Earthy-Smelling Substance from a Blue-Green Alga
s. Safferman, Aaron A. Rosen, Charles I. Mashni, and Mary E. Morris U. S. Department of the Interior, Federal Water Pollution Control Administration, Cincinnati, Ohio 45226 Robert
rn The blue-green alga Symplocu inuscorum produces an earthy-smelling metabolite at an estimated concentration of 0.6 mg. per liter of culture medium. The substance has been isolated by preparative gas chromatography and identified as geosmin on the basis of direct comparison with an actinomycete-produced standard. The results suggest that organisms other than actinomycetes are possible sources of earthy taste and odor problems in water supplies.
and egg albumen media (Safferman and Morris, 1962); starch-casein medium (Kuster and Williams, 1964); neopeptone-dextrose and diamalt media (Cooke, 1963). An authentic sample of geosmin was supplied by N. N. Gerber, Rutgers, The State University, New Brunswick, N.J.
Culture Methods
The alga was routinely cultured in 250-ml. Erlenmeyer flasks containing 100 ml. of modified Chu No. 10 nutrient medium. Each flask was inoculated with 5 ml. of stock culture, the matted filaments of which had been first dispersed by aseptically homogenizing them for 5 seconds in a Waring ctinomycetes produce a characteristic earthy odor Blendor. Incubation was carried out for 15 to 20 days at 20" C. (Waksman, 1959) which led to their early indictment "Cool white" fluorescent tubes provided illumination of 160 as a cause of earthy taste and odor problems in water supplies to 180 foot-candles. Large-scale culturing for isolation of (Adams, 1929; Thaysen, 1936). Recent studies (Silvey, geosmin was carried out aseptically in 5-gallon carboys Russell, et a/., 1950; Silvey and Roach, 1956) have again which were illuminated at 400 to 500 foot-candles and vigdirected attention to the effect of these organisms on water orously aerated. quality. Gerber and Lechevalier (1965) recently isolated a Bacterial contamination of the unialgal culture could be specific compound having this earthy odor from the metabolic readily demonstrated by use of several media. However, no products of several Streptomyces strains. The compound, actinomycetes or fungi could be detected microscopically or which was named geosmin, was characterized as a colorless isolated from the algal culture. Attempts to free the organism neutral oil with an indicated molecular formula of Cp2HYP0 from bacteria by isolating the algal filaments on agar (Lewin, and an approximate boiling point of 270" C. (Gerber and 1959) removed only part of the bacterial contaminants; Lechevalier, 1966). consequently, an examination was undertaken of the odor During routine transfer of a stock culture of the filamentous contribution of the contaminating bacteria originally present. blue-green alga Symploca niuscorum strain I U 617 (Indiana These bacteria were isolated on agar and each was cultured University culture collection), an earthy odor was perat 22" to 25" C. for 14 days. The cultures were examined ceived, indistinguishable from that of an actinomycete periodically for development of odor. In no instance was a culture studied previously (Romano and Safferman, 1963). perceptible earthy odor produced in any of the test media. The odorous substance, which was isolated and shown to Evidently, the bacterial contaminants could not be a primary be identical to the geosmin produced by actinomycetes, is source of the earthy odor. apparently the first example of such a compound in an alga.
