EPA-670/2-74-094 November 1974 Environmental Protection Technology Series MICROBIOLOGICAL PRODUCTION OF GEOSMIN National Environmental Research Center Office of Research and Development U.S. Environmental Protection Agency Cincinnati, Ohio 45268 ------- EPA-670/2-74-094 November 1974 MICROBIOLOGICAL PRODUCTION OF GEOSMIN By Nancy N. Gerber Institute of Microbiology Rutgers - The State University of New Jersey New Brunswick, New Jersey 08903 Program Element No. 1CB047 Project Officer Alan A. Stevens Water Supply Research Laboratory National Environmental Research Center Cincinnati, Ohio 45268 NATIONAL ENVIRONMENTAL RESEARCH CENTER OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO 45268 For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 ------- REVIEW NOTICE The National Environmental Research Center - Cincinnati has reviewed this report and approved its publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. * » 11 ------- FOREWORD Man and his environment must be protected from the adverse effects of pesticides, radiation, noise and other forms of pollution, and the unwise management of solid waste. Efforts to protect the environment require a focus that recognizes the interplay between the components of our physical environment — air, water, and land. The National Environmental Research Centers provide this multidisciplinary focus through programs engaged in • studies on the effects of environmental contaminants on man and biosphere, and • a search for ways to prevent contamination and to recycle valuable resources. This research has provided increased understanding of aspects of the microbiological production of a chemical compound which causes objectionable tastes and odors in water supplies, and has resulted in the availability of standard samples of that compound. This new understanding and the samples themselves can now be used by other researchers in monitoring of water treatment unit processes and development of appropriate new control technology. A. W. Breidenbach, Ph.D Director National Environmental Research Center, Cincinnati 111 ------- Abstract Geosmin, trans-1, 10-dimethyl-trans-9-decalol, is produced by numerous actinomycetes and some blue-green algae. It has been shown to be responsible for earthy/musty odors in public water supplies. Samples of purified geosmin were needed by the U.S. Environmental Protection Agency and by others for use as odor standards and for use in water treat- ment unit process development studies. This work was carried out to fulfil this need. / The microorganism, Streptomyces sp. CWW3, was grown for 5 days at 28° with shaking in a broth medium. The mixture was steam distilled and the clear distillate extracted with methylene chloride. The geosmin containing methylene chloride extract was separated and the geosmin purified by column and gas chromatography. Seven hundred nine milligrams of geosmin was prepared. This report was submitted in fulfillment of Research Grant No. R-801630 by the Institute of Microbiology, Rutgers - The State University of New Jersey under the sponsorship of the Environmental Protection Agency. Work was completed as of June 15, 1974. IV ------- Table of Contents Page Introduction 1 Experimental 2 Sterilization 2 Media 2 Gas chromatography 2 Actinomycetes in sterile soil 3 Production of geosmin 3 Purification of geosmin 4 Preparation of samples 5 Summary of samples sent 5 Discussion 6 References 7 ------- Conclusions It is possible to prepare pure geosmin in milligram amounts from Streptomyces sp. CWW 3. Recommendations Milligram or gram amounts of geosmin could probably be produced by any one of the American pharmaceutical companies who presently make antibiotics by fermentation from Streptomyces, VI ------- I n't r oduc t ion "osme" Geosmin from the Greek "geos" meaning earth and meaning odor is the name which has been given to the substance responsible for the characteristic " earthy JJodor of the soil. It is.,produced by numerous actinomycetes I and some blue-green algae ' . Geosmin and methylisoborneol ' (Fig. 1) have been shown to be responsible for earthy/musty odorSpin public water supplies in North America » Europe , and Japan . The threshhold odor of geosmin is 0.1 ppb . It is probably geosmin which accumulates inthe flesh of fish which inhabit earthy/musty tainted water making the fish unpalatable and unmarketable. 