyces, Trichosporon and other yeast genera are widespread in the dry valleys (Atlas et al., 1978, Vishniac & Hempling 1979, Vishniac & Beharaeen 1982) and coastal Antarctica locations (Goto et al. 1969). With the establishment of the Bulgarian base on Livingston Island in 1993, an intensive investigation of the Antarctic microflora and fauna was launched for the purpose of studying the biodiversity and ecosystem in the region (Chipev & Veltchev 1996). In the course of their development, yeasts are in constant interchange with their surroundings and accumulate substances which differ in their chemical composition. The biological potential of psychrophilic yeasts can be studied for the production of biologically active substances (enzymes, lipides, polysaccharides, etc.) through the setting up of suitable conditions and substrates for culture development and the accumulation of the desired metabolites. Following the isolation, identification and study of the metabolic properties of Antarctic yeasts, a collection was made of representatives of the Sporobolomyces, Cryptococcus, Rhodotorula, and Candida genera and strain producers of the b-glucosidase and protease enzymes were selected out of it (Pavlova et al. 2001; Pavlova et al., 2003) and strains rich in lipides (Zlatanov et al., 2001). Yeasts belonging to the Sporobolomyces, Cryptococcus, Rhodotorula genera have been reported to possess the ability to synthesize different polysaccharides: mannan, glucan, glucomannan, galactomannan, phosphomannan (Adami & Cavazzoni, 1990; Chiura et al., 1982; Elinov et al., 1992; Peterson et al., 1990). The subject of this paper is the selection of Antarctic yeast strains for exopolysaccharide biosynthesis, their identification and the study of polymer pro-duction on media with different carbon sources. MATERIALS AND METHODS The samples (moss, lichen and soil) were taken from different sites of Livingston Island by the Bulgarian Antarctic Expedition in the summer of 2001-2002. Suitable diluted suspensions were prepared in order to obtain single morphologically different colonies on malt plate agar. The cultivation was carried out at 4°C for 3 to 14 days. The isolated colonies were reinoculated several times for purity, maintained on malt slant agar and stored at 4°C. The media for the morphological, cultural and physiological studies were prepared according to the prescriptions of Kreger van Rij (1987) and Barnett et al.

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(1990). The strains were inoculated in a YPD liquid medium for the determination of the cultural characteristics (sediment, ring, and film). The carbon source assimilation was studied using a yeast nitrogen base (YNB, Sigma) with the addition of a 1.0 % (w/v) source, and incubation at 22°C was carried out. Nitrate, ethylamine and cadaverine were used as a sole nitrogen source. The fermentation of sugars was tested using Durham tubes. Records were taken for up to three weeks. The diazonium blue B (DBB) test was used for differentiating ascomycetes from basidiomycetes yeasts. The basal medium for exopolysaccharide biosynthesis for the testing of carbon sources contained in (%): (NH4)2SO4 – 0.20, KH2PO4 – 0.1, MgSO4.7H20 – 0.05, NaCI – 0.01, CaCI2.2H2O and 0.1 of yeast extract. Glucose, sucrose and fructose were tested as carbon sources and supplemented to the basal medium in 4.0% concentration. The inoculum was obtained from yeast on a rotary shaker (220 min-1) in 500 ml Erlenmeyer flasks containing 50 ml of Sabouraud media (Merck, Germany) at 22°C for 48 h. The fermentation media were inoculated with 1.0% w/v inoculum. The cultivation was carried out in 500 ml Erlenmeyer flasks containing 50 ml of the tested medium on a rotary shaker (220 min-1) at 22°C for 120 h. Whole cell cultures were centrifuged at 6000g for 30 min to separate cells from the supernatant. The exopolysaccharides in the culture supernatants were precipitated with two volumes of 96% ethanol at 4°C for 18-24h. The precipitate was recovered by centrifugation at 6000g for 10 min, washed with ethanol, dried and weighed. RESULTS AND DISCUSSION Antarctic yeast strains AL114 and AL118 were isolated from the Punta Hesperides sample consisting of moss, lichen and soil. The strains grew well at a temperature of 4-8° C and within the 25-30°C temperature range. The morphological data on the yeast strains studied are presented in Table 1. There are considerable differences in the surface, margins and colony colours of the two strains, as well as their cell shapes. They also differ in the pseudo and real mycelium formation. Physiological and biochemical studies were carried out using tests for the assimilation of carbon and nitrogen sources (Table 2). The strains assimilated D-glucose, D-galactose, sucrose, maltose, lactose, sorbose, cellobiose, trehalose, rafinose, melezitose, D-xylose, ethanol, D-glicerol, D-glucoronate, nitrite. None of them utilized methanol and cadaverine. Strain AL114 was non- fermentative and did not hydrolize arbutin. Strain AL118 weakly fermented D-glucose and hydrolyzed arbutin. Neither strain produced acid or grew on vitamin-free media. The AL114 strain was characterized according to the Kurtzman and Fell determiner as being closest to the Pseudozyma antarctica genera (syn. Thrichosporon oryzae, Sporobolomyces antarcticus). What

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K. PAVLOVA, A. GUSHTEROVA, I. SAVOVA, M. NUSTOROVA

Table 1. Morphological and cultural characteristics of yeast strains from Livingston Island. Characteristics AL114

AL118

Strains AL101

Even, smooth even

AL107

AL108

Creased, frosted uneven

Smooth

In large creases, frosted Slightly wavelike

Creamy, dull Spherical and single, in pairs

Reddish pink, semiglossy Elliptic, more rarely oval

Orange red, semiglossy Elongated elliptic, oval to cylindrical

Reddish pink to coral, dull Elliptic, rarely oval

+ Rudimental -

Rudimental -

-

-

+ + + + 13.64

+ Well shaped Heavy W 72.73

Broken Thin + 9.23

Islands Strong Heavy 18.18

Colonia Surface

Rough, wrinkled Margins Uneven, with a pseudomycelial halo Colour Dark creamy, semiglossy Cells Elongated, cylindrical, septate and nonseptate, hyphae Ascospores Teliospores Pseudomycelium + True mycelium + Growth in a liquid medium Film + Ring Strong Sediment Off-white Grown at 25°C + Grown at 30°C + Grown at 35°C + Grown at 37°C + Survival, % 4.85

even

(-) – negative, ( +) positive

made it different was the assimilation of the carbon sources D-glucosamine, inulin and citric acid. Following the criteria of the same determiner, strain AL118 was identified as Debariomyces hansenii. The AL101, AL107 and AL108 Antarctic strains were isolated from the Caleta Argentina sample consisting of soil and dark green moss. The three strains are reddish pink and orange pink in colour, which is indicative of their pigment-producing ability. The microflora of different Antarctic specimen includes pigment forms which definitely play a protective role under extreme conditions by increasing the strength of microorganisms (Abyzov, 1993).

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Table 2. Biochemical and physiological characteristics of yeast strains. Characteristics D-Glucose D-Galactose Sucrose Maltose Lactose L-Sorbose Cellobiose Trehalose Melibiose Raffinose Melezitose D-Xylose L-Arabinose D-Arabinose D-Ribose D-Glucosamine Salicin Me-á-D-glucoside Starch Inulin Ethanol Methanol Mannitol Myo-Inositol Erythritol Ribitol Galactitol D-Glucuronate Succinate Citrate Nitrate Cadaverin Ethylamin Fermentation Hydrolysis arbutin DBB Acetic acid production Urease Starch formation 50 % glucose

AL114

AL118

+ + + + + + + + + + + W, D + + + + W + + + + W + + + + + + + ND

+ + + + + + + + + + + + + W, D + ND ND ND ND + + ND + + + + W + +

Strains AL101 + W, D + + + + + + + W D + D + + + + D + + + + -

AL107

AL108

+ + + + D ND + + + D, W + + ND + + ND + + + + + + + + + ND

+ + + + D ND + + + D, W ND ND ND + ND ND + ND + + + + ND

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K. PAVLOVA, A. GUSHTEROVA, I. SAVOVA, M. NUSTOROVA

Table 2. Continued. Characteristics AL114 10 % NaCl + 5% glucose ND Vitamin-free medium + 0,1% cyclohexane -

AL118 ND -

Strains AL101

AL107

AL108

+ + -

ND + -

ND + -

W –weak growth; D-delayed; ND-not determined; (-)-negative; (+)-positive

The three strains are facultative psychrophiles: strain AL101 grew slightly at 25oC and did not grow at 30oC, 35oC and 37oC, strain AL107 grew well at 25°C and did not grow at the other temperatures, whereas strain AL108 did not grow at any of the temperatures mentioned. The morphological, physiological and biochemical characteristics of the strains are presented in Tables 1 and 2. Over 60 tests were used in the taxonomic investigations of the cultures. The colonial and cell morphology and the reproduction type were studied. The properties which were found to be common to all three strains were that they assimilated glucose, sucrose, maltose, raffinose, melezitose and failed to assimilate lactose, melibiose, ramnose. Me-D-glucoside, D-glucosamine, methanol, inositol, galactitol. Out of the nitrogen sources, they assimilated nitrate and did not assimilate cadaverine and ethylamine. None of the three strains fermented glucose. The results of the DBB test, arbutin hydrolysis, urease activity and growth in a vitamin-free medium were positive. The strains were not cyclohexamide-resistant. After their morphological, cultural, physiological and biochemical properties had been determined, the strains were identified as follows: strain AL101 as Sporobolomyces roseus, strain AL107 as Rhodotorula glutinis and strain AL108 as Sporobolomyces roseus, according to the Kurtzman and Fell determiner, 1998. The strains were examined for exopolysaccharide biosynthesis on culture media containing glucose, sucrose and fructose as carbon sources in 4% concentrations. The results indicated extracellular polysaccharide synthesis by the cultures (Table 3). The Sporobolomyces roseus AL107 and Sporobolomyces roseus AL108 strains reached a high exopolysaccharide yield on a culture medium containing 4% of sucrose: 4.98g/l and 4.88g/l respectively. On the 4% fructose containing medium they produced 4.35 g/ l and 4.28 g/l of polysaccharide, whereas glucose suppressed the biosynthesis. The rest of the strains synthesized polysaccharides within the 3.83 g/l – 3.96 g/l range on a sucrose containing medium and 3.10 g/l – 3.77 g/l in a medium containing fructose. In the process of polysaccharide biosynthesis the yeasts changed the initial pH 5.3 to pH 1.65 – 2.10 and the latter was preserved till the end of fermentation. This

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Table 3. Biosynthesis of polysaccharides by Antarctic yeast strains on media with different sugars. Strains

Pseudozyma antarctica AL104 Debariomyces hansenii AL118 Rhodotorula glutinis AL101 Sporobolomyces ro-seus AL107 Sporobolomyces roseus AL108

Polysaccharides g/l Sucrose - 4% Glucose - 4% pH PolysacpH Polysaccharides, g/l charides, g/l

Fructose - 4% pH Polysaccharides g/l

1.75 2.05 1.90 1.88 1.96

1.65 2.08 2.00 2.01 2.01

3.90 3.96 3.83 4.98 4.88

1.68 2.10 2.00 2.01 1.98

3.05 2.26 3.02 2.35 2.17

3.26 3.10 3.77 4.35 4.28

specific feature of polysaccharide formation by yeast proves a regulating factor for the polysaccharide biosynthesis. REFERENCES ABYZOV S.S. 1993. Antarctic Microbiology. Ed. Fridmann. I.N. Willey Liss Inc. 265-295. ADAMI A., V. CAVAZZONI. 1990. Exopolysaccharides produced by some yeast strains. Annali di Microbiologia ed Enzymologia 40: 245-253. ATLAS R.M., M.E.di MENNA, R.E. CAMERON. 1978. Ecological investigation of yeast in Antarctic soils. Antarct. Res. Ser. 30: 27-34. BARNETT J.A., R.W. PAYNE, D. YARROW. 1990. Yeasts: Characteristics and Identification. Cambridge University Press. CHIPEV N., K. VELTCHEV. 1996. Livingston Island: an environment for Antarctic life. Bulgarian Antarctic Research. Life Sci. I: 1-6 CHIURA H., M. IIZUKA, T. YAMAMOTO. 1982. A glucomannan as an extracellular product of Candida utilis. I. Production and characterization of a glucomannan. Agric Biol Chem 46: 1723-1731. ELINOV N.P., E.P. ANANYEVA, G.A. VITOVSKAYA. 1992. Peculiarities of biosynthesis and characteristics of exoglucans in yeasts of Sporobolomyces genus.: irobiologia, 4: 615-621. GOTO, S., Sugiyama J. & LIZUKA, H. 1969. Taxonomic study of Antarctic yeasts. Mycologia 61: 748-774. MENNA M.E. di. 1960. Yeasts from Antarctica. J.Gen.Microbiol. 23: 295-300. MENNA M.E. di. 1966a. Three new yeasts from Antarctic soils: Candida nivalis, Candida gelida and Candida frigida. Antonie van Leeuwenhoek. J. Microbiol. Serol. 32: 25-28. MENNA M.E. di. 1966b. Yeasts in Antarctic soils. Antonie van Leeuwenhoek. J. Microbiol. Serol. 32: 29-38.

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PAVLOVA K., G. ANGELOVA, I. SAVOVA, D. GRIGOROVA, L. KUPENOV. 2002. Studies of Antarctic yeast strains for the production of b-glucosidase. World J. Microbiol. & Biotechnol. 18: 569-573. PAVLOVA K., D. GRIGOROVA, T. HRISTOZOVA, A. ANGELOV. 2001. Yeast strains from Livingston Island, Antarctica. Folia Microbiol. 46, 5: 397-401. VISHNIAC H.S., S. BAHARAEEN. 1982. Five new basidiomycetous yeast species segregated from Cryptococcus vishniacii ement.auct an Antarctic yeast species comprising four new varieties. Int. J. Syst. Bacteriol. 32: 437-445. VISHNIAC H.S., H.W.P. HEMPLING. 1979. Evidence of an indigenous microbiota yeast in the dry valley of Antarctica. J. Gen. Microbiol. 112: 301-314. ZLATANOV M., K. PAVLOVA, D. GRIGOROVA. 2001. Lipid composition of same strains from the Livingston Island, Antarctica. Folia Microbiol. 46, 5: 401-405.

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© PENSOFT Publishers Bulgarian Antarctic Research BIOCHEMICAL CHARACTERISTIC OF ANTARCTIC YEASTS 35 Sofia – Moscow Life Sciences, vol. 4: 35-46, 2004

Biochemical Characteristic of Antarctic Yeasts K. PAVLOVA1*, M. ZLATANOV2, L. KOLEVA3, I. PISHTIYSKI3 1

Department of Microbial Biosynthesis and Biotechnologies, 26 Maritza Blvd, 4002 Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Chemical Technology, University of Plovdiv, 4000 Plovdiv, Bulgaria

2

Department of Biochemistry and Molecular Biology, HIFFI,

3

26 “Maritza Blvd, 4002 Plovdiv, Bulgaria *Author for corresspondence: Tel.: +359-32-603-831, E-mail:[email protected]

ABSTRACT Five yeast strains Cryptococcus albidus 16-1, Cryptococcus laurentii 16-2, Rhodotorula minuta 16-3, Candida oleophila 23-1 and Rhodotorula mucilaginosa 23-2, isolated from Antarctic soils and mosses were investigated. They were cultivated in YPD medium with addition of 0,17M or 0,85M NaCI at temperature 4°C and 15°C for determination of biomass concentration and its chemical composition. The effect of these factors on the fatty acid composition of the yeast lipids was studied and it was found that the strains Cryptococcus albidus 16-1, Cryptococcus laurentii 16-2 and Rhodotorula mucilaginosa 23-2 synthesized lipids with unsaturated fatty acids 78.6%, 76.9% and 66.5%, respectively, at temperature 4°C. The strain Rhodotorula minuta 16-3 synthesized 54.1% unsaturated fatty acids at 4°C and 66.7% at 15°C. The amounts of unsaturated fatty acids in lipids of Candida oleophila 231 were 64.3% and 70.9% at temperatures of cultivation 4°C and 15°C, respectively. The availability of 0.17M NaCl in the culture medium stimulated the synthesis of palmic acid (C16-0) and suppressed oleic acid (C18:1) in the yeasts of genera Cryptococcus and Rhodotorula. At this concentration of NaCl, the amount of linoleic acid (C18:2) increased in the strains of genus Rhodotorula. Availability of 0.85M NaCl in the culture medium for cultivation of Rhodotorula mucilaginosa 23-2 strongly influenced the synthesis of stearinic (C18-0) 64.2% and suppressed the oleic acid (24.2%). This NaCI concentration increased the amount of linoleic acid (C18:3) in Cryptococcus albidus 16-1 from 0.3% to 13.6% and linoleic acid (C18:2) of Rhodotorula minuta 16-3 was from 23.9% to 30.3%. *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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36 KEY WORDS

Antarctica, yeasts, proteins, nucleic acids, carbohydrates, lipids, fatty acids.

INTRODUCTION The biodiversity of yeasts in terrestrial Antarctic ecosystem has been the subject of scientific publications related to taxonomic study, biochemical and biological characteristics and activity by a large number of researchers (Di Menna, 1966b; Friedmann, 1993; Goto et al., 1969; Hu et al., 1993; Ratledge, 1988; Ray et al., 1989; Vincent & Williams, 1989; Vishniac, 1999; Wynn-Williams 1996b). Microorganisms regulate membrane lipid composition in response to environmental temperature which plays an important role in the regulation of their fatty acid component (Brenner, 1984; Hasel,1995). The maximum growth temperatures, which vary from >15 and <20°C to >20 and <25°C for biotypes of yeast strain Cryptococcus antarcticus, affect fatty acid composition (Vishniac & Kurtzman, 1992). Investigators reported that microorganisms, including yeasts at higher environmental temperature, generally produced an increase in the degree of saturation of membrane lipids (Arthur & Watson, 1976; Suutari et al., 1990; Vani Saxena, 1998; Watson, 1987). The temperature limits of growth of three psychrophilic yeasts, Leucosporidium frigidum, L.gelidum and L.nivalis, were examined by Watson et al. (1976). At temperatures lower than – 1°C and ethanol as a substrate, 90% of the total fatty acids were unsaturated with a predomination of linolenic (C18:3) from 35 to 50% and linoleic (C18:2) from 25 to 30% acids. At a temperature close to the maximum for growth 18°C linolenic acid constituted less than 20% of the total fatty acids, while oleic (20 to 40%) and linoleic (30 to 50%) acids were the major components. The membrane lipid composition changed depending on the temperature of growth of the psychrophilic yeasts. Ninety percent of the ten mitochondrial membrane lipids of Leucosporidium frigidum, grown at – 0.5°C, were unsaturated: 27% linoleic and 53% linolenic acids (Arthur & Watson, 1976). The strain Rhodotorula minuta 11 P-33 exhibited a temperature-dependent degree of unsaturation, similar to L.starkeyi which showed a max degree of unsaturation 1.2 at 20°C, which decreased to 0.8 at 32°C (Suutari et al., 1990). The fatty acids of Cr.antarticus grown at 13°C were 87% to 93% unsaturated and those of Cr.vishniacii were 91% unsaturated. Neither species produced linoleic acid under these conditions (Vishniac & Kurtzman, 1992). Fatty acid cmpositions, phospholipid sterols and tocopherols were determined in separated lipid fractions after fermentation of 5 Antarctic yeast strains in a medium containing glucose, peptone and yeast extract. Unsaturated fatty acids, mainly oleic (51.0-65.0%) and linoleic (9.5-16.8%) predominated in triacilglicerols (Zlatanov et al., 2001). Vani Saxe-

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na et al. (1998) found out that the general fatty acid profile of Rh.minuta grown at different temperatures showed a wide range of fatty acids (C7 to C18). Synthesis of long-chain fatty acid, e.g. oleic and linoleic acids, were predominant at 30-32°C, whereas short-chain acids, e.g. C7, C8, C9, were predominantly synthesized at 38°C. In spite of the numerous reports for microorganisms which confirm this phenomenon, it should not be accepted as a rule applied to all microorganisms without any exceptions. There were researchers who did not observe this universally known effect. Some differences in the fatty acid composition or in the degree of unsaturation were observed in Sacch.cerevisiae, grown at 15°C and 30°C (Hunder & Rose, 1972). Fatty acid composition of lipids of Lipomyces starkney, cultivated at temperatures 20°C and 30°C, showed that the temperature of cultivation did not affect the correlation of fatty acids in the lipid composition – 39.3% saturated and 61.9% unsaturated at 30°C and 38.1% saturated and 60.7% unsaturated at 20°C (Lukashin & Pshenichnaya, 1973). The effect of sodium chloride on the composition of yeast fatty acids was examinated by Combs et al. (1968), Watanable and Takakuwa (1984) and Yoshikawa et al. (1995). These authors established that the main components were palmitic, oleic and linoleic acids. Depending on the salt concentration and yeast species an increase or a decrease in the fatty acids presence could occur. Celligoi et al. (1997) studied three yeast strains – Candida parakrusei, Hansenula suaveloenus and an unidentified yeast “a” for lipid production utilizing sugar-cane molasses and molasses supplemented with sodium chloride and found out that, the addition of NaCl raised the percentage of palmitic acid in the three strains, as well as linoleic acid in yeast strains Candida parakrusei and Hansenula suaveloenus. The aim of this paper is to study the effect of the temperature and sodium chloride, addition to the culture medium, on the growth, biochemical characteristic and fatty acid composition of five Antarctic yeast strains. METHODS Five yeast strains were isolated from moss and soil samples taken by the Bulgarian Antarctic Expedition from Livingston Island, Antarctica. They were identified according to the morphological, cultural and physiological characteristics as Cryptococcus albidus 16-1, Cryptococcus laurentii 16-2, Rhodotorula minuta 16-3, Candida oleophila 23-1 and Rhodotorula mucilaginosa 23-2 and were maintained on malt slant agar and stored at 4°C (Pavlova et al., 2001). The strains were deposited at the National Bank for Industrial Microorganisms and Cell Cultures, Bulgaria. Yeast strains were cultivated in a media containing (g/l): peptone (Fluka), Chemie AG, Buchs, Switzerland ) – 10.0, yeast extract (Difco, Laboratories, Detroit, Michi-

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gan, USA ) – 3.0, glucose (Fluka, Chemie AG, Buchs, Switzerland ) – 20.0 and basal medium with addition of 0.17M NaCI or 0.85M NaCI. The initial pH was adjusted to 5.3-5.6 and the media were sterilized at 112°C for 30 min. The inoculum was prepared by growing cells from malt agar and was obtained on a rotary shaker (23 rad/s) in 500 ml Erlenmeyer flasks containing 50 ml of Sabourand dextrose broth (Merck, Darmstadt, Germany) at 15°C for 48 h. The fermentation medium was inoculated with 10% (v/v) unoculum. The cultivation was carried out in 1000 ml Erlenmeyer flasks containing 100 ml of a growth medium on a rotary shaker (23 rad/s) at 4°C and 15°C for 72 h. The biomass was separated by centrifugation at 4000 x g for 20 min, and washed twice with distilled water then its dry weight was determined by direct weighing of cells after drying it at 105°C for 24h. The nonpolar lipids were extracted in a Soxhlet apparatus with n-hexane for 8 h. After rotation vacuum distillation of the solvent the extracted oils were weighed. Methyl esters of fatty acids as standards were supplied by Merck, Darmstadt, Germany. The fatty acid composition of triacylglucerols was identified by capillary gas chromatography of its methyl esters. The esterification was carried out by Metcalfe and Wang Lechnique (Metcalfe & Wang, 1981). Methyl esters were purified by a thin-layer chromatography. The identification was carried out on a Pay Unicam 304, provided with a flame-ionization detector, 30-m capillary column Innowax (Scotia Pharmacenticals) impregnation and conditions as follows: column temperature 165-225°C, at a gradient of 4 K per min, detector temperature 300°C, injector temperature 280°C, carrier gas N2. Proteins were determined by the methods of Lowry et al. (1951) and by Kjeltec Auto 1030 Analyzez (Tecator, Sweden), following the instructions of the manufacturers. The amounts of nucleic acids (Spirin, 1958), carbohydrates (Dubois, 1956) and lipids (Folch et al. 1957) were measured according to published procedures. The results represent the averages of the three simultaneous experiments, performed in duplicate. RESULTS AND DISCUSSION The production and the chemical composition of biomasses of Antarctic yeasts, cultivated at different temperatures 4°C and 15°C, are presented in (Table 1). Biomass concentrations for strains were higher at 15°C compared to those at 4°C, while the chemical composition was influenced differently at the change of cultivation temperature. Protein content of Cryptococcus albidis 16-1 and Cryptococcus laurentii 16-2 increased from 24.30% and 32.98%, respectively at 4°C to 36.6% and 40.38% at 15°C. At the same time, in strain C. albidus 16-1 the quantity of carbohydrates decreased, as did the quantity of lipids in C. laurentii 16-2. A very slight change

4 15 4 15 4 15 4 15 4 15

Criptococcus albidus 16-1

Rhodotorula mucilaginosa 23-2

Candida oleophila 23-1

Rhodotorula minuta 16-3

Criptococcus laurentii 16-2

°C

Strains 5.01 ± 0.25 6.60 ± 0.30 5.10 ± 0.25 5.73 ± 0.28 6.80 ± 0.35 8.03 ± 0.50 8.53 ± 0.45 10.72 ± 0.55 5.36 ± 0.30 6.72 ± 0.35

Biomass, g/l 24.30 ± 0.30 36.67 ± 0.38 32.98 ± 0.35 40.38 ± 0.42 35.06 ± 0.35 33.58 ± 0.35 41.14 ± 0.40 39.76 ± 0.40 39.11 ± 0.40 29.60 ± 0.25

Proteins, % 2.27 ± 0.13 5.02 ± 0.15 5.17 ± 0.15 6.04 ± 0.16 4.43 ± 0.14 4.38 ± 0.14 5.51 ± 0.15 5.99 ± 0.16 3.82 ± 0.14 2.72 ± 0.13

Nucleic acids, %

Table 1. The effect of temperature on chemical composition of biomass from Antarctic yeasts.

