Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 8, Num. 3, 1997

IDENTIFICATION OF 2-METHYLISOBORNEOL (MIB) AND GEOSMIN AS VOLATILE METABOLITES OF STREPTOMYCES VIOLACEUSNIGER

I. SAADOUN, K.K. SCHRADER^1 and W.T. BLEVINS^2

Dept. of Biological Sciences, Jordan University of Science & Tech., Irbid-22110, Jordan,
^1 USDA-Agricultural Research Service, Greenville, MS 38703 and
^2 Nuclear Sciences Bldg, Auburn University, Auburn, AL 36849

Code Number: AC97006
Sizes of Files:
    Text: 11.3K
    Graphics: Line drawings (gif) - 10.4K

ABSTRACT. An actinomycete isolate recovered from the sediment of a local stream was identified as Streptomyces violaceusniger. The isolate was shown to produce the two common off-flavour compounds, 2-methylisoborneol (MIB) and geosmin.

Geosmin (trans-1,10-dimethyl-trans-9-decalol) (Fig. 1A) and 2-methylisoborneol (1,2,7,7-tetramethyl-exo-bicyclo-heptan-2:ol or MIB) (Fig. 1B) are odorous metabolites produced in the aquatic environment by actinomycetes (Blevins, 1980; Gerber, 1969, 1979, 1983; Gerber & Lechevalier, 1965; Izaguirre et al., 1982; Rosen et al., 1968, 1970; Schrader & Blevins, 1993; Thysen, 1936) and cyanobacteria (Kikuchi et al., 1973). The occurrence of these compounds in drinking water and fish is a world-wide problem (Aschner et al., 1967; Bays et al., 1970; Brown & Boyd, 1982; Cees et al., 1974; Person, 1979, 1980; Piet et al., 1972; Romano & Safferman, 1968; Schrader & Blevins, 1993; Thysen, 1936).

Figure 1. Chemical structures of geosmin (A) and 2-methylisoborneol (2-MIB) (B).

MATERIALS and METHODS

Isolation of actinomycetes. Sediment samples, collected from a local stream, Auburn, AL, were serially diluted and spread-plated onto 1.0% yeast extract - 10% dextrose (YD) agar (pH 7.5), supplemented with cycloheximide (50-75 ug/ml) to inhibit growth of fungi. Inoculated YD plates were incubated at 29 C for 3 dd. Leathery and/or chalky colonies were identified as actinomycetes and purified by streaking onto YD plates. Selected isolates were speciated by gas chromatography (GC) fatty acid methyl ester analysis (Microbial Identification System, MIDI, Newark, Del., USA).

Detection of geosmin and MIB production. Purified isolates were screened for production of geosmin and MIB. Individual isolates were streaked onto YD plates to obtain confluent growth and a single depression slide containing 0.1 ml of dibutylphthalate (DBP) (Eastman Kodak, Rochester, N.Y.) was placed in the lid of each inverted Petri dish. At 5, 7, and 14 dd incubation at 29 C, 0.2 ul of the dibutyl phthalate (DBP) was injected into a Perkin-Elmer model 8500 gas chromatograph (Perkin-Elmer Corp., Norwalk, Conn.). The absorption of geosmin and MIB by DBP permits a quantitative detection of off-flavour compounds produced by microbial isolates (Durham 1975).

A bonded Carbowax 20m fused silica capillary column (50m x 0.25mm i.d. x 0.25 um film thickness; Perkin-Elmer) was used with a flame ionisation detector (FID). Helium served as carrier gas at a linear velocity of 22cm/s and a split ratio of 5:1. The column temperature was set at 100 C for 1 min then programmed to 160 C at 20 C/min and iso-time of 1 min, 210 C at 10 C/min and iso-time of 4min and finally to 230 C at 5 C/min and iso-time of 22 min. The injector and detector temperatures were set at 300 C and the gauge pressure was set at 20psi (1 psi = 6.895kPa).

To confirm geosmin and MIB production, strains were grown in YD broth (pH 7.5) at 29 C in shaken cultures (200rpm) for 7 days. Cultures were concentrated (20%) and extracted with 20% and 10% volumes of methylene chloride. Combined extracts were concentrated to 0.1ml for further analysis by GC.

Odour metabolite detection was accomplished using the above gas chromatograph equipped with a FID. Analysis was carried out on a stabilwax fused silica capillary column (30m x O.25mm i.d. x 0.25 um film thickness; Supelco, Inc., Bellefonte, PA). The column temperature was equilibrated at 80 C for 2 min, then programmed to 200 C at 6 C/min and iso-time of 1 min and finally to 250 C at 10 C/min and iso-time of 7 min. The injector and detector were set at 300 C, the average linear gas velocity was set at 20 cm/s with helium used as the carrier gas and the split ratio was set at 50:1 (Schrader & Blevins, 1993).

Borneol and different concentrations of authentic standard of geosmin (Wako Chemicals U.S.A., Inc., Dallas, TX) and MIB (Dr. R.T. Lovell, Department of Fisheries and Allied Aquaculture, Auburn University, AL) were used to make cubic fit standard curves from PE Nelson Omega System hardware (Perkin-Elmer). These curves were used to identify and quantify geosmin and MIB in culture samples. The GC peaks corresponding to geosmin and MIB were confirmed by GC-mass spectrometry using a model VG70E mass spectrometer (VG Analytical, Manchester, England) at the Auburn University Mass Spectrometry Center.

