Jun 5, 1991 - Food Flavor Quality Unit, Southern Regional Research Center, Agricultural ResearchService, United States Department. ofAgriculture, 1100 ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1991, p. 3429-3432
Vol. 57, No. 12
0099-2240/91/123429-04$02.00/0 Copyright C) 1991, American Society for Microbiology
Effects of Farnesol and the Off-Flavor Derivative Geosmin on Streptomyces tendae CHRISTOPHER P. DIONIGI,* DAVID F. MILLIE, AND PETER B. JOHNSEN Food Flavor Quality Unit, Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee Boulevard, P.O. Box 19687, New Orleans, Louisiana 70179 Received
5
June 1991/Accepted 12 September 1991
Effects of the sesquiterpene farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-l-ol) and the sesquiterpene derivative geosmin (1,10-trans-dimethyl-trans-9-decalol) were investigated in a geosmin-producing actinomycete, Streptomyces tendae. Exposure to 300 ,uM farnesol reduced biomass (fresh matter) accumulation by 97% compared with biomass accumulation by controls, whereas an equal amount of geosmin did not affect biomass accumulation. Increasing exposure to farnesol corresponded with reduced optical density of the culture, reduced levels of geosmin, and reduced metabolic heat production compared with controls, while exogenous geosmin did not affect these parameters. Geosmin dissipated from uninoculated medium more rapidly than farnesol, indicating that in addition to the lower toxicity of geosmin, the actual exposure to geosmin over time may be less than exposure to an equal amount of farnesol. Cultures grown on Actinomyces-B medium contained 99.5% less geosmin and were more sensitive to farnesol than those grown on Hickey-Tresner medium, indicating that geosmin synthesis was associated with reduced sensitivity to farnesol. Consumption of farnesyl moieties during geosmin synthesis may reduce the potential for farnesol-induced inhibition of growth and metabolism. Geosmin (1,10-trans-dimethyl-trans-9-decalol) (Fig. 1) is produced by certain cyanobacteria, actinomycetes, and fungi that inhabit aquatic and soil environments (10, 12, 15, 25, 26). Low concentrations of geosmin (ca. 0.01 to 0.1 pug/liter) can impart musty or earthy "off-flavors" to food, potable water, and aquaculture-raised fish, causing economic losses in these and related industries (10, 19, 22). Surface waters have been reported to contain 1 to 2 ,ug of geosmin per liter (14). Pesticides currently available to control aquatic microorganisms are not selective for offflavor-producing taxa. These agents can cause a massive die-off of the microbial standing crop. Subsequent biomass decomposition can increase biological oxygen demand and release nutrients that may stimulate additional population blooms (24). Control strategies that selectively reduce offflavor-producing taxa may result in less environmental impact and benefit industries that experience off-flavor-related problems. Increased information concerning microbial sources of geosmin may facilitate the development of such strategies. Existing evidence indicates that geosmin synthesis involves a sesquiterpene precursor (5, 21) and possibly mixedfunction oxidase activity (8) and L-methionine (2) (Fig. 1). Although the intermediates involved in geosmin synthesis have not been reported, the pyrophosphate ester of farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol [FPP]) is considered the universal precursor of the sesquiterpene-derived metabolites (7). Farnesyl moieties are also involved in the synthesis of steroids, photosynthetic pigments, and many other essential and secondary metabolites (3, 7). The involvement of FPP in these pathways coupled with a relatively high susceptibility to phosphohydrolysis (18) suggests that farnesol may also occur widely. The antimicrobial activity of farnesol has been investigated in several taxa (4, 9, 16, 17). However, the effects of farnesol on an off-flavor*
producing taxon have not been reported. Although geosmin has been found in several environments (10, 12, 26), its effects on microbial populations have not been reported. The objectives of this investigation were to determine the effects of farnesol and geosmin on the growth and metabolism of the geosmin-producing actinomycete Streptomyces tendae Ettlinger (ATCC 31160). MATERIALS AND METHODS