32.87 ± 1.00 29.54 ± 0.95 30.98 ± 1.00 30.50 ± 1.00 30.80 ± 1.00 28.84 ± 0.95 27.84 ± 0.90 29.51 ± 0.95 32.81 ± 1.00 32.58 ± 1.00

Carbohydrates, %

15.95 ± 1.15 15.29 ± 1.10 14.02 ± 1.25 8.56 ± 0.75 14.17 ± 1.15 9.03 ± 0.80 17.25 ± 1.30 13.05 ± 1.05 10.73 ± 0.90 13.95 ± 1.10

Lipids, %

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K. PAVLOVA, M. ZLATANOV, L. KOLEVA, I. PISHTIYSKI

in the content of proteins and carbohydrates in strains Rhodotorula minuta 16-3 and Candida oleophila 23-1 was observed. However, the quantity of lipids decreased considerably when the temperature was raised – in strain Rh. minuta 16-3 from 14.7% to 9.03%, and in C. oleophila 23-1 from 17.25% to 13.05%. The results of strain Rh. micilaginosa 23-2 showed a considerable decrease in the protein content at 15°C compared to 4°C (from 39.11% to 26.60%), while lipids content increased from 10.73% to 13.95%. The quantity of nucleic acids is equal to that of the protein for all strains. The data in Table 2 present the study of the effect of the addition of sodium chloride in culture medium on the biomass concentration and chemical composition of the yeasts. These data showed that the addition of 0.17M NaCI in the culture medium stimulated the growth of Antarctic yeasts while the accumulation of biomass and its value decreased with the addition of 0.85M NaCl. The maximum biomass production (14.53 g/l) showed strain C.oleophila 23-1 under cultivation in medium YPD with 0.17M NaCl at 15°C. The strains Rh. minuta 16-3 and Rh. mucilaginosa 23-2 synthesized 8.82 g/ l and 7.3 g/l biomass. The suitable NaCl concentration for biomass accumulation, protein and nucleinic acids was 0.17M NaCl. This concentration also referred to carbohydrate content, but only for C. albidus 16-1 and C. oleophila 23-1. Lipid content was high in all biomasses at addition of 0.85M NaCl in the medium. From the results described it became clear that cultivation temperature, as well as the adding of NaCl in the medium influenced the chemical composition of the examined strains. This was also the conclusion of Celligoi et al. (1997), who studied the effect of NaCl on the quantity and content of biomasses of three different strains. Lipids were extracted from the yeast strains when they reached the stationary phase of growth in order to get a stable fatty acid composition. The fatty acid profile of the lipids of the five yeast strains, depending on growth temperature, are presented in Table 3. Oleic, palmitic and linoleic acids were the predominant fatty acids in lipids of all five strains. Three of them grown at 4°C synthesized a high quantity of unsaturated fatty acids: C. albidus 16-1-78.6% of the overall quantity of fatty acids were unsaturated: 55.4% oleic, 21.3% linoleic, 1.4% palmoleic and 0.5% linolenic acid; C. laurentii 16-2 synthesized 76.9% unsaturated fatty acids – 56.9% oleic, 16.1% linoleic, 2.8% linolenic and 1.1% palmoleic acids; the fatty acids of Rh. mucilaginosa 23-2 strain grown at 4°C synthesited 66.5%-46.7% oleic, 26.9% palmoleic, 16.1% linoleic and 2.4% linolenic acid. The fatty acid profiles of Rh. minuta 16-3 and Candida oleophila 23-1 indicated that these strains produced mainly palmitic, stearic and oleic acids. The strain Rh. minuta 16-3 synthesized 54.1% unsaturated at 4°C, and 66.7% unsaturated fatty acids at 15°C. The amount of unsaturated fatty acids of the strain Candida oleophila 23-1 at 4°C was 64.3%, whereas under cultivation at 15°C it was 70.9%. At low temperature both strains synthesized 21.9% and 15.4% stearic acid respectively, which distinguished them from the other three strains. The linolenic acids were synthesized in negligible

NaCI

Control 0.17 M 0.85 M Criptococcus laurentii 16-2 Control 0.17 M 0.85 M Rhodotorula minuta 16-3 Control 0.17 M 0.85 M Candida oleophila 23-1 Control 0.17 M 0.85 M Rhodotorula mu-cilaginosa 23-2 Control 0.17 M 0.85 M

Criptococcus Albidus 16-1

Strains 6.60 ± 0.45 6.64 ± 0.45 6.07 ± 0.40 5.73 ± 0.40 6.25 ± 0.45 5.80 ± 0.40 8.03 ± 0.55 8.82 ± 0.50 8.10 ± 0.50 10.72 ± 0.55 14.53 ± 0.50 8.44 ± 0.50 6.72 ± 0.40 7.36 ± 0.45 4.46 ± 0.35

Biomass, g/l 36,67 ± 0,35 38,39 ± 0,40 38,16 ± 0,40 40,38 ± 0,45 52,01 ± 0,55 37,42 ± 0,42 33,58 ± 0,35 38,59 ± 0,42 24,65 ± 0,28 39,76 ± 0,45 40,82 ± 0,42 37,56 ± 0,40 29,60 ± 0,30 41.56 ± 0.45 34.47 ± 0.38

Proteins, % 5.02 ± 0,05 5.43 ± 0,05 5.32 ± 0.05. 6.04 ± 0.16 7.56 ± 0,17 6.00 ± 0.16 4.38 ± 0.14 4.73 ± 0.15 2.35 ± 0.13 5.99 ± 0.16 5.44 ± 0.15 6.75 ± 0.20 2.72 ± 0.13 4.78 ± 0.15 4.28 ± 0.14

Nucleic acids, %

Table 2. The effect of sodium chloride on biomass composition from Antarctic yeasts.

29.54 ± 0.82 34.11 ± 0.92 29.47 ± 0.90 30.50 ± 1.00 25.98 ± 0.85 29.74 ± 0.92 28.84 ± 0.92 26.94 ± 0.90 30.78 ± 1,10 29.51 ± 0.95 36.90 ± 1.20 29.67 ± 0.90 32.58 ± 1.00 28.50 ± 0.92 29.56 ± 0.90

Carbohydrates, %

15.29 ± 1.45 16.29 ± 1.50 16.51 ± 1.50 8.56 ± 0,85 7.13 ± 0,60 12.75 ± 1.25 9.03 ± 0,85 14.29 ± 1.30 14.98 ± 1.20 13.05 ± 1.20 10.37 ± 0.85 14.55 ± 1.15 13.95 ± 1.15 14.09 ± 1.20 16.25 ± 1.25

Lipids, %

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ND – not detected

ND 2.3 24.2 0.3 ND 3.4 57.7 11.9 0.2 70.1 29.9

0.2 0.1 15.9 1.1 0.5 6.4 56.9 16.1 2.8 76.9 23.1

0.1 0.1 17.7 1.4 0.4 3.1 55.4 21.3 0.5 78.6 21.4

C12:0 C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 Unsaturated Saturated

ND 0.3 18.5 0.2 ND 4.5 61.0 15.5 ND 76.7 23.3

Cryptococcus laurentii 16-2 4 ºC 15 ºC

Criptococcus albidus 16-1 4 ºC 15 ºC ND ND 21.5 0.5 2.5 21.9 51.8 1.2 0.6 54.1 45.9

ND 6.3 24.4 0.4 ND 2.6 51.1 15.1 0.1 66.7 33.3

Rhodotorula minuta 16-3 4 ºC 15 ºC ND ND 19.8 2.1 0.5 15.4 61.7 0.5 ND 64.3 35.7

ND 0.6 25.0 0.4 ND 3.5 54.0 16.5 ND 70.9 29.1

Candida oleophila 23-1 4 ºC 15 ºC

0.2 0.1 26.9 1.3 1.0 5.3 46.7 16.1 2.4 66.5 33.5

ND 4.3 31.4 0.7 ND 2.7 51.0 9.7 0.2 61.6 38.4

Rhodotorula mucilaginosa 23-2 4 ºC 15 ºC

42

Fatty acids, %

Table 3. The effect of temperature on fatty acid composition of Antarctic yeast lipids.

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amounts. Both yeast strains had greater quantities of unsaturated than saturated fatty acids in lipid fractions at 15°C and they are considered exceptions to the predominant conclusions of many researchers that microorganisms synthesize greater quantity of unsaturated fatty acids under low temperature. Similar results regarding the oleic acid and palmitic acid content in oleoginous yeast strains have been reported by other researchers. Ykema et al. (1989) reported 55.4% oleic acid and 16.9% palmitic acid in the triacylglycerol fraction of lipids isolated from oleaginous yeast Apiotrichum curvatum ATCC 20509. Akhtar et al. (1998) reported oleic and palmitic acids as the major constituents of lipids, synthesized by strains Apiotrichum curvatum ATCC 10567, which synthesized 17.5% palmitic acid and 57.1% oleic acid and Lipomyces starkeyi ATCC 12659 synthesized triacylglucerols containing 37.7% palmitic and 46.6% oleic acid. Lipid content and composition can be altered by the addition of lipogenic factors in the culture medium although this depends on the concentration of the factor utilized and the microbe type studied (Celligoi et al., 1997; Watanabe & Takakuwa, 1984). The influence of NaCl on the fatty acid content of Antarctic yeast strains was examined as a lipogenic factor. The fatty acid composition of the lipid fractions of the Antarctic yeast strains, as dependent on the concentration of sodium chloride, is presented in Table 4. The availability of 0.17M NaCl in the nutritious medium stimulated palmic acid synthesis and suppressed oleic acid of the yeast of genera Cryptococcus and Rhodotorula. The lipid fractions of C.albidus 16-1 and C.laurentii 16-2 contained 29.5% and 30.7% palmic acid, respectively, and 41.2% and 35.2% oleic acid, respectively; the lipid fractions of Rh.minuta 16-3 and Rh.mucilaginosa 23-2 contained 27.1% and 32.5% palmic acid and 36.5% and 34.8% oleic acid. The increased level of linoleic acid in comparison with the control test was obvious in both strains. The only exception was strain C. oleophila 23-1, where the presence of 0.17M NaCl in the medium resulted in a decrease of palmic acid from 25.0% to 21.0% and an increase of oleic acid from 53.7 to 57.1%. The presence of 0.85M NaCl in the culture medium of C.albidus 16-1 had a strong influence on the synthesis of linoleic and linolenic acid – the latter from trace in the control test reached the level of 13.6%. Lipid fraction of yeast strain C.laurentii 16-2 cultivated under this concentration of NaCl contained palmitic acid 41.0%, equal to its content in palm oil (41.6%) while the quantity of palmooleic acid considerably increased – from 0.3% to 27.7%. NaCl influence on the composition of lipids of Rh.minuta 16-3 which expressed in the double increase of linoleic acid (15.1 to 30.3%) compared to the control test. The level of stearinic acid significantly increased in strain Rh.mucilagenosa 23-2 from 2.7% in the control test to 64.2% in the presence of 0.85M NaCl, and from 3.5% to 33.6% in C.oleophila 23-1. There was a positive correlation between the increase of NaCl concentration and the degree of fatty acid saturation of the cell lipids in four

ND – not detected; Tr. – traces.

1.1 Tr. 5.7 3.4 ND ND 57.2 19.0 13.6 93.2 6.8

ND 0.3 18.5 0.2 ND 4.5 61.0 15.5 Tr. 76.7 23.3

C12:0 C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 Unsatu-rated Saturated

2.7 5.0 29.5 4.0 0.3 2.7 41.2 14.3 0.3 59.8 40.2

Criptococcus albidus 16-1 Con- 0.17M 0.85M trol NaCl NaCl ND 2.3 24.2 0.3 ND 3.4 57.7 11.9 0.2 70.1 29.9

1.0 1.0 30.7 17.0 ND 7.2 35.2 7.7 0.2 60.1 39.9

0.2 0.3 41.5 27.7 ND ND 28.5 1.8 Tr. 58.0 42.0

Cryptococcus laurentii 16-2 Con- 0.17M 0.85M trol NaCl NaCl ND 6.3 24.4 0.4 ND 2.6 51.1 15.1 0.1 66.7 33.3

1.3 0.9 27.1 2.7 0.7 4.4 36.5 23.9 2.5 65.6 34.4

4.9 2.5 15.6 2.5 0.9 10.7 28.8 30.3 3.8 65.4 44.6

Rhodotorula minuta 16-3 Con- 0,17 M 0.85M trol NaCl NaCl ND 0.6 25.0 0.4 ND 3.5 53.7 16.8 Tr. 70.9 29.1

0.8 0.7 21.0 2.0 1.0 4.3 57.1 13.1 Tr. 72.2 27.8

0.7 0.4 3.2 0.1 0.6 33.6 58.4 0.1 2.9 61.5 38.5

Candida oleophila 23-1 Con- 0.17M 0.85M trol NaCl NaCl ND 4.3 31.4 0.7 ND 2.7 51.0 9.7 0.2 61.6 38.4

0.9 0.5 32.5 1.0 1.9 5.6 34.8 19.9 2.9 58.6 41.4

2.1 0.9 4.7 2.4 Tr. 64.2 24.2 0.2 1.3 28.1 71.9

Rhodotorula mucilaginosa 23-2 Con- 0.17M 0.85M trol NaCl NaCl

44

Fatty acids,%

Table 4. The effect of sodium chloride on fatty acid composition of Antarctic yeast strains.

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yeast strains, with the exception for strain Cr.albidus 16-1. The amount of unsaturated fatty acids in this strain at medium of 0.85M NaCl was 93.2%. NaCl concentration influenced the fatty acid composition of Antarctic yeasts, and stimulated the synthesis of particular fatty acids, depending on the individual strain characteristics. ACKNOWLEDGMENTS The authors thank the National Foundation for Research at the Ministry of Education and Science of the Republic of Bulgaria for financing this research. REFERENCES AKHTAR P., J.I. GRAY, A. ASGHAR. 1998. Chemical haracterization and stereospecific analysis of lipids synthesized by certain yeast strains. J. Food Lipids 5: 299-311. ARTHUR H., K. WATSON. 1976. Thermal Adaption in Yeast: growth temperatures, membrane lipid and cytochrome composition of psychrophilic, mesophilic yeasts. J. Bacteriology 128: 56-68. BRENNER R.R. 1984. Effect of unsaturated acids on membrane structure and enzyme kinetics. Prog. Lipid Res. 23: 69-73. CELLIGOI M.A., D.F. ANGELIA, Y.B. BUZATO. 1997. Application of sugar – cane molasses in production of lipids by yeast. Arg.Biol.Technol. 40: 693-698. COMBS T.J., J.J. GUARNER, M. AKADISANO. 1968. The effect of sodium chloride on the lipid content and fatty acid composition of Candida albicans. Mycologia 60: 1233-1239. DI MENNA M.E.1966b. Yeasts in Antarctica. Antonie Van Leeuwen-hock, 32: 29-38. DUBOIS M., K.A. GILLES, J.K. HAMILTON, P.A. REBERS, F. SMITTH. 1956. Calorimetric method for determination of sugars and related substances. Anal.Chem. 28: 350-355. FOLCH J., M. LOES, G. SLOANE-STANLEY. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J.Biol.Chem. 226: 497-500. FRIDMANN E. 1993. Antarctic Microbiology,Wiley-Liss, U.S., ISBN 0-471-50776-8. GOTO S., J. SUGIYAMA, H. IZUKA. 1969. A taxonomic study of Antarctic yeasts. Mycologia 61: 748-774. HAZEL J.R. 1995. Termal adaptation in biological membranes: Is homeoviscons adaptation the explanation? Annu.Rev.Physiol. 57: 19-42. HU J., Y. LI, L. WANG, Y. XUE. 1993. Biochemical characteristics and biological activity of soil microorganisms from Antarctic King George Island. Acta Microbiol.Sin. 33: 151-156. HUNDER K., A.H. ROSE. 1972. Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature. Biochemica et biophysica acta 260: 639-653. LOWRY O., N. ROSEBROUGH, A.L. FARR, & R.J. RANDAL. 1951. Protein measurement with the Folin phenol reagent. J.Biol.Chem. 193: 265-275.

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LUKASHIN A.A., S.N. PSHENICHNAYA. 1973. The effect of aeration and temperature of cultivation on the lipid composition of yeasts Lipomyces starkey and Sporobolomyces roseus. In Microorganisms – producers of biological-active substances. Publ.”Science and Tech-niques” Minsk. METCALFE L., C. WANG. 1981. Rapid preparation of fatty acid mathyl esters using organic base catalyzed transesterification. J.Chromatogr.Sci. 19: 530-534. PAVLOVA K., D. GRIGOROVA, T. HRISTOZOVA, A. ANGELOV. 2001. Yeast strains from the Livingston Island,Antarctica. Folia Microbiol. 46: 397-401. RATLEDGE C. 1988. Yeast for lipid production. Biochem.Soc.Trans. 16: 1088-1091. RAY M.K., S. SHIVAJI, R.N. SHYAMALA, P.M. BHARGAVA. 1980. Yeast strains from the Schimarcher oasis Antarctica. Polar Biol. 9: 305-309. SUUTARI M., K. LIUKKONEN, S. LAAKSO. 1990. Temperature adap-tation in yeasts the role of fatty acids. J. Gen.Microbiol. 136b: 1469-1474. SPIRIN A.S. 1958. Spectrophotometric determination of the nucleic acids content. Biochimia. 23: 656-661. SAXENA V., C.D. SHARMA, S.D. BHAGAT, V.S. SAINI, H.D.K. ADHIKARI. 1998. Lipid and fatty acid biosynthesis by Rhodotorula minuta. JAOCS. 75: 501-505. VINCENT W., C. WILLIAMS. 1989. Microbial communities in southern Victoria land streans (Antarctica). II. The effect of low temperature. Hydrobiologia. 172: 39-49. VISHNIAC H., C.P. KURTZMAN. 1992. Cryptococcus antarcticus sp. Nov. and Cryptococcus albidosimilis sp. Nov., Basidioblas- tomycetes from Antarctic soils. International Journal of Systematic Bacteriology 42: 547-553. VISHNIAC H.C. 1999. In J. SECKBACH (Ed.): Enigmatic Microorga-nisms and Life in Extreme Environments. Kluwer Academic Publishers, Dordrecht (Netherlands). WATANABE Y., M. TAKAKUWA. 1984. Effect of sodium chloride on lipid composition of Saccharomyces rouxii. Agr. Biol. Chem. 48: 2415-2422. WATSON K. 1987. In The yeasts. Vol.2. Edited by A.H. Rose and J.H. Harrison. Academic Press London. pp. 41-71. WATSON K., H. ARTHUR, W.A. SHIPTON. 1976. Leucosporidium yeasts: Obligate psychrophiles which alter membrane-lipid and cytochrome composition with temperature. J.Gen.Microbiol. 97: 11-18. WYNN-WILLIAMS D.D. 1996b. Antartic microbial diversity: the basis of polar ecosistem process. Biodiv. Conservat. 5: 1271-1293. YKEMA A., M.M. KARTAR, H. SMITH. 1989. Lipid production in whey permeate by an unsaturated fatty acid mutant of the oleaginous yeast Apiotrichum curvatum. Biotech. Lett. 11: 477-482. YOSHIKAWA S., N. MITSUI, K.I. CHIKARA, H. HASHIMOTO, M. SHIMOSAKA, M. OSAZAKI. 1995. Effect of salt stress on plasma membrane permeability and lipid saturation in the salt tolerant yeast Zygosaccharomyces rouxii. J. Ferment. and Bioeng. 80: 131-135. ZLATANOV M., K. PAVLOVA, D. GRIGOROVA. 2001. Lipid composition of some yeast strains from the Livingston, Antarctica. Folia Microbiologia 46: 402-408.

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© PENSOFT Bulgarian TEMPERATURE AND SODIUM CHLORIDE ON THE BIOMASS AND FATTYAntarctic ACIDS ...Research EFFECT OFPublishers 47 Sofia – Moscow Life Sciences, vol. 4: 47-53, 2004

Effect of Temperature and Sodium Chloride on the Biomass and Fatty Acids Composition of Antarctic Yeast Strain Sporobolomyces roseus AL8 L.KOLEVA1*, K.PAVLOVA2, M.ZLATANOV3 1*

Department of Biochemistry and Molecular Biology, UFT, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria

2

Department of Microbial Biosynthesis and Biotechnologies, 26 Maritza Blvd, 4002 Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Chemical Technology, University of Plovdiv, 4000 Plovdiv, Bulgaria

3

* Author for corresspondence: E-mail: lkoleva@abv. bg

ABSTRACT The yeast strain Sporobolomyces roseus AL8 was isolated from Antarctic moss samples from the region of the Bulgarian base on Livingston Island. The effect of temperatures (4°C, 18°C and 24°C ) and sodium chloride ( 0,17M and 0,85M) on the biomass and fatty acids composition of Sporobolomyces roseus AL8 were investigated. The temperature of cultivation has a significant effect on the biomass quantity obtained from Sporobolomyces roseus AL8 and it’s composition. The biomass yield at 4°C is about two times lower than at 18°C and 24°C. The lower temperature inhibits the nucleic acid and protein biosynthesis and stimulates the synthesis of carbohydrates and expecially lipids. Unsaturated fatty acids, mainly oleic, linoleic and linolenic acids predominated in triacylglicerol factions. The strain Sp. roseus AL8 synthesizated 75.4% unsaturated acids at 4°C and 74.0% at 24°C without NaCl and 80.0% at 4°C and 81.4% at 24°C with NaCl. KEY WORDS Antarctica, Carbohydrates, Fatty acids, Lipids, Nucleic acids, Protein, Sporobolomyces roseus AL8.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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INTRODUCTION Microorganisms living at temperatures below 10°C, high moisture, rough winds, u.v. stress, continuous light and dark periods have to adapt to these environments in such a way that metabolic processes, reproduction and survival strategies are optimal for their natural biotopes (Margesin & Schiner, 1999a). There is an increasing interest in the potential of cold-adapted organisms, since they play a major role in the processes of nutrient turnovel and primary biomass production in cold ecosystems (Margesin & Schiner, 1999b). The biodiversity of yeast in terrestrial Antarctic ecosystem is the subject of scientific publications related to taxonomic study, biochemical and biological characteristics and biothechnological application (Di Menna, 1996; Goto et al., 1969; Ratledge, 1988; Ray et al., 1989; Friedmann, 1993; Hu et al., 1993; WynnWilliams, 1996b; Pavlova, 2001). Microorganisms regulate membrane lipid composition in response to environmental temperature, which plays an important role in the regulation of their fatty acid content (Brenner, 1984; Hazel, 1995). The maximum growth temperatures, which vary from >15 and < 20° to >20 and < 25°C for biotypes of the yeast strain Cryptococcus antarcticus, have an effect on the fatty acid composition. Vishniac and Kurtzman studied fatty acid composition of yeast strains from Antarctica. Cryptococcus albidus, C. albidosimilis, C. antarcticus and C. vishniacii grown at 13°C were found to contain 34-54% oleic acid, 31-51% linoleic acid and 5.917.7% palmitic acid (Vishniac, 1992). Investigators reported that at higher enviromental temperatures, yeasts generally produced an increase in the degree of saturation of membrane lipids (Arthur and Watson, 1976; Watson, 1987; Suutari et al., 1990; Vani Saxena et al., 1998). The effect of sodium chloride on the composition of yeast fatty acids was examined by Watanable & Takakuwa, 1984 and Yoshikawa et al., 1995. The authors established that the main components were palmitic, oleic and linoleic acids. Depending on the salt concentration and yeast species, an increase or a decrease in the fatty acids presence could occur. The aim of the paper is to study the effect of the temperature and the concentration of the sodium chloride added to the culture medium on the growth, biochemical characteristic and fatty acid composition of the antarctic strain Sporobolomyces roseus AL8. MATERIALS AND METHODS Microorganism The Antarctic yeast stain Sporobolomyces roseus AL8 was isolated from a moss sample taken by the Bulgarian Antarctic Expedition from Livingston Island. The strain was identified using the criteria of yeast classification proposed by Kurteman & Fell,

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1998 and was deposited at the National Bank for Industrial Microorganisms and Cell Cultures, Bulgaria. Media The yeast strain was cultivated in medium containing (g/l): pepton (Fluka) – 10,0; yeast extract (Difco) – 3,0; glucose (Fluka) – 20,0. The initial pH was adjusted to 5,3 – 5,6 and the media were sterilized at 112°C for 30 min. Yeast growth The inoculum was prepared by growing cells from malt agar and was obtained on a rotary shaker (220 rpm) in 500 ml Erlenmeyer flasks containing 50 ml of Sabourand dextrose broth (Merck) at 18°C for 48h. The fermentation medium was inoculated with 10 v/v inoculum. The cultivation was carried out in 1000 ml Erlenmeyer flasks containing 100 ml of a growth medium on a rotary shaker (220 prm) at 4°C, 18°C and 24°C for 72h. The biomass separated by centrifugation at 4000 min-1 for 20 min, washed twice with distilled water, resuspended and disintegrated with glass beads in a Braun apparatus (B. Braun Melsungen AG, Melsingen, Germany). The cell suspension was centrifuged at 10 000 × g for 20 min and biomass was liophilization on – 64°C. Fatty acid composition The fatty acid composition of triacylglycerols was identified by capillary gas chromatography of their methyl esters. The esterification was carried out by the Metcalfe & Wang technique, 1981. Methyl esters were purified by thin-layer chromatography. Determination was accomplished on a Pay Unicam 304 unit, provided with a flame-ionization detector, 30 m capillary column Innowax impregnation (Scotia Pharmacenticals Ltd) and conditions as follows: column temperature 165°C to 225°C with a change 4°C/min, detector temperature 300°C, injector temperature 280°C, gascarrier (N2). Analytical methods Proteins were determined by the methods of Lowry et al., 1951 and by Kjeltec Auto 1030 Analyzez (Tecator, Swedwn), following the instructions of the manifacturers. The amounts of nucleic acids, carbohydrates and lipids were measured according to published procedures (Spirin, 1958; Dubois et al., 1956; Folch et al., 1957). RESULTS AND DISCUSSION The effect of the temperature of cultivation on the amount and composition of the biomass accumulated by the Antarctic yeast strain Sporobolomyces roseus AL8 is pre-

