APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1982, p. 708-7140099-2240/82/030708-07$02.00/0

Vol. 43, No. 3

Geosmin and 2-Methylisoborneol from Cyanobacteria in ThreeWater Supply Systems

GEORGE IZAGUIRRE,* CORDELIA J. HWANG, STUART W. KRASNER, AND MICHAEL J.McGUIRE

Water Quality Branch, The Metropolitan Water District of Southern California, La Verne, California 91750

Received 16 July 1981/Accepted 10 November 1981

Three Oscillatoria strains and one Anabaena species were isolated from threedifferent water supply systems in California that experienced earthy-musty tasteand odor problems in their drinking water. Unialgal cultures, free of actinomy-cetes, were purged using the Grob closed-loop stripping analysis method, and theresulting methylene chloride extracts were analyzed on a gas chromatograph/massspectrometer. Geosmin was produced by Oscillatoria simplicissima and Ana-baena scheremetievi, and 2-methylisoborneol was produced by 0. curviceps and0. tenuis. These compounds are the two major causes of earthy-musty tastes andodors in water. In three instances, the major odorant found in culture waspreviously identified in the water or sediment sample from which the respectiveorganism was isolated. 0. curviceps was implicated in a taste and odor episodeinvolving 2-methylisoborneol in a major reservoir. Geosmin and 2-methylisobor-neol were easily detected with culture samples of only 4 to 25 ml.

The occurrence of objectionable tastes andodors in drinking water is a common and wide-spread problem. The most troublesome odorsare usually those described as muddy or earthy-musty. Two organic compounds which havebeen implicated as the cause of earthy-mustyodor problems in water are geosmin and 2-methylisoborneol (MIB) (19, 21).Geosmin, an earthy-smelling substance, was

isolated in 1964 by Gerber and Lechevalier (5).MIB, a musty- or camphorous-smelling com-pound, was reported in 1969 by Medsker et al.(16) and independently by Rosen et al. in 1970(21). Both compounds are saturated cyclic ter-tiary alcohols and are thus resistant to oxidationby conventional water treatment methods.Geosmin and MIB have extremely low thresholdodor concentrations, 10 and 29 ng/liter, respec-tively (2, 18). These are average values, andmany people are able to detect the compounds atlower concentration levels.Geosmin is a metabolite of many actinomy-

cetes of the genus Streptomyces, and most pre-vious work on geosmin has focused on theseorganisms (for a recent review, see reference 4).However, geosmin is also known to be producedby at least 11 cyanobacteria in the genera Oscil-latoria, Lyngbya, Symploca, and Anabaena (15,17, 22). MIB was known as a metabolite of onlycertain actinomycetes until 1975, when a Lyng-bya species capable of releasing MIB was re-ported from an experimental fish-farming lake inManitoba (25).

Most of the earthy-musty taste and odor prob-lems reported by water supplies have been at-tributed to actinomycetes (9, 21, 23). In mostcases in which blue-green algae (cyanobacteria)were implicated, the odorous compound was notidentified, nor was its concentration determined(3, 13, 20). Undoubtedly, there have been otherinstances in which cyanophyta were responsiblefor odor problems, but for a variety of reasonsthis has rarely been documented. Various watersupply systems have experienced elusive tasteand odor episodes not attributable to planktonicalgae or actinomycetes. The purpose of thisstudy was to identify the causative agents of twosuch occurrences and quantitate the metabolitesinvolved.

MATERIALS AND METHODSMicroorganisms. Four different filamentous cyano-

bacteria were isolated from three water supply sys-tems in California (Table 1). The first, an Oscillatoriaspecies, was isolated from a widespread infestation inthe littoral zone of Lake Mathews of The MetropolitanWater District of Southern California. It has beenprovisionally assigned to Oscillatoria curviceps. Thetrichomes are normally rigid and straight, 9 to 11 p.m indiameter, with one or both ends bent to one side, andcontain cytoplasmic granulation (Fig. 1). The habitat ison the surface of mud or rocks in shallow water nearshore, intermingled with other algae.A second strain, identified as 0. tenuis var. levis

Gardner, was isolated from a sediment sample takenfrom Phoenix Lake, Marin Municipal Water District,in northern California. It has been implicated in a

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TABLE 1. Cyanobacteria from three water supply systems

Alga Source Trichome diam Cell lengthAlga Source ~~~~ ~~~~~~~~(pLm)(pm)

0. curviceps Littoral growth, Lake Mathews 9-11 30. tenuis var. levis Gardner Sediment, Phoenix Reservoir 7.5-8 30. simplicissima Gomont Attached growth, raw water pipeline 8 7A. scheremetievi Elenkin Bloom, Tinemaha Reservoir 9.5-11 7

FIG. 1. A variant of 0. curviceps isolated from Lake Mathews, California. Bright field, x400. Filaments werefixed with Formalin to prevent movement. Bar = 10 ,um.