A
Materials
Results nnd Discussion
Symploca niuscorum I U 617 was cultured in a modified Chu No. 10 nutrient medium: Ca(N0J2.4H20, 0.232 gram; K2HP01, 0.01 gram; MgS04.7H20, 0.025 gram; Na2C03, 0.02 gram; NazSiO3.5H~0,0.044 gram; ferric citrate, 3.5 X loV3 gram; citric acid, 3.5 X 10-3 gram; CuC12.2H20, 1 X lo-: gram; CoC1.2.6H20, 2 X loF5 gram; ZnCln, 4 X gram; MnC12.4H20, 1.4 X gram; H3B03,2.4 X gram; distilled water, 1000 ml. The purity of the algal culture was determined employing the following media: modified Chu No. 10 nutrient medium (Chu) 4- 0.1% sodium glutamate; Chu 0.1% glucose; Chu 0.1 proteose; Chu 0.2% maltose; Chu 0.2% starch; soil extract medium: 4 garden soil-water supernatant in Bristol's solution (Starr, 1964); yeast extract, nutrient,
No apparent differences in geosmin production were shown between the original and partially purified algal cultures. In the latter cultures, traces of bacterial contaminants were revealed as minute pellets, present only in glutamate- or peptone-supplemented Chu medium. These bacteria apparently constitute an infinitesimal portion of the biomass, since microscopic examination of the culture consistently failed to reveal their presence. These facts strongly imply that the alga is responsible for the earthy odor. Twenty-four liters of culture were pooled and immediately distilled at atmospheric pressure until about 20 % of the volume was collected. The distillate was then extracted six times with a total volume of 4 liters of anhydrous ethyl ether. Only negligible quantities of the earthy odor remained in the
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Volume 1, Number 5, May 1967 429
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TEMFERATU?E
Figure 1. G a s chromatogram of 1.0 p l . of earthy odor concentrate from Symploca ~nuscoriim Column (1.8 meters x 3.2 mm.) containing 20% SE-30 on 60- to 80mesh Chromosorb W ; temperature programmed at 6’ C. per minute; dual flame ionization detector
water. The odor material was further concentrated by evaporating the ether extract to a final volume of 0.5 mi. under a stream of dry air. Gas chromatography (Figure 1) showed that the concentrated extract contained many detectable components. By peak-area analysis, however, the single strong peak of geosmin eluting at 153” C. represented 75% of the total metabolites recovered. The principal component, isolated by preparative gas chromatography, had an intense earthy odor. An odor panel determined that its threshold odor concentration in water was 0.2 p.p.b., which accounted for most of the odor of the concentrated extract. The isolate was identified as geosmin on the basis of the following comparisons with an authentic sample: An odor panel judged the odor qualities of both substances to be identical. Their threshold odor concentrations of 0.16 cs. 0.2 p.p.b, (based on estimated weights determined by relative peak areas) were equal within the precision of this measurement. The retention times of the algal isolate matched those of the geosmin reference on three different gas chromatographic columns (SE-30, Apiezon L, and diethylene glycol adipate). The infrared spectrum of the algal isolate matched that of geosmin (Gerber and Lechevalier, 1966)-points of siiiiilarity were the absence of hydroxyl, carbonyl, olefinic, and aromatic bands and the presence of identical prominent bands at 6.9, 7.25, 7.95, 8.6, 9.15, 9.5, 9.8, 10.0: 10.3, 10.6, 10.9. 11.3, and 11.8 microns. The odor intensity of the algal isolate was reduced by a factor of 2000 when it was treated with HC1 in methylene chloride in a procedure which is known to destroy geosmin (Gerber and Lechevalier, 1965). Based on peak-area analysis of the ether concentrate, the yield of geosmin was 0.6 mg. per liter of pooled algal culture. This yield is comparable with that obtained from various species of Streptomjws (Gerber and Lechevalier, 1965). Accordingly SJ*/np/OCCl ~nuscoru~nand possibly other algae are potential sources of earthy taste and odor problems in water supplies. Literature Cited
Adams, B. A., Water Writer Eng. 31, 327 (1929). Cooke, W. B., Public Hedrh Sercice Publ. No. 999-WP-1, 55-7 (1963). Gerber, N. N., Lechevalier, H. A., Appl. Microhiol. 13, 935 (1965). Gerber, N. N., Lechevalier, H. A , , Division of Microbial Chemistry and Technology, 152nd Meeting, ACS, New York, N. Y . ,September 1966. Kuster, E., Williams, S. T., Nurure 202, 928 (1964). Lewin, R. A,, Rec. Algologique 5, 181 (1959). Romano. A. H.. Safferman. R. S.. J . Am. Wtrtor. Works Assoc.’55, 169 (1963). Safferman, R. S., Morris, M. E., Appl. Microhiol. 10, 289 (1962). Silvey, J. K. G., Roach, A. W., Public Works 87, 103 (1056). Silvey, J. K. G., Russell, J. C., Redden, D. R., McCormick, W. C., J . Am. Writer Works Assoc. 42,1018 (1950). Starr, R. C., Am. J . Botanq 51, 1013 (1964). Thaysen, A. C., Ann. Appl. Biol. 23,99 (1936). Waksman, S. A , , “The Actinomycetes. Vol. I. Nature, Occurrence, and Activities,” p. 156, Williams & Wilkins, Baltimore, Md., 1959. ~~
Receiced f i r recienl AliriIl4, 1967. Accepted Mu), I I , 1967.
430 Environmental Science and Technology