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L. KOLEVA, K. PAVLOVA, M. ZLATANOV

sented in Table 1. The yield of biomass was strongly affected by the temperature. It was twice lower at 4°C in comparison with the temperature of cultivation of the strain at 18°C and 24°C. In the same direction the contents of protein and nucleic acids were changed. Their amounts were quite low in the biomass accumulated at 4°C – only 22,24% protein and 2,21% nucleic acids. When the strain was cultivated at 18°C, the contents of protein and nucleic acid dramaticly increased – 53,6% and 7,7%, respectivly. In contrast to that the amounts of lipids and catbohydrates in the biomass accumulated at temperature 4°C were 27,9% lipids and 25,8% carbohydrates, while their values strongly decreased to 11,46% lipids and 18,9% carbohydrates at temperature 18°C. The increasing of temperature from 18°C to 24°C did not affect the growth of the strain – only some negligible increase in the biomass concentration and the protein content occurred, but a decrease in that of the lipids. These results are of interest because of the fact that none of the Antarctic yeast strains investigated in our laboratory regestered so remarkably the effect of temperature of cultivation on biomass composition (data not shown). Therefore, the methabolism of the strain Sporobolomyces roseus AL8 reacted more effectively to the surrounding temperature which contributed for its surviving in an extreme environment. The effect of the concentration of sodium chloride in the culture medium on the yield and the composition of the biomass from Sporobolomyces roseus AL8 is presented in Table 2. It was not so considerable as was the temperature. 0,17M NaCI did not affect biomass concentration but increased the amount concentration of protein in it from 55,98% to 61,20%. The amounts of carbohydrates and lipids decreased negligibly. After cultivation of the strain in a medium with the addition of 0,85M NaCI, the biomass concentration decreased from 8,36 g/l to 7,39 g/l but the contents of protein, nucleic acids and carbohydrates did not change. The effects of temperature of cultivation and concentration of sodium chloride on fatty acid composition in the lipid fraction of the strain Sporobolomyces roseus AL8 are presented in Table 3. After cultivation of the strain at temperature 4°C the amounts Table 1. Effect of temperature on the production and chemical composition of biomass from Sporobolomyces roseus AL8 Parameters Biomass,g/dm3 Proteins, % Nucleic acids, % Carbohydrates, % Lipids, %

4

Temperature, °C 18

24

4,37±0,20 22,25±0,15 2,21±0,11 25,82±0,80 27,90±0,95

8,50±0,25 53,16±0,40 7,05±0,15 18,90±0,70 11,46±0,80

9,25±0,30 54,89±0,42 6,68±0,15 19,39±0,70 9,90±0,80

Each experiment was realized in triplicate

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Table 2. Effect of sodium chloride on the production and chemical composition of biomass from Sporobolomyces roseus AL8 at 24°C Parameters Biomass,g/dm Proteins, % Nucleic acids, % Carbohydrates, % Lipids, % 3

K

Sodium chloride, M 0,17

0,85

8,36 ± 0,28 55,98 ± 0,35 6,14 ± 0,18 18,52 ± 0,75 11,14 ± 0,85

8,48 ± 0,31 61,20 ± 0,42 6,28 ± 0,18 17,25 ± 0,81 9,78 ± 0,92

7,39 ± 0,25 61,33 ± 0,48 6,73 ± 0,22 17,20 ± 0,89 8,51 ± 0,85

Each experiment was realized in triplicate Table 3. Effect of incubation temperature and sodium chloride on the fatty acid composition of Sporobolomyces roseus AL8 Fatty acid

Laurinic (C12:0) Myristic (C14:0) Palmitic(C16:0) Palmoleic(C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic(C18:2) Linolenic(C18:3) Saturated Unsaturated

Temperatur, °C 4°C 24°C without NaCl without NaCl 0.5 Trace 19.9 1.2 4.2 60.1 10.9 3.2 24.6 75.4

0.4 0.2 10.2 1.7 14.7 57.8 12.9 1.6 26.0 74.0

Sodium chloride, M 0.17M NaCl 0.85M NaCl 24 °C 24 °C 0.6 0.3 10.9 1.1 7.2 70.5 7.0 1.4 20.0 80.0

0.8 0.5 13.9 3.4 1.4 68.8 9.1 0.1 18.6 81.4

Each experiment was realized in triplicate

of palmitic and linoleic acids were twice higher in comparison with those at 24°C. The synthesized oleic acid was 60,1% at 4°C and 57,8% at 24°C but the content of stearic acid was 3,5 times higher at 24°C. The differences in the amounts of unsaturated fatty acids synthesizer by the strain Sporobolomyces roseus AL8 was insignificant – 75,4% at 4°C and 74,0 at 24°C. From the data it could be seen that the strain synthesized the highest amounts of oleic, palmitic and linoleic acids. The addition of sodium chloride in the culture medium stimulated mainly the synthesis of stearic acids (Table 3). Its content increased from 57,8% to 70,5%, so the percentage of unsaturated fatty acids increased from 74,0% to 80,0%, with 0,17M NaCI and 81,4% with 0,85M NaCI at temperature 24°C. This showed that the addition of NaCI affected the synthesis of unsaturated fatty acids in the Antarctic strains

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Sporobolomyces roseus AL8. The increase of concentration of NaCI added in the medium strongly affected acids, which correlated with the results of other authors (Watanabe & Takamuwa, 1984, Celligoi, 1997). In conclusion, it could be noticed that the strain Sporobolomyces roseus AL8 is an interesting object for investigation. It reacted to the temperature changes in a different manner in comparison with the other Antarctic yeast strains that we had. This represents a repression of protein and nucleic acids biosynthesis but stimulation of the biosynthesis of carbohydrates and lipids which helps it to survive at extremely low temperatures. The total content of saturated and unsaturated fatty acids in lipid fraction changes insignifficantly with the increase in temperature from 4°C to 24°C. Therefore, yeast cells do not survive by changing the ratio of saturated / unsaturated fatty acids but by increasing in the total lipid content. REFERENCES ARTHUR A.H., K. WATSON. 1976. Thermal Adaptation in Yeast: Growth Temperatures, Membrane Lipid, and Cytochrome Composition of Psychrophilic, Mesophilic and Termophilic Yeasts, J. Bacteriology 128, 1, 56-68. BRENNER R.R. 1984. Effect of Unsaturated Acids on Membrane Structure and Enzyme kinetics, Prog. Lipid Res., 23, 69-73. CELLIGOI M.A., D.F. ANGELIS, Y.B. BUZATO. 1997. Application f sugar-cane molasses in the production of lipids by yeast. Arg. Biol. Technol. 40, 3, 693-698. DI MENNA M.E. 1996b. Yeast in Antarctica. Antonie van Leenwenhock 32, 29-38. DUBOIS M., K.A.GILLES, J.K.HAMILTON, P.A.REBERS, F. SMITH. 1956. Calorimetric method for determination of sugars and related substances. Anal.Chem. 28, 350-355. FOLCH J., M. LOES, G. SLOANE-STANLEY. 1957. A simple method for isolation and purification of total lipids from Anienal tissues. J. Biol. Chem. 226, 497-500. FRIEDMAN E. 1993. Antarctic Microbiology, Wiley-Liss, U.S., ISBN 0-471-50776-8 GOTO S., J. H. SUGIYAMA, LIZUKA. 1969. Taxonomic study of Antarctic yeasts. Mycologia 61, 748-774. HAZEL J.R. 1995. Thermal adaptation in biological membranes: Is homeoviscons adaptation the explanation? Annu. Rev. Physiol. 57, 19-42. HU J., Y. LI, L. WANG, Y. XUE. 1993. Biological characteristics and biological activity of soil microorganisms from Antarctic King George Island, Acta Microbiol. Sin. 33, 151-156. KURTZMAN C.P., J.W. FELL. 1998. The Yeasts: Tasonomic Study, 4th ed. Elsevier Scientific Publishers, Amsterdam (Netherlands). LOWRY O., N. ROSEBROUGH, A.L. FARR, R.J. RANDAL. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193; 265-275.

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MARGESIN R., F. SCHINER. 1999a. Cold-Adapted Organisms. Springer, Heidelberg, Germany, ISBN 3-540-64973-5. MARGESIN R., F. SCHINER. 1999b. Biotechnological Applications of Cold-Adapted Organisms. Springer, Heidelberg, Germany, ISBN 3-540-64972-7. METCALFE L., C.WANG. 1981. Rapid preparation of fatty acid methyl esters using organic base catalysed transes lerification. J. Chromatogr. Sci., 19, 530-534. PAVLOVA K., D. GRIGOROVA, T. HRISTOZOVA, A. ANGELOV. 2001. Folia Microbiol. Yeast strains from Livingston Island, Antarctica. 46, 5, 397-401. RATLEDGE C. 1988. Yeast for lipid production. Biochem. Soc. Trans. 16, 1088-1091. RAY M., S. SHIVAJI, RAO, N. SHYAMALA, P. BHARGAVA. 1989. Yeast strain from the Schirmacher Oasis Antarctica. Polar Biol., 9, 305-309. SPIRIN A.S. 1958. Spectrophotometric determination of the nucleic acids content. Biochimia, 23; 656-661. SUUTARI M., K. LIUKKONEN, S. LAAKSO. 1990. Temperature adaptation in yeasts the role of fatty acids. J. Gen. Microbiol. 136b 1469-1474. VANI, SAXENA, C.D. SHARMA, S.D. BHAGAT, V.S. SAINI, H.D.K. ADHIKARI. 1998. Lipid and Fatty acid Biosynthesis by Rhodotorula minuta JAOCS 75, 4, 501-505. VISHNIAC, H., C.P. KURTZMAN. 1992. Cryptococcus antarcticus sp.nov. and Cryptococcus albidosimilis sp.nov. Basidioblastomycetes from Antarctic soils. International Journal of Systematic Bacteriology 42, 4. 547-553. WATANABE Y., M. TAKAKUWA. 1984. Effect of sodium chloride on lipid composition of Saccharomyces rouxii. Agr. Biol. Chem. 48, 10, 2415-2422. WATSON K. 1987. Temperature relations. In. The yeasts. Vol.2. Edited by A.H. Rose and J.H.Harrison. Academic Press London. pp. 41-71. WYNN-WILLIAMS D.D. 1996b. Antarctic microbial diversity: the basis of polar ecosistem process. Biodiv. Conservat. 5, 1271-1293. YOSHIKAWA S., N. MITSUI, K.I. CHIKARA, H. HASHIMOTO, M. SHIMOSAKA, M. OSAZAKI. 1995. Effect of salt stress on plasma membrane permeability and lipid saturation in the salt tolerant yeast Zygosaccharomyces rouxii, J. Ferment. and Bioeng. 80, 2, 131-135.

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©A PENSOFT Bulgarian Antarctic LKALOIDS Publishers FROM THE ANTARCTIC STRAIN MICROBISPORA AERATA SUBSP . NOV. IMBAS -11A Research ... 55 Sofia – Moscow Life Sciences, vol. 4: 55-64, 2004

Alkaloids from the Antarctic Strain Microbispora aerata subsp. nov. Imbas-11a. Isolation, Separation and Physico-chemical Properties V. IVANOVA1, U. GRAEFE2, R. SCHLEGEL2, A. GUSTEROVA1, K. ALEKSIEVA1, M. KOLAROVA1, R. TZVETKOVA1 Institute of Microbiology, Bulgarian Academy of Sciences,

1

26 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria Hans-Knoll-Institute of Natural Product Research, Beutenbergstrasse,

2

11, D – 07745 Jena, Germany

ABSTRACT Alkaloids were discovered by a chemical screening in the cultural broth of Microbispora aerata subsp. nov. IMBAS-11A. The strain was isolated from penguin excrements collected on the Antarctic Livingstone Island. It was established that the isolated alkaloids possessed in the structure indole chromophore and belonged to the family of indole alkaloids (tryptamine derived amides). KEY WORDS Microbispora aerata subsp. nov. IMBAS-11A, alkaloids, N-acetyltryptamine.

INTRODUCTION During the course of our screening programme for bioactive secondary metabolites from Antarctic strains we found that the strain Microbispora aerata subsp. nov. IMBAS-11A produced tryptamine alkaloids. From the petrol extract of plant material (seeds of Annonia reticulata) it was isolated N-fatty acyl derivatives of tryptamine – a fraction, which contained nitrogen and longchain acyl moiety (Maeda et al., 1993). The compounds possess insecticidal properties.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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Riemer et al., 1997 report on the isolation and structure, elucidation of four new tryptamine derived amides (madugin, methylmadugin, prebalamide and balasubramide) which were found as major components in the methanolic leaf extracts of Clausena indica. Anderton et al., 1994 report on alkaloids such as N-methyltyramine, N,N-dimethyltryptamines and 1,2,3,4-tetrahydro-b-carbolines from Phalaris arundinacea. Also Anderton et al., 1998 were found in the leaf extract with 0.1N HCl of Phalaris coerulescens the alkaloid (-) – coerulescine. From the South East Asian medicinal plant Horsfieldia superba, indole alkaloid horsfiline (Jossang et al.,1991) was isolated. This paper describes the production, isolation, separation and physico-chemical properties of the alkaloids from Microbispora aerata subsp. nov. IMBAS-11A and the identification of the main compound of complex. MATERIAL AND METHODS Producing Organism and growth conditions The strain Microbispora aerata subsp. nov. IMBAS-11A was isolated from penguin excrements collected on the Antarctic Livingstone Island. Sampling had been performed in the course of the Third Bulgarian Expedition to Antarctica (1995-1996). The strain Microbispora aerata subsp. nov. IMBAS-11A was deposited in the collection of the Institute of Microbiology of the Bulgarian Academy of Sciences. Based on the effect of temperature on its growth rate, it is a thermophil, optimal temperature for growth at 50°C. The mature slant culture of Microbispora aerata subsp. nov. IMBAS-11A was inoculated into Erlenmeyer flasks (500 ml), containing 50 ml of seed medium consisting of 0.5% peptone, 0.5% corn steeped in liquor, 1.0% starch, 0.5% NaCl and 0.5% CaCO3, pH 7.2 after autoclaving. The flasks were cultivated on a rotary shaker at 325 rpm for 18 hours at 50°C. The seed culture (5%) was inoculated into Erlenmeyer flasks (1000 ml), containing 400 ml of the production medium. The fermentation medium contained the same ingredients as the seed medium. The cultivation was carried out for 48 hours at 50°C on a rotary shaker. Isolation of crude alkaloid complex from a strain Microbispora aerata subsp. nov. IMBAS-11A After 48 hours the fermentation broth (10.00 liters) was adjusted to pH 8.0-8.5 and centrifuged. The mycelium (approx. 100 g) was extracted by 3 x 100 ml acetone and the combined acetone extract was concentrated to aqueous layer. The residual watery phase was re-extracted by 2 x 300 ml ethyl acetate. The culture filtrate was extracted 2 times by 5.0 liters of ethyl acetate. The combined ethyl acetate extract from the mycelium and culture filtrate, respectively, was filtered, dried over Na2SO4

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and evaporated to dryness (yield 1.40 g). A chloroform solution of the crude product was chromatographed on a silica gel 60 (70-230 mesh) column, equilibrated with chloroform. The compounds in the crude product were eluted from the column with chloroform, chloroform-methanol (2:1) and methanol. The fractions were combined and evaporated in vacuo to dry residue. The complete separation and purification of the compounds of complex could be achieved by preparative high-performance liquid chromatography (HPLC). Preparative HPLC Column: Lichrospher 100, RP-18 (10mm) (250 x 10 mm). Mobile phase: gradient of 12% to 90% acetonitrile in 2 mM NH4OAc buffer, pH 5.0. Flow rate: 5.0 ml/ min. Detection: 220 nm. After concentracion and drying of the fractions 36, 10, 25, 9, 20 and 12 mg of pure compounds, 3, 4, 5, 6, 7 and 8 were obtained. Separation and isolation of the compound 9 by TLC The complete separation and purification of the compound 9 of the complex could be achieved by preparative thin-layer chromatography (TLC) on a silica gel plates with the following mobile phase: chloroform-methanol (85:15, v/v). The compound was eluted with ethyl acetate. After concentration and drying, 15 mg of compound 9 was obtained. Separation of the alkaloid complex with analytical HPLC Column: Symmetry C 18, 5 mm (150 × 3.9 mm). Mobile phase: gradient of 20% to 90% acetonitrile in 2 mM NH4OAc buffer, pH 5.0. Flow rate: 1 ml/min. Detection: 220 nm. Thin-layer chromatography (TLC) TLC was carried out on silica gel plates (Merck 60, F254) with the following mobile phases: chloroform; chloroform-methanol (9:1; 8.5: 1.5; 8:2 by vol.). The chromatographic spots were visualized by spraying with 0.5% solution of vanilline in sulphuric acid /acetic acid/ methanol and heating at 120°C for 3-5 minutes. General experimental procedures UV-VIS and IR-spectra were recorded on a Beckman DU 601 and Shimadzu IR scanning spectrophotometers. Mass-spectra (HREI-MS) were recorded on a double focusing sector field mass spectrometer AMD-402 (AMD Intectra, Harpstedt, Germany). Electrospray mass spectra were recorded on a triple-quadrupole instrument Quattro (VG Biotech, Altrincham, England). 1H and 13C-NMR spectra were recorded on a Bruker Avance DRX 500 NMR spectrometer. 1H-NMR spectra were recorded in

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ET AL.

CD3OD at 300 MHz; 13C-NMR spectra were recorded in CD3OD at 75.46 MHz. The chemical shifts are expressed in values (ppm) with TMS as an internal standard. RESULTS AND DISCUSSION The isolation of the alkaloid complex from the culture broth of Microbispora aerata subsp. nov. IMBAS-11A and the preparative chromatographic separation of its compounds is presented in Scheme 1. The homogenity of the isolated product after silica gel chromatography was confirmed by analytical HPLC. Figure 1 presents the HPLC-chromatogram of the alkaloid complex and Table 1 the quantitative analysis 7 8

1 075 000 µV

5

100

4 3

80

6 9

60

2

40

1

30

3`

0

75 000

1`

1``

0.0

40.0

minutes

Fig. 1. Analytical-HPLC profile of the alkaloid complex. Column: Symetry C18, (150 × 3.9 mm; 5 µm); mobile phase: gradient of 20% to 90% acetonitrile in 2 mM NH4OAc buffer, pH 5.0; flow rate: 1 ml/min; detection: UV 220 nm. Compounds (1-8) Table 1. HPLC quantitative analysis of compounds 1-8. Peak No 1 2 3 4 5 6 7 8

Rt-time (minutes)

Height (µv)

Area (µv-sec)

Area (%)

6.94 7.74 12.61 17.22 20.98 24.01 26.16 27.24

93737 158772 266865 293613 1933691 247718 868299 539704

1384866 2870814 7339630 7463511 129066968 5229442 19889484 10482278

0.713 1.478 3.778 3.842 66.452 2.692 10.240 5.397

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Culture broth (10.00 liters) adjusted to pH 8.0-8.5 centrifugation Mycelium extracted with acetone, filtered

Filtrate extracted with ethyl acetate

Acetone extract concentrated in vacuo

Aqueous layer (discarded)

Ethyl acetate extract

Aqueous layer extracted with ethyl acetate Ethyl acetate extract dried with Na2SO4, filtered and concentrated in vacuo Crude product Silica gel chromatography eluted with a chloroform; chloroform-methanol (2:1); methanol Alkaloid complex Preparative HPLC Column: Lichrospher (250x10 mm)

100,

RP-18,

eluted with a gradient of 12 % to 90 % acetonitrile in 2 mM NH4OAc buffer, pH 5.0. Flow rate: 5.0 ml/min. Detection:

210 nm Fractions concentrated under reduced pressure compound 3 compound 4 compound 5

compound 6 compound 7 compound 8

Scheme 1. Isolation procedure of the alkaloid complex from Microbispora aerata subsp. nov. IMBAS-11A

0.46

218,270

DMSO, MeOH, CHCl3

220,276 272sh,290sh 0.55

DMSO, MeOH, CHCl3

waxy solid

waxy solid

7 waxy solid

8 waxy solid

9

DMSO, MeOH, CHCl3, EtAc 222,280 272sh,290sh 0.64

201.1(M-H)202.10940 C12H14N2O DMSO, MeOH, EtAc, CHCl3 222,278 272sh,290sh 0.72

357.2(M-H)358.0001 C21H18N4O2 DMSO, MeOH, EtAc, CHCl3 224,280 272sh,290sh 0.76

355.2(M-H)356.11969 C21H16N4O2

DMSO, MeOH, EtAc, CHCl3 224,280 272sh,290sh 0.81

314.2(M-H)315.10751 C19H13N3O2

DMSO, MeOH, EtAc, CHCl3 224,280 272sh,290sh 0.86

312.2(M-H)313.08950 C19H11N3O2

203.0(M+H)+ 359.1(M+H)+ 357.5(M+H)+ 314.0(M+H)+ + + + + 225.0(M+Na) 381.1(M+Na) 379.1(M+Na) 338.1(M+Na) 336.1(M+Na)+ + + + 426.9(2M+Na) 739.3(2M+Na) 734.7(2M+Na) 653.3(2M+Na)+ 649.4(2M+Na)+

waxy solid

5

V. IVANOVA

UV-VIS(λmax) nm Rf on TLC Silica gel CHCl3-MeOH (8:2)

waxy solid

194.0(M+H)+ 284.1(M+H)+ 216.0(M+Na)+ 306.1(M+Na)+ 409.2(2M+Na)+ 567.6(2M+H)+ 589.6(2M+Na)+ 192.1(M-H)282.2(M-H)193.09950 C11H15NO2

waxy solid

4

60

ES-MSHREI-MS Molecular formula Solubility

Appearance Molecular Weight ES-MS+

3

Compounds 6

Table 2. Physico-chemical properties of compounds 3-9 from Microbispora aerata subsp. nov. IMBAS-11A

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ET AL.

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of HPLC-separation of the complex. As a result, eight peaks (1-8) were obtained. Compound 5 with retention time (Rt) at 20.89 min is the main compound (66.45%), the compounds 7, 8, 4, 3 and 6 are (10.24%, 5.39%, 3.84%, 3.77% and 2.69%), respectively, of the complex. The physico-chemical properties of the compounds of complex are summarized in Table 2. The compounds (3-9) were isolated as waxy solids and were soluble in lower alcohols, dimethylsulfoxide, pyridine, ethyl acetate and chloroform. The molecular weight and the elemental composition of the compounds 3, 5, 7, 8 and 9 were determined by high resolution electron impact mass spectrum (HREI-MS), see Table 2. The data showed that the compounds (3-9) were nitrogen-containing compounds with pH 8.0-8.5. The compounds (5-9) had identical UV-spectra with lmax (MeOH) at 222-224; 278-280 and 272 sh, 290 sh nm, typical of an indole chromophore, but compound 3 had UV-spectrum at 218, 270 nm, typical of a benzene ring chromophore. This was further confirmed by strong IR absorptions at 3405-3476 cm-1 for 5-9, indicating the N-H vibration in the indole moiety. The characteristic signal at 3423 cm-1 of 5 is indicative of > N-H stretching of secondary amides and the strong bands at 1652-1690 cm-1 in compounds 3, 5 and 7 were typical of the N-C=0 stretching region of amides. The main compound 5 (Fig. 2) by thin- layer chromatography on silica gel plates with chloroform-methanol (8:2, v/v) as the mobile phase had Rf = 0.64 and demonstrated positive colour reaction to 0.5% vanillin /sulphuric acid/acetic acid (pink).

O

HN 9 8 4 3

4a

5

2 1

6

7a

N

7

H Fig. 2. Structure of N-acetyltryptamine (compound 5)

10

11

CH 3

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ET AL.

The EI-mass spectrum (Fig. 3) of 5 exhibited a strong ion peak at m/z 202.1, corresponding to a C12H14N2O, together with several other peaks at m/z 143, 130, 115, 103, 77. The typical fragmentation peaks at m/z 143 and 130 were observed also by the compounds 6-9. In the 1H-NMR spectrum for 5 (Fig. 4), one methyl signal at d 100.00 %

143.0

130.0

77.0 103.1 115.0 50

60

70

80

90

100

110

120

130

140

150

m/z

100.00 %

202.1

160

170

180

190

200

M 210

220

230

240

250

m/z

ppm

8

7

6

5

Fig. 4. 1H-NMR spectrum of compound 5

4

3

3.4458

2.4031

2.6200

2.4056

3.0322

0.9726

1.0000

integral

Fig. 3. Electron impact mass spectrum of compound 5

2

1

0

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1.89; methylene protons at d 2.93, 3.46 and five aromatic protons at d 7.54, 7.29, 7.06, 7.04 and 6.98 were observed. The 13C-NMR and DEPT spectra (Fig. 5 and 6) of 5 indicated twelve carbons: one methyl carbon at 22.6 ppm (quartet); two methylene carbons at 26.2 and 41.5 ppm (triplets); five aromatic carbons at 112.21, 119.23,

ppm

180

160

140

120

100

80

60

40

20

ppm

120

100

Fig. 6. DEPT 135 of compound 5

80

60

40

22.587

26.196

41.542

49.844

112.202

123.338 122.292 119.563 119.223

ppm

Fig. 5. 13C-NMR spectrum of compound 5

20

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ET AL.

119.57, 122.30 and 123.34 ppm (doublets); three quaternary carbons at 113.3, 128.8 and 138.1 ppm (singlets) and one carbonyl carbon at 173.2 ppm. These data indicated that the compound 5 is identical with (3-acetyl amino ethyl) indole or N-acetyltryptamine. The first report about N-acetyltryptamine was from Maeda et al., 1993. This compound was isolated from the ground seeds of Annona reticulata. The metabolite was reported also to occur in Myxobacteria but never in thermophilic strains. The other compounds (6-9) of the complex possessed in the structure also indole chromophore. The compounds (5-9) are natural products, directly isolated from the culture broth of the antarctic strain Microbispora aerata subsp. nov. IMBAS-11A and belonged to the family of the indole alkaloids. ACKNOWLEDGEMENTS This study was supported by DFG (project 436 BUL 113/107/0). We express our thanks. REFERENCES ANDERTON N., P. A. COCKRUM, D. W. WALKER, J. A. EDGAR. 1994. In: Plant-associated Toxins: Agricultural, Phytochemical and Ecological Aspects. eds S. M. Colegate and P. R. Dorling. CAB International, Wallingford, p. 269. ANDERTON N., P. A. COCKRUM, S. M. COLEGATE, J. A. EDGAR, K. FLOWER, I. VIT, R. I. WILLING. 1998. Oxindoles from Phalaris coerulescens. – Phytochemistry, 48: 437-439. JOSSANG A., P. JOSSANG, H. A. HADI, T. SEVENET, B. BODO. 1991. Horsfiline, an Oxindole Alkaloid from Horsfieldia superba. – J. Org. Chem., 56: 6527-6530. MAEDA U., N. HARA, Y. FUJIMOTO, A. SRIVASTAVA, Y. K. GUPTA, M. SAHAI. 1993. NFatty acyl tryptamines from Annona reticulata. – Phytochemistry, 34: 1633-1635. RIEMER B., O. HOFER, H. GREGER. 1997. Tryptamine derived amides from Clausena indica. – Phytochemistry, 45: 337-341.