Bacterial culture. Cultures of S. tendae were established in polystyrene petri dishes (100-mm diameter) containing 20 ml of Hickey-Tresner (HT) (11) or Actinomyces-B (AB) medium (1) solidified with 1.2% (wt/vol) bacteriological-grade agar. Each dish was inoculated with a 100-pul aliquot of spores suspended in sterile water, and the spores were then incubated in the dark at 28°C. Geosmin and farnesol solutions. Stock solutions of farnesol (95%; Aldrich Chemical Co., Inc., Milwaukee, Wis.) or geosmin (99%; Givaudan Corp., Clifton, N.J.) yielding 0, 50, 100, 200, and 300 ,uM farnesol and 0, 10, 100, 1,000, and 54,600 jig of geosmin per kg were prepared in 100% ethanol. Forty microliters of each stock solution was added to 100 ml of medium. Biomass accumulation. Cultures were grown on polycarbonate membranes (0.05-,um pore size, 90 mm in diameter; Costar/Nucleopore, Cambridge, Mass.) centered on the surface of the medium. Cells and membranes were removed from the medium 48 h after inoculation, and biomass accumulation was calculated by subtracting the membrane weight from the total. Optical density of culture. Increases in pigments and cell production in agar-grown cultures can be determined with a Hunter D25-PC2 colorimeter (8, 23). Forty-eight hours after inoculation, culture dishes were placed over the measurement port (19-mm inner diameter) of the colorimeter, and the optical density was determined at four random locations on
Corresponding author. 3429
3430
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DIONIGI ET AL.
MVA
100
MVAPP IPP
GPP
-- *
DMAPP
-
o 75
0
' 0
3
.
-* Monoterpenes
0 50
(n
o
LL-
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0
IOH
GSM FPP
Pite
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r Other Metabolites FIG. 1. Schematic representation of the initial steps of the terpene pathway showing the possible derivation of the musty-earthy off-flavor microbial metabolite geosmin. Abbreviations: MVA, mevalonic acid; MVAPP, IPP, DMAPP, GPP, and FPP, pyrophosphate esters of mevalonic acid, isopentenol, dimethylallyl alcohol, geraniol, and farnesol, respectively; OPP, pyrophosphate; Pi, inorganic phosphate; GSM, geosmin.
50
100
200
300
Farnesol (,uM) FIG. 2. Effects of farnesol on fresh-matter accumulations by S. tendae cultures. Data are expressed with ±1 standard error of a dosage mean (n = 4).
)
each culture. The means of these four L values were then subjected to statistical analysis. Geosmin accumulation. Cells and membranes were removed from the medium, weighed, extracted in hexane and internal standard (1.25 pLg of 2-undecanone per sample), and assayed by gas chromatography-flame ion detection (8, 13). Geosmin and internal standard peaks were identified by comparing retention times with those of authentic standards and mass spectrometry. The concentration of geosmin in uninoculated controls was subtracted from the concentrations of geosmin in the samples before statistical analysis was done. Metabolic-heat production. Metabolic-heat production was determined by differential isothermal heat conductance calorimetry (6) at maximal culture metabolic-heat generation (24 h after inoculation) with a Hart Scientific model 7707 calorimeter. Specific-heat conductance values (base lines) were determined for each ampoule before a preweighed (ca. 0.15-g) 7-mm disk of inoculated medium was sealed inside each sample ampoule. The reference ampoule contained sterile medium. Ampoules were allowed to temperature equilibrate for 3,680 s before data acquisition. Base lineadjusted reference values were subtracted from sample values prior to statistical analysis. Dissipation from uninoculated medium. Stock solutions of farnesol and geosmin were combined with sterile medium and incubated in petri dishes as described above for microbial cultures. Medium samples were obtained immediately following solidification and at 24, 48, and 72 h. Separate plates were used at each time point. Farnesol and geosmin concentrations were determined by gas chromatography as described above. Statistical analysis. The effects of farnesol and geosmin on optical density of the culture, metabolite heat production, and geosmin and biomass accumulation were determined in
a series of randomized complete-block experiments. Calorimeter treatments could not be replicated within an experiment; therefore, replication was achieved by repeating each experiment. Replication for all other treatments was achieved by establishing at least three complete randomized blocks within each experiment and by repeating each experiment. Data were combined, and sources of variation were analyzed according to McIntosh (20).