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series of odor problems since 1967. The trichomes ofthis organism are also conspicuously granular.A third strain, 0. simplicissima Gomont, was isolat-

ed from a raw water pipeline downstream from LakeMathews, growing among a tangled mass of deadCladophora. The fourth organism, Anabaena schere-metievi Elenkin, was isolated from a mixed bloom inTinemaha Reservoir of the Los Angeles Department ofWater and Power. There was an intense, musty odor inthe water at the time of the bloom. The filaments aresolitary and straight, and a prominent feature of thisorganism is the presence of numerous pseudovacu-oles.

Isolation. The blue-green algae were isolated fromthe respective environmental samples by a combina-tion of accepted methods (1). Filaments taken from asample were subjected to cycloheximide (J. T. BakerChemical Co.) at a concentration of 90 to 100 mg/literfor at least 24 h to eliminate eucaryotic contaminants(26). Filaments were then transferred to an agar plateas an aid in separation of the organisms and removal ofattached particles. There was no growth on the agar; itserved only as a temporary physical support. Thefilaments were later picked from the agar with aDrummond 25-,ul micropipette and given severalsuccessive rinses in 1 or 2 ml of sterile phosphatebuffer in oven-sterilized watch glasses. After this, oneto three filaments were placed in a suitable liquidmedium. Eventually three of the organisms were iso-lated from a single filament derived from the primaryisolation cultures. 0. simplicissima was isolated bygrowth on the agar medium and transfer of filamentswhich had migrated across the plate. The resultingcultures were unialgal but were bacterially contam-inated.Media and growth conditions. The medium used for

the Oscillatoria species was medium 11 of Hughes etal. (10), modified as follows. Ferric ammonium citratewas substituted for the less-soluble ferric citrate (afterStanier et al.) (24). The trace element solution includedonly the salts supplying B, Mn, Zn, Mo, Co, and Cu; alower NaNO3 concentration was used for all threespecies, i.e., 50 mg/liter for 0. curviceps and 0. tenuisand 170 mg/liter for 0. simplicissima. More recently,the medium for 0. curviceps has included NaCl at 150mg/liter to more closely parallel the salinity of thenatural habitat. The agar medium was the modifiedmedium 11 with 1% agar (wt/vol) and NaNO3 at 0.2 g/liter. For isolation of single filaments, 1% agar in tapwater was also used. The Anabaena species wasgrown in ASM-1 medium of Gorham et al. (6), with theaddition of Na2MoO4 2H2O at 0.39 mg/liter.The liquid cultures were grown as shallow layers in

125- to 1,000-ml Erlenmeyer flasks illuminated byoverhead cool white fluorescent light, warm whitelight placed to one side, or daylight from a northwindow. Incident light values ranged from 590 to 1,780lux, as measured by a Gossen Luna-Pro sbc lightmeter.

Cultures to be analyzed were tested for the presenceof actinomycetes by plating duplicate 0.2-ml portionson starch-casein agar and incubating at 28°C and roomtemperature for 10 days (12). A positive control con-sisted of a plate inoculated with a dilute suspension ofspores from a streptomycete stock culture. The cul-tures were also examined microscopically.

Analytical method. Chemical analyses for geosmin

and MIB in water were performed by a Grob closed-loop stripping analysis (CLSA) method (7, 8). In thistechnique, semivolatile organic compounds werestripped from an aqueous sample by a recirculatingvolume of air and adsorbed from the gas phase onto a1.5-mg activated carbon filter. The filter was thenremoved and extracted with carbon disulfide. Identifi-cation and quantification of the odorants were com-pleted by injecting a sample of the extract into aFinnigan model 4023 gas chromatograph/mass spec-trometer/computer system with a 30-m SE-52 fusedsilica capillary column. The detection limit for geos-min and MIB in 1 liter of water by CLSA was 2 ng/litereach. The use of this technique for water and sedimentsamples has been described in detail elsewhere (11).The high sensitivity of the CLSA method coupled