1 12 Since our initial isolation and structure determination of geosmin as trans-1, 10-dimethyl-trans-9-decalol we have had many requests for authentic samples of geosmin. Twenty three requests from 1967-1972 were listed in our grant application. The majority of the requests involved research on water or fish. Since then our supplies of geosmin have been exhausted. Because of the number of requests received by us and by EPA, financial assistance was provided to our laboratory by EPA for production of purified geosmin to be distributed to other researchers by EPA and used in EPA water treatment unit process development studies. R%» _ AO^ MS" geosmin methylisoborneol Fig. 1. The structures of geosmin and methylisoborneol, ------- The yields of isolated geosmin have always been about 1 mg per liter of whole broth. This meant that the typical 40 flask, 10 1 batch which is the largest that a technician can conveniently handle yields about 10 mg of geosmin. A reasonable production schedule considering the amount of shaker space available at 28° for the 2 1 production flasks is one batch per week. The preparation of seed cultures and media, sterilization and innoculation as well as work up of the batch takes about half the work time of one technician. Therefore, we contracted to prepare 10 mg of geosmin per week after a start-up delay of 1 month. This suggests 480 mg of geosmin as the year's output. Because during the year we were able to increase the yields slightly, we actually sent 709 mg of pure geosmin in four quarterly shipments. We also sent pure methylisoborneol (16 mg) and some mixtures of geosmin and methylisoborneol (71 mg). Experimental Sterilization. Normal sterilization procedure is in an autoclave 20 min. at 121° using 15 pounds steam pressure. Media. The recipes given are for liquid media (broth). For solid media add 15 g agar per liter. Cerelose is a commercial grade of glucose. Soy-Bean. 10 g Staley's soybean meal, 20 g Cerelose, 5 g Bacto-peptone or 10 g Wilson Co. meat peptone 851C, 5 g sodium chlorine per liter of tap water, pH adjusted to 7.5 with 40% aqueous NaOH before sterilization, about 500 mg of CaCO., added to each 2 liter flask. Yeast-Dextrose. 10 g Difco yeast-extract, 10 g Cerelose per liter of tap water, pH adjusted to 7.0-7.2 with 40% aqueous NaOH. Gas Chromatography (GC). GC was carried out with an F & M model 700 dual column instrument with a thermal conductivity detector. The coiled copper column was 6 ft by 1/8 inch and contained 10% SE-30 in Diatoport W, 60-80 mesh. This is a non-polar column which separates substances roughly in order of the volatility. Carrier gas was helium at 50 ml/min. ------- Actinomycetes in Sterile Soil. To 100 g of garden soil which has been autoclaved for 1-2 hours is added 1 g CaCO- and 259 mg. of dried blood. Next day the mixture is autoclaved again for 1-2 hours, and then 20 ml sterile distilled water and about 4 ml of packed cells is added. The cells are obtained by centrifuging a freshly grown broth culture and are usually washed with sterile water. The soil-cells mixture is incubated for 2-3 days at 28° then stored in the refrigerator. Production of Geosmin. A small portion of soil containing Streptomyces sp CWW3 was taken with a sterile spatula and innocu- lated into yeast-dextrose broth, 50 ml/250 ml flask. After 24 hours incubation at 28 on a rotary shaker at about 200 rpm, the resulting cell suspension was pipetted aseptically into 14 similar flasks of yeast-dextrose broth (2 1/2 ml of cell suspension per flask). After 24 hours shaking at 28° these flasks were used to innoculate 40 flasks of soy-bean medium, 250 ml/2 liter flask (1/3 of the contents of a small flask poured aseptically into each large flask.) After 5 days on a reciprocal shaker, 80 strokes per min. at 28 the contents of the flasks were combined and distilled at atmospheric pressure until about 2 liters of distillate had collected. Boiling chips and several milliliters of polyglycol-2000 antifoaming agent were added to minimize foaming (Hodag Chem. Co., Chicago, 111. or Dow Chemical Co., Midland, Mich.). The distillate was extracted twice with 10-20% of methylene chloride and the methylene chloride extracts concen- trated in a stream of warm air. The extracts of the distillate from one such 10 liter batch after concentration to 10 ml was suitable for gas chromatographic (hereafter called GC) assay and was stable to storage at 5°. Typical yields, deduced from the areas of the peaks in the GC assay were: methylisoborneol 16 mg, geosmin 23 mg and sesquiterpenol 13 mg (hereafter called MIB, geos and SQ). Table 1. RETENTION TIMES OF MIB AND GEOS Initial temp. 80° 100° 120° Rate of temp. increase 5°/min n .. Retention MIB 14 10 7 time GEOS 20.5 16 12 (min.) of SQ 27 21.5 18 ------- Purification of Geosmin. In a typical purification experiment