RESULTS AND DISCUSSION Biomass accumulation. Increasing farnesol exposure coincided with decreased (P 2 0.0001) biomass accumulation, indicating that S. tendae growth is inhibited by farnesol (Fig. 2). This observation agrees with reports of antimicrobial and phytotoxic activity of farnesol in a variety of other taxa (4, 9, 16, 17). To compare the effects of geosmin and farnesol, cultures were grown on medium containing 300 ,uM concentrations of either compound. Cultures grown on farnesol exhibited nearly complete (97%) inhibition of biomass accumulation (P 2 0.0001) compared with untreated controls, whereas 300 ,uM geosmin did not affect (P 2 0.85) biomass accumulation compared with untreated controls, indicating that, at this dosage, geosmin was less inhibitory than farnesol. Geosmin at 300 ,uM corresponds to ca. 54,600 ,ug/kg, which is much greater than geosmin concentrations reported in aquatic environments (e.g., 1 to 2 ,ug/liter) (14). Therefore, it is unlikely that cultures of S. tendae would be inhibited by the concentrations of geosmin that have been observed in aquatic environments. Metabolic heat, optical density, and geosmin accumulation. Cultures grown on medium containing 100 ,M farnesol produced 30.16% less (P 2 0.0011) metabolic heat than untreated controls (Table 1). Cultures grown on medium containing either 200 or 300 ,uM farnesol exhibited very little growth and were not assayed. Growth on medium containing 1,000 ,ug of exogenous geosmin per kg did not inhibit (P 2 0.4618) metabolic-heat generation compared with untreated controls. Dosages of farnesol greater than 50 ,uM coincided with decreasing optical density of the cultures and decreasing geosmin accumulation (Fig. 3 and 4). In contrast, the optical density and geosmin accumulation in cultures grown on
EFFECTS OF FARNESOL AND GEOSMIN ON S. TENDAE
VOL. 57, 1991
TABLE 1. Effects of farnesol on metabolic-heat generation by S. tendae cultures (n = 3)
I
100
Metabolic-heat generation
Farnesol concn
,uW/g (±2 SE)
% of untreated cultures (±2 SE)
1,133.31 (+69.66) 1,175.46 (+48.26) 791.52 (±16.84)
100.00 (+6.15) 103.72 (±4.26) 69.84 (±1.49)
(t,M) 0 50 100
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75
c
E
C) cJ
gn
0 U,
50
0
0 %-Ol
media containing concentrations of geosmin that ranged from ones similar to those reported in natural environments (14) to several orders of magnitude greater did not differ (P . 0.1330 and .0.6972, respectively) from those of untreated controls. Because farnesyl moieties are involved in geosmin (5, 21) and pigment (3, 7) synthesis, it might be expected that exogenous farnesol would increase geosmin production and optical density of the culture. However, terpenes such as farnesol have been reported to disrupt membrane function, ultimately reducing cell viability (4, 17). These toxic effects may offset any increases in geosmin or pigment synthesis associated with exogenous farnesol. Dissipation from uninoculated medium. The concentration of farnesol in uninoculated medium did not change (P 0.2449) after 72 h of incubation (Fig. 5). However, a reduction (P 2 0.0001) in geosmin concentration was observed, indicating that the actual exposure to geosmin may be less than the exposure to an equal initial dosage of farnesol over time (Fig. 5). The rapid dissipation of geosmin may functionally reduce the potency of geosmin relative to farnesol. Differential sensitivity to farnesol. FPP is considered the universal precursor of the sesquiterpenes (7). FPP and other farnesyl moieties occur in a wide variety of taxa, especially in photoautotrophic organisms (3). Geosmin production indicates the presence of farnesyl moieties in S. tendae (5). Farnesyl moieties are relatively susceptible to phosphohydrolysis (18), and farnesyl moieties released by cell lysis from other populations could contribute to farnesol exposure. For example, farnesol and other sesquiterpenes were found in the aqueous fraction isolated from water nutgrass (Cyperus serotinus) (9). Conceivably, cells capable of utilizing endogenous or exogenous farnesyl moieties for geosmin
25
0
0 -1
0
50
1 00
Farnesol
200
-1
300
(,AM)
FIG. 4. Effect of farnesol on the geosmin concentrations of S. tendae cultures grown on agar. Data are expressed with + 1 standard error of a dosage mean (n = 6).
production may be less susceptible to farnesyl exposure than cultures that do not produce geosmin. The relatively greater production of geosmin on HT medium compared with production on AB medium allowed the examination of this issue.