with the abundant geosmin and MIB production inlaboratory cultures required modification of the meth-od for these analyses. Before stripping in a 125-mlbottle, a 4- to 25-ml sample of liquid culture wasdiluted to 60 ml with the appropriate algal growthmedium and further diluted with 65 ml of "organic-free" water (Millipore Corp., Super-Q water pre-stripped in the CLSA apparatus). Likewise, calibra-tion standards were prepared by spiking a mixture of60 ml of medium and 65 ml of water with geosmin andMIB. Preparing samples and standards with the samematrix ensured consistent stripping efficiencies.The sample was prestripped for 10 s with an auxilia-

ry carbon filter in place to flush air contaminants out ofthe CLSA system. The auxiliary filter was then ex-changed for a clean one, and the culture was strippedfor 2 h. A 5-mg activated carbon filter was used forculture analyses rather than the 1.5-mg filter used withwater samples to provide a higher adsorptive capacityfor the higher organic compound concentrations. The5-mg filter was successively extracted with 30-, 25-,and 20-,ul portions of methylene chloride.

RESULTS

Metabolites in culture and in environmentalsamples. The results of the analyses of the fouralgal isolates are summarized in Table 2. Themajor odorous compound identified in the corre-sponding environmental samples is also listed.Figure 2 is a chromatogram of an 0. curvicepsculture run by the CLSA method showing thepeak for MIB.No actinomycetes were detected in any of the

cultures, either by microscopic examination orby plating on starch-casein agar. Contaminatingbacteria in the cultures did not produce thecharacteristic earthy-musty odor when grown ona variety of agar media.Both 0. curviceps and 0. tenuis generated

considerable MIB in culture and were markedby an unusual moldy-musty odor encounteredonly with these two organisms. MIB was alsofound in the sediments from which these twoorganisms were isolated.

In the case of 0. simplicissima, it was notpossible to analyze the original sample. Howev-er, the smell of geosmin was quite evident in the

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TABLE 2. Algal culture analyses

Odorous compound in:Alga Environmental Culturca

sample

0. curviceps MIBb MIB (12, 26)MIB (20, 42)

0. tenuis var. levis MIB MIB (60, 24)Gardner MIB (90, 31)

0. simplicissima Geosminc Geosmin (60, 21)Gomont Geosmin (100, 61)

A. scheremetievi Geosmind Geosmin (9, 25)Elenkin Geosmin (18, 33)

Geosmin (80, 78)a Each entry represents a separate culture. The first

number in parentheses represents the concentration inmicrograms per liter, and the second number repre-sents the age of the culture in days.

b The level of MIB was 25 ng/liter in water and>1,000 ng/liter in sediment.

c Geosmin was suspected.d The level of geosmin was 100 ng/liter.

patch of filaments from which the organism wasultimately isolated. The Anabaena species gave

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a strong response for geosmin, which was alsofound in the water of origin at 100 ng/liter.Lake Mathews case history. The potential of 0.

curviceps for affecting the aquatic environmenton a large scale was demonstrated in LakeMathews in September 1980. MIB was found inthe reservoir effluent at a concentration of 16 ng/liter, at the same time that an earthy-musty odorwas noticed in the water. The MIB levels in-creased to around 25 ng/liter in the water columnby late September and early October (Fig. 3) andgradually subsided after that to about 3 ng/literby late November. A depth profile indicated thatthe MIB was concentrated above the thermo-cline (at 18 to 21 m below the surface) and wasnegligible below the thermocline.The MIB was traced to a shoreline infestation

of 0. curviceps. Most of the growth occurred atdepths ranging from 2 to 9 m and was not visiblefrom the shore. The samples of littoral algalmaterial contained a wide variety of blue-greenalgae, diatoms, green algae, and protozoa, butonly those samples having many filaments of 0.curviceps were marked by the intense odor ofMIB; those samples without this organism didnot have a strong earthy-musty odor. Actinomy-cetes were not found in significant numbers in

17:20 19:30Retention Time (min:sec)

FIG. 2. Chromatogram (corrected for background peaks) from the CLSA of an 0. curviceps culture.

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712 IZAGUIRRE ET AL.

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FIG. 3. MIB levels and temperature variation for Lake Mathews effluent.

the samples and in any case did not produceodors in culture.

Concentrations of MIB in Lake Mathewswere highest when water temperatures were 24to 25°C. This corresponds to the temperaturerange at which optimal growth of 0. curvicepsoccurred in vitro. The affected reservoir has amaximum storage capacity of 182,000 acre-feet(225 x 106 m3), with a mean maximum depth of56 m. This taste and odor episode is discussed ingreater detail by McGuire et al. (14).

