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

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                                       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

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                      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

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                         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

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                           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

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                     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

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                           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

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                         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,

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      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.

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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

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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.

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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.

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           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.

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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.

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 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.

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                                   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

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