Cultures grown on AB medium contained 118 + 21 ig of geosmin per kg, which was 99.5% less than the geosmin content of cultures grown on HT medium. This reduced geosmin accumulation was associated with an increased (P -0.0001) sensitivity to farnesol in AB medium-grown cultures compared with HT-grown cultures (Fig. 6). For example, cultures grown on HT medium containing 100 ,uM farnesol accumulated as much biomass as untreated controls, whereas cultures grown on AB medium with 100 ,uM farnesol exhibited a nearly complete inhibition of biomass accumulation (Fig. 6). However, 200 and 300 ,uM farnesol produced a nearly complete inhibition of biomass production on both AB and HT media (Fig. 6). Geosmin-induced off flavors cause economic losses in several industries. Managers of water resources have few viable options for controlling off-flavor-producing taxa. Selective control agents for off-flavor-producing taxa are not 125
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Farnesol (j,sM) FIG. 3. Effects of farnesol on optical densities of S. tendae cultures grown on agar. Data are expressed with ±+1 standard error of a dosage mean (n = 6).
c 0 0
Geosmin 0
0 0
24
48
72
Time (hr) FIG. 5. Dissipation of farnesol and geosmin from sterile agar at 28°C. Data are expressed with + 1 standard error of a dosage mean. n = 5 for dosage at 24 h; n = 6 for all other means.
APPL. ENVIRON. MICROBIOL.
DIONIGI ET AL.
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N 1
\
HT Medium
8.
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0) 0
c 0
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Farnesol (,uM) FIG. 6. Effects of farnesol on fresh-matter accumulations by S. tendae cultures grown on HT or AB medium. Data are expressed with 1 standard error of a dosage mean (n = 4).
available, and the use of nonselective agents can have negative environmental impacts (24). This work suggests that inhibition of geosmin synthesis may selectively suppress geosmin-producing taxa by promoting an accumulation of potentially toxic precursors. ACKNOWLEDGMENTS We thank D. A. Greene-Ingram for her help with the growth and maintenance of microbial cultures and data acquisition; S. W. Lloyd for his mass-spectrum analysis; G. A. Johnson for his help with the microcalorimetry; and B. G. Montalbano, T. B. Moorman, M. A. Klich, B. M. Lawrence, S. A. Harris, A. M. Altazan, and V. I. Sullivan for their help with the preparation of the manuscript. REFERENCES 1. Ajello, L., L. K. Georg, W. Kaplan, and L. Kaufman. 1963. Laboratory manual for medical mycology. U.S. Department of Health, Education and Welfare Public Health Service publication no. 994. Communicable Disease Center, Atlanta. 2. Aoyama, K. 1990. Studies on the earthy-musty odors in natural water. IV. Mechanism of earthy-musty odor production of Actinomyces. J. Appl. Bacteriol. 68:405-410. 3. Banthorpe, D. V., and B. V. Charlwood. 1980. The isoprenoids, p. 185-220. In A. Bell and B. V. Charlwood (ed.), Encyclopedia of plant physiology, vol. VIII. Secondary plant products. Springer-Verlag, New York. 4. Bard, M., M. R. Albrecht, N. Gupta, C. J. Guynn, and W. Stillwell. 1988. Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23:534-538. 5. Bentley, R., and R. Meganathan. 1982. Geosmin and methylisoborneol biosynthesis in Streptomyces, evidence for a isoprenoid pathway and its absence in non-differentiating isolates. FEBS Lett. 125:220-222. 6. Criddle, R. S., R. W. Breidenbach, E. A. Lewis, D. J. Eatough, and L. D. Hansen. 1988. Effects of temperature and oxygen depletion on metabolic rates of tomato and carrot cell cultures and cuttings measured by calorimetry. Plant Cell Environ. 11:695-701. 7. Croteau, R. 1987. Terpenoid natural products: a biosynthetic