the distillate-extract concentrate from 2 batches was further concentrated and applied to a chromatography column made up of 'Baker, analyzed1 reagent aluminum oxide powder (12g) in methylene chloride. The aluminum oxide had previously been stirred several hours with distilled water, filtered,and air dried for several days at room temperature. The 3 desired volatile alcohols passed readily through the alumina column and were detected in the first three 10 ml eluate fractions, but acidic and dark impurities were retained on the column. The 3 eluate fractions were combined and applied to a narrow (1.4 x 36 cm) 20 g column of 'Baker analyzed1 reagent silica gel powder in methylene chloride. Elution was with methylene chloride, 10 ml fractions. GC assay revealed the products, and the amounts were estimated from peak areas. The retention times of the 3 volatile products are given in Table 1. The results of a typical purification experiment are summarized in Table II. Table II. SUMMARY OF SILICA GEL CHROMATOGRAPHY Fraction # Total Vol Product and Amount 1-3 60 nothing 4 10 35 mg geos. 5 10 21 mg geos & 7 mg MIB 6 10 21 mg MIB 7 10 10 mg MIB 8-12 50 42 mg SQ Subsequent purifications of up to ten combined batch extracts were carried out in exactly the same way with appropriate increases in amounts of reagents. ------- Preparation of Samples. Three types of samples were prepared. Mixed Geosmin and Methylisoborneol. Fractions #4-7 (Table II) were combined then dispensed into 3 glass ampoules. The methylene chloride solvent was removed in a stream of air and the last traces of solvent were removed by placing the ampoules in a vacuum oven for 2 minutes at 25-27 inches vacuum. Although fractions 4-7 assayed 94 mg of geosmin and MIB the weight in the 3 ampoules after solvent removal was 71 mg of this mixture. This loss of the desired volatile products during solvent removal was unadvoidable and was sometimes as much as 50%. Purified Geosmin. In later experiments eluate fractions containing geosmin only, were used and 70% ethanl was chosen as the solvent for shipment. Most ampoules were shipped containing 20-35 mg geosmin and 0.5 ml 70% ethanol, and the exact weight of geosmin was recorded on each ampoule. The 70% ethanol solvent was chosen because it preserved the sterility of the sample without dehydration. This solvent was also compatible with the aqueous phase in eventual dilution for studies in water. GC Pure Geosmin and MIB. In another experiment, the relevant eluate fractions from the alumina column were taken to dryness and redissolved in a small volumn of cyclohexane for preparative GC. In solution were 16 mg MIB, 23 mg geosmin and 13 mg SQ,but the amounts recovered from the preparative GC were 8 mg MIB, 15 mg geosmin and 8 mg SQ. These losses during preparative GC were typical and unavoidable under the conditions used. GC pure materials were collected in capillary tubes 1.6 - 1.8 x 100 mm which fit snugly into the exit port of the GC. The tubes were sealed at both ends before shipment. Summary of Samples Sent. A summary of samples sent to EPA is presented in Table III. ------- Table III. SUMMARY OF SAMPLES SENT TO EPA Date Samples Amount and Form 9/28/73 10/1/73 11/1/73 3/5/74 6/20/74 geos. MIB geos & MIB mixture geos, 20 small sealed tubes each 1.5 mg 20 small sealed tubes each 0.8 mg 25 mg in 1 ml H20 & 3 drops CHC13, sealed ampoule 21 mg in 70% ethanol, sealed ampoule 25 mg no solvent, sealed ampoule 10 sealed ampoules total 184 mg 10 sealed ampoules total 308 mg 5 sealed ampoules total 187 mg Total geosmin 709 mg Discussion Streptomyces sp CWW3 had been isolated from Lake Michigan water. It was received from Miss Lilia McMillan of the Central Water Filtration Plant, Chicago, Illinois and was considered, along with several other strains which were sent at the same time, to be implicated in odor problems for that department. In order to preserve this culture with the minimum of change it was immediately put into sterile soil. Frequently when a micro- organism is taken from its natural environment and placed on laboratory media it ceases to produce the substance or feature which one desires to investigate. However, CWW3 stored in soil, during 3 years was a stable and reliable producer of geosmin, methylisoborneol and the sesquiterpene alcohol C,cH2gO. Other geosmin producing actinomycetes are listed in references 1 and 2, however, it should be emphasized that many of them were tried only once. In the cases where repeated fermentations were done, the yields of geosmin were variable and low. ------- 1. Gerber, N. N. and H. A. Lechevalier. Geosmin, an Earthy- Smelling Substance Isolated from Actinomycetes. Applied Microbiology, 13^ (6):935-938, November 1965. 