Cultivation of 0. curviceps. Three of the fourcyanobacteria grew readily in culture, but manydifficulties were encountered in the cultivationof 0. curviceps. In many cultures of this orga-nism, growth was erratic, sometimes ceasingaltogether for no apparent reason long beforereaching maturity. Although nutrient require-ments have not been studied systematically, it isclear that this strain cannot tolerate the nitratelevels commonly used in media for blue-greenalgae (100 mg of NaNO3 per liter was inhibi-tory), nor can it tolerate light intensities muchhigher than about 1,400 lux.

Precision and accuracy of analytical method.For a 5-ml sample of the liquid culture, both

geosmin and MIB had a linear range of 0.4 to 60,ug/liter and a working range extended to 120 ,ug/liter with the use of a nonlinear calibrationcurve. Duplicate analyses of an Anabaena cul-ture yielded 9.1 and 8.5 ,ug of geosmin per liter,whereas an 0. curviceps culture (from LakeSkinner) yielded 110 and 120 ,ug ofMIB per liter.Recoveries from spiked cultures ranged from 73to 116% for MIB and 96 to 124% for geosmin.

DISCUSSIONThe production of MIB by cyanobacteria in

water supplies is a virtually unrecognized phe-nomenon. This study contains the first reportedinstance of a functioning drinking water supplybeing affected by MIB originating from a blue-green alga. The only previous reference to acyanophyte producing MIB was that cited earli-er, from a lake in Manitoba used for the farmingof rainbow trout. There is no indication that thiswater was ever used for drinking purposes, andsince MIB levels for the lake water were notdetermined (J. Tabachek, personal communica-tion), it is not certain that the organism by itselfeven had a measurable impact on the water.

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GEOSMIN AND MIB FROM CYANOBACTERIA

Based on the field and laboratory data, it isclear that 0. curviceps was the prime contribu-tor to the MIB problem in Lake Mathews fromSeptember to November 1980. Other cyanobac-teria from Lake Mathews have failed to produceMIB in culture. The absence of actinomycetes inthe 0. curviceps cultures indicated that the MIBwas a product of the alga and not of an actino-mycetic contaminant.With regard to the Marin strain of 0. tenuis,

the finding that it is a strong MIB producer lendssupport to the long-held view that it has been themain source of taste and odor problems in thereservoirs where the organism is found. It hasnot been confirmed that it is the primary causeof the problems since the major odorous com-pound in the water during an odor episode inMarin's reservoirs has not been determined.However, it appears likely that 0. tenuis playsthe same role as has been shown for 0. curvi-ceps in Lake Mathews.

Ironically, strains of 0. tenuis reported previ-ously have produced geosmin, and not MIB,with essentially the same medium employed inthis work. An explanation may be that produc-tion of the compounds is a very strain-specificproperty. The variant discovered by Tabachekand Yurkowski (25) is clearly not identical to theone described in this report.

It is significant that both MIB-producing Os-cillatoria species are nonplanktonic taste andodor producers and are thus not readily detect-able in water samples. This fact is of greatpractical importance to water utilities. It is pos-sible that in past taste and odor problems ofunknown origin, similar attached organisms mayhave been responsible, which endless watersamples for plankton analysis would not reveal.The significance of 0. simplicissima as a

geosmin producer is uncertain, as its knowndistribution is restricted to a relatively smalllocation. A. scheremetievi is a classic planktonicnuisance organism, unlike the other organismsdiscussed. The identification of geosmin in acti-nomycete-free unialgal cultures of this organismplaces it alongside A. circinalis as a confirmedgeosmin producer among bloom-forming cyano-bacteria and, thus, as only the second strictlyplanktonic cyanophyte in which this propertyhas been clearly demonstrated chromatograph-ically. However, it is not certain whether A.scheremetievi was the only component of thebloom which released geosmin, since attemptsto isolate three other species were unsuccessful.

In culture, all four organisms initiate the pro-duction of their respective odorous compoundsduring active growth. In the case of 0. curvicepsand 0. tenuis, odor was evident even before thecultures were dense enough to exhibit any color,and the 'organisms displayed the gliding and

rotational movement typical of healthy filamentswell into the odorous period. Under the condi-tions of culture, odor production was sometimesnoticeable after as little as 3 days (0. tenuis and0. simplicissima) and no later than 18 days forAnabaena species, long before the cultures be-gan the death phase. Moreover, the MIB level ina culture of 0. curviceps killed by the addition ofa copper complex was not significantly higherthan in the same culture before treatment.Therefore, the process of geosmin or MIB pro-duction for these four species did not appeardependent on death and decay of the organisms.