RESULTS and DISCUSSION

Isolates of actinomycetes were tentatively assigned to the genus Streptomyces on the basis of morphological and cultural characteristics.

Isolate C4-S produced both MIB and geosmin, as confirmed by GC analysis. Colonies of the isolate initially appear leathery but later develop a grey, chalky appearance with production of diffusible dark brown pigments in YD and oatmeal agar. Strain C4-S was identified as Streptomyces violaceusniger by fatty acid methyl ester analysis.

Mass spectra of MIB and geosmin produced by S. violaceusniger were essentially identical to those of their respective standards, with major ion peaks at 43, 55, 69, 95, 108, 135 and 150m/e and at 41, 55, 69, 97, and 112m/e respectively (Figs. 2 and 3).

Figure 2. Mass spectra of (A) standard 2-MIB (see text) and (B) of the metabolite produced by S. violaceusniger.

Figure 3. Mass spectra of (A) geosmin from Wako Chemicals USA, Inc. and (B) of the metabolite produced by S. violaceusniger.

Further experiments are needed to study the physiology of MIB production by S. violaceusniger and to correlate the abundance of the isolate and MIB accumulation. In fact the isolation of the actinomycete from mud does not imply that its propagules are abundant in the habitat or that the isolate itself is the primary contributor of off-flavour problems in drinking water.

ACKNOWLEDGEMENTS.

This research was financed in part by grants from the U.S. Department of the Interior's Geological Survey, Washington, D.C., as authorised by the Water Resources Research Act of 1984 (P.L 98-242), through the Water Resources Research Institute at Auburn University. I. Saadoun was supported by a scholarship from the Jordan University of Science & Technology.

REFERENCES

Aschner, M., C. Laventer & L Chorin-Kirsch (1967). Off-flavor in carp from fish ponds in the coastal plain and the Galilee. Bamijdgeh, Bull. Fish Cult. in Israel., 19:23-25

Bays, L R., N.P. Burman & M.W. Lewis (1970). Taste and odour in water supplies in Great Britain: A survey of the present position and problems for the future. Water Treat. Exam., 19:136-160

Blevins, W.T. 1980. Geosmin and other odorous metabolites of microbial origin, In: F.E. Guthrie & J.J. Perry (eds.) Introduction to Environmental Toxicology. Elsevier-North Holland, pp. 350-357

Brown, S.W. & C.E. Boyd. (1982). Off-flavor in channel catfish from commercial ponds. Trans. Am. Fish. Soc., 111:379-383

Cees, B., J. Zoeteman & J.W. Soboslai (1974). Cause and identification of taste and odour compounds in water. Sci. Total Environ., 3:103-115

Gerber, N.N. (1969). A volatile metabolite of actinomycetes, 2-methylisoborneol. J. Antibiot., 22:508-509

Gerber, N.N. (1979). Volatile substances from actinomycetes: their role in odor pollution of water. Crit. Rev. Microbiol., 7:191-214

Gerber, N.N. (1983). Volatile substances from actinomycetes: their role in odor pollution of water. Water Sci. Technol., 15:115-125

Gerber, N.N. & H.A. Lechevalier (1965). Geosmin, an earthy-smelling substance isolated from actinomycetes. Appl. Microbiol., 13:935-938

Izaguirre, G., C.J. Hwang, S.W. Krasner & M.J. McGuire (1982). Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems. AppI. Environ. Microbiol., 43:708-714

Kikuchi, T., T. Mimvra, Y. Masada & T. Inoue (1973). Odorous compounds in water supplies. Identification of geosmin from water of lake Inbanuma by mass spectrometry combined with gas chromatography. Chem. Pharm. Bull., 21:1847-1848

Person, P.E. (1979). The source of muddy odor in bream (Abramus brama) from the Porvoo Sea Area (Gulf of Finland). J. Fish Res. Board. Can., 36:883-890

Person, P.E. (1980). Sensory properties and analysis of two muddy odour compounds, geosmin and 2-methylisoborneol in water and fish Wt. Res., 14: 1113-1118

Pier, G.J., B.C.J. Zoetman & A.J.A. Krayeveld (1972). Earthy smelling substances in surface waters of the Netherlands. Water Treat. Exam., 21:281-286

Romano, A.H. & R.S. Safferman (1968). Studies on actinomycetes and their odors. J. AWWA, 55:169-176

Rosen, A.A., R.S. Safferman, C.I. Mashni & A.H. Romano (1968). Identity of odorous substances produced by Streptomyces griseoluteus. Appl. Microbiol. 16:178-179

Rosen, A.A., C.I. Mashni & R.S. Safferman (1970). Recent developments in the chemistry of odour in water the cause of earthy/musty odour. Water Treat. Exam., 19:106-110

Schrader, K.K. & W.T. Blevins (1993). Geosmin-producing species of Streptomyces and Lyngbya from aquaculture ponds. Can. J. Microbiol., 39: 834-840

Thysen, A.C. (1936). The origin of an earthy of muddy taint in fish. I. The nature and isolation of the taint. Ann. Appl. Biol., 23: 99-104.

Copyright 1997 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia

The following images related to this document are available:

Line drawing images