OCCURRENCE OF VOLATILE NITROGEN-CONTAINING COMPOUNDS IN NITROGEN-FIXING CYANOBACTERIUM Aphanizomenon flos-aquae

Transcription

1 Journal of Chemical Ecology, Vol. 26, No. 6, 2000 OCCURRENCE OF VOLATILE NITROGEN-CONTAINING COMPOUNDS IN NITROGEN-FIXING CYANOBACTERIUM Aphanizomenon flos-aquae VALERY M. DEMBITSKY, * ILIA SHKROB, and OVADIA LEV Division of Environmental Sciences The Fredy and Nadine Herrmann Graduate School of Applied Sciences P.O. Box 39074, The Hebrew University of Jerusalem Jerusalem 91390, Israel (Received February 17, 1999; accepted January 23, 2000) Abstract The occurrence of volatile nitrogen-containing compounds in the cyanobacterium Aphanizomenon flos-aquae has been studied. Twenty compounds belonging to four groups amides of carboxylic and fatty acids, heterocyclic compounds and their derivatives, aromatics, and alkoxyamines and amides were identified by gas chromatography mass spectrometry that used serially coupled capillary columns with different polarity of stationary phases. Distribution of bioactive non-toxic and toxic nitrogen-containing metabolites in cyanobacteria are discussed. Key Words Aphanizomenon flos-aquae, cyanobacteria, volatile nitrogen-containing compounds. INTRODUCTION Cyanobacterial blooms occasionally occur in many marine, freshwater, and brackish natural and man-made lakes and reservoirs around the world (reviewed in Plinski and Codd, 1997; Sellner, 1997; Paerl, 1996; Falconer, 1994). Most undesirable are the microalgae blooms and the drift to cyanobacterial dominance in the phytoplankton community structure (Paerl, 1996). Biological and chemical characteristics of nitrogen containing toxins, mainly those found in Aphanizomenon and other cyanobacteria, have been reviewed previously (Carmichael, 1997; Sivonen, 1996; Codd and Poon, 1988; Slater and Blok, 1983). *To whom correspondence should be addressed / 00/ $18.00/ Plenum Publishing Corporation

2 1360 DEMBITSKY, SHKROB, AND LEV Many cyanobacterial biotoxins (neurotoxins, hepatotoxins) have been isolated and identified (Sivonen, 1996; Shimizu, 1993). Previously isolated toxins from Aphanizomenon spp. include nitrogen-containing compounds such as saxitoxin and neosaxitoxin (Ikawa et al., 1982), and cylindrospermopsin, the sulfateguanidinium zwitterion in Aphanizomenon ovalisporum (Banker et al., 1997). This toxin also was isolated from the cyanobacterium Cylindrospermopsis raciborskii and from Umezakia natans (Codd and Poon, 1988). The cyanobacterium Alphanizomenon flos-aquae contains some nontoxic pterin glycosides (Ikawa et al., 1995). This report describes the composition of volatile nitrogen-containing compounds that were identified by gas chromatography mass spectrometry (GC-MS) from cyanobacteria Aphanizomenon flos-aquae. METHODS AND MATERIALS Biological Material. Lyophilized cells of two strains of Aphanizomenon flos-aquae were used for extraction of organic volatile compounds. Strain 1 was collected from a cyanobacterial bloom in Klamath Lake (Oregon, USA), and strain 2 in Upper Klamath Lake (Oregon), obtained from Prof. I. Dor. Extraction Procedure. Compounds were isolated from lyophilized cells of Aphanizomenon flos-aquae. Cells were sequentially extracted with organic solvents and collected in four fractions: pentane, acetone, dichloromethane, and ethylacetate, as described by Dembitsky et al. (1999). All fractions were evaporated under nitrogen at 10 C and stored at 10 C before GC-MS analysis. GC-MS Analysis. Volatile compounds extracted by acetone (fraction 2), dichloromethane (fraction 3), and ethylacetate (fraction 4) were separated by using a gas chromatograph Hewlett-Packard 5890 (series II), which was modified to accept a glass capillary column. A Hewlett-Packard GC-mass selective detector (5971B MSD) was used. Volatile fractions were analyzed by GC on serially coupled capillary columns with consecutive nonpolar and semipolar stationary phases (Dembitsky et al., 1999): RTX-1 (Restek) 30 m 0.32 mm ID, 1 mm film thickness, coupled with a secondary capillary column RTX-1701 (Restek) 30 m 0.32 mm ID, 0.25 mm film thickness. The GC oven was programmed at 40 C for 2 min, then 2 C/ min to 300 C and held 20 min at 300 C. Injector temperature was kept at 180 C (splitless). The flow rate of carrier gas (helium) was 25 cm/ sec. The mass detector was operated at 194 C with an ionization energy of 70 ev. Scan range was from 30 to 650 m/ z with a scan rate of 0.9 scan/ sec. Solvent delay was 12 min. Volatile nitrogen-containing compounds were identified from a search of mass spectral libraries (NBS49, NBS75, Wiley 138 & 275).