2. Gerber, N. N. Geosmin, an Earthy-Smelling Substance Isolated from Actinomycetes. Biotechnology and Bioengineering 9_(3) :321-327. 3. Safferman, R. S., A. A. Rosen, C. I. Mashni and M. E. Morris. Earthy-Smelling Substance from a Blue-Green Alga. Environmental Science and Technology 1:429-430, May 1967. 4. Kikuchi, T., T. Mimura, K. Harimaya, H. Yano, T. Arimoto, Y. Masada and T. Inoue. Odorous Metabolite of Blue- Green Alga: Schizothrix muelleri Nageli collected in the Southern Basin of Lake Biwa. Identification of Geosmin. Chemical and Pharmaceutical Bulletin (Tokyo) 23^(10) :2342-2343, 1973. 5. Gerber, N. N. A Volatile Metaobolite of Actinomycetes, 2- methylisoborneol. The Journal of Antibiotics (Tokyo) 22:508-509, October 1969. 6. Rosen, A. A., C. I. Mashni and R. S. Safferman. Recent Developments in the Chemistry of Odour in Water. The Cause of Earthy/Musty Odour. Water Treatment and Examination 10_: 106-119, 1970. 7. Piet, G. J., C. J. Zoeteman and J. A. Kraayeveld. Earthy Smelling Substances in Surface Waters of the Netherlands Water Treatment and Examination 2^(4):281-286, 1972. 8. Kikuchi, T., T. Mimura, Y. Itoh, Y. Moriwaki, K. Negoro, Y. Masada and T. Inoue. Odorous Metabolites of Actinomyces Biwako-C and -D Strain Isolated from Bottom Deposits of Lake Biwa. Identification of Geosmin, 2-methylisoborneol and Furfural. Chemical and Pharmaceutical Bulletin (Tokyo). 2M10) : 2341, 1973. ------- 9. Thaysen, A. C. The Origin of an Earthy or Muddy Taint in Fish. Annals of Applied Biology. 2:3:99, 1936. 10. Aschner, M., Ch. Laventer and I. Chorin-Kirsch. Off Flavour in Carp from Fish Ponds in the Coastal Plain and the Galil. Bamidgeh, Bulletin of Fish Culture in Israel, 19_(1) : 23-25, 1967. 11. Cherry, A, E. Use of Potassium Permanganate in Water Treat- ment. Journal of the American Water Works Association. £4_(4) :417-424, April 1962. 12. Gerber, N. N. Geosmin, from Microorganisms is trans-J, 10- dimethyl-trans-9-decalolC Tetrahedron Letters (25):2971- 2974, 1968. ------- TECHNICAL REPORT DATA (Please read Instructions on the reverse before completing) . REPORT NO. EPA-670/2-74-094 3. RECIPIENT'S ACCESSION-NO. 4. TITLE AND SUBTITLE MICROBIOLOGICAL PRODUCTION OF GEOSMIN 5. REPORT DATE November 1974; Issuing Date 6. PERFORMING ORGANIZATION CODE 7. AUTHOR(S) Nancy N. Gerber 8. PERFORMING ORGANIZATION REPORT NO 9. PERFORMING ORGANIZATION NAME AND ADDRESS Institute of Microbiology Rutgers - The State University of New Jersey New Brunswick, New Jersey 08903 10. PROGRAM ELEMENT NO. 1CB047; ROAP 21AQB; Task 9 11.S«MX«*SKGRANT NO. R-801630 12. SPONSORING AGENCY NAME AND ADDRESS National Environmental Research Center Office of Research and Development U.S. Environmental Protection Agency Cincinnati, Ohio 45268 13. TYPE OF REPORT AND PERIOD COVERED Final 14. SPONSORING AGENCY CODE 15. SUPPLEMENTARY NOTES 16. ABSTRACT Geosmin, trans-1, 10-dimethyl-trans-9-decalol, is produced by numerous actinomycetes and some blue-green algae. It has been shown to be responsible for earthy/musty odors in public water supplies. Samples of purified geosmin were needed by the U.S. Environmental Protection Agency and by others for use as odor standards and for use in water treatment unit process development studies. This work was carried out to fulfil this need. The microorganism, Streptomyces sp. CWW3, was grown for 5 days at 28 with shaking in a broth medium. The mixture was steam distilled and the clear distillate extracted with methylene chloride. The geosmin containing methylene chloride extract was separated and the geosmin purified by column and gas chromato- graphy. Seven hundred nine milligrams of geosmin was prepared. This report was submitted in fulfillment of Research Grant No. R-801630 by the Institute of Micro- biology, Rutgers - The State University of New Jersey under the sponsorship of the Environmental Protection Agency. Work was completed as of June 15, 1974. 17. KEY WORDS AND DOCUMENT ANALYSIS a. DESCRIPTORS b.lDENTIFIERS/OPEN ENDED TERMS COS AT I Field/Group *0dors Odor control *Water treatment Microorganisms Fermentation Purification Chromatograpy *Actinomyces * S treptomyce s *Geosmin *Methylisobornepl *Actinomycetes *Standard samples *Geosmin production 13B 18. DISTRIBUTION STATEMENT RELEASE TO PUBLIC 19. SECURITY CLASS (ThisReport)' UNCLASSIFIED 21. NO. OF PAGES 15 20. SECURITY CLASS (Thispage) UNCLASSIFIED 22. PRICE EPA Form 2220-1 (9-73) U. S. GOVERNMENT PRINTING OFFICE: 197'i-657-588/5322 Region No. 5-11 ------- |