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overview, p. 59-64. In D. W. Newman and K. G. Wilson (ed.), Models in plant physiology and biochemistry, vol. II. CRC Press, Inc., Boca Raton, Fla. Dionigi, C. P., D. A. Greene, D. F. Millie, and P. B. Johnsen. 1990. Mixed function oxidase inhibitors affect production of the off-flavor microbial metabolite geosmin. Pestic. Biochem. Physiol. 38:76-80. Elakovich, S. D. 1987. Sesquiterpenes as phytoalexins and allelopathic agents, p. 93-109. In G. Fuller and W. D. Nes (ed.), Ecology and metabolism of plant lipids. American Chemical Society, Washington, D.C. Gerber, N. N. 1983. Volatile substances from actinomycetes: their role in the odor pollution of water. Water Sci. Technol. 15:115-125. Hickey, R. T., and H. D. Tresner. 1952. A cobalt-containing medium for sporulation of Streptomyces species. J. Bacteriol. 64:981. Izaguirre, G., C. J. Hwang, S. W. Kranser, and M. J. McGuire. 1982. Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems. Appl. Environ. Microbiol. 43:708714. Johnsen, P. B., and J. W. Kuan. 1987. Simplified method to quantify geosmin and 2-methylisoborneol concentrations in water and microbiological cultures. J. Chromatogr. 409:337-342. Juttner, F. 1984. Dynamics of the volatile organic substances associated with cyanobacteria and algae in a eutrophic shallow lake. Appl. Environ. Microbiol. 47:814-820. Kikuchi, T., S. Kadota, H. Suehara, A. Nishi, and K. Tsubaki. 1981. Odorous metabolites of a fungus, Chaetomium globosum Kinze ex Fr. Identification of geosmin, musty-smelling compound. Chem. Pharm. Bull. 29:1781-1784. Knoblock, K., A. Pauli, and B. Iberl. 1989. Antibacterial and antifungal properties of essential oil components. J. Ess. Oil. Res. 1:119-128. Knoblock, K., A. Pauli, B. Iberl, N. Weis, and H. Weigand. 1988. Mode of action of essential oil components on whole cells of bacteria and fungi in plates tests, p. 288-299. In P. Schreier (ed.), Bioflavor '87. Walter de Gruyter, Berlin. Koyama, T., H. Fujii, and K. Ogura. 1985. Hydrolysis of polyprenyl pyrophosphates. Methods Enzymol. 110:153-155. Lovell, R. T., I. Y. Lelana, C. E. Boyd, and M. S. Armstrong. 1986. Geosmin and musty-muddy flavors in pond-raised channel catfish. Trans. Am. Fish. Soc. 115:485-489. McIntosh, M. S. 1983. Analysis of combined experiments. Agron. J. 75:153-155. Naes, H., H. C. Utkilen, and A. F. Post. 1989. Geosmin production in the cyanobacterium Oscillatoria brevis. Arch. Microbiol. 151:407-410. Persson, P. E. 1980. Sensory properties and analysis of two muddy odour compounds, geosmin and 2-methylisoborneol, in water and fish. Finn. Fish Res. 4:1-13. Pomeranz, Y., and C. E. Meloan. 1978. Color of foods, p. 72-83. In Food analysis: theory and practice. AVI Publishing Co., Westport, Conn. Tucker, C. S. 1985. Water quality, p. 135-227. In C. S. Tucker (ed.), Channel catfish culture. Elsevier Science Publishing Co., Inc., New York. Wu, J. T., and F. Juttner. 1988. Effects of environmental factors on geosmin production by Fischerella muscicola. Water Sci. Technol. 20:143-148. Yagi, O., N. Sugiura, and R. Sudo. 1981. Odorous compounds produced by Streptomyces in Lake Kasumigaura. Verh. Int. Ver. Limnol. 21:641-645.