It is known that under artificial cultivation,cyanobacteria, like other organisms, may losesome of the characteristics found in nature. Databased on laboratory cultures must be interpretedwith great caution, since the natural environ-ment is very different from that of a syntheticmedium in a flask. At least in one respect,however, there was good correlation betweenthe properties of these organisms in culture andin the field. In three of the four cases, the majorodorant found in culture had previously beenidentified in the water or sediment sample fromwhich the respective organism was isolated. Thecharacteristic of the odor in culture was virtuallyidentical with that of the environmental sample.Moreover, the ability to produce geosmin orMIB was not lost in subculture over manymonths, suggesting that it is a persistent featureof their metabolism.The CLSA method, coupled with gas chro-

matograph/mass spectrometer/computer systemanalysis, was an integral part of this study.Unlike other methods which require large vol-umes of sample and cumbersome, time-consum-ing concentration steps, this method made possi-ble the rapid detection of microgram-per-literlevels of odorous compounds like geosmin andMIB with relatively small volumes of culture(generally around 5 ml). It is a very sensitive andconvenient method which will play an increasingrole in future research on taste- and odor-caus-ing substances in the aquatic environment.

ACKNOWLEDGMENT

We thank G. W. Prescott for his kindly assistance inidentifying three of the algal isolates.

LITERATURE CITED

1. Allen, M. M. 1973. Methods for cyanophyceae, p. 127-138. In J. R. Stein (ed.), Handbook of phycological meth-ods: culture methods and growth measurements. Cam-bridge University Press, Cambridge.

2. Cees, B., J. Zoeteman, and G. J. Piet. 1974. Cause andidentification of taste and odour compounds in water. Sci.Total Environ. 3:103-115.

3. Clare, L. G., and N. E. Hopson. 1975. Algae problems ineastern Lake Erie. J. Am. Water Works Assoc. 67:131-134.

4. Gerber, N. N. 1979. Volatile substances from actinomy-

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cetes: their role in the odor pollution of water. Crit. Rev.Microbiol. 7:191-214.

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6. Gorham, P. R., J. S. McLachlan, U. T. Hammer, andW. K. Kim. 1964. Isolation and culture of toxic strains ofAnabaena flos-aquae (Lyngb.) de Breb. Verh. Int. Ver.Theor. Angw. Limnol. 15:796-804.

7. Grob, K. 1973. Organic substances in potable water and inits precursor. Part I. Methods for their determination bygas-liquid chromatography. J. Chromatogr. 84:255-273.

8. Grob, K., and F. Zurcher. 1976. Stripping of trace organicsubstances from water. Equipment and procedure. J.Chromatogr. 117:285-294.

9. Henley, D. E., W. H. Glaze, and J. K. G. Silvey. 1969.Isolation and identification of odor compound producedby selected aquatic actinomycete. Environ. Sci. Technol.3:268-271.

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11. Krasner, S. W., C. J. Hwang, and M. J. McGuire. 1981.Development of a closed-loop stripping technique for theanalysis of taste- and odor-causing substances in drinkingwater, p. 689-710. In L. H. Keith (ed.), Advances in theidentification and analysis of organic pollutants in water,vol. 2. Ann Arbor Science Publishers, Ann Arbor, Mich.

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Izaguirre. 1980. Closed-loop stripping analysis at theparts-per-trillion level as a tool for solving taste and odorproblems, p. 377-396. In Proceedings of the 8th AnnualAWWA Water Quality Technology Conference. Ameri-can Water Works Association, Denver.

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16. Medsker, L. L., D. Jenkins, J. F. Thomas, and C. Koch.1969. Odorous compounds in natural waters. 2-Exo-hy-droxy-2-methylbornane, the major odorous compoundproduced by several actinomycetes. Environ. Sci. Tech-nol. 3:476-477.

17. Narayan, L. V., and W. J. Nunez III. 1974. Biologicalcontrol: isolation and bacterial oxidation of the taste-and-odor compound geosmin. J. Am. Water Works Assoc.66:532-536.

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20. Rebhun, M., M. A. Fox, and J. B. Sless. 1971. Chlorina-tion of odorants from algal blooms. J. Am. Water WorksAssoc. 63:219-224.

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