Odorous Compounds in Natural Waters 2-Exo-Hydroxy-2-Methylbornane, the Major Odorous Compound Produced by Several Actinomycetes Lloyd L. Medsker, David Jenkins, and Jerome F. Thomas College of Engineering, Sanitary Engineering Research Laboratory, University of California, Berkeley, Richmond Field Station, Richmond, Calif. 94804
C. Koch Department of Chemistry, University of California, Berkeley, Calif.
The camphor-smelling compound 2-exo-hydroxy-2-methylbornane has been isolated from the culture broth of the organisms Streptomyces antibioticus No. 5324, Streptomyces praecox ATCC 3374, and Streptomyces griseus ATCC 10137, and has been shown to be the major odorous component produced by these organisms.
R
ecent literature has reported work on the isolation and identification of putrid and musty-earthy-smelling compounds produced by Actinomycetes which are capable of causing objectionable odors in water supplies (Collins and Gaines, 1964; Dougherty, Campbell, et al.? 1966; Gerber, 1968; Medsker, Jenkins, et a/., 1968). This paper describes the isolation and identification of a camphor-smelling compound 2-exo-hydroxy-2-methylborCH3
\;"""HI
2-EXO-HYDROXY
-2-METHYL
B O R N A N E I11
nane (I) which is the major odorous constituent produced by three out of 28 species of Actinomycetes surveyed. Materials and Methods Streptomyces antibioticus N o 5234, obtained from J. K. G. Silvey, North Texas State University, Denton, Tex., was maintained onslantsof Emerson's Medium (grams per liter: glucose, 10.0; yeast extract, 10.0; beef extract, 4.0; peptone, 4.0; NaCl, 2 . 5 ; agar, 20.0). The slants were incubated a t room temperature in a humid incubator until growth was well established, then stored at 10" C. until needed. For batch growth, about 300 ml. of Emerson's Medium with 15 to 18 grams of agar per liter was layered in the bottom of a 2.5-liter culture flask; when growth was abundant (three o r four days), about 300 ml. of the medium without agar was introduced, and the vessels were left undisturbed until they were harvested after six or seven days. Streptomyces praecox, ATCC 3374 and Streptoin>ws griseus, ATCC 10137, both obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, were maintained and batch cultured in the same manner as S. antibioticus. The yield of I was between 1 to 3 mg. per culture vessel for all three of these organisms. I n addition, an extract from the 476 Environmental Science & Technology
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fermentation broth of S. griseus, ATCC 10137. containing a yield of I was provided by L. H. Sarett. Merck, Sharpe and Dohme Research Laboratories, Rahway, N. J. 07065. Analytical gas chromatography was carried out with a Varian Aerograph Model 1520-B gas chromatograph. A Varian Aerograph Model 700 Autoprep was used for preparative work. Instrumental analyses were carried out with a Varian Model HA-100 nuclear magnetic resonance spectrometer. a Consolidated Electrodynamics Corp. Model 110 mass spectrometer, and a Cary-White Model 90 infrared spectrometer. Sample material from the Actinomycetes was collected by steam distillation of the combined culture medium and mycelium. The distillates were salted with about 20% NaCl and extracted twice with 5 % (v./v.) methylene chloride. The combined extracts were slowly evaporated to 0.3 to 0.5 ml. at room temperature under a reduced pressure of 20 mni. Hg. Purification was accomplished by preparative gas chromatography, using a 4.7-mm. i.d. X 5-meter AI column packed with 1 % Carbowax 20 M on 100- to 120-mesh Chromosorb G, AW. The peak of interest tluted in about 90 minutes, using 20 ml. per minute helium carrier gas flow and a column temperature of 95" C. The compound was trapped in a 30-cm. Y I-mm. i.d. glass capillary tube fitted against the exit port of the instrument at the time of peak elution. A sample of I was prepared by a Grignard synthesis originally proposed byZelinsky (1901); 4.9 grams of M g was reacted with 30.7 grams of CHJ in 100 ml. ether. The 25 grams of dcamphor in 100 ml. ether was added rapidlq and allowed to react for 30 minutes. The yield of I was about 30,x. and was quite pure except for the residual camphor which was removed by passing the mixture through an alumina column. Further purification was done with gas chromatography on the same column used for the preparative separation of I. The identity of this product and a sample of I obtained from S. griseus was established by comparison of m.p. and N M R spectra. The identical nature of the camphor-smelling material from the three species of Actinomycetes reported here was established by mass spectrometry. Results, Discussion und Conclusions
Compound I is an extremely volatile white solid with 11i.p. 158 to 160" C.; retention volume index 1179, and threshold odor 0.10 pg, per liter; Compound I reacts readily with Lucas reagent. A mass spectrum (Figure I ) gave the empirical formula CnH,,O with the parent peak at m.ie. 168 and base peak at 95. The proposed fragmentation of I in the mass spectrometer is shown in Figure 2. The N M R spectrum showed unsplit methyl peak absorptions at 6 = 1.20, 1.08, 0.85, and 0.82. The infrared spectrum showing hydroxyl absorbances at 3620 and 3480 cm.-' is shown in Figure 3.
Ioci
c r
80iL.
I
1 EO-
:[ 40
t
f
I
2cL p
, ?
-2
Ill 40
lli
60
li
AH
?=IO8
7
1 1 1
80 100 m/e
I20
140
I60
180
J
+=
+=I25
Figure 1. Low-resolution mass spectrum of I Source temperature, 125' C.; ionizing voltage, 70v.
-
L
Figure 2. Proposed fragmentation of I in mass spectrometer
-
-
-~12
xc
Figure 3.
I07
Infrared spectrum of I, approximatel) 1
~b-
in CCll
From studies with many species of Actinomycetes, evidently the characteristic odor produced by these organisms is due largely to the presence of geosmin, identified by Gerber (1968). However. the over-all odor is always the result of a combination of many odorous compounds and in some species, such as those discussed here, the major odorous component is not geosmin but a somewhat similar compound present in about 10 times higher concentration. Geosmin and I have many structural features in common. Thus both contain a tertiary and highly sterically hindered OH group as their sole functionality. Both compounds have saturated ring systems with methyl substituents. A further study of earthy-musty odors might reveal that compounds with similar structural features to I and geosmin are responsible for the earthy-musty odor.
U. S. Department of Agriculture, Albany, Calif., for their assistance with instrumental analysis and interpretation; J. K. G. Silvey for providing many of the Actinomycete cultures surveyed; T. S. Coughlin for bacteriological assistance; and J. A. Eliason who performed much of the analytical work.
Acknobt /edgrrrmr
Receiced for reciew August 28, 1968. Accepted January 24, 1969. The research w,us supported in purt by the U.S. Public Health Grant N o . VI-00168.
The authors thank R. E. Ludin and James Sherer of the
Literuture Cited
Collins. R. P., Gaines, H. D., Appl, Microbiol. 12, 335-6 (1964). Dougherty, J. D., Campbell, R. D., Morris, R. L.. Science 152, 1372(1966). Gerber, N. N., Tet. Lit. No. 25,2971-4 (1968). Medsker, L. L.. Jenkins, D., Thomas, J. F., ENVIRON. ScI. TECHNOL. 2,461-4 (1968). Zelinsky, N., Ber. Drsch. Cheni. Ges. 34,2877 (1901).
Volume 3, Number 5, Mal 1969 477