3 NITROGEN-CONTAINING COMPOUNDS IN CYANOBACTERIUM 1361 RESULTS AND DISCUSSION Chemical Composition. Chemicals identified in the two strains of A. flosaquae are shown in Figures 1 3. Although the analysis yielded more than 150 peaks, the structures of only 20 compounds could be confirmed at >90% confidence level. Compounds are divided into a few groups, the most common being amides of carboxylic acids (Figure 1). The following compounds are represented: derivatives of acetic acid (N-methyl-acetamide, N-(2-phenylethyl)-acetamide, and N-ethyl-N-phenyl-acetamide); derivatives of formic acid (N,N-dimethyl-for- FIG. 1. Amides of carboxylic acids identified from cyanobacterium Aphanizomenon flosaquae.

4 1362 DEMBITSKY, SHKROB, AND LEV FIG. 2. Heterocyclic compounds identified by GC-MS in the cyanobacterium Aphanizomenon flos-aquae. mamide, N-(2-phenylethyl)-formamide); and amides of fatty acids [9(Z)-octadecenamide, 3-methyl-butanamide, N-acetyl-propanamide, and benzen-acetamide]. All compounds of this first group were found in both strains. The second group consists of heterocyclic compounds and their derivatives (Figure 2): 2-piperidinone, 2,3,4,5-tetrahydropyridazine, 3-ethyl-4-methyl-1H-pyrrole-2,5-dione, and 2-methyl-5-methoxy-4-pyridinol (found in strain 1 only), and derivatives of 2- pyrrolidinone both methyl and trimethyldodecyl (found in both strains, Figure 2). The third group consists of amines of aromatic compounds such as 3- methoxy-benzenamine and 8-amino-2-naphthalenol (found in both strains). The fourth group is represented by 2,2-dimethoxy-N-methyl-ethanamide (found in both strains) and two derivatives of hydroxylamine (Figure 3). A majority of the toxins isolated from cyanobacterium are nitrogen-containing compounds (Carmichael, 1997; Patterson et al., 1994). Many investigations of cyanobacterial toxicity in aquatic environments have been promoted by the occurrence of poisoning incidents affecting agricultural livestock, wild animals, birds, fish, and other water invertebrates (Sellner, 1997; Christoffersen,

5 NITROGEN-CONTAINING COMPOUNDS IN CYANOBACTERIUM 1363 FIG. 3. Aromatic amines and other compounds identified from cyanobacterium Aphanizomenon flos-aquae. 1996; Falconer, 1994). Besides toxins, the blue-green algae are known to synthesize pharmacologically active secondary products that have potential roles as drugs. Cyanobacterium have been investigated in the last decade with growing intensity (Underdal, 1995; Patterson et al., 1994). Substances isolated include amides, polyketides, peptides, alkaloids, and mycosporins (Bandaranayke, 1998; Weckesser et al., 1996; Namikoshi et al., 1992; Paul, 1992; Teuscher et al., 1992). The cyanobacteria invesigated specifically for amines is Microcystis aeruginosa (Herrmann and Jüttner, 1977). Only 2-amino-propane has been identified, although trimethylamine, 1-aminopropane, and ethanolamine are found in the culture of the golden brown alga Ochromonas danica and in the green alga Chlamydomonas (Herrmann and Jüttner, 1977). Amides of carboxylic and fatty acids, named malyngamides, are widespread in nature and are found in marine cyanobacteria, algae, and some invertebrates (Thaker et al., 1997; Pennings et al., 1996; Todd and Gerwick, 1995; Orjala et al., 1995). Amides of fatty acids are often incorporated into lipid molecules such as ceramides, sphingolipids, N-acylated lipids, and bacterial lipoproteins. They act as lipid bioregulators formed from long-chain saturated and unsaturated fatty acids via amidation by the corresponding amines (Bezuglov et al., 1998; Di Marzo, 1998).