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© PENSOFT PublishersRETIRUGA (BULL.: FR.) REDHEAD VAR. ANTARCTICA Bulgarian Research HORAC Antarctic ... ARRHENIA 65 Sofia – Moscow Life Sciences, vol. 4: 65-68, 2004

Arrhenia retiruga (Bull.: Fr.) Redhead var. antarctica Horac – One Agarical Fungus from Livingston Island, South Shetlands (The Antarctic) M. GYOSHEVA1, R. METCHEVA2 1

Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria

Institute of Zoology, Bulgarian Academy of Sciences, Sofia, Bulgaria

2

ABSTRACT This paper reports one agarical fungus – Arrhenia retiruga (Bull.: Fr.) Redhead var. antarctica Horak found in the South Bay region on Livingston Island during the Eight Bulgarian Antarctic Campaign (1999-2000). KEY WORDS Arrhenia retiruga var. antarctica, Basidiomycetes, Agaricales, Livingston Island, Antarctica.

INTRODUCTION The investigation on the species diversity and distribution of fungi on Livingston Island is part of the Bulgarian biological research activity in Antarctica. Arhenia salina (Høiland) Gulden was the first macromycete species found on Livingston Island during the Bulgarian Antarctic expeditions (Gyosheva & Chipev, 1999). This species was collected during the Antarctic summer of 1996-97. The present paper reports on the finding of another taxon of genus Arrhenia (Agaricales, Tricholomataceae) – Arrhenia retiruga (Bull.: Fr.) Redhead var. antarctica Horak on Livingston Island. The material reported here was collected by the second author (R. M.) during the Eighth Bulgarian Antarctic Campaign (1999-2000) on Livingston Island.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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MATERIALS AND METHODS The fungal specimens examined were collected during the Antarctic summer of 1999-2000, on the Hurd Peninsula of Livingston Island (62°58’S and 60°21’W). The localities were situated along the coast of South Bay and have a NNW exposition. The specimens were identified after Agerer (1984), Barrasa & Ricco (2003), Høiland (1976), Horak (1966, 1982). Voucher specimens are kept in the Mycological Collection of the Institute of Botany (SOM). RESULTS AND DISCUSSION Morphological description and chorological information on the taxon Basidiomycetes Agaricales Tricholomataceae Arrhenia retiruga (Fr.) Rocken var. antarctica Horak). (Fig. 1) (Leptoglossum retirugum (Fr.) Ricken var. antarcticum Horak). Basidiomata cyphelloid (like inverted cup), almost round to discoid. Pileus 5-10 mm in diameter, cupulare, dorsal attached, pale brown to yellowish, brown grayish, higrophanous, margin lobed, irregular wavy, glabrous. Hymenophor consisting of slightly reticulate veins, concolorous with pileys. Stipe absent. Spores (4) 7-9´4-7.5 µm, lacrymoid or pyriform, smooth, hyaline. Basidia 4-spored. Cheilocystidia and Pleurocystidia absent. Localities: South Shetlands, Livingston Island, South Bay region, Svetilishteto locality, on rocks covered with mosses (Polytrichum sp., Drepanocladus sp.), 16 Feb 2000.

Fig. 1. Arrhenia retiruga var. antarctica - basidiomata.

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The fruit bodies were observed in small groups (2-3 specimens) and numerous amongst mosses on higher places of the rocks, near old nests of birds. Remarks: The examined taxon has been described from the Antarctic region by Horak (1966). The specimens examined have been collected on Deception Is. and on Half Moon Is (Soth Shetlands). The fungus has been described by Horak as Leptoglossum lobatum (Pers.: Fr.) Ricken var. antarcticum. The author in a later publication (Horak, 1982) based on Kühner & Lamoure (1972) revised the materials and transferred the variety to the species Leptoglossum retirugum (Bull.: Fr.) Ricken. According to contemporary taxonomy and nomenclature of fungi (Kirk et al., 2001), L. retirugum var. antarcticum is a synonym of Arrhenia retiruga (Fr.) Ricken var. antarctica Horak. In both localities, Horak (1966) observed the fungus growing closely attached amongst mosses and lichens in numerous colonies. After Horak (1982) this fungus is probably common in its habitats. Arrhenia retiruga have a very broad ecological amplitude. This species occurs in the alpine zone and in the Arctic areas (Høiland, 1976) Arrhenia salina - the another taxon of genus Arrhenia found on Livingston Island (Gyosheva & Chipev, 1999) was observed during previous Bulgarian Antarctic expeditions (in the summer seasons of 2001 and 2003). The fungal specimens were collected from two new localities in the coastal zone of South Bay: Svetilishteto (62 °58’ and 60°21’W), amongst mosses, 14 Feb 2001; Kaleta Argentina (62°40’S and 60°24’W), amongst mosses, 19 Feb 2003. ACKNOWLEDGEMENTS This investigation was supported by grant, BA-504 from the National Fund Scientific Investigation. REFERENCES AGERER R. 1984. Leptoglossum omnivorum sp. nov. from Antarctica. – Trans. Brit. Mycol. Soc., 82 (1): 184-186 BARRASA M., V. RICO. 2003. The non omphalinoid species of Arrhenia in the Iberian Peninsula. – Mycologia, 95 (4): 700-713. GYOSHEVA M., N. CHIPEV. 1999. Arrhenia salina (Høiland) Gulden – new macromycete species to the Livingston Island, South Shetlands, Maritime Antarctic. – Bulgarian Antarctic Research. Life sciences, 2: 25-31. HØILAND K. 1976. The genera Leptoglossum, Arrhenia, Phaeotellus and Cyphellostereum in Noray. and Svalbard. – Norw. J. Bot., 23(4): 201-212

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HORAK E. 1966. Sombre dos nuevos especies do hongos recolectadas en el Antartico.- Contr. Inst. Antart. Arg., 104: 1-13. HORAK E. 1982. Agaricales in Antarctica and Subantarctica : distribution, ecology and taxonomy. Arctic and alpine Mycology, edite by G. A. Laursen & J. E. Ammirati. Univ. Washington Press, Seattle, London: 82-122. KIRK P. M., P. F. CANNON, J. C. DAVID, J. A. STALPERS (Eds), 2001. Dictionary of the fungi. 9th edn. CAB International. Oxon. KÜHNER R., D. LAMOURE. 1972. Agaricales de la zone alpine. Pleurotacées. – Le Botaniste, 55: 7-37.

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©DPENSOFT Publishers Antarctic ISTRIBUTION OF FRESHWATER ALGAE ON LIVINGSTON ISLAND, SOUTH SBulgarian HETLANDS ISLANDSResearch ... 69 Sofia – Moscow Life Sciences, vol. 4: 69-82, 2004

Distribution of Freshwater Algae on Livingston Island, South Shetlands Islands, Antarctica .II. (Cyanoprokaryota) D. TEMNISKOVA-TOPALOVA1, R. ZIDAROVA1 Faculty of Biology, St. Kliment Ohridsky University of Sofia,

1

8 Dragan Tzankov St., 1164 Sofia, Bulgaria

ABSTRACT Preliminary data on blue-green algae, distributed in diverse aquatic and terrestrial biotops in the region of the Bulgarian Antarctic and Spanish Stations on Livingston Island are reported. Determined are 32 species and one form from the genera Anabaena Bory, Cyanothece Komárek, Cylindrospermum Kützing, Gloeocapsa Kützing, Lyngbya Agardh, Merismopedia Meyen, Mycrocystis Kützing, Nostoc Adanson, Nodularia Mertens, Oscillatoria Vaucher, Phormidium Kützing, Spirulina Turpin and Synechoccocus Nägeli. Each species is supplied with a concise description, dimensions and distribution. KEY WORDS Freshwater, blue-green algae, continental aquatic and terrestrial habitats, Hurd Peninsula, Livingston Island.

INTRODUCTION This publication, which offers preliminary data on blue-green algae, is a continuation of our investigations on freshwater eukaryotic (Chlorophyta, Chrysophyta, Botrydiophyta, Euglenophyta etc) and prokaryotic (Cyanophyta) algae on Livingston Island (Temniskova-Topalova & Kirjakov, 2002). So far 35 species and 1 form of green algae have been reported and their distribution in aquatic and terrestrial biotops situated in the region of the Bulgarian Antarctic station on Hurd Peninsula, Livingston Island, and some around the Spanish station is analyzed. It has been established that *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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snowy and glacier biotops have a specific flora; pond biotops have a higher diversity of representatives from the class Zygnematophyceae, while stream biotops have a higher diversity of Ulothrix spp., Zygnema spp. st., and Prasiola calophylla (Cormichel) Meneghini. On rocks, at places inhabited by penguins, Prasiola crispa (Lightfoot) Meneghini has a significant distribution. (Temniskova-Topalova & Kirjakov, 2002). STUDY AREA AND MATERIALS The region of the Bulgarian St. Kliment Ohridski Antarctic station was studied, as well as, partially, the region of the Spanish Station on Livingston Island, south Shetlands Islands. The Bulgarian Station is situated at South Bay, in the northwestern part of Hurd Peninsula. The physic-geographical characteristic of the island is given by Chipev & Veltchev (1996). In a short version, incl. the climate conditions, it is given also by Temniskova-Topalova & Chipev (2000). The samples were taken during the Bulgarian Antarctic Expeditions in the astral summers of the December 1994/95 – February 2003 period, by biologists Dr. N. Chipev, Dr. R. Mecheva, Dr. I. Pandurski and A. Kovachev). The algae were collected from various continental aquatic and terrestrial habitats. The aquatic habitats cover glacier ponds and streams, the Todorina Bouza lake, rockenclosed puddles of different size and duration, well-distanced from the sea coast in the littoral of South Bay, fed by thawing snow and glacier streams, snow and ice. The terrestrial habitats are rocks, sea coastal stripes of gravel, soil, lichens (Alectoria ochroleuca (Hoffm.) Mass and Cladonia sp.), moss, grass and moss-grass turfs (bryophytes Sanionia georgico-uncinata (Müll. Hal.) Ochyra et Hädenast*, Polytrichastrum alpinum (Hedw.) G.L. Sm.*, Polytrichum junipernum Hedw.*, Bryum urbanskyi Broth in Dryg.*, Bryum sp., Batrania patens Brid.*, Usnea sp. and the grasses Deschampsia antarctica Desv. and Colobanthus sp. Different ecological groups of algae have been covered: planktonic, benthic – epilithic, epipelic, epiphytic and aerophilic. The biotops studied are situated in different places of the peninsula. GPS coordinates have been applied, the determination of which started in 2001. – Playa Bulgara (Bulgarian beach): – PB-N – South Bay under the Bulgarian station, sea coast rock with bird nests and guano, northern exposure. GPS: 62058’07.3"S, 60021’21.8"W. Turfs from: Sanionia georgico-uncinata, Deschampsia antarctica + S. georgico-uncinata, Colobanthus sp., Bryum sp. + Colobanthus sp. – The end of the Bay – GPS: 62038’41.8"S 60022’23.7"W. * The species bryophytes are determened by Dr. A. Ganeva

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– The Big puddle, fed by thawing snow, situated above the sea level, without any direct contact to the sea. GPS: 62038’25.9"S, 60022’03.4"W. – Massive Punta Hesperides and its surroundings: – Smooth on the rock of Punta Hesperides, turned to Playa Bulgara, above the place of debarkation. GPS: 62038’36.0"S, 60022’14.2"W. – Skua’s nest above the Bulgarian station, northern exposure, 50 m a.s.l., GPS: 62038’36"C, 60022’14"W. Turfs from Deschampsia antarctica and from bryophytes Sanioinia georgico-uncinata + Polytrichastrum alpinum and from Cladonia sp.+Polytrichastrum alpinum + Sanionia georgico-uncinata. – Rocks on slopes of Punta Hesperides. GPS: 62038’22.9"S; 60022’15.1"W. – Hesperides Point, HPP-S1, GPS: 620 38’41.8'’S, 600 22' 23.7" W. Turfs from Sanionia georgico-uncinata + Bryum urbanskyi (rare). – HPP-S2, GPS: 62038’52'’S, 60022’17.9"W and 62038’52.6"S, 60022’24.2"W. Turfs from Sanionia georgico-uncinata + Deschampsia antarctica. – By Todorina Bouza Lake – 84 m a.s.l. Near by birds are nesting. GPS: 62038’49"S, 60022’17.3"W. – Todorina Bouza Lake in the hollow behind Punta Hesperides. – stream flowing out of Todorina Bouza Lake – below the lake at 40-50 m from flowing out (before going down to the level of the coast), GPS: 62038’47.7"S, 0022’20.2"W. Turfs from: Polytrichum junipernium + Deschampsia antarctica + Colobanthus sp.; Polytrichum juniperinum + Sanionia georgico-uncinata. – The Svetilishteto glacier stream, formed by thawing snow, under a scree. GPS: 62058’07.3"S, 60021’21.8"W. Turfs from Deschampsia antarctica+Colobanthus sp. – above the rock Green-Peace – group of three lakes, situated above one another -100 m a.s.l. – Caleta Argentina (Argentinean beach). A quiet and sheltered bay after the Spanish Station. There is a colony of penguins papua there. Turfs from Polytrichum alpinum and Bryum sp. RESULTS AND DISCUSSION 13 genera of blue-green algae have been determined, represented by 32 species and 1 form. The genus Oscillatoria has the highest species diversity – 9 species and 1 form (28.1%), the genus Lyngbya has 5 species (15,6%), followed by the genera Merismopedia (12.5%) and Phormidium (9.4%), with 4 and 3 species, respectively. The rest of the genera are represented by 1 or 2 species. In terms of the distribution of the blue-green algae in the region studied, on the data-base that we have, it was determined that the following species have the widest distribution: Lyngbya, (mainly L. aerugineo-coerula, etc), famous as a widely distributed

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species in Antarctica (Tell et al., 1995 etc), Oscillatoria (O. brevipes f. capitata, O. beggiatoiformis, O. mougeotia, O. pseudogeminata etc), Cyanothece aeruginosa, Gloeocapsa kütziagiana, Phormidium retzii, Merismopedia tennuissima. The species Oscillatoria, of a relatively large quantity, is developing and it is mainly in the second half of the summer. Developing comparatively abundantly are Lyngbya aerugineo-coerulea (Caleta Argentina, 2001), and Synechocystis salina in the coast puddle (in 2001) are in large numbers. Species composition of the algae Each species is given a concise description, including data on its size, comments on some characteristics, its distribution on Hurd Peninsula and in other Antarctic locations registered in the available literature. Anabaena laxa (Rabenh.) Ar. Braun Trichomes are straight, parallel, united in groups. Their width is 3.6-4.2-4.8 µm, with a scarcely visible mucilage sheath. The cells are butty or almost spherical. Heterocysts are ellipsoidal with a length of 5.2-7.4 µm, width 4-5.8 µm, or almost spherical, 5.6-8.2 µm in diameter. The spores are oval-cylindrical, 11.5-15.8×5.5-6.8 µm. Samples: three ponds amidst a rock massif (II-3/20.01.1996); a small lake above the Todorina Bouza Lake (11./21.01. 2001); Punta Hesperides near Todorina Bouza Lake (2"/02 2003); Svetilishteto (7/02.2003); the end of the Bay (3/02.2003 and 4"/02.2003). Cyanothece aeruginosa (Nägeli) Komárek Cells are solitary or in twos during division, rarely four together. Cells are ellipsoidal to cylindrical, width 6.2-10.5 µm, length twice more than the width. Samples: the coastal puddle (II-4/14/12/1994); a group of ponds (II-3/20.01.1996); a lake above the Bulgarian Station (152/26.12.1997); a puddle near the Birds’ Rock (170/29.12. 1997); lithotelm of the massive Punta Hesperides, northern exposure (18/ 12.12. 1998); the end of the Bay (3/02.2003, 4/02.2003 and 4"/02. 2003). Antarctica: Ongul Islands (Akiyama, 1967 – acc. to Pankow et al., 1987); Canada Glacier, Southern Victoria Land (Wharton et al., 1981); Dronning-Maud-Land (Pankow et al., 1987); Gondwana Lake, Northern Victoria Land (Fumanti et al., 1995). Cylindrospermum muscicola Kützing Trichomes are 3.2-4 µm wide. Cells are cylindrical or almost square, length 4.0-4.6 µm µm. Heterocysts are elongated, length 6.0-7.5 µm, width 3.8 µm. Spores are ellipsoidal, with a smooth episporium, size 14-15×7.8-8.8 µm. Samples: among mosses by three small nearby ponds (II-3A/20.01. 1996); a small puddle on the coast fed by water from a glacier stream (II-27/19.01.1996); among mosses at the outflow of the lake, on the rock southwards from Todorina Bouza Lake (129/21/12/1998).

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Gloeocapsa kützingiana Nägeli Colonies are almost spherical to ellipsoidal, with homogenous mucilage. Colonies are usually 2-4 celled, rarely more. Cells 3.7 µm in diameter. Samples: on the rock in the bottom of South Bay (II-5/21.01.1996); lithotelm on the rock southwards from Svetilishteto (77/18.12. 1998); Skua’s nest – wet soil with moss turfs: Polytrichastrum alpinum + Sanionia georgico-uncinata (6/29.01. 2001); Caleta Argentina – among wet moss – Polychastrum alpinum (8/02.02. 2001). Antarctica: Dronning-Maud-Land (Hirano, 1979 – acc. to Broady, 1982); Vestfold Hills, epilithes from Prinzess Elizabeth Land and Mac Robertson Land (Broady, 1981 a); Taylor Valley, Victoria Land (Broady, 1982); ponds and rock surfaces, Ross Island (Broady, 1989); cf. Gloeocapsa kützinigiana, Taylor Valley (McKnight et al., 1998); Gloeocapsa cf. kützinigiana, on wet soil, La Gorce Mountains (Broady & Weinstein, 1998). Gloeocapsa sp. Colonies are irregular, solitary. Cells are spherical with layered sheaths of mucilage. Sample: along a vertical rock (II-3/6.12.1994). Antarctica: unidentified species Gloeocapsa were reported for the Western part of East Antarctica (Bardin et al. 1969); Southern Victoria Land (Friedmann & Ocampo, 1976); salinå lakes (Wright & Burton, 1981); Southern Victoria Land (Howard-Williams & Vincent, 1989); continental Antarctica, in coastal zones on meltwater irrigate rockfaces; Taylor Valley (McKnight et al., 1998); King George Island (Unrein & Vinocur, 1999); Schirmacher Oasis (Pandey et al., 2000); periphyton in lake Boeckella, Hope Bay (Pizarro et al., 2002). Lyngbya aerugineo-coerulea (Kütz.) Gomont Filaments are solitary, 6.0-6.5 µm wide. The mucilage sheaths are homogenous, fine and colourless. The trichomes are unpressed, 5.5-6.0 µm wide. Cells length 2.5-5.0 µm. Samples: Hesperides Point HPP: Soil sample on Sea gravel (S1/27.01.2001); often on wet soil with moss turfs Polytrichastrum alpinum + Sanionia georgico-uncinata, Skua’s nest,(29.01.2001); Caleta Argentina (8/02.02.2001); Punta Hesperides near the Todorina Bouza Lake (2'/02.2003). Antarctica: lakes and ponds Boeckella, Esperanza, Flora, Encantado, Escondido, Hope Bay (Tell et al., 1995); Gondvana Lake, Northern Victoria Land (Fumanti et al., 1995); – periphyton in lake Esperanza, Hope Bay (Pizarro et al., 2002). Lyngbya cryptovaginata Nägeli Filaments are straight, solitary, 5.2 µm wide. Cells are almost square, apical cell rounded.

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Sample: wet soil with moss turfs, Skua’s nest (6/29.01.2001). Lyngbya kützingii (Kütz.) Schmidle Filaments are curved, with fine colourless mucilage sheaths. Trichomes width 3.2 µm, slightly pressed at transverse walls near the ends. Cells are square with granules along the transverse walls. Samples: glacial lake on the rocks above the Bulgarian Station (152/26.12. 1997); small kale above Todorina Bouza Lake (3A/21.01.2001). Lyngbya nigra Agardh Filaments are straight, with fine homogenous mucilage sheaths. Trichomes width 7.6 µm. Cells length 2.8 µm, width 1.2 µm. Apical cells with round-conical calyptra. Sample: Caleta Argentina (8/02.02.2001). Lyngbya scotti F. E. Fritsch Filaments are curved, width 3.8-4.0 µm. Mucilage sheaths are colourless. Trichomes width 2.4-2.6 µm. Cells are square to slightly elongated. Sometimes the transverse walls have granules. Apical cells are conical, without calyptra. Samples: among Oscillatoria spp., Birds Market (18/6.12. 1994 and 19/6.12. 1994); upon turf of Bryum sp., Playa Bulgara PB-N (3/21.01.2001). Antarctica: on the surface or in the mucilage of other blue-green algae, Antarctica (Gollerbach et al. 1953). Reported for Antarctica are also many unidentified species Lyngbya: Lyngbya sp., sublithic species, found under quartzstones at Vestfold Hills (Broady, 1981b) and Lyngbya sp. in ponds and on rock surfaces, Ross Island (Broady, 1989) etc. Merismopedia arctica (Kosinskaja) Komárek et Anagnostidis Colonies are large, with densely arranged cells. Cells have colourless thick mucilage envelopes. Cells are more or less spherical, about 2 µm in diameter. Samples: amoung moss-grass turfs, Birds Market (18/6.12.1994); of the group of small ponds (II-41a/20.01. 1996). Merismopedia minima G. Beck Colonies are small, with 4 to 24-30 cells. Cells are spherical or slightly ellipsoidal, about 0.5 µm in diameter. Samples: strongly wet moss-grass turf, Birds Market (II-37/18.12.1994); – on the rock, along which water flows from the thawing snow, to the from Green-Peace rock (63/17.12. 1997). Merismopedia punctata Meyen Coloinies consist of loosely arranged cells. Cells are spherical, some of them ellipsoidal, 2.4- 3.0 µm in diameter.

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Samples: amoung moss-grass turfs, Birds Market (18/6.12.1994); group small ponds (II-41a/20.01. 1996). Merismopedia tennuissima Lemmermann Coloinies consist of numerous cells (50-60), densely arranged. Cells are spherical to slightly ellipsoidal, 1.5-2.0 µm in diameter. Samples: Birds Market (II – 18/6.12. 1994 and II – 19/6.12.1994); a pond on the hill to the south from Todorina Bouza Lake (125/21.12.1998); a puddle beside Todorina Bouza Lake (22'/01. 2001). Antarctica: lake Boeckella, Hope Bay (Tell et al., 1995). According to Tell et al. (1995) the species is found at cape Adare, McMurdoOrkneys, Victoria Land. Microcystis sp. Colonies formless. Cells spherical, without shaping mucilage sheath, all being embedded in a single homogenous mass of mucilage. Sample: a stream flowing out out of a glacial lake (II-7/18.12. 1994). Antarctica: unidentified species Mycrocystis in salt lakes (Wright & Burton, 1981); Microcystis sp., Rothera Point, Adelaide Island (Priddle & Belcher, 1981). Nostoc punctiforme (Kütz.) Hariot Colonies are elongated. Trichomes are numerous, densely interlaced, width 3.24.5-4.8 µm µm. Heterocysts re round, almost spherical, 5.0-5.8 µm in diameter. Spores have a smooth colourless episporium, length 7.0 µm, width 4.8 µm. Samples: from a group of ponds above Green-Peace rock (II-3/20.01.1996); the end of the Bay (4/02.2003 and 4'/02. 2003); the Svetilishteto glacier stream (7/ 02.2003). Antarctica: Ross Island (Fritsh, 1912 – according to Wharton et al.,1981); Nostoc punctiforme (?), Novolazerevskaya Station (Komarek & Ruzicka, 1996); Lake Bonney (Seaburg et al.,1979 – according to Wharton et al., 1981); Southern Victoria Land (Wharton et al.,1981). Nostoc sp. juv. Thalli are microscopic, dense, flat, irregular. Samples: on moss from Monte Charua (II-11/26.01. 1996); among wet mosses near the runoff of the small pond, northwards from the Bulgarian Station (86/ 20.12.1998). Antarctica: Unidentified species Nostoc were pointed at: Wright Dry Valley (Holm – Hansen, 1964); under the ice cover of Lake Verkhneye, Schrimacher Oasis (Saag, 1979 – acc. to Kaup, 1994); salt lakes (Wright & Burton, 1981); Southern Victoria Land (Seaburg et al., 1981); under quartz stones at Vestfold Hills (Broady, 1981 â); Taylor Valley (Broady, 1982); ponds and rock surfaces, Ross Island (Broady, 1989);

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Southern Victoria Land (Howard-Williams & Vincent, 1989); Taylor Valley (Mc Knight et al., 1998); in meltwater ponds on Mc Murdo Ice Shelf (Hitzfeld et al. 2000). Nodularia sp. Thalli are formless, muscilaginous. Filaments dark and muscilaginous. Samples: in ponds formed by thawing snow and ice (II-42/22.12.1994); a small lake above Todorina Bouza Lake (11/21.01. 2001). Antarctica: Unidentified species Nodularia were established under quartz stones at Vestfold Hills (Broady, 1981 â); salt lakes (Wright & Burton, 1981); in meltwaters ponds on Mc Murdo Ice Shelf (Hitzfeld et al., 2000). The species Nodularia harveyana Thur. was indicated for Canada Glacier, Southern Victoria Land (Wharton et al., 1981); Taylor Valley (Broady, 1982); Vestfold Hills (Broady, 1986-acc. to Broady, 1996); Gondwana Lake, Northern Victoria Land (Fumanti et al., 1995); Nodularia cf. harveyana, Taylor Valley (Mc Knight et al., 1998). Oscillatoria beggiatoiformis Gomont Trichomes are slightly spiral curved, width 4.3-4.8 µm, narrowed at the end. Cells almost square, with granules at transverse walls. Terminal cells capitated with calyptra. Samples: group ponds (II-3/20.01. 1996); on rock in the bottom of South Bay (II-5/21.01.1996); on stones in a stream (II-10/26.01.1996); on the bottom of a pond (II-36 A/26.01.1996 and II-36 A/26.01.1996); Punta Hesperides by the Todorina Bouza Lake (2'/02. 2003); the end of the Bay (3'/02. 2003, 4/ 02.2003 and 4'/02.2003). Oscillatoria brevipes (Kütz.) Gomont Table I, fig. 7 Trichomes are straight, width 4.0-4.6 µm, narrowed at the end. Cells length 1.53.0 µm µm, 2-3 times shorter than wide, with granules at transverse walls. Calyptra broadly conical. Samples: on rocks in the bottom of South Bay (58/16.12. 1998); on rock, to the south from Svetilishteto (76/18.12.1998); Hesperides Point HPP, soil sample on sea gravel, (S1/27.01.2001); by Todorina Bouza Lake (2'/02.2003). Oscillatoria brevipes (Kütz.) Gomont f. capitata Claus Table I, fig. 6 Trichomes’ width 5.4-6.0 µm. Cells length 1.7-2.0 µm, with granules at transverse walls. Calyptra capitated. Samples: soil sample on sea gravel, HPP-S1 (27.01.2001); Playa Bulgara, PB-N3/ 21.01.2001); water flowing out Todorina Bouza Lake (99/02.2001 and 100/02.2001); among wet mosses near Todorina Bouza Lake (33 and 60/02.2001); Kaleta Argentina (8/02.02.2001).

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Oscillatoria gracilis Böcher Trichomes are thin, fine, width 1.8 µm. Cells’ length 3.0-3.7 µm. Apical cells rounded. Samples: puddle at the Todorina Bouza Lake (02.2001); Hesperides Point – HPP, Snails in the bag (S2 /27.01.2001). Oscillatoria mougeotii (Kütz.) Forti Trichomes are straight or slightly curved, unpessed, width 6.0-7.5 µm. Cells length 2.0-2.5 µm with visible gas vacuoles. Apical cells rounded. Samples: straining mosses from run-off of the lake, southwards from the Todorina Bouza Lake (129/21.12.1998); puddle at Todorina Bouza Lake (22/02.2001); among wet mosses near Todorina Bouza Lake (33 and 60/02.2001). Oscillatoria nigra Vaucher Table I, fig. 1-5 Trichomes are blue-violet, straight or curved in various degrees, width 9.0-10.0 µm. Cells length 2.5-3.0 µm. Sample: Hesperides Point – HPP, soil sample on sea gravel (S1/27.01. 2001). Oscillatoria pseudogeminata G. Schmid Trichomes are strongly curved, width 18 µm. Cells are usually square, or length 11.5 times larger that width, length 3.0-3.7 µm. Transverse walls rather thick. Apical cells rounded. Table I

1

2

3

6

7

5 4

(scale bars=10 µm.)

Fig. 1-5. Oscillatoria nigra Vaucher Fig. 6. Oscillatoria brevis (Kütz.) Gomont f. capitata Claus Fig. 7. Oscillatoria brevis (Kütz.) Gomont

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Samples: Hesperides Point – HPP: soil sample on sea gravel (S1/27.01.2001) and S2/27.01. 2001; puddle at Todorina Bouza Lake (22/02.2002). Oscillatoria rupicola Hansgirg Trichomes are straight or slightly curved, width 4.5-5.0 µm. Trichomes solitary or seldom united in small sheafs. Cells are usually square, or length 1-1.5 times smaller that the width, length 2.5-3.0 µm. Apical cells rounded. Sample: Skua’s nest (6/29.01.2001). Oscillatoria tenuis Agardh Trichomes are slightly pressed, straight, sometimes curved at the ends, width 5.57.8 µm. Terminal cells hemispherical. Samples: puddles between rocks in the bottom of South Bay (II-4/21.01.1996); puddle at the colony of penguins, Caleta Argentina (157/24.12.1998); the end of the bay (3/02.2003 and 4'/02.2003). Antarctica: Dronning-Maud-Land (Pankow et al., 1987); King George Island (Unrein & Vinocur, 1999). Oscillatoria spp. Samples: II-3/6.12.1994; II-7/18.12.1994; II-9/29.12.1994; II-13/5.12.1994; II14/5.12. 1994; II-18/6.12.1994; II-19/6.12.1994; II-26/8.12.1994; II-27A/9.12.1994; II-27B/9.12.1994; II-27C/9.12.1994; II-29/10.12.1994; II-33/14.12.1994; II-37/ 18.12.1994; II-38/18.12.1994; II-42/22.12.1994; II-44C/29.12. 1994; II-2/19.01. 1996; II-4/21.01.1996;II-11/26.01.1996;II-33/21.01.1996;II-36a/26.01.1996; II-4/ 24.01.1997;18/8.12.1997;117/21.12.1997;152/26.12.1997;170/29.12.1997;123/ 21.12.1998;58/16.12.1998;100/20.12.1998; 2/02.2003; 2"/02.2003; 4/02.2003; 4"/ 02.2003; 5/02.2003; 7/02.2003; 8/02.2003; 9/02.2003. Antarcica: Unidentified species Oscillatoria were pointed at Wright Dry Walley (HolmHansen,1964); Lake Verkhneye, Schirmacher Oasis (Saag, 1979 – acc. to Kaup, 1994); salt lakes (Wright & Butron, 1981); meltstream from Don Juan pond (Goldmann et al., 1972 – cit. after Wright & Burton, 1981); Victoria Land (Howard-Williams & Vincent, 1989); in ponds and on rock surfaces Ross Island (Broady, 1989); King George Island (Unrein & Vinocur, 1999); Lake Fryxell, Taylor Valley, Southern Victoria Land (McKnight et al., 2000). Phormidium foveolarum (Montagne) Gomont Trichomes are upright, pressed, width 1.8-2.0 µm. Cells length 1.5-1.8 µm, almost equal to the width. Calyptra absent. Terminal cells rounded. Sample: on soil beside a spring, at the northern slope of the Third Peak, northwards from the Bulgarian Station (103/20.12.1998); on wet soil, Punta Hesperides (10/8.02.2001); at Todorina Bouza Lake (2'/02.2003); the end of

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the Bay (3/02.2003; 3/02.2003; 4/02.2003;4'/02.2003; 4"/02.2003; 5/02.2003; 7/02.2003). Phormidium retzii (Ag.) Gomont Trichomesare straight, sometimes slightly pressed, width 5.8-7.0 µm. Transverse walls of the cells without granules. Apical cells in the upper part slightly narrowed, obtuse, with thick external covering. Samples: the group of the three ponds among a rock massif (II-3/20.01.1996); stream (II-10/26.01.1996); a pond on the hill northwards from the Bulgarian Station (87/20.12.1998); a stream flowing out of Todorina Bouza Lake (130/21.12.1998). Antarctica: at Mirnoi Station (Vialow & Sdobnikova, 1961); Dronning-MaudLand (Pankow et al., 1987); McMurdo Ice Shelf (James et al., 1995). Phormidium spp. Samples: II-8/29.12.1994; II-36/18.12.1994; II-3/20.01.1996; II-11/26.01.1996; II-26/23.01.1996;II-33/21.01.1996;II-46/29.01.1996;152/26.12.1997;170/ 29.12.1997; 3/20.12.1998; 123/21.12.1998. Antarctica: Wright Dry Valley (Holm-Hansen,1964); soils from the west part of East Antarctica (Bardin et al., 1969) – living stromatolites in Lake Hoare, Southern Victoria Land (Seaburg et al., 1980); Rothera Point, Adelaide Island (Priddle & Belcher, 1981); salt lakes (Wright & Burton, 1981); Mawson Rock, MacRobertson Land and Pr. Elizabeth Land (Broady 1981a). Signy Island (Priddle & Belcher, 1982); ponds and rock surfaces, Ross Island (Broady,1989); Southern Victoria Land (HowardWilliams & Vincent, 1989); Southern Victoria Land (Vincent & Howard-Williams, 1989); Lake Fryxell, Taylor Valley, Southern Victoria Land (McKnight et al., 2000); periphyton in lakes Esperanza, Boeckella and Pingui, Hope Bay (Pizarro et al., 2002). Spirulina laxa Smith Trichomes are regular spiral curved, width 3.5-4.5 µm. Samples: On snow near glacier streams at the Bulgarian Station (II-1/19.01.1996); puddle above Morski Luv Lake (II-4/24.01.1997). Synechocystis salina Wislouch Cells solitary or more in number, spherical 2.5-3.0 µm in diameter. Sample: coastal puddle (02.2001). The blue-green algae reported here are cosmopolits. In terms of their destribution at habitats, it turned out that there is no clear difference between the species inhabiting aquatic and terrestrial habitats. Some of the species occur in aquatic as well as in terristrial habitats. That fact about blue-green algae confirms the oppinion of Vicents James (1996 – acc. to Broady, 1996) that the difference between aquatic and

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terristrial habitats is not absolutely clear and, undoubtedly, some of the representatives inhabiting them coincide. ACKNOWLEDGEMENTS The autors are grateful to their colleagues Dr. N. Chipev, Dr. R. Mecheva, Dr. I. Pandurski and A. Kovachev for the material placed at our disposal. This study was funded by the Ministry of Education, Project BA/801. REFERENCES BARDINV., L. GERASIMENKO, M. POUSHEVA. 1969. Nekotorie dannie î rasprostranenii vodoroslei v zapadnoi chasti Vostochnoi Antarctidi. Bull. Sov. antarct. Exp., 54: 41-49 (in Russian). BROADY P. 1981a. Ecological and taxonomic observations on subaerial epilithic algae from Princess Elizabeth Land and MacRobertson Land, Antarctica. Br. Phycol. J., 16: 267-266. BROADY P. 1981b. The ecology of sublithic terrestrial algae at the Vestfold Hills, Antarctica. Br. Phycol. J., 16: 231-240. BROADY P. 1982. Taxonomy and ecology of algae in a freshwater stream in Taylor Valley, Victoria Land, Antarctica. Arch. Hydrobiol. Suppl. 63,3, 331-349. BROADY P. 1989. Broadscale patterns in the distribution of aquatic and terrestrial vegetation at three ice-free regions on Ross Island, Antarctica. Hydrobiologia, 172: 77-95. BROADY P. 1996. Diversity, distribution and dispersal of Antarctic terrestrial algae. Biodiversity and Conservation 5: 1307-1335. BROADY P., R. WEINSTEIN. 1998. Algae, lichens and fungi in La Gorce Mountains, Antarctica. Antarctic Science, 10(4): 376-385. CHIPEV N., K. VELTCHEV. 1996. Livingston Island: an Environment for Antarctic Life. Bulg. Antarctic Res., Life Sc.,1: 1-6. FRIEDMANN E., R. OCAMPO. 1976. Endolithic Blue-Green Algae in the Dry Valleys: Primary Producers in the Antarctic Desert Ecosystem. Science, Vol. 193, 1247-1249. FUMANTI B., S. ALFINITO, P. CAVACINI. 1995. Floristic studies on freshwater algae of Lake Gondwana, Northern Victoria land (Antarctica). Hydrobiologia, 316: 81-90. GOLLERBACH M., E. KOSSINSKAJA, V. POLJANSKII. 1953. Blue-green algae. In: Guide to the freshwater algae of USSR. 2. Moscow, Sovetskaja nauka, 652p. (in Russian). HITZFELD B., C. LAMPERT, N. SPAETH, D. MOUNTFORT, H. KASPAR, D. DIETRICH. 2000. Toxin production in cyanobacterial mats from ponds on the McMurdo Ice Shelf, Antarctica. Toxicon, 38(12): 1731-1748. HOLM-HANSEN O. 1964. Isolation and Culture of Terrestrial and Freshwater Algae of Antarctica. Phycologia, 4(1): 43-51.

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DISTRIBUTION OF FRESHWATER ALGAE ON LIVINGSTON ISLAND, SOUTH SHETLANDS ISLANDS ... 81 HOWARD-WILLIAMS C., V. VINCENT. 1989. Microbial communities in southern Victoria Land streams (Antarctica). I. Photosynthesis. Hydrobiologia, 172: 27-38. JAMES M., R. PRIDMORE, V. CUMMINGS. 1995. Planktonic communities of melt ponds on the McMurdo Ice Shelf, Antarctica. Polar Biol., 15: 557-567. KAUP E. 1994. Annual primary production of phytoplankton in Lake Verkhneye, Schirmacher Oasis, Antarctica. Polar Biol., 14: 433-439. KOMAREK J., J. RUZICKA. 1966. Freshwater alge in a Lake in Proximity of Novolazerevskaya Station, Antarctica. Preslia (Praha), 38: 237-244. KONDRATEVA N., O. KOVALENKO, L. PRIHODKOVA. 1984. Cyanophyta. In: Viznachnik prisnovodnih vodorostei Ukrainskoi RSR. I. Kiev, Naukova dumka, 388p.( in Ucrainian). MCKNIGHT D., A. ALGER, C. TATE, G. SHUPE, S. SPAULDING. 1998. Longitudinal patterns in algae abundance and species distribution on meltwater streams in Taylor Valley, Southern Victoria Land, Antarctica.-Ecosysytem dynamics in a polar desert: The McMurdo Dry Valleys, Antarctica, 109-127. MCKNIGHT D., B. HOWES, C. TAYLOR, D. GOEHRINGER. 2000. Phytoplankton dynamics in a stably stratified Antarctic lake during winter darkness. J. Phycol., 36: 852-861. PANDEY K., A. KASHYAP, R. GUPTA. 2000. Nitrogen-fixation by non-heterocystous cyanobacteria in an antarctic ecosystem. Israel Journal of Plant Sciences, 48(4): 267-270. PANKOW H., P. HAENDEL, W. RICHTER, U. WAND. 1987. Algologische Beibachtungen in der Schirmacher- und Unterseeoase (Dronning-Maud-Land, Ostantarktika).Arch. Protistenkd., 134: 59-82. PIZARRO H., A. VINOCUR, G. TELL. 2002. Peryphyton on artificial substrata from three lakes of different trophic status at Hope Bay (Antarctica). Polar Biol., 25: 169-179. PRIDDLE J., J. BELCHER. 1981. Freshwater Biology at Rothera Point, Adelaide Island: II. Algae. Br. Antarct. Surv. Bull., 53, 1-9. PRIDDLE J., J. BELCHER. 1982. An annotated list of benthic algae (excluding diatoms) from freshwater lakes on Signy Island. Br. Antarct. Surv. Bull., 57, 41-53. SEABURG K., B. PARKER, R. JR. WHARTON, W. VINYARD, G. SIMMONS, G. LOVE. 1980.Distribution and species composition of attached algal mats (living stromatolites) in Lake Hoare, Antarctica. In: Intern. Phycological Society Meetings 1980, p. 113. SEABURG K., B. PARKER, R. JR. WHARTON, G. JR. SIMMONS. 1981. Temperaturegrowth responses of algal isolates from Antarctic oases. J. Phycol., 17: 353-360. STARMACH K. 1966. Cyanophyta – Sinice. Glaucophyta – Glaukofity. In: K. Starmach (ed.), Flora Sladkowodna Polski, Warszawa, PWN, 807 p. TELL G., A. VINOCUR, I. IZAGUIRRE. 1995. Cyanophyta of lakes and ponds of Hope Bay, Antarctic Peninsula. Polar Biol., 15: 503-509. TEMNISKOVA-TOPALOVA D., N. CHIPEV. 2001. Diatoms from Livingston Island, the South-Shetland Islands, Antarctica. Proc.16th Intern. Diatom Symposium (A. EconomouAmilli Ed.), Univ. of Athens, Greece: 291-314.

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TEMNISKOVA-TOPALOVA D., I. KIRJAKOV. 2002. Distribution of Freshwater Algae on Livingston Island, South Shetland Islands. Bulg. Antarctic Res., Life Sc.,3: 53-67. UNREIN F., A. VINOCUR. 1999. Phytoplankton structure and dynamics in a turbid Antarctic Lake (Potter Peninsula, King George Island).Polar Biol., 22: 93-101. VIALOW, O. & SDOBNIKOVA, N. 1961. Sweet-water algae of Antarctica. Acta Societatis Botanicorum Polonie, Vol. XXX (3-4), 765-773. VINCENT W., C. HOWARD-WILLIAMS. 1989. Microbial communities in southern Victoria Land sterams (Antarctica). II. The effects of low temperature. Hydrobiologia, 172: 39-49. WHARTON R., W. VINYARD, B. PARKER, G. JR. SIMMONS, K. SEABURG. 1981. Algae in cryoconite holes on Canada Glacier in Southern VictoriaLand, Antarctica. Phycologia, 20(2): 208-211. WHRIGHT S., H. BURTON. 1981. The biology of antarctic saline lakes. Hydrobiologia, 82: 319-338.

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© PENSOFT Publishers DATA AND SUMMARIZED CHECK-LIST ON THE RHIZOPODS Bulgarian Antarctic Research ... ADDITIONAL 83 Sofia – Moscow Life Sciences, vol. 4: 83-93, 2004

Additional Data and Summarized Check-list on the Rhizopods (Rhizopoda: Amoebida &Testacea) from Livingston Island, South Shetlands, the Antarctic V. GOLEMANSKY, M. TODOROV Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel Blvd., 1000 Sofia, Bulgaria

ABSTRACT A total of 48 taxa of Protozoa (3 naked rhizopods and 45 testate rhizopods) were found in 82 moss, soil and aquatic samples collected from Livingston Island, South Shetlands, during the period 1994-2002. The testacean fauna found in the various habitats of Livingston Island is mainly composed of and dominated by cosmopolitan ubiquitous species, e. g. Trinema lineare, Centropyxis aerophila, Euglypha rotunda, E. laevis, Corythion dubium, Assulina muscorum, Difflugia lucida, Microchlamys patella etc. The highest species diversity was established in the moss-communities of Polytrichum sp. and Drepanocladus sp. (38 species, 17 genera) whereas the testacean fauna in the aquatic and soil habitats is twice as poorer (22 species, 10 genera and 21 species, 12 genera, respectively). It is noteworthy that in about 50% of the investigated aquatic and soil samples no testate amoebae have been established. Trinema lineare was the most frequent species and played a dominant role in all investigated habitats. Its frequency of occurrence (pF) ranged between 27.5% in aquatic habitats and 77.0% in moss habitats. Euglypha rotunda, Centropyxis aerophila, Assulina muscorum and Corythion dubium were the other comparatively widespread and dominant testate amoebae in moss and soil habitats. Comparison of our data with those from other sub-Antarctic and maritime Antarctic locations shows that the number of taxa found in Livingston Island is lower than those of some well-studied sub-Antarctic islands and is higher than those of the locations with the same or higher latitude. These results confirm the hypothesis that there is a clear trend of decreasing species-richness in Antarctic testate rhizopod communities with increasing latitude. *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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KEY WORDS Rhizopods, Testate amoebae, Biotopic distribution, Livingston Island, Antarctic.

INTRODUCTION The rhizopods of the southern temperate, Antarctic and Arctic zones have focused the attention of scientists increasingly and have been of great interest during the last 2-3 decades. A lot of new data about the biodiversity, the biogeography and the ecology of the protozoan fauna of these areas were published. The ecological conditions of the Antarctic are very extreme and only a few animal groups can endure them and survive. Rhizopods are one of those groups, but their species diversity is very limited in the Antarctic and Arctic zones (Beyens et al., 1986a, 1986b; Smith, 1982a, 1984, 1992, 1996; Smith and Wilkinson, 1987; Trappeniers et al., 1999, 2002; Van Kerckvoorde et al., 2000; Wilkinson, 1994, etc.). In two previous publications we presented our preliminary results of investigations of the species composition and biotopic distribution of the freshwater, mossand soil-inhabiting testate amoebae from the Livingston Island, carried out during the period from 1994 to 1997 (Todorov and Golemansky, 1996, 1999). We have received some new living material (freshwater, moss and soil samples), collected during the Bulgarian Antarctic expedition from 1998 to 2003. That has permitted us to complete our knowledge about Rhizopods from the Antarctic region. The aim of the present study is to report new data about naked and testate amoebae from Livingston Island and to give a summarized list of all Rhizopods found to date in the region of the Bulgarian Antarctic station. MATERIAL AND METHODS The new materials for the present study were collected by Dr. I. Pandursky and Dr. R. Mecheva during the Bulgarian Antarctic campains carried out in the 1998 – 2003 period. Sampling sites were located in the Punta Hesperides area and Punta Polaka area of Hurd Peninsula of Livingston Island (lat 62º35'-62º45' S, long. 60º32'60º43' W). For the proposed generalizing publication a total of 82 moss, soil and freshwater samples were studied for the whole period of our investigation – from 1994 to 2003. Samples were collected from the following 3 habitats: (1) Moss communites dominated by Polytrichum sp. and Drepanocladus sp. :30 samples; (2) Shallow soils under tufts of the grass Deschampsia antarctica Desv. : 23 samples; (3) Benthos from some small glacial lakes: 29 samples.

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A biocenological analysis was fulfilled by using the indices of occurrence frequency (pF) and dominance frequency (DF) (De Vries, 1937). The frequency of occurrence (pF) of the particular species was calculated by the formula: pF = (m/n).100 , where m is the number of the samples in which the species was found and n is the total number of the samples. Depending on their pF index, the species were classified in 3 categories, as follows: (1) constant, recorded in more than 50% of samples; (2) incidental, recorded in 25-50% of samples; (3) accidental, found in less than 25% of the samples. The dominance frequency (DF) of the particular species was calculated by the formula: DF = (d/n).100 , where d is the number of the samples in which the species dominates, and n is the total number of the samples. RESULTS A total of 48 taxa, belonging to 3 genera of naked amoebae and 17 genera of testate amoebae, were established in the habitats studied. Ten of them (3 naked amoebae and 7 testate amoebae) have not been established in our previous investigations (Todorov and Golemansky, 1996, 1999). The list of the taxa, their frequency of occurrence (pF) and dominance frequency (DF) in the terrestrial and freshwater habitats studied are presented in Table 1. Species are listed in taxonomic sequence of orders and in alphabetical sequence of generic names within orders. The species diversity of the protozoan fauna in moss communities of Polytrichum sp. and Drepanocladus sp. is twice as big as that in the soil and aquatic habitats (41, 21 and 22 taxa, respectively). A total of 41 taxa (3 naked amoebae and 38 testate amoebae) were recorded in 30 investigated samples of this terrestrial habitat. The genera Euglypha (11 taxa) and Centropyxis (6) were represented by most of the species, and 14 of the remaining 18 genera were represented by one species only (Fig. 1). Trinema lineare, Euglypha rotunda and Centropyxis aerophila were the constant species in moss habitats and had the highest frequency of occurrence (pF): 77,0%, 60,0% and 57,0%, respectively. These species also had the highest dominance frequency (DF), e.g. T. lineare (20,0%), E. rotunda (13,3%) and C. aerophila (13,3%). Six of the other species in this habitat had a frequency of occurrence (pF) between 25% and 50% and were classified as incidental. These are: E. laevis (47,0%), A. muscorum (37,0%), T. pulchellum (33,3%), E. compressa f. glabra (30,0%), M. patella (30,0%), and D. lucida (30,0%). The

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Table 1. Summarized check-list of the rhizopods and their frequency of occurrence (pF) and dominance frequency (DF) in the continental habitats on the Livingston Island, South Shetlands, Antarctic. ¹ Biotopes Moss Soil Aquatic (30 samples) (23 samples) (19 samples)

Taxa 1 Order Euamoebida * Hartmanella vermiformes Page * Thecamoeba striata (Penard) * Vannella simplex (Wohlfarth-Bottermann) Order Arcellinida Arcella arenaria Greeff * A. arenaria var. sphagnicola Deflandre Centropyxis aculeata (Ehrenberg) Stein C. aerophila Deflandre C. aerophila var. sphagnicola Deflandre C. cassis Deflandre C. constricta (Ehrenberg) Deflandre C. elongata (Penard) Thomas * C. minuta Deflandre C. sylvatica (Deflandre) Bonnet & Thomas * Cyclopyxis eurystoma Deflandre Difflugia ampullula Playfair D. lacustris (Penard) Ogden D. lucida Penard D. penardi Hopkinson D. pristis Penard Heleopera sylvatica Penard Microchlamys patella (Clap.& Lachm.) Cockerel Microcorycia flava Greeff Nebela lageniformis Penard * Plagiopyxis callida var. grandis Thomas * P. declivis Thomas Order Reticulolobosa Cryptodifflugia compressa Penard Difflugiella oviformis (Penard) Bonnet & Thomas Phryganella acropodia (Hertw.& Less.)Hopkinson Order Gromiida Assulina muscorum Greeff Corythion aerophila Decloitre C. dubium Taranek

2

3

4

3.3/0.0 6.6/3.3 10.0/0.0

-

-

13.3/0.0 3.3/0.0 57.0/13.3 6.6/0.0 3.3/0.0 3.3/0.0 3.3/0.0 10.0/0.0 10.0/0.0 3.3/0.0 30.0/3.3 3.3/0.0 6.6/0.0 30.0/6.6 6.6/0.0 6.6/0.0 3.3/0.0 -

56.6/8.6 8.6/0.0 4.3/0.0 4.3/0.0 8.6/0.0 4.3/0.0 4.3/0.0

6.9/0.0 6.9/0.0 3.4/0.0 6.9/0.0 3.4/0.0 3.4/0.0 3.4/0.0 3.4/0.0 17.2/3.4 3.4/0.0 3.4/0.0 3.4/0.0 3.4/0.0 -

10.0/0.0 13.3/3.3 10.0/3.3

4.3/0.0 21.6/0.0

3.4/0.0 -

37.0/3.3 20.0/3.3 23.3/6.6

21.6/0.0 8.6/0.0 20.0/3.3

6.9/0.0 3.4/0.0

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Table 1. Continued. 1 Order Gromiida Euglypha bryophila Brown E. ciliata (Ehrenberg) Leidy E. ciliata f. glabra Wailes E. compressa Carter E. compressa f. glabra Wailes E. cristata Leidy E. denticulata Brown E. laevis Perty E. rotunda Wailes & Penard E. strigosa (Ehrenberg) Leidy * E. strigosa f. glabra Wailes E. tuberculata Dujardin Trachelocorythion pulchellum (Penard) Bonnet * Trinema complanatum Penard T. enchelys (Ehrenberg) Leidy T. lineare Penard T. lineare var. truncatum Chardez T o t a l t a x a : 48

2

3

4

3.3/0.0 10.0/0.0 6.6/0.0 6.6/0.0 30.0/0.0 6.6/0.0 13.3/0.0 47.0/6.6 60.0/13.3 13.3/0.0 3.3/0.0 33.3/3.3 3.3/0.0 13.3/0.0 77.0/20.0 41

8.6/0.0 8.6/0.0 13.0/0.0 4.3/0.0 13.0/0.0 16.6/0.0 4.3/0.0 60.6/17.3 10.0/0.0 21

3.4/0.0 6.9/0.0 6.9/0.0 3.4/0.0 3.4/0.0 27.5/6.9 22

¹ Numenator - frequency of occurrence (pF); denominator - dominance frequency (DF). * New rhizopods to the protozoan fauna of Livingston Island.

other thirty-two testaceans (about ¾ of all species recorded in moss habitats) are rare, with a frequency of occurrence below 25%, and appear accidental. A total of 21 species, belonging to 12 genera were recorded in soil samples. Two genera Euglypha and Centropyxis were represented by the greatest number of species (6 and 3, respectively). Both Corythion and Trinema were represented by two species each, and the remaining 8 genera were represented by 1 species only. Among all species found in this biotope, only two were frequently occurring and played a dominant role. These are T. lineare (pF=60.0%, DF=17.3%) and C. aerophila (pF=56.6%, DF=8.6%). All the remaining species recorded in soil samples were accidental, with a frequency of occurrence less than 25% and low dominance frequency (DF from 0% to 3.3%). A total of 22 species belonging to 10 genera were recorded in aquatic habitats. About ¾ of the species recorded here were the representatives of three genera: Centropyxis (6 species), Difflugia (5) and Euglypha (4). Each of the remaining 7 genera was represented by one species only. There were no constant species with a pronounced dominant role in the aquatic habitats. Only T. lineare and D. lucida were relatively frequently found (pF= 27.5% and 17.2%, respectively) and their population

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Number of taxa

10

8 Moss Soil Aquatic

6

4

2

A rce A lla ssu Ce lin ntr a op C yx i Cr oryt s yp hio tod n iff Cy lugia clo py D xis iffl D ugia iffl ug i Eu ella g l H yp ar ha tm an H ella e l M eop icr er och a M lam icr ys oco ryc i N a e Ph bel ryg a a Pl nell ag a iop T y Tr heca xis ach m elo oeb cor a yth i Tr on ine V ma an ne lla

0

Genera

Fig. 1. A comparison of the species richness of the rhizopod genera established in moss, soil and aquatic habitats from Livingston Island.

densities were relatively highest (DF = 8,3% and 5.3%, respectively). All the remaining 20 species were accidental, found only in one or two of the 29 aquatic samples investigated. DISCUSSION It may be concluded that the testacean fauna of Livingston Island is comparatively poor and is mainly composed of and dominated by cosmopolitan ubiquitous species, e. g. Trinema lineare, Centropyxis aerophila, Euglypha rotunda, E. laevis, Corythion dubium, Assulina muscorum, Difflugia lucida, Microchlamys patella etc. Some genera, e.g. Difflugia, Centropyxis, Plagiopyxis and Nebela, which are represented with dozens or even hundreds of species in temperate and tropical regions, are presented by a few species in the studied habitats of Livingston Island and most of them are with a low population density. In general, the species poorness of these genera in maritime Antarctic islands (in particular in Livingston Island) can be explained by the fact that many of these islands are at great distances from the continents and, on the other hand, most of the representatives of these genera are larger (typically 100-200 µm) and heavier species. These are may be two reasons (together with local microclimatic conditions) which limited the ubiquitous dispersion of the species with large and heavy shells by the wind, by the floiting vegetation or by the birds.

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The data of our study confirms to a great extent the hypothesis of Smith (1982a, 1986, 1987) and Smith and Wilkinson (1986) that there is a clear trend of decreasing species richness in Antarctic testate rhizopod communities with increasing latitude. Comparison of the data of testacean fauna of Livingston Island with those from other sub-Antarctic, maritime Antarctic and continental Antarctic locations shows that the number of taxa found in this study (45) is lower than those of some well-studied sub-Antarctic islands, e.g. Marion Island – 53 taxa (Grospietsch, 1971) and Îles Kerguelen – 50 taxa (Bonnet, 1981), but is higher than those of the locations with the same or higher latitude, e.g. South Georgia – 29 (Beyens et al., 1995; Richters, 1908; Sandon and Cutler, 1924; Smith, 1978, 1982a, 1982b; Smith and Headland, 1983), South Orkney Islands – 27 (Heal, 1965; Smith, 1973, 1974a, 1984), Elephant Island – 15 (Smith, 1972), Deception Island – 4 (Smith, 1974b, 1985), Ile de la Possession – 9 (Smith, 1975), Brabant Island – 7 (Smith, 1987), Adelaide Island – 9 (Smith, 1986), Continental Antarctic – a total of 26 (Decloitre, 1960; Penard, 1911; Smith, 1992; Sudzuki, 1964). We agree with Smith (1982b) and Smith and Wilkinson (1987) that this trend of faunal pauperization of testate amoebae with latitude can be explained by the progressive absence of habitats provided by angiosperm vegetation and increasingly unfavourable microclimatic conditions, i.e. the local environment is an important factor determining the geographical distribution in the Southern Hemisphere. The distribution of the species of genus Nebela is an illustrative example confirming the hypothesis of Smith a. Wilkinson (1987). The geographical barriers play an important role in Nebela biogeography, modifying the relationship between species richness and climate. Till now in the Southern Hemisphere, south of 40º S, 38 taxa of the genus Nebela have been found, and about half of them (18) have restricted distribution to the Gondwanaland continents or Southern Hemisphere islands, and 14 taxa may be endemic to single locations (Smith and Wilkinson, 1987; Wilkinson, 1994). In all investigated samples of different habitats from Livingston Island, only one species of the genus Nebela was found. This is N. lageniformis, which, as a whole, is a cosmopolitan species with a circumpolar distribution. Similarly, low species-richness of the genus Nebela has been observed in other maritime Antarctic islands, Antarctic Peninsula and Continental Antarctic, too, e.g. in the faunas of two studied islands of the South Shetland Islands – Elephant and Deception Islands, the representatives of genus Nebela are missing at all, in South Orkney Islands were found 2 species, in Graham Coast (2), in Marguerite Bay Islands (4), in Terre Adélie (1) etc. (Decloitre, 1964; Heal, 1965; Penard, 1913; Smith, 1972, 1973, 1978, 1985, 1986). These data are in contrast with the higher species-richness of the genus Nebela in temperate and cold temperate regions of the Southern Hemisphere, e.g. Southern Chile (31 taxa), New Zealand (15), Terra del Fuego (14) etc. (Bonnet, 1966; Certes, 1891; Gracia and Gadea,

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1982; Hoogenraad and de Groot, 1948, 1955; Jung, 1942; van Oye, 1956; Penard, 1911; Richters, 1907, 1908; Vucetich, 1974; Wailes, 1913). As a whole, the established species diversity of testaceans in the different habitats on Livingston Island studied confirms the important role of the local environment and the local microclimat in the distribution of testate amoebae. The highest species diversity was established in the moss-communities of Polytrichum sp. and Drepanocladus sp. (38 species, 17 genera) whereas the testacean fauna in the aquatic and soil habitats is twice poorer (22 species, 10 genera and 21 species, 12 genera, respectively). It is noteworthy that in about 50% of the aquatic and soil samples investigated, no testate amoebae have been established. Where the testaceans were established, the species diversity and population density were extremely low. The present study confirms our previous observation that T. lineare has the widest distribution and plays a dominant role in all investigated terrestrial and aquatic habitats from Livingston Island (Todorov and Golemansky, 1996, 1999). Its frequency of occurrence (pF) ranged between 27.5% in aquatic habitats and 77.0% in moss habitats. This species also had the highest dominance frequency (DF) which ranged between 6,9% in aquatic habitats and 20.0% in moss habitats. The high abundance and the dominant role of this species in aquatic and moss habitats have been reported in some studies on testaceans from arctic regions, too (Beyens et al., 1986a, 1986b; Trappeniers et al., 1999, 2002; Van Kerckvoorde et al., 2000). Surprisingly, T. lineare is missing in the check-list of testate amoebae in some of the other studies on the testaceans of maritime Antarctic islands and Continental Antarctic (Smith, 1972, 1975, 1986, 1987, 1992, etc.). Summarizing the data of these study, one can see that the species C. dubium, A. muscorum, P. acropodia, C. aerophila and D. lucida have been more frequently pointed out as dominats. In our study, all these species also had high frequency of occurrence and dominance frequency, but they were always lower than those of T. lineare. ACKNOWLEDGEMENTS We are grateful to Dr. N. Chipev Dr. R. Mecheva and Dr. I. Pandursky for providing the samples and the environmental data used as a basis for this work. The investigation was supported by the National Scientific Research Foundation of the Republic of Bulgaria (Grant A-801/1999). REFERENCES BEYENS L., D. CHARDEZ, R. DE LANDTSHEER. 1986a. Testate amoebae populations from moss and lichen habitats in the Arctic. – Polar Biol., 5: 165-173.

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BEYENS L., D. CHARDEZ, R. DE LANDTSHEER. 1986b. Testate amoebae communities from aquatic habitats in the Arctic. – Polar Biol., 6: 197-205. BEYENS L., D. CHARDEZ, D. DE BAERE, C. VERBRUGGEN. 1995. The aquatic testate amoebae fauna of the Stromness Bay area, South Georgia. – Antarctic Science, 7(1): 3-8. BONNET L. 1966. Le peuplement thécamoebien de quelques sols du Chili (1). – Protistologica, 2 (2): 113-141. BONNET L. 1981. Thécamoebiens (Rhizopoda, Testacea). – Publ. Comité Fançais Rech. Ant., C. N. F. R. A., 48: 23-32. CERTES A. 1891. ‘Protozaires’. – Mission Scientifique du Cap Horn, 1882-1883, 24: 165-178. DECLOÎTRE L. 1960. Thécamoebiens de la 8-ème Expédition Antarctique Française. – Bull. Mus. Hist. Nat., 2-ème série, 32: 242-251. DECLOÎTRE L. 1964. Thécamoebiens de la Douxième Expédition Antarctique Française en Terre Adélie. – Exp. Pol. Franç., 259: 47 p. DE VRIES M. 1937. Methods used in plant sociology and agricultural botanical grassland research. – Herbage Rev., 5: 76-82. GRACIA M.P., E. GADEA. 1982. Moss-inhabiting Thecamoebae of Chilöe Island, Chile. – J. Protozool., 29 (2): 305. GROSPIETSCH T. 1971. Beitrag zur Ökologie der Testaceen Rhizopoden von Marion Island. – In: van Zuideren-Bakker E.M., Winterbottom J.M. & Dyer R.A. (eds.). Marion and Prince Edward Islands. Balkema, Cape Town, pp. 411-419. HEAL O.W. 1965. Observation on the testate amoebae (Protozoa: Rhizopoda) from Signy Island, South Orkney Islands. – Bull. Br. Antarct. Surv., 6: 43-47. HOOGENRAAD H.R., A.A. DE GROOT. 1948. Thecamoebous moss rhizopods from New Zeland. Hydrobiologia, 1: 28-44. HOOGENRAAD H.R., A.A. DE GROOT. 1955. Thecamoebe moss-rhizopoden aus Südamerica. – Arch. f. Hydrobid., 45: 346-366. JUNG W. 1942. Südchilenische Thecamöben. – Arch. Protistenkd., 95 (3): 253-256. OYE P. VAN. 1956. On the thecamoeban fauna of New Zeland with a description of four new species and biogeographical discussion. – Hydrobiologia, 8 (1-2): 16-37. PENARD E. 1911. Sarcodines. Rhizopodes d’eau douce. – British Antarctic Exp., 1907-1909. Rep. Sci. Investig., Biol., 1(6): 203-262. PENARD E. 1913. Rhizopodes d’eau douce. Deuxième Expédition Antarctique Française (19081910). – Sciences Naturelles: Documents Scientifiques: 1-16. RICHTERS F. 1907. Die Fauna der Moosrasen des Gaussbergs und einiger südlicher Inseln. – Deutsche Südpolar-Expedition 1901-1903, 9: 259-302. RICHTERS F. 1908. Moosbewohner. – Wissenschaftliche Ergebnisse der Schwedischen Südpolar-Expedition, 1901-1903, 6(2): 1-16. SANDON H., D.W. CUTLER. 1924. Some Protozoa from the soils collected by the QUEST Expedition, 1921-1922. – J. Linn. Soc. (Zool.), 36: 1-12.

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SMITH H. G. 1972. The terrestrial Protozoa of Elephant Island, South Shetland Islands. – Br. Antarct. Surv. Bull., 31: 55-62. SMITH H. G. 1973. The Signy Island terrestrial reference sites II: The Protozoa. – Br. Antarct. Surv. Bull., 33/34: 83-87. SMITH H. G. 1974a. A comparative study of Protozoa inhabiting Drepanocladus moss carpet in the South Orkney Islands. – Br. Antarct. Surv. Bull., 38: 1-16. SMITH H. G. 1974b. The colonisation of volcanic tephra on Deception Island by protozoa. – Br. Antarct. Surv. Bull., 38: 49-58. SMITH H. G. 1975. Protozoires terricoles de l’île de la Possession. – Rev. Écol. Biol. Sol, 12(2): 523-530. SMITH H.G. 1978. The distribution and ecology of terrestrial Protozoa of sub-Antarctic and maritime Antarctic Islands. – Sci. Rep. Br. Antarct. Surv., 95: 104 p. SMITH H. G. 1982a. A comparative study of the terrestrial Protozoa inhabiting moss turf peat on Iles Crozet, South Georgia and the South Orkney Islands. – In: Colloque sur les Ecosystèmes Subantarctique, Paimpont, C. N. F. R. A., 51: 137-145. SMITH H. G. 1982b. The terrestrial protozoan fauna of South Georgia. – Polar Biology, 1: 173-179. SMITH H. G. 1984. Protozoa of Signy Island fellfields. – Br. Antarct. Surv. Bull., 64: 55-61. SMITH H. G. 1985. The colonization of volcanic tephra on Deception Island by protozoa: longterm trends. – Br. Antarct. Surv. Bull., 66: 19-33. SMITH H. G. 1986. The testate rhizopod fauna of Drepanocladus moss carpet near Rothera Station, Adelaide Island. – Br. Antarct. Surv. Bull., 72: 77-79. SMITH H. G. 1987. A species-poor testate Rhizopod fauna on Brabant Island. – Br. Antarct. Surv. Bull., 77: 173-176. SMITH H. G. 1992. Distribution and ecology of the testate rhizopod fauna of the continental Antarctic zone. – Polar Biol., 12: 629-634. SMITH H. G.1996. Diversity of Antarctic terrestrial protozoa. – Biodiversity and Conservation, 5: 1379-1394. SMITH H. G., R.K. HEADLAND. 1983. The population ecology of soil testate rhizopods on the sub-Antarctic islands of South Georgia. – Rev. Écol. Biol. Sol, 20(2): 269-286. SMITH H. G., D.M. WILKINSON. 1987. Biogeography of testate rhizopods in the Southern Temperate and Antarctic zones. – In: Colloque sur les Ecosystèmes Subantarctique, Paimpont, C. N. F. R. A., 58: 83-96. SUDZUKI M. 1964. On the microfauna of the Antarctic region. 1. Moss-water community at Langhovde. – JARE Sci. Rep. E, 19: 41 pp. TODOROV M., V. GOLEMANSKY. 1996. Notes on testate amoebae (Protozoa: Rhizopoda) from Livingston Island, South Shetland Islands, Antarctic. – In: Golemansky, V. & N. Chipev (eds.). Bulgarian Antarctic Research. Life Sciences. Pensoft Publ., Sofia-Moscow: 70-81.

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TODOROV M., V. GOLEMANSKY. 1999. Biotopic distribution of testate amoebae (Protozoa: Testacea) in continental habitats of the Livingston Island (Antarctic). – In: Golemansky, V. & N. Chipev (eds.). Bulgarian Antarctic Research. Life Sciences. II. Pensoft Publ., SofiaMoscow: 48-56. TRAPPENIERS K., A.VAN KERCKVOORDE, D. CHARDEZ, I. NIJS, L. BEYENS. 1999. Ecology of testate amoebae communities from aquatic habitats in the Zackenberg area (NorthEast Greenland). – Polar Biology, 22: 271-278. TRAPPENIERS K., A.VAN KERCKVOORDE, D. CHARDEZ, I. NIJS, L. BEYENS. 2002. Testate amoebae assemblages from soils in the Zackenberg area, Northeast Greenland. – Arctic, Antarctic, and Alpine Research, 34 (1): 94-101. VAN KERCKVOORDE A., K. TRAPPENIERS, D. CHARDEZ, I. NIJS, L. BEYENS. 2000. Testate amoebae communities from moss habitats in the Zackenberg area (North-East Greenland). – Acta Protozool., 39: 27-33. VUCETICH M.C. 1974. Tecamebionas du turberas de Tierra del Fuego. – Neotropica, 20 (61): 27-35. WAILES G.H. 1913. Freshwater rhizopods from North and South America. – J. Linn. Soc. Zool., 32: 201-218. WILKINSON D.M. 1994. A review of the biogeography of the protozoan genus Nebela in the southern temperate and Antarctic zones. – Area, 26 (2): 150-157.

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© PENSOFT Publishers Antarctic Research ) ... REVIEW ON THE FREE-LIVING COPEPODS (CRUSTACEABulgarian 95 Sofia – Moscow Life Sciences, vol. 4: 95-99, 2004

Review on the Free-living Copepods (Crustacea) from the Region of the Bulgarian Antarctic Base, Livingston Island IVAN PANDOURSKI1, APOSTOL APOSTOLOV2 1

Institute of Zoology, 1, Tzar Osvoboditel Bd., 1000 Sofia, Bulgaria 2

Izgrev, Bl. 35, entr. R, 8008 Bourgas, Bulgaria

ABSTRACT Fourteen Copepod species are reported from various freshwater and marine biotopes in the region of the Bulgarian Antarctic Base at Livingston Island, Antarctica. Short zoogeographical remarks and notes on habitat preferences for each species are given. KEY WORDS Free-living Copepods, zoogeography, Livingston Island, Antarctica.

INTRODUCTION The article presents the summarized results on the species composition, the habitat and general zoogeographical distribution of free-living Copepods from the region of the St. Kliment Ohridsky Bulgarian Antarctic Base, 62o 38‘ S and 60o 22‘W, Livingston Island. This is the most abundant meiobenthic faunistic group, inhabiting various freshwater and marine biotopes on the coast and the littoral zone of the Island. The question of formation of Antarctic copepod community and their enrichment with new immigrants, influenced by human impact and the global climate changes, is still unsolved. The Copepod Crustaceans can be used as bioindicators and biomonitors of environmental changes.

*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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MATERIALS AND METHODS The material for this review was collected during the 6th (1997/98), 7th (1998/99) and 8th (1999/2000) Bulgarian Antarctic Expeditions. Original and published data (Apostolov & Pandourski, 1999, 2000, 2002; Pandourski & Chipev, 1999; Pesce & Pandourski, 2002) were discussed. The faunistic samples originate from different freshwater and marine habitats (glacial lakes, lithotelms, interstitial beach water, pelagic zone of South Bay, coastal lithotelms, marine algae, etc.). The material of marine zooplankton was collected with a plankton net with a mesh of 0,125 mm. The samples from the littoral interstitial waters were collected using the Karaman-Chappuis method and the phytophilous species by repeated rinsing of marine algae and then filtering the water through a hand net. SPECIES COMPOSITION AND REMARKS Crustacea Copepoda Calanoida Fam. Centropagidae Boeckella Guerne (de) & Richard, 1889; Bayly, 1964 Boeckella poppei Mrazek, 1901 This calanoid is a common species in Patagonia and in glacial or volcanic freshwater lakes of the islands of the South Atlantic, including the South Shetland Archipelago in Antarctica. This is the only planktonic crustacean species in glacial lakes near the Bulgarian Antarctic base, where it reaches high abundance in the austral summer. The variation in some basic morphometric and morphological characters in male and female of B. poppei is analyzed and discussed in Pandourski & Chipev (1999). Cyclopoida Fam. Cyclopinidae Pseudocyclopina Lang, 1946 Pseudocyclopina livingstoni Pesce and Pandourski, 2002 The all known six species of Pseudocyclopina can be considered endemic to Antarctic waters. Up to now, P. livingstoni is found only in a planktonic community and interstitial littoral waters of the South Bay of Livingston Island. According to Pesce & Pandourski (2002), on account of the absence of antennary exopodite, the species is closely related to P. belgicae (Giesbrecht) and P. guentheri Elwers, Martinez Arbizu et Fiers.

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Harpacticoida Fam. Harpacticidae Harpacticus Milne-Edwards, 1840 Harpacticus furcatus Lang, 1936 The studied specimens have large morphological variability. On Livingston Island H. furcatus is abundant and inhabits lithotelms with algae covered bottoms close to the sea or up to 10-12 m inland. It is known also from interstitial waters of sand banks, dry at the time of low-tide. Rarely found among red sea algae. The species has circum-antarctic distribution. Fam. Tisbidae Scutellidium Claus, 1866 Scutellidium longicauda (Philippi, 1840) Only one specimen of this phytophilous species was found among the red sea algae. It is known from the Mediterranean region (including the Black Sea), the Atlantic Ocean and Livingston Island in Antarctica. Scutellidium sp. The distinctive signs of our specimens from the coastal zooplankton do not allow us to refer them to one of the already described species from the genus. Tisbe Lilljeborg, 1853 Tisbe sp. The only one specimen found on Livingston Island inhabits a marine interstitial biotope: large-grained sand and fine gravel in Marine Lion Bay. The absence of swimming legs and a barely visible genital field does not allow us to refer our specimen to one of the known species. Fam. Peltidiidae Alteutha Baird, 1845 Alteutha sarsi Monard, 1924 The species is described from the Mediterranean Sea. Our finding is the first one from the Antarctic region. The specimen from Livingston Island originates from zooplankton. This phytophilous species probably separates from algae and appears in free water. Fam. Thalestridae Paradactylopodia Lang, 1948 Paradactylopodia brevicornis (Claus, 1866) This species with a wide morphological variability is a permanent inhabitant of phytal. P. brevicornis and is found in the interstitial waters of sand banks of the Black Sea coast. A Cosmopolite species.

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Fam. Parastenhellidae Parastenhelia Thompson & Scott, 1903 Parastenhelia hornelli Thompson & Scott, 1903 This is a very rare phytophilous species. It has wide distribution in the World Ocean: the Mediterranean basin, the Indian Ocean, the Antarctic region. On Livingston Island it is found in a sand bank with many red marine algae around it. Fam. Diosaccidae Amphiascus Sars, 1905 Amphiascus elongates Ito, 1972 For the first time the species was described as a commensal on the cancer Telmessus cheiragonus (Tilesius) in the Pacific (Hokkaido Island, Japan). On Livingston Island this species is found in a marine interstitial biotope in a sand bank. Fam. Ameiridae Ameira Boeck, 1864 Ameira parvula (Claus, 1866) A widely distributed species from the tropical to the polar zones of the World Ocean. It inhabits different marine biotopes: sandy bottom, interstitial beach waters Ameira sp. Fam. Laophontidae Heterolaophonte Lang, 1948 Heterolaophonte livingstoni Apostolov & Pandourski, 2000 This endemic species for Antarctic coastal ecosystems inhabits small lithotelms and interstitial waters on Livingston Island. Frequently, these habitats are highly eutrophicated because of excrements of abundant sea birds. Paralaophonte Lang, 1948 Paralaophonte livingstoni Apostolov, 2004 A limited number of specimens was found in the coastal zooplankton community of Livingston Island. CONCLUSIONS In total, 14 copepod species from 10 families were established in the coastal and littoral zone near the Bulgarian Antarctic Base in Livingston Island. The only found calanoid (B. poppei) inhabits the freshwater glacial lake. The other 13 species are part of brakish and marine communities. Three copepods can be considered as endemic species for the region of Livingston Island: Pseudocyclopina livingstoni, Paralaophonte livingstoni and Heterolaophonte livingstoni. H. furcatus has circum-antarctic distribution. The

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group of cosmopolite species consists of six species. The specimens of four genera are not determined at the species level, because of insufficient faunistic material. ACKNOWLEDGEMENTS This work was supported by grants B-A-504 and B-A-801 from the National Fund for Scientific Investigations of the Bulgarian Ministry of Education, Science and Technologies. REFERENCES APOSTOLOV A. 2004. Paralaoponte livingstoni n.sp. (Crustacea: Copepoda: Harpacticoida) un nouveau représentant de l’île de Livingston, Antarctique. – Historia naturalis bulgarica, 16: 59-67. APOSTOLOV A., I. PANDOURSKI. 1999. Marine Harpacticoids (Crustacea : Copepoda) from the littoral of the Livingston Island (the Antarctic). – Bulgarian Antarctic Research, Life Sciences, 2: 68-82. APOSTOLOV A., I. PANDOURSKI. 2000. Heterolaophonte livingstoni sp. n. (Crustacea, Copepoda, Harpacticoida) de la zone littorale de l’île de Livingston, Antarctique. – Annali del Museo Civico di Storia Naturale « G. Doria », XCIII : 239-252. APOSTOLOV A., I. PANDOURSKI. 2002. Marine Copepods (Crustacea) from Livingston Island (Antarctica). – Bulgarian Antarctic Research, Life Sciences, 3: 70-81. PANDOURSKI I., N. CHIPEV. 1999. Morphological variability in a Boeckella poppei Mrazek, 1901 (Crustacea: Copepoda) population from a glacial lake on the Livingston Island (the Antarctic). – Bulgarian Antarctic Research, Life Sciences, 2: 83-92. PESCE G. L., I. PANDOURSKI. 2002. Pseudocyclopina livingstoni sp. n. (Copepoda, Cyclopinidae) from the Livingston Island (Antarctica). – Biologia, Bratislava, 57/2: 133-137.

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© PENSOFT Publishers Research METALS AND TOXIC ELEMENTS CONTENT IN GENTOO Bulgarian PENGUINSAntarctic ... HEAVY 101 Sofia – Moscow Life Sciences, vol. 4: 101-106, 2004

Heavy Metals and Toxic Elements Content in Gentoo Penguins (Pygoscelis papua) Feathers During Moult R. METCHEVA1, L. YURUKOVA2 1

Institute of Zoology, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria Institute of Botany, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

2

ABSTRACT The concentrations of the 11 microelements (Al, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd and Pb) have been determined in feathers of moulting Gentoo penguins (Pygoscelis papua), from the Antarctic region, Livingston Island. The results show that the area of Antarctica studied is still an environment less affected by emissions. Moulting plumage accumulates these toxicants during one year of living, and it can be used successfully for biomonitoring purposes. KEY WORDS Pygoscelis papua, feathers, heavy metals, toxic elements, Livingston Island, Antarctica.

INTRODUCTION Marine birds are exposed to a wide range of pollution because they spend most of their time in aquatic environment where they are exposed by external contact, by inhalation and particularly by ingestion of food and water. An important aspect of the pollution effect lies in the exposure assessment (Petering, 1974, Osborn, 1978, Walsh, 1990). Penguin’s feathers are a good subject for monitoring, as bioindicators of the state of the environmental trace element contamination because they are rich in disulfide bonds that are readily reduced to sulfhydryl groups that bind metals. As feather protein is laid down, it becomes a chelator that binds and removes metals from the blood supply. Metal levels in feathers reflect on blood circulation during feather forming. *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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This is the reason why many studies have reported various irregularities of heavy metal concentrations in different feathers of individual birds (Muller et al., 1984; Furness et al., 1986; Braune & Gaskin, 1987). They are interpreted as resulting from the moulting pattern (Goede & Bruin 1984, 1986; Strugler et al., 1987). The purpose of this study was to determine the concentration of some heavy metals and toxic elements in feathers of Gentoo penguins during moult for no breeding birds. In order to obtain more general results a spectrum of 11 microelements was analyzed, with different biological functions and behavior. MATERIAL AND METHODS Feather samples of Gentoo penguins (Pygoscelis papua) were collected from a total number of 28 individuals at the end of moult at Kaleta Argentia 62º 40´ 09.8´´ S and 60º 24´ 0.8.1´´ W – Livingston Island from the South Shetland Islands on February 16 th of 2002, during the Antarctic summer. The penguins were of an unknown age, and the state of moult was defined as middle. All samples were stored in polyethylene collectors. The samples were dried at 80ºC till constant weight and then wet-ashed. About 1 g of the material was treated with 15 ml nitric acid overnight. The wet-ashing procedure was continued with heating on a water bath, followed by the addition of 2 ml of hydrogen peroxide. This treatment was repeated till full digestion. The filtrate was diluted with double distilled water to 25 ml. The solutions were stored in plastic flasks. Duplicates of each feather sample were prepared independently. The elements Al, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd and Pb were determined by atomic emission spectrometry with inductively coupled plasma (ICP-AES). The instrument used was SPECTROFLAME (Germany) with 3 air polychromators, 1 vacuum polychromator and a scanning monochromator. The deviation between the duplicates was below 5% in all cases. The analytical precision was ensured with blanks and stock standards (Merck) used for the preparation of working aqueous solutions. The quality control was proved with standard reference materials (CRM 281 and CRM 142R). The concentrations are expressed as mg/kg of dry weight. Statistical processing included the maximum, minimum and average value, as well as the standard deviation for all the elements. In addition, the uncertainties (EURACHEM/CITAC Guide, 1999), including type of sampling, sample cleaning, homogenization procedures, etc. were calculated. The correlative analysis was done searching for significant positive and negative relationships between pairs of microelements according to a different level of reliability (p≤0.05, p≤0.01, p≤0.001).

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RESULTS AND DISCUSSION The inorganic content of bird feathers is available in some papers (Weyers & Glück 1988; Janiga et al.,1990; Altmeyer et al., 1991). The first attempt to study penguin feathers (Pygoscelis antarctica) in the Antarctic region was published by Metcheva & Yurukova (2002). This study for Gentoo penguins‘ feathers is the next step from the biomonitoring program of the Bulgarian Antarctic Biological Program. Minimum, maximum, average concentrations and uncertainty of analyzed microelements in the penguin’s feathers are given in Table 1. The elements Pb, Se, As, Cd and Co were found very often below the detection limits, 1.0, 0.8, 0.6, 0.2 and 0.2 mg/kg, respectively. The elements iron (10 times), lead and arsenic (above 7 times), aluminum, nickel and manganese (5 times), cadmium and cobalt (above 2 times) accumulated in the feathers varied among 28 different individuals. The changes in the concentrations of copper and zinc were in the narrowest range of values. The average concentrations of the microelements in the Gentoo penguin’s feathers studied were found in the descending order of: Zn>Fe>Al>Cu>Mn>Ni>Pb, Se, As, Co, Cd. The mean element distribution in the feathers is shown as polygons in Fig. 1. Table 1. Concentrations of microelements (mg/kg dry weight) in feathers of Pygoscelis papua. n=28

Al

Mn

Fe

Co

Ni

Cu

Zn

As

Se

Cd

Pb

min max mean uncertainty

25 129 46 22

1.4 6.5 2.6 1.0

24 236 53 40

<0.2 0.50

0.89 4.3 2.2 0.97

13 19 16 2

78 101 89 8

<0.6 4.0

<0.8 4.9

<0.2 0.43

<1.0 6.8

Pb

Fe

4

120 80

Mn

40

Zn

0

2 0

Al As

Cu

Cd

Ni Co

mg/kg d.w.

Fig. 1. Polygons of element distribution in the feathers of Gentoo penguin (Pygoscelis papua), mg/kg dry weight.

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The results of the correlation analysis are presented in Table 2. Positive correlations with the highest reliability (p≤0.001) were established for manganese, iron and aluminum. The positive relationship with the lowest reliability (p≤0.05) was obtained for the couples: Zn-Cu, Zn-Ni, Mn-Ni. The extremely toxic element cadmium appears in healthy wild birds and varied widely among species populations, with average contents of below 0.1 to 32 mg/kg of wet weight in the liver, and below 0.3 to 137 mg/kg in the kidney (Walsh, 1990). Because of that, special attention for this heavy metal should be paid. Cadmium concentrations were consistently several orders of magnitude higher in pelagic seabirds than in terrestrial birds in supposedly unpolluted areas and, among seabirds, values in squid eating or insect eating species are generally highest (Bull et al., 1977; Cheng et al., 1984; Elliot et al., 1992). The highest individual concentrations of cadmium measured in the kidneys of apparently healthy adult birds were 275 mg/kg in a Greenland Kittiwake, 183 mg/kg in a Greenland Glaucous gull (Nielsen & Dietz, 1989), 166 mg/kg in a Macaroni penguin (Northeim, 1987) Despite enormous variation in cadmium levels among species, intraspecific variation tent to be lower for other nonessential metals, such as mercury and lead, for populations of seabirds. Walsh (1990) suggested that this could be an evolutionary consequence of long term exposure to high natural levels of Cd in oceanic food chains, leading to seabirds evolving greater ability to regulate tissue concentrations of Cd. The toxic heavy metal Cd was accumulated in kidney and liver, 2.6 and 0.33 mg/kg of wet weight, respectively, in an adult individual of Gentoo penguin (Pygoscelis papua), collected in the same Antarctic region – Livingston Island (Mecheva & Yurukova, in press). In the Gentoo plumage, the concentration of this very toxic metal seems to be low and the maximum was 0.43 mg/kg (Table 1). This concentration is lower (around 2 times) in comparison with other published results concerning feathers in small insectivorous birds from polluted regions in Europe, where the content of Cd reached 0.9 mg/kg and higher (above 2 times) as compared to Chinstrap’s penguin feathers Table 2. Correlative analysis for significant positive and negative relationships between pairs of microelements Al Al Mn Fe Ni Cu Zn

Mn

Fe

Ni

Cu

Zn

0.64***

0.93*** 0.80***

0.09 0.39* 0.24

0.11 0.35 0.22 0.08

0.24 0.33 0.31 0.40* 0.44*

* – p ≤ 0.05; *** – p ≤ 0.001

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(Metcheva & Yurukova, 2002). The published data for European regions showed large amounts for some metals in feathers – 28.5 mg/kg Pb, 173.0 mg/kg Zn and 363.3 mg/kg for Fe (Sawicka-Kapusta & Kozlovski, 1984) in comparison with the concentrations of these metals in Gentoo’s feathers which, respectively, were: maximum 6.8 mg/kg Pb, average values of 89 mg/kg Zn and 53 mg/kg of Fe (Table 1). The concentrations of Cu, Co are similar to the amounts found in the moulting feathers of passerine birds. CONCLUSIONS The annually moulting plumage of Gentoo penguins proves the successful use of the subject for biomonitoring assessments. Data for different heavy metals and toxic elements accumulated in Gentoo penguins feathers confirmed that the region of Antarctica, especially the studied area of the South Bay on Livingston Island, is an environment less affected by anthropogenic influence. ACKNOLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. # 2001 – 0517 REFERENCES ALTMEYER M., DITTMANN J., DMOWSKI K., WAGNER G., MULLER P., 1991. Distribution of elements in flight feathers of a White-tailed eagle. Sci. Total. Environ., 105: 157-164. BRAUNE B., GASKIN D., 1987. Mercury levels in Bonaparte’s gulls (Larus philadelphia) during autumn moult in Quoddy region, New Brunswick, Canada. Arch. Environ. Contam. Toxicol., 16: 539–549. BULL K., MURTON R., OSBORN D., WARD P., CHENG L., 1977. High levels of cadmium in Atlantic seabirds and sea-skaters. Nature (Lond.), 269: 507–509. CHENG L., SCHULZ–BALDES M., HARRISON C., 1984. Cadmium in ocean skaters, Halobates sericeus (Insecta), and in their seabird predators. Mar. boil. (N. Y.), 79: 321–324. ELLIOT E., SCHEUHAMMER A., LEIGHTON F., PEARCE P., 1992. Heavy metal and metallthionein concentrations in Antlantic Canadian seabirds. Arch. Environ. Contam. Toxicol., 22: 63–73. EURACHEM/CITAC GUIDE, 1999. Quantifying uncertainty in analytical measurement. Eurachem Workshop, Helsinki. June 1999, Second Edition, 119. FURNESS R. R., MUIRHEAD S., WOODBURN M., 1986. Using bird feathers to measure mercury in the environment: relationships between mercury content and moult. Mar. Pollut. Bull., 17: 27–30.

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GOEDE A., DE BRUIN A., 1984. The use of bird feather parts as a monitor for metal pollution. Environ. Pollut. Ser., B., 8: 281–298. GOEDE A., DE BRUIN A., 1986. The use of bird feather for indicating heavy metal pollution. Environ. Monit. Assess., 7: 249–256. JANIGA M., MANKOVSKA B., BOIBALOVA M., DURCOVA G., 1990. Significance of concentrations of lead, cadmium and iron in the plumage of the feral pigeon. Arch. Environ. Contam. Toxicol., 19: 2-6. METCHEVA R., L. YURUKOVA, 2002. Toxic and heavy metals concentration in Chinstrap penguin’s (Pygoscelis antarctica) feathers during molt. In: Bulgarian Antarctic Research. Life Sciences, 3: 101-105. MECHEVA R., L. YURUKOVA (in press). Major essential and trace elements in Gentoo Penguin (Pygoscelis papua) from Livingston Island, Antarctica. – Acta Zoologica Bulgarica. MULLER P., DMOVSKI K., GAST F., HAHN E., WAGNER G., 1984. Zum Problem der Bioindikation von standortspezifischen Schwermetallbelastungen mit Vogelfedern. Wiss. Umvelt, 3: 139-144. NIELSEN O., DIETZ R., 1989. Heavy metals in Greenland seabirds. Medd. Gronl. Biosci. Rep. N 29. NORTHEIM G., 1987. Levels and interactions of heavy metals in sea birds from Svalbard and the Antarctic. Environ Pollut. 47: 83–94. OSBORN D., 1978. Toxic and essential heavy metals in birds. Inst. Terr. Ecol., Nat. Environ. Res. Counc., Ann. Rep., 53-56. PETERING H. G., 1974. Trace ålement Metabolism in Animals. Univ. Park. Press, Baltimore. SAWICKA-KAPUSTA K., KOZLOVSKI J., 1984. Flow of heavy metal through selected homeothern consumers. In: Forest Ecosystems in Industrial Regions (Ed. W. Grodzinski, Weiner J. & Maycock P.). Ecol. studies 49, Springer Verlag, Berlin, Heidelberg, New York, Tokyo. STRUGLER J., ELLIOTT J., WESELOH M., 1987. Metals and essential elements in herring gulls from the Great Lakes. J. Great Lakes Res., 13: 43–55. WALSH P., 1990. The use of seabirds as monitors of heavy metals in the marine environment. In: Heavy Metals in the Marine Environment (Ed. R. Furness & Rainbow P.). CRC Press, Boca Raton, Flo. WEYERS B., GLÜCK E., 1988. Investigation of the significance of heavy metal contents of blackbird feathers. Sci. Total Environ. 77, 1: 61-67.

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© PENSOFT PublishersSTUDIES AND DIFFERENTIAL COUNTING OF LEUCOCYTES Bulgarian Antarctic Research IN G ENTOO ... BLOOD CHEMISTRY 107 Sofia – Moscow Life Sciences, vol. 4: 107-113, 2004

Blood Chemistry Studies and Differential Counting of Leucocytes in Gentoo Penguins (Pygoscelis papua) in Relation of Moulting and Non-moulting Stages E. TRAKIJSKA¹, K. STOJANOVA², R. METCHEVA¹ ¹Institute of Zoology, Bulgarian Academy of Sciences, 1, Tzar Osvoboditel Blvd., 1000 Sofia, Bulgaria ²N. Pirogov National Polyprofile Hospital for Active Treatment and Emergency, 21 Totleben Blvd, 1606 Sofia, Bulgaria

ABSTRACT Analyses of 13 blood chemistry parameters from 52 individuals Pygoscelis papua have been carried out. Means, standard deviations and differences between non-moult and moult adults, as well as for juveniles were obtained for each parameter investigated. Non-moult birds exhibited greater mean values of ASAT, ALAT, CPK, LDH, HBDH, AP and lower mean values of GGT and AMS. The mean values of total protein, albumin and globulin were lowest in moult birds. Significant differences in ASAT ALAT, CPK, TPRO, ALB and GLO between non-moult and moult adults were found. Moult juveniles displayed significant differences in the concentration of CPK and AP in comparison with non-moult and moult adults. Microscope differential counting of leucocytes (leucogramme) has been accomplished. KEY WORDS Pygoscelis papua, biochemical parameters, leucogramme, non-moult, moult, juveniles.

INTRODUCTION The study of the levels of some blood chemistry constituents has been carried out with birds of Pygoscelis papua. The main aim of the research was to determine the limits of some blood chemistry parameters, to estimate significant differences be*Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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tween adults (non-moult, moult) and juveniles birds, as well as to compare our data with other data obtained for gentoo. Penguins have developed different mechanisms of adaptation to extreme climatic conditions, such as maintaining a high level of energy in metabolism and in relation to that the investigations of the penguin physiological status, particularly hematological parameters, became an important task. MATERIALS AND METHODS The samples were collected on Kaleta Argentina, (62° 40' 09.8" S and 60° 24' 80" W), Livingston Island, South Shetland Islands, during the Antarctic summer 2001/02. Blood samples from 52 individuals were obtained: 17 non-moult adults, 27 moult adults and 8 moult juveniles. 52 blood slides were analyzed as well. Blood samples up to 1.5 ml were collected from the cubital vein of the flipper into heparinized plastic containers. All samples were collected between 2 pm and 6 pm to eliminate possible diurnal fluctuations, caused by circadium rhythms (Garcia– Rodriguez et al., 1987; Ferrer, 1990). Blood samples followed the normal laboratory procedures. Heparinized and separated plasma was spun down for 10 min at 3000 rpm for obtaining serums, which were stored frozen and transported to the laboratory. Blood slides were fixed by methanol for 5 minutes and colored by standard laboratory procedures. They were observed by a Carl Zeiss Jena microscope (immersion lens HI 100/ 1.25 and lens 10/0.25). Analyses of 13 blood parameters were carried out on a computer autoanalyser Cobas Mira and Human with the reagents, recommended by Merck. The methods employed were: aspartate aminotransferase (ASAT) – IFCC photometric determination; alanin transferase (ALAT) – IFCC photometric determination; creatin phosphokinase (CPK) – UV NAC; laktat dehydrogenase (LDH) – standard method DGKCh photometric determination; α hydroxy butirat dehydrogenase (HBDH) – standard method DGKCh photometric determination; γ -glutamil transferase (GGT) – kinetic colorimetric method; alkaline phosphatase (AP) – colorimetric determination test; α -amylase (AMS) – CNPG3 photometric determination method; Triglyceride (TG) – GPO PAP; Total protein (TPRO)– Biuret reaction; Albumin (ALB) – BGG; Globulin (GLO) – estimated by difference TPRO/Alb; Magnesium – Xylidil blue reaction. The Student’s t-test was employed to investigate significant differences of biochemical values between non-moult and moult birds, as well as the differences between juveniles and adults.

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RESULTS AND DISCUSSION The present investigation showed that non-moult and moult phases are characterized by significantly different concentrations of plasma enzymes, metabolites and microelements. The differences between non-moult and moult adults, as well as between moult juveniles and adults were displayed because of mobilization of the stored nutritients of biochemical processes, pertaining to the diverse physiological requirements. The obtained values for non-moult, moult and juveniles gentoo penguins are shown in Table 1. Significant differences (p< 0,05) between two phases of physiological cycles were found in 6 biochemical parameters – ASAT, ALAT, CPK, TPRO, ALB and GLO from 13 investigated parameters. Significant differences between nonmoult or moult adults and moult juveniles were established in CPK and AP (p< 0.05). It is important to notice the stable values of TG and Mg in different physiological phases in adults and juveniles as well. The obtained results for the other parameters showed no significant differences between the groups compared (Aguilera et al., 1993). In this investigation, previous data from other studies have been considered in relation to comparison (Ghebremeskel et al., 1989, 1992; Aguilera et al., 1993; Metcheva Table 1. Biochemistry values of 13 analyzed hematological parameters in non-moult, moult and juveniles gentoo penguins (Pygoscelis papua) Parameters

non-moult (mean ± SD) n = 17

moult (mean ± SD) n = 27

juveniles (mean ± SD) n=8

*ASAT U/l *ALAT U/l *CPK U/L** LDH U/l HBDH U/l GGT U/l **AP U/l AMS U/l *ALB g/l *TPRO g/l * GLO g/l TG mmol/l Mg mmol/l

121 ± 34.9 170.9 ± 93 283.9 ± 121.4 312 ± 93.5 98.1 ± 40.9 2.2 ± 1.1 201.4 ± 53.7 1153.2 ± 307.7 16.8 ± 2.6 46 ± 4.2 29.2 ± 2.8 1.6 ± 0.36 1.9 ± 0.18

110.8 ± 42.7 132.6 ± 51.4 250.9 ± 74.2 243.3 ± 97.4 90.7 ± 34.1 3 ± 0.98 190.4 ± 63.7 1392.1 ± 492.2 11.9 ± 2.1 34.7 ± 2.1 22.8 ± 1.8 1.4 ± 0.21 2.1 ± 0.17

104.3 ± 46.7 137.2 ± 31.1 498.2 ± 121.3 493.7 ± 127.4 158.3 ± 37.2 2.2 ± 1.3 257.2 ± 32.8 1227.4 ± 287.2 20.4 ± 1.2 44.3 ± 3.4 23.9 ± 2.7 1.6 ± 0.35 1.8 ± 0.06

*Significant differences (p<0.05) in ASAT, ALAT, CPK, ALB, TPRO and GLO between nonmoult and moult adults. ** Significant differences (p<0.05) in AP and CPK between juveniles and adults.

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and Trakiiska, 2002). However, the main aim of this study was to determine the limits in biochemical parameters characterizing the different physiological stages. A significant decrease was observed in the values of ASAT, ALAT and CPK in late moult birds, compared with non-moult (p< 0.05). Fig1. It might be explained with increased amino acid transport activities in pre-moult stage – because the proteins comprise over 90% of feathers and their protective sheets, which are the major products of moulting activity (Ghebremeskel et al., 1992; Metcheva and Trakiiska, 2002; Aguilera et al., 1993.). Abnormally high values of ASAT and ALAT were found in pre-moult penguins, about 10 times more than normal values in man, in order to meet efficiently the nutritional demand in the moulting phase (Rosa et al., 1993). Significantly different higher concentrations of total protein, albumin and globulin (p< 0.05) in non-moult and pre-moult penguins (24 h before starting to lose feathers) were found in comparison with post-moult Fig 2. Those high values coincided with the beginning of the period of plumage formation, due to the mobilization of tissue proteins and the utilization of the resulting amino acids for feather synthesis (Ghebremeskel et al., 1992). Comparing the data obtained in this study, it is obvious that the values of AP and CPK in juveniles are higher than in non-moult and moult adults and that is confirmed

Fig. 1. Values of ASAT, ALAT and CPK in non-moult and moult birds

Fig. 2. Values of TPRO, ALB and GLO in non-moult and moult birds

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by other investigations (Aguilera et al., 1993; Ghebremeskel et al., 1989; Metcheva and Trakiiska, 2002). Significant differences of AP and CPK (p< 0.05) were observed between juveniles and adults (non-moult and moult birds) Fig 3. Those higher concentrations of AP and CPK in juveniles appeared to be related to osteoblastic activities, as well as to the growth and secondary ossification of osseous tissues (Vinuela et al., 1991). It is important to notice that the values of plasma concentration for TG and Mg were very stable in the three groups compared, almost identical for non-moult, moult and juveniles. That fact is supported by other studies as well (Ghebremeskel et al., 1992; Metcheva and Trakiiska, 2002). The differences in leucogramme between non-moult and moult gentoo penguins are shown in Table 2. Higher number of lymphocytes and eosinophyles (normal values: Lympho 26–54; Eo 1 -7) in moult birds was observed in comparision with non-moult. At the same time the number of heterophyles (normal values: Hetero 41–70) in moult birds is lower than in non-moult. Probably, it appears to be related to some diseases as a consequence of parasytes in intestines. It is known that in the moult stage a remarkable loss of weight is observed, which demonstrates the nutritional stress that the birds endure. During the moult phase, probably the immune system is in a higher activity, and this is obvious from the number of lymphocytes rate.

Fig. 3. Values of AP and CPK in juveniles and adults

Table 2. Leucogramme (% total leucocytes) of gentoo penguins. Leucocytes Heterophyles Lymphocytes Monocytes Eosinophyles

non-moult n=17

moult n=27

juvenils n=8

12 – 43 12 – 78 1–4 1 - 17

1 - 37 47 – 84 1–5 2 – 43

4 – 18 20 – 68 1–7 4-7

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In non-moult birds, the number of heterophyles is lower, but close to the normal rate; the number of monocytes found in non-moult penguins is in normal limits (normal values Mo 0–4). The normal values, used for comparison with data obtained in this research, were established by Clarke and Kerry (1994) and Hawky et al. (1989). Previous data for juveniles was not found in order to compare them with the results obtained in this study. The values of leucocytes in juveniles were very close to the normal limits of adults, except for heterophyles – the number was lower than normal. CONCLUSION This research showed that the three compared groups of studied penguins (nonmoult, moult adults and juveniles) displayed significant differences among them. The results obtained indicate that the penguins change the levels of certain biochemical parameters during the moult phase in order to meet the nutritional demand of that physiological stage and efficiently to allocate the diminishing reserve to the various requirements. Significant differences were found in the values of the biochemical parameters related to cellular and tissue requirements during the different physiological stages. The results of the differential counting of leukocytes confirmed the considerable physiological differences in non-moult and moult phases. ACKNOWLEDGMENTS This research is supported by a grant from INTAS 2001 – 0517. We are particularly grateful to Dipl. Ing. J. Jankov, who helped considerably in the field work. Special thanks to M.Sc. E. Blajeva, from the Biochemical Laboratory of N. Pirogov Hospital for Active Treatment and Emergency, who provided the laboratory facilities. We are also grateful to M.Sc. S. Bichev and Ph.D. A. Savov for their support. REFERENCES AGUILERA E., J. MORENO, M. FERRER. 1993. Blood chemistry values in three Pygoscelis penguins. Comp. Biochem. Physiol. V. 105A, 3:471-473. CLARKE I., K. KERRY. 1993. Deseases and parasites in penguins. Korean Journal of Polar Research. 4: 79-96. GARCIA–RODRIGUES T., FERRER M., RECIO F., CASTROVIEJO J. 1987. Circadian rhythms of determined blood chemistry values in buzzards and eagle owls. Comp. Biochem. Physiol. 88A, 633-669.

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GHEBREMESKEL K., G. WILLIAM, I. KEYMER, H. HORSLEY, D. GARDNER. 1989. Plasma chemistry of rockhopper (Eudyptes crestatus), magellanic (Spheniscus magellanicus) and Gentoo (Pigoscelis papua) wild penguins in relation to molt. Comp. Biochem. Physiol. V 92A(1):43-47. GHEBREMSKEL K., T. WILLIAMS, G. WILLIAMS, D. GARDNER, M. CRAWFORD. 1992. Dynamics of plasma nutrients and metabolites in moulting Macaroni (Eudyptes chrysolophus) and Gentoo (Pygoscelis papua) penguins. Comp. Biochem. Physiol. V.101A(2):301-307. HAWKEY C., D. HORSLEY, I. KEYMER. 1989. Hematology of wild penguins (Sphenisciformes) in Falkland Islands. Avian Pathology. 18 : 495-502. METCHEVA R., E., TRAKIISKA. 2002. Preliminary morphophysiological investigations of Gentoo penguins (Pygoscelis papua) on Livingston Island. Bulgarian Antarctic Research. V.3:97-101. ROSA R., E. RODRIGUES, M. BACILA. 1989. Blood glucose partition and levels of glycolytic enzymes in erythrocytes and somatic tissues of penguins. Comp. Biochem. Physiol. 92B:307-312. ROSA C., R. ROSA, E. RODRIGUES, M. BACILA. 1993. Blood constituents and elecrophoretic patterns in antarctic birds: penguins and skuas. Comp. Biochem. Physiol. V104 (1):117-123. VINUELA J., FERRER M., RECIO F. 1991. Age-related variatations in plasmatic levels of alkaline phosphatase, calcium and inorganic phosphorus in chicks of two species of raptors. Comp. Biochem. Physiol. 98A, 49-54.

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© PENSOFTGPublishers Bulgarian Antarctic Research ENTOO PENGUIN COLONY ESTIMATES USING DIGITAL PHOTOGRAPHY 115 Sofia – Moscow Life Sciences, vol. 4: 115-121, 2004

Gentoo Penguin Colony Estimates Using Digital Photography R. METCHEVA, P. ZEHTINDJEV, Y. YANKOV Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel Boul., 1000 Sofia, Bulgaria Bulgarian Antarctic Institute, 15 1 Tzar Osvoboditel Boul., 1504 Sofia, Bulgaria

ABSTRACT Gentoo Penguins (Pygoscelis papua) are a sensitive indicator of change in the Antarctic environment and have been monitored intensively. One-year observations of nesting Gentoo carried out at Livingston Island (South Shetlands) with the help of digital photography for mapping the nests are presented. This method was used because it produces as good results as ground counting, but is much less intrusive and more convenient. KEY WORDS Gentoo, Livingston Isl., digital photography, monitoring, population.

INTRODUCTION Gentoo penguins Pygoscelis papua have a circumpolar distribution in the Sub-Antarctic zone, breeding usually in small colonies on islands in the South-Atlantic and Indian Oceans in the Arc and on the Antarctic Peninsula (Harrison, 1983). Penguins are intensively monitored as part of an international effort to understand the role of apex predators in the Southern Oceans (Trivelpiece et al. 1990; Agnew, 1997). Among the key variables annually assessed by these monitoring programs are measures of population change such as breeding population size (CCAMLR, 1992), which provide the essential data necessary to determine trends within and among regions. This paper describes a one-year study of nesting Gentoo carried out at Livingston Island, South Shetlands. *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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MATERIAL AND METHODS Fieldwork was carried out in the austral summer between December 2002 and February 2003 with some additional data from 2001 and 2002 on the same colony located on Caleta Argentina – 62° 40´ 088´´ S; 60° 23´ 040´´ W (Fig. 1). The terrain associated with this colony from relatively level terraces on about 10 m above s. l. with steep ridges up to 100 m above the sea level (Fig. 2).

Fig. 1. Location of the studied colony.

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Fig. 2. Gentoo colony at Caleta Argentina, Livingston island.

The objectives were to test the feasibility of using photos of Gentoo penguin colonies taken from a constant point from about 50 m above s. l. with a digital camera Olympus C4000Z as an alternative census method. There are only a few publications describing the use of digital photography to census penguin colonies (Greenfield and Wilson, 1991, Fraser et al. 1999). The pictures were taken on 01 of Jan. 4, 10, and 17 between 10 and 11 a.m. The air temperature varied between 0° and 5° C. When the pictures were taken, constant borders on the terrain had to be taken into account. Their format had to be equal. Then a simple grid was overlapped (Figs. 3, 4, 5, 6). RESULTS AND DISCUSSION On Figures 3, 4, 5, 6, Gentoo nests are mapped and it is possible to count the breeding couples in an easer way. The number of Gentoo pairs breeding on Caleta Argentina varied in the three years (2000, 2001, 2002) – between 84 and 101 couples, and changed markedly in the last breeding season (2003). The population decreased by about 70 %, being lowest at the end of the season. The reason for such an unsuccessful breeding season can be explained through the difficulties in finding sufficient food. Such a decrease in Gentoo breeding pairs was reported by Croxall and Prince (1979) – 75 % at Bird Island in 1978. The authors

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Fig. 3. Number of nests in the colony on 01.01.2003. N=55

J H G F E D C B A 1

2

3

4

5

6

7

8

Fig. 4. Number of nests in the colony on 04.01.2003. N=38

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J H G F E D C B A 1

2

3

4

5

6

7

8

9

10

11

12

9

10

11

12

Fig. 5. Number of nests in the colony on 10.01.2003. N=34

J H G F E D C B A 1

2

3

4

5

6

7

8

Fig. 6. Number of nests in the colony on 17.01.2003. N=16

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speculated that the birds may have had difficulties in finding sufficient food to come into breeding conditions, although no data on food availability was available. Another more reasonable cause for the decrease in the number of penguins is the delaying of egg-laying (Haywood et al. 1985; Priddle et al., 1988). Gwynn (1953) suggested that the availability of snow-free ground was an important factor in the annual variation in the timing of breeding in Gentoo penguins. At South Georgia, 1987 was the most severe winter since 1980 (B.A.S. (British Antarctic Survey, unpublished data), with snow and ice covering the breeding areas until early November, and brash-ice filling the coves, preventing normal patterns of colony attendance. At some colonies, therefore, the physical presence of snow and ice may have delayed breeding (e.g. Johnson Cove), but at other colonies (e.g. Square Pond) nesting areas were clear of ice by early October and egg laying was still delayed by about 30 days. The climate conditions at Caleta Argentina were very similar for the Gentoo breeding pairs and egg-laying in comparison with the previous season was delayed with two weeks. First chick clutched at January 1, 2003. There was also a big pressure of predators – skuas and leopard seals. These are very significant factors influencing the population decrease. CONCLUSIONS Using digital photography provides a cost-effective and efficient alternative for population counting for monitoring purposes. This method gives quick results, completely eliminating the need of a photo-laboratory. The main advantage of using this method is that the nesting penguins are completely undisturbed. ACKNOLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. # 2001-0517. REFERENCES AGNEW D.J. 1997. The CCAMLR ecosystem monitoring programme. 9: 235-242. CCAMLR. 1992. Standard methods for monitoring studies. Convention for the conservation of Antarctic marine living resources, Hobart, Tasmania, Australia. CROXALL J. P., P. A. PRINCE. 1979. Antarctic seabird and seal monitoring studies. Polar Rec. 19: 573-595. FRASER W.R., J.C. CARLSON, P.A. DULEY, E.J. HOLM, D.L. PATTERSON. 1999. Using kite-based Aerial Photography for Conducting Adelie Penguin Censuses in Antarctica. Waterbirds, 22 (3): 435-440.

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GREENFIELD L.G., K.J. WILSON. 1991. Adelie Penguin Colony Estimations from aerial Photography and Ground Counts.Polar Record 27, 161, 1991: 129-130. GWYNN A.M. 1953. The egg-laying and incubation periods of rockhooper, macaroni and Gentoo penguins. A.N.A.R.E. Rep. (Zool.) (B) 1: 1-29. HARRISON P. 1983. Seabirds. An identification guide. London. Croom Helm. HAYWOOD R.B., I. EVERSON, J. PRIDDLE. 1985. The absence of krill from the South Georgia zone, winter 1983. Deep-sea Res. 32: 369-378. PRIDDLE J., J. P. CROXALL, W. R. HAYWOOD, E. J. MURPHY, P. A. PRINCE, S.B. SEAR. 1988. Large scale functions in distribution and abundance of krill – A discussion of possible causes. In: Antarctic Ocean and resources variability. (Ed.). Springer Verlag, Berlin; pp. 169-182. TRIVELPIECE W. Z., G. R. TRIVELPIECE, G. R. GENPEL, J. KJELMYR, N.J. VOLKMAN.1990. Adelie and Chinstrap penguins: their protection as monitors of the Southern ocean marine ecosystem. In: Antarctic ecosystems, ecological change and conservation. (Ed. K. R. Kerry & G. Hempel). Springer Verlag, Berlin; pp. 191-202.

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© PENSOFT Publishers STUDIES ON THE CONTENTS OF CHEMICAL ELEMENTSBulgarian IN SOIL CAntarctic OVER ... Research COMPARATIVE 123 Sofia – Moscow Life Sciences, vol. 4: 123-128, 2004

Comparative Studies on the Contents of Chemical Elements in Soil Cover from Livingston Island, Antarctica MARIA G. SOKOLOVSKA1, JAUME BECH2 1

Forest Research Institute; BAS; St. Kliment Ohridski Blvd., 132; BG-1756 Sofia 2

University of Barcelona, Av. Diagonal, 645, Barcelona 08028, Spain

ABSTRACT The study is a part of long-term ecological surveys of the environment on Livingston island, one of the group of the South Shetland Islands, Antarctica. The natural level of six trace elements (Pb, Cu, Zn, Cd, Mn, Sr) has been analysed as well as for Fe and Al, their variability depending on the situation of the study sites on the Antarctic Land. Analyses have been performed mainly using atomic absorption spectrometry and ICP. The studied samples are from the surface layers of the primary soil cover of the island. The acidity of the soil substrata varies within the limits of pH 5,40-6,50. The organic matter contents has a big amplitude (from 0,26 to 8,04). The comparative juxtaposing of these characteristics permit the classification of a number of regularities as a result of the integral action of all ecological factors. KEY WORDS Antarctic soils, natural level, heavy metals, acidity, organic matter.

INTRODUCTION The chemical elements are distributed and dispersed relatively evenly and everywhere in the Earth crust (Vernadskiy, 1954). The dispersed distribution is normal, common status of the elements. All types of natural formations (rocks, soils, waters, air, plants, etc.) are characterised by their relatively sustainable levels of contents of the elements. Under the influence of the various energetic processes, a re-distribution *Grant B. A. 801 / 1998 of National Fund for Scientific Researches of Bulgaria

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could be carried out, as well as accumulation of the chemical elements. The concentrations of elements, from a geochemical point of view, are anomalies. According to the genesis the anomalies in the environment are natural and anthropogenic (technogenic in particular). The nature on Antarctica, the last discovered continent, is still virgin, with no lasting impact of human presence and following the basic ecological structures, supporting a balance of nature. As part of this setting, soil cover is also subordinated to these conditions and follow, their course. This study deals with problems, which have arisen in the course of the study of the environment of Livingston Island, as part of the long term ecological studies of Antarctica. The aim of the study was to outline specific ecological issues present under contemporary ecological conditions in the region. on the basis of comparative analysis. MATERIALS AND METHODS Livingston Island (southern latitude 62°27’-62°48’ and western longitude 59o45`61°15`) is second in size of the group of Southern Sheltland islands. The islands are a non-stable tectonic zone of a very big importance (Hobbs, 1968). The age of the rocks is from Paleosoic to medium Tertiary. The different authors (Zheng et al.,1996) suppose a complicated process of the vulcanism on Livingston Island, where the magmas are generated from different sources. The formation of the upper rocks is a result of the melting of different magma sources with different degrees of melting. The possible crust source (“the pollution”) could be the Miers Bluff formation (MBF) from early Triace age, which is the local fundament of Livingston Island. From a chemical point of view, the rocks show strong alkaline affinity, which is unique for this island (Smellie et al., 1984). One of the features of the Antarctic environment is the combination between low temperatures and high light intensity during the Antarctic summer and a minimal quality of light during the winter. The climate is cold, dry and stormy. Plants can grow only during the few summer weeks when there is sufficient water with dissolved mineral substances. Land ecosystems are characterized by the turnover of few participants and short ecological food chains. Research began with the processing of material, given to us by members of Bulgarian expeditions in Antarctica (during the period 1994 -1997). Considering the specific natural conditions of the environment, questions treating the peculiarities of the humus formation in the soil cover on Livingston Island were examined (Sokolovska et al., 1996), as well as meso- and micromorphological characteristics of the soil cover on the island (Ilieva, Grozeva, 1999), biogenity and its relationship with the basic physical-and-chemical properties of the soil cover (Nustorova, Grozeva, Gush-

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terova, 2002). This joint study with colleagues from the University of Barcelona, Spain, on the contents of different chemical elements (macro- and trace) in soil samples from the Antarctic will allow to complete and extend the studies after the scientific programme. Surface samples from the primary soil cover of the island came from locations in the central sector, known as Punta Hesperides and Punta Polaka. The selection of the sampling sites through digging or profiles was done according to the terrain (a slope, a depression, or even ground), the type of vegetation (lichen, mosses grass cover) and the habitants of various birds, base ground or rock, with additional secondary influences (erosion, movement of land mass). According to ISO standard procedures (ISO 1991), an aqua regia extraction was carried out - an extract of high quality reagents (Merck and Carbo Erba). The elements content (Pb, Cu, Zn, Cd, Mn, Fe, Sr, Al) was determined through the use of a ICP- POLYSCAN 61E spectrometer at the Barcelona University soil laboratory. The same elements, for comparison, were determined for the Perkin Elmer - nuclear-absorbtion spectrometer (AAS) in Sofia. The results given in ppm, are shown in table 1. Soil samples were analysed aditionally for the content of organic substances (C%) and acidity of soil solutions (pH in H2O). RESULTS AND DISCUSSION The content of elements in the soil cover of the island corresponds to the concrete conditions of soil-formation. At the same time, under conditions such as soil forming materials, climate, relief, vegetation and specific physical-and-chemical properties of the different elements, the chemical result from the changes is “leaching” and re-sedimentation of Fe, Ca, K and other elements. And this causes quite significant differences in their distribution. Thus the studied objects on Livingston Island can be grouped depending on the variety in the vegetation cover and according to their designation to: objects i) with grass cover (object 5, 9, 18); ii) a surface with tree lichens (object 8); iii) screes (object 3,10); iv) bottom sediments on old snow lakes (object 47); v) habitats of birds or their nutritive stations (object 1). The accumulation of greater quantities of elements, such as Pb, Cu, Zn, as well as Sr in substrate below grass communities (objects 5, 9, 18) has a biogenetic character as well (table 1). A decisive role is played by the enrichment with organic mater (78%). In the course of its transformation, more P is separated (130-227 ppm). This group is represented by primitive soils, formed at higher altitudes (300-400 m a.s.l.), whose soil substrata are highly active and with potential acidity. The processes of soil formation are rather young, which does not allow the creation of normal conditions where humus substances reach their maturity in order to form stronger organic mineral complexes (Sokolovska et al., 1996).

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Accumulation of higher quantities of Fe and Mn in samples of fragmented soil cover in screes and among rock complexes (object 3 and 10) is the result of an opposite effect. The processes of shift and transformation, which are of importance with alluvial landscapes and determine the character of distribution of microelements, yield particularly good results. Owing to the stronger influence of the geochemical parameters of the base rock, in this case mechanical sediments, the soil substrate reaches 7-8 cm thickness. It is enriched with alkaline soil cations. The presence of organic substances is weak (0.34%-0,26%). Lower values of pH contribute to the greater migration of heavy metals. Therefore acidity determines the fate of these elements in soil substrates, however only when we consider the leading elementary soil formation processes, resulting in both soil acidity and other soil features in soil variety, including the general regularities of heavy metals. The enrichment of surface layers of primary soil cover of Livingston Island, Antarctica, with microelements once again raises the question whether it was also due to atmospheric emissions.The familiar concept of the quantity of heavy metals, occuring on the land surface through emissions, is given in studies of polar snow (Boutron, 1979). Such studies are not numerous, and the conclusions are not lacking in contradictions due to considerable difficulties, chiefly owing to problems in methodology. Inspite of this they could well represent the natural aerosol background layer. Thus, Cadmium (Cd), which is known to be volatile and an element with a low concentration in the litosphere, is characteristic with the considerable enrichment of part of the studied objects. It is generally believed that volcanic activity was chiefly responsible for chalcophyllic elements - Pb, Cu, Zn, in the course of formation of the natural aerozol background. Their concentration is from 10 to 100 times higher than that of the lithosphere (Herron et al., 1977). In the case of the other elements like Fe and Al, concentrations in aerozols and the surface are approximately identical. This is sufficient ground to consider that these elements could have secondary origins. We came to this conclusion comparing the results of our studies with similar ones, conducted in Europe (Bowen, 1966). This comes to show, that during the last few decades as a result of anthropogenic activities (industry, transport etc), serious pollution with heavy metals has been observed. Fine aerozols can also reach the surface in the form of dry sediments, however selfpurification of the atmosphere is done through precipitation. Therefore, enrichment of the soil cover with these toxic elements depends on the nature of rainfall (snow or rain), the composition and properties of the soil cover, the relief, erosion processes etc. Thus in our case, most of the precipitation presupposes any stronger change of the composition of heavy metals from the soil cover of Livingston Island. A comparison of the results of the concentrations of microelements, obtained according to the two analytical methods (AAS and ICP), notes substantial differences with Pb and Sr (Table 1). Qualitative values, obtained under the ICP method, com-

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Table 1. Concentration of elements (ppm), acidity and organic matter in the soil cover of Livingston Island Objects/ Element Pb Cu Zn Cd Sr Mn Fe Al* P* pH(H2O) C (%)

1

3

10

8

5

9

18

47

42,5 8,7 19,5 26,0 70,0 86,4 1,0 1,3 58,6 4,4 380 229 25252 18567 16616 94 5,80 3,36

38,1 17,5 17,7 18,7 91,8 95,0 0,1 1,3 46,4 4,4 495 364 25539 22222 14967 40 6,35 0,34

54,2 17,5 20,5 17,7 104,0 145,0 0,8 1,0 68,3 5,9 560 317 28734 17543 19595 31 6,40 0,26

37,9 8,7 21,4 18,8 85,6 97,4 0,8 1,0 48,3 7,4 467 313 23322 17398 14043 88 5,90 3,82

23,9 8,7 25,4 29,2 79,1 176,8 0,6 1,8 65,4 14,7 342 242 14642 10672 9418 130 6,00 7,41

53,9 43,9 26,7 14,2 143,0 174,2 0,4 1,3 48,6 1,5 507 184 25799 10234 15947 25 6,50 0,90

58,7 43,9 43,0 39,6 81,5 90,2 0,5 1,1 51,4 4,4 320 201 22882 16959 13266 227 3,75 8,04

36,0 8,8 16,3 15,6 67,6 60.9 1,1 62,9 5,9 327 173 21544 14766 15541 29 5,40 0,97

Numerator - determined by ICP Nominator - determined by AAS * The results of Al and P are determined by AAS only

pared to AAS, are higher for most elements with the exception of Zn and Cd. Generally the tendency in variability of the content of microelements were confirmed by both methods. AAS is a typical monoelemental method which requires more time in individual sampling of the elements, while with ICP the method offers high thresholds of discovery, a high level of automation, and is relatively free of matrix influence. Probably the second method is more suitable for most of the analysed elements. CONCLUSION Antarctica is a remote and secluded region with specific plant and animal communities developing there. The environment of this continent remains unknown. The study of the contents and peculiarities of chemical elements and their distribution in

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the soil cover under the conditions of arctic regions not only enriches and expands existing information on concrete soils, but also provides information about the biogeo-chemical circulation of the studied macro and trace elements including the contribution of the regional and global processes in their migration as fundamental task of the soil monitoring. ACKNOWLEDGMENTS The authors express their gratitude for the collaboration of Dr. Nesho Chipev and assistant Zlatil Vergilov, who carried out the sampling during the scientific expeditions to Livingston Island, Antarctica. REFERENCES VERNADSKIY V.I. 1954. Selected works. Moskva, AN SSSR, vol. 1, 396-410, 519-527. (In Russian) BOUTRON C. 1979. Trace elements content of Greenland snows an east-west transect. - Geochim. Cosmochim. Acta, 32, ¹8, 1253-1258. BOWEN H.J.M. 1966. Trace elements in biochemistry. - London, New York, p. 157. HERRON M.H., C.C. LANGWAY, H.V. WEISS, J.H. CRAGIN. 1977. Atmospheric trace metals and sulphate in the Greenland ice sheet.- Geochim. Cosmochim. Acta, 41, 915-920. HOBBS G.J. 1968. The geology of the South Shetland Islands. - Scientific Report, 47, British Antarctic Survey, London, 1-34. ILIEVA R., M. GROZEVA 1999. Morphology of Soils from the Livingston Island, South Shetland Islands (the Antarctic). - Bulgarian Antarctic Research: Life Sciences, vol. 2, 97-105. NUSTOROVA M., M. GROZEVA, A. GUSHTEROVA 2002. Study on Soils from the Region of Livingston Island (Antarctica). - Bulgarian Antarctic Research: Life Sciences, vol.3, 21-28. SMELLIE J.L., R.J. PANKHURST, M.R. THOMSON, R.E.S. DAVIES. 1984. The geology of the South Shetland Islands: VI Stratigraphy, Geochemistry and Evolution. - Scientific reports, British Antarctic Survey, 87, 1-85. SOKOLOVSKA M., L. PETROVA, N. CHIPEV. 1996. Particulars of Humus Formation in Antarctic Soils: Factors, Mechanisms, Properties. - Bulgarian Antarctic Research: Life Sciences, vol. 1, 7-12. ZHENG X., F. SAABAT, J.L. SMELLIE. 1996. Mesozoic-Cenozoic volcanism on Livingston Island, South Shetland Islands, Antarctica: Geochemical evidences for multiple magma generation processes. - Korean Journal of Polar Research, v.7, 1/2, 35-45.

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Other titles published by Pensoft in 2003-2004 Patrikeev, M. 2004. The Birds of Azerbaijan. Pensoft Series Faunistica No 38, ISSN 1312-0174. ISBN 954642207X, Pensoft Publishers, Sofia-Moscow, 170x240, 250 distribution maps, 6 graphs, 70 colour and black and white photographs, complete bibliography, index. In English. Hardback, 500pp. Evans, W., Yablokov, A. 2004. Noninvasive Study of Mammalian Populations. ISBN 9546422045, Pensoft Publishers, Sofia-Moscow, 165x240, 53 figs, bibliography. In English. Hardback, 118pp. Fedorov, LA., Yablokov, AV. 2004. Pesticides - The Chemical Weapon that Kills Life. The USSR‘S Tragic Experience. Pensoft Environmental Series No 4. ISBN 9546422053, Pensoft Publishers, Sofia-Moscow, 165x240, tables, bibliography. In English. Hardback, 136pp. Pruitt, W.O., Baskin L.M. 2004. Boreal Forest of Canada & Russia. ISBN 9546421995, Pensoft Publishers, Sofia-Moscow, 165x235, 84 figures, tables, index, references. In English & Russian. Hardback, 160pp. Mironov, O. 2004. The Environment and the Violations of Human Rights. Special Report of the Commissioner on Human Rights in the Russian Federation. ISBN 9546422126, Pensoft Publishers, Sofia-Moscow, 140x225. In English. Paperback, 95 pp. Ingrisch, S, Willemse, F. 2004. Bibliographia Systematica Orthopterorum Saltatoriorum. Systematic Bibliography of Saltatorial Orthoptera from Linnaean Times to the End of the 20th Century (about 1750 to 2000). With a CD.. Pensoft Series Faunistica No 37, ISSN 1312-0174. ISBN 9546422061, Pensoft Publishers, Sofia-Moscow, 170x240, list of references, extended CD version. In English. Hardback, 480pp. Mikhaljova, E. 2004. The Millipedes (Diplopoda) of the Asian part of Russia. Pensoft Series Faunistica No 39, ISSN 1312-0174. ISBN 9546422037, Pensoft

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Publishers, Sofia-Moscow, 165x235, 35 maps, 675 b/w figs, color photos, references, index. In English. Hardback, 292pp. Kuzmin, SL (ED). 2004. Amphibians of the Russian Far East. Advances in Amphibian Research, vol.8, ISSN 1310-8840. ISBN 9546421952, Pensoft Publishers, Sofia-Moscow, 165x235, color plates, figures, tables, references, index. In English. Hardback, 462pp. Kuzmin, S, Altig, R. (ED). 2003. Ecological Specificity of Amphibian Populations. Advances in Amphibian Research in the Former Soviet Union, Volume 7, ISSN 13108840. ISBN 9546421782, Pensoft Publishers, Sofia–Moscow, In English. Paperback, 220pp. Legakis, A, Sfenthourakis, S, Polymeni, R, Thessalou-Legaki, M. 2003. The New Panorama of Animal Evolution. Proceedings of the XVIII International Congress of Zoology, Athens, Greece, September, 2000. ISBN 9546421642, Pensoft Publishers, Sofia & Moscow, 170x240, graphs, tables, references. In English. Hardback, 724pp. Michev T., Profirov L. 2003. Mid-winter Numbers of Waterbirds in Bulgaria (19772001). Results From 25 Years of Mid-winter Counts Carried Out at the Most Important Bulgarian Wetlands. ISBN 9546421758, Pensoft Publishers, Sofia-Moscow, 210x290, tables, graphs, excellent b/w drawings, index, bibliography. In English. Paperback, 168 pp. Krassilov, VA. 2003. Terrestrial Paleoecology and Global Change. Russian Academic Monographs, No 1. ISBN 9546421537, Pensoft Publishers, Sofia-Moscow, 170x240, graphs, photos, tables, bibliography. In English. Hardback, 480pp. Golemansky, V., Chipev, N. 2002. Bulgarian Antarctic Research.Life Sciences, Volume 3. ISBN 9546421596, Pensoft Publishers, Sofia & Moscow, 165x235, tables, morphological drawings, figures and photos. In English. Paperback, 105pp. Romanenko, EV. 2002. Fish and Dolphin Swimming. Russian Academic Monographs, No 2. ISBN 9546421502, Pensoft Publishers, Sofia-Moscow, 170x240, graphs, tables, photos, extended bibliography of over 800 references. In English. Hardback, 430pp.