6 1364 DEMBITSKY, SHKROB, AND LEV The cyanobacterium Microcoleus lyngbyaceus, which produces a variety of nitrogen-containing secondary metabolites (malyngamide A and B) is eaten by the sea hare Stylocheilus longicauda, which sequesters them and transforms some into related compounds (Pennings et al., 1996). The cyanobacteria as reported by Paul (1992) produce aromatic compounds that enter into toxic structures (Carmichael, 1997; Sivonen, 1996; Shimizu, 1993; Codd and Poon, 1988), and some volatile aromatics are found in other cyanobacteria (Slater and Blok, 1983; Tsuchiya et al., 1981). For instance, naphthalene and chloro-, bromo-, methyl-, and ethyl-benzene derivatives were isolated from cultures of blue-green algae and microalgae (Jüttner and Wurster, 1979; Tsuchiya et al., 1979; Carmichael, 1986). The wide range of secondary metabolites from cyanobacteria also includes cyclic depsipeptides and cyanopeptolins, which may serve as biochemical and pharmacological tools because most of them exhibit protease-inhibitory and/ or cytotoxic activities (Weckesser et al., 1996). The study of freshwater and marine toxins and nontoxic nitrogen-containing components is likely to contribute to our understanding of the process of transformation of nontoxic compounds to toxins under extreme ecological conditions. We are interested in other lipophilic components, such as dioic and fatty acids, hydrocarbons of the genus Aphanizomenon that are dependent on ecological factors in pools (Murata and Nishida, 1987). Further study may also lead to development of new pharmacological agents. REFERENCES BANDARANAYKE, W. M Mycosporins: Are they nature s sunscreens? Nat. Prod. Report. 15: BANKER, R., CARMELI, S., HADAS, O., TELTSCH, B., PORAT, R., and SUKENIK, A Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. J. Phycol. 33: BEZUGLOV, V. V., BOBROV, M. Y., and ARCHAKOV, A. V Bioactive amides of fatty acids. Biochemistry (Moscow) 63: CARMICHAEL, W. W Algal toxins. Adv. Bot. Res. 12: CARMICHAEL, W. W The cyanotoxins. Adv. Bot. Res. 27: CHRISTOFFERSEN, K Ecological implications of cyanobacterial toxins in aquatic food webs. Phycologia 36(Suppl. 6): CODD, G. A., and POON, G. K Cyanobacterial toxins, pp , in L. J. Rogers and J. R. Gallon (eds.). Biochemistry of the Algae and Cyanobacteria, Clarendon Press, Oxford. DEMBITSKY, V. M., SHKROB, I., and DOR, I Separation and identification of hydrocarbons and other volatile compounds from cultured blue-green alga Nostoc sp. by gas chromatography mass chromatography using serially coupled capillary columns with consecutive nonpolar and semipolar stationary phases. J. Chromatogr. 682: DI MARZO, V Endocannabinoids and other fatty acid derivatives with cannabimimetic properties: Biochemistry and possible physio-pathological relevance. Biochim. Biophys. Acta 1392:

7 NITROGEN-CONTAINING COMPOUNDS IN CYANOBACTERIUM 1365 FALCONER, I. R Mechanism of toxicity of cyclic peptide toxins from blue-green algae, pp , in I. R. Falconer (ed.). Algal Toxins in Seafood and Drinking Water. Academic Press, Cambridge. HERRMANN, V., and JÜTTNER, F Excretion products of algae. Identification of biogenic amines by gas-chromatography and mass spectrometry of their trifluoroacetamides. Anal. Biochem. 78: IKAWA, M., WEGENER, K., FOXALL, T. L., and SASNER, J. J., JR Comparison of the toxins of the blue-green alga Aphanizomenon flos-aquae with the Gonyaulax toxins. Toxicon 20: IKAWA, M., SASNER, J. J. JR., HANEY, J. F., and FOXALL, T. L Pterins of the cyanobacterium Aphanizomenon flos-aquae. Phytochemistry 38: JÜTTNER, F., and WURSTER, K Einfach Anordnung zur Adsorption von Geruchsstoffen aus Algen an Tenax GC und deren Uberfuh rung in Gaschromatographie-System. J. Chromatogr. 175: MURATA, N., and NISHIDA, I Lipids of blue-green algae (Cyanobacteria), pp , in P. K. Stumf (ed.). The Biochemistry of Plants, Vol. 9. Academic Press, New York. NAMIKOSHI, M., SIVONEN, K., EVANS, W. R., SUN, F., CARMICHAEL, W. W., and RINEHART, K. L Isolation and structures of microcystins from a cyanobacterial water bloom (Finland). Toxicon 30: ORJALA, J., NAGLE, D., and GERWICK, W. H Malyngamide H, an ichthyotoxic amide possessing a new carbon skeleton from the Caribbean cyanobacterium Lyngbya majuscula. J. Nat. Prod. 58: PAERL, H. W A comparison of cyanobacterial bloom dynamics in freshwater, estuarine and marine environments. Phycologia 35(Suppl. 6): PATTERSON, G. M., LARSEN, L. K., and MOORE, R. E Bioactive natural products from bluegreen algae. J. Appl. Phycol. 6: PAUL, V. J Ecological Roles of Marine Products. Comstock Publ., Ithaca, New York. PENNINGS, S. C., WEISS, A. M., and PAUL, J. J Secondary metabolites of the cyanobacterium Microcoleus lyngbyaceus and the sea hare Stylocheilus longicauda: Palatability and toxicity. Mar. Biol. 126: PLINSKI, M., and CODD, G. A Cyanobacteria a factor of animal intoxification: A review. Medyc. Weteryn. 53:8 10. SELLNER, K. G Physiology, ecology, and toxic properties of marine cyanobacteria blooms. Limnol. Oceanogr. 42: SHIMIZU, Y Microalgal metabolites. Chem. Rev. 93: SIVONEN, K Cyanobacterial toxins and toxin production. Phycologia 35(Suppl 6): SLATER, G. P., and BLOCK, V. C Volatile compounds of the cyanophyceae a review. Water Sci. Tech. 15: TEUSCHER, E., LINDEQUIST, U., and MUNDT, S Blue-green algae, sources of natural active agents. Pharm. Ztg. Wiss. 137: THACKER, R. W., NAGLE, D. G., and PAUL, V. J Effects of repeated exposures to marine cyanobacterial secondary metabolites on feeding by juvenile rabbitfish and parrotfish. Mar. Ecol. Prog. Ser. 147: TODD, J. S., and GERWICK, W. H Malyngamide I from the tropical marine cyanobacterium Lyngbya majuscula and the probable structure revision of stylocheilamide. Tetrahedron Lett. 36: TSUCHIYA, Y., SHUDO, K., and OKAMOTO, T Odorous compounds of the blue green algae Oscillatoria sp., and river water. Identification of 2-methyl-isoborneol, geosmin, p-cresol, indole, and 3-methyl-indole. Eisei Kagaki 25: TSUCHIYA, Y., MATSUMOTO, A., and OKAMOTO, T Identification of volatile metabolites pro-

8 1366 DEMBITSKY, SHKROB, AND LEV duced by blue-green algae, Oscillatoria splendida, O. amoena, O. geminata, and Aphanizomenon species. J. Pharm. Soc. Jpn. Yakugaku Zasshi 101: UNDERDAL, B Water pollution-health aspects. Norsk Vet. Tidsskr. 107: WECKESSER, J., MARTIN, C., and JAKOBI, C Cyanopeptolins, depsipeptides from cyanobacteria. Syst. Appl. Microbiol. 19: