Agricultural and Biological Chemistry

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Studies on Utilization of Hydrocarbons by Yeasts

Kei Arima, Shigeo Ogino, Keiji Yano & Gakuzo Tamura

To cite this article: Kei Arima, Shigeo Ogino, Keiji Yano & Gakuzo Tamura (1965) Studies on
Utilization of Hydrocarbons by Yeasts, Agricultural and Biological Chemistry, 29:11, 1004-1015,
DOI: 10.1080/00021369.1965.10858509

To link to this article: https://doi.org/10.1080/00021369.1965.10858509

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 (Agr. Biol. Chern., Vol. 29, No. 11, p. 1004~1008, 1965]

Studies on Utilization of Hydrocarbons by Yeasts

Part I. Studies on Cultural Conditions of Yeasts

By Kei ARIMA, Shigeo 0GINo,* Keiji YANO and Gakuzo TAMURA

Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo
Received June 8, 1965

The production of microbial cell substances from hydrocarbons has been attracting
attention of people for many years. Production of bacterial cell from hydrocarbons is
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disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell pro-
duction. The effect of medium composition on yeast growth and the utilization of in-
dividual hydrocarbon by yeast, strain Y-3, were investigated.
As a nitrogen source, urea was more effective than ammonium nitrate. When a very
small amount of corn steep liquor was added, yeast growth was very improved. Aliphatic
series of hydrocarbon lower than C 9 were not or very slightly assimilated by this yeast.
Generally speaking, series of even-number hydrocarbons were more effective than
those of odd-number hydrocarbons.

Recently, hydrocarbon fermentation has been Broth (1 ~4 ml)

widely investigated by many researchers. 1 - 31 -0.0025% Aerosol OT
solution was added
Especially, the production of microbial cell (0.2 ml/ml broth)
substance from hydrocarbons has become a -agitated and centrifuged
(4000r.p.m. for 15 min.)
great deal of interest.
In this paper, the effect of cultural condi- I
ppt. sup.
tions on growth of Y-3 strain which is similar
to Pichia sp. and the results of experiments ~-washed with petroleum ether

on the utilization of individual pure alkanes !-centrifuged

by this yeast are reported. I

sup. ppt.

EXPERIMENTAL AND RESULTS -suspended in 0.8% NaC!

solution
!
Yeast growth was determined as follows: sample
cell suspension was prepared as Fig. 1 and the FIG. I. Preparative Procedure of Cell Suspension
optical density at 650 m,u was measured by for Determination of Growth.
spectrophotometer.
Effects of nitrogen sources on yeast growth
As nitrogen sources, urea and ammonium
* Present address; Research Department, Pharmaceutical nitrate were employed. Compositions of
Division of Sumitomo Chemical Co., Ltd. Kasugade-cho,
Konohana-ku, Osaka.
media are shown in Table I.
1) "New Aims for Biosynthesis", Chemical Week, Feb. 2, Hydrocarbon was a mixture of an equal
49 (1963).
2) "Paraffin Feedstock for BP Bugs", European Chemical amount of kerosene and liquid paraffin.
News, Dec. 28, 26 (1962). One hundred ml of four kinds of media
3) ]. Takahashi, K. Kobayashi, Y. Kawabata and K.
Yamada, This Journal, 27, 836 (1963). shown in Table I, in 500 ml shaker flasks,
 Studies on Utilization of Hydrocarbons by Yeasts. Part I 1005

TABLE I. COMPOSITIONS OF MEDIA EMPLOYED IN The results are shown in Fig. 2. These
TEST OF CULTURAL CONDITIONS growth curves show that, as a nitrogen source,
AN* U* urea is more suitable than ammonium nitrate
Na 2HP04·l2 H 20 2.5 g 2.5 g in case of 10% hydrocarbon, and that, when
KHzP04 3.5 g 3.5 g 5% hydrocarbon was employed, the growth
MgS04·7 H20 l.Og 1.0 g is almost equal.
NaCI 0.5 g 0.5 g
(NHz)2CO Effects of C/N ratio on yeast growth
3.8g
NH 4NOa 5.0g Effects of C/N ratio (hydrocarbon concentra-
Hydrocarbon 50.0 or 50.0 or tion W/V% per urea concentration W/V %)
100.0 g lOO.Og on yeast growth were tested.
Tap Water 1.0 I 1.0 I The composition of media was the same as
pH 6.5 6.5 U-media in Table I except for CjN ratio of
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*U : the medium containing urea as nitrogen source
AN: the medium containing ammonium nitrate as nitro-
gen source.
were inoculated with 2 ml of seed culture
after fifteen min. steam-heat sterilization at
120°C.
Composition of seed culture medium was
hydrocarbon mixture 5.0%, disodium phos-
phate 12 aq. 0.25%, monopotassium phosphate
0.35%, magnesium sulfate 7 aq. 0.10%, sodium
chloride 0.5%, ammonium nitrate 0.50% and
tap water.
Flasks were shaken on the reciprocal shaker
at 30°C.
Time of cultivation (days)
FIG. 2. Effects of Nitrogen Sources of Yeast Growth.
CNH 2 )2CO; 6 - 6 Hydrocarbon 10% (w/v)
CNH2l2CO; 0 - 0 Hydrocarbon 5% (w/v)
NH 4 NOa ; 6---6 Hydrocarbon 10% (w/v)
NH4 NOa ; 0---0 Hydrocarbon 5% (w/v)
C!N ratios based on urea (see text) are 26.3 in case of
10% hydrocarbon and 13.2 when 5% hydrocarbon is
employed.
nitrogen source and hydrocarbons. The ex-
periments were carried out as follows: first
group included three flasks into which hydro-
carbon was added at concentration of 2.5%.
The C/N ratios of each flask were 10, 20 and
30, respectively.
The second group included also three flasks
into which hydrocarbon was added at con-
centration of 5.0%. The C/N ratios of each
flask were the same as the first group.
The third group and fourth group included
the same number of flasks into which hydro-
carbon was added at concentration of 7.5%
and 10.0%, respectively. The C/N ratios of
each flask of two groups were the same as
above.
All flasks contained 100 ml of media and
were inoculated with 2 ml of seed culture,
the composition of which was hydrocarbon
mixture 5.0%, disodium phosphate 12aq. 0.25;?6',
monopotassium phosphate 0.35%, magnesium
sulfate 7 aq. 0.10%, sodium chloride 0.05%,
urea 0.38% and tap water.
These flasks were shaken on the reciprocal
shaker at 30oC.
The results are shown in Fig. 3. These
results show that the CjN ratio 30 is most
suitable for growth of this yeast at all con-
centrations of hydrocarbon tested.
Effects of corn steep liquor on yeast growth
Effects of corn steep liquor on yeast growth
were tested by the same cultural procedures
as described above.
Into three 500 ml shaker flasks which con-
 1006 Kei ARIMA, Shigeo OGINO, Keiji YANO and Gakuzo TAMURA

--;;:_
E 2.+ ~

"-
8
fiS fiS 3.0
"'
~ "'
~

~ ~
..':'

1:~.
·;;; ..':'
·;;;
2.0-

"
v "
...""'-~ ...""'-~'"
P. I I I
P.
0 0
2 3

Time of cultivation (days)

Time of cultivation (days)
FIG. 3a. Effects of CjN Ratio on Yeast Growth.
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FIG. 3d. Effects of CfN Ratio on Yeast Growth.

Hydrocarbon concentration·2.5°-b
0 - 0 C/N Ratio 10 X - X C/N Ratio 20 Hydrocarbon-10.0%
t:,-t:, C!N Ratio 30 0 - 0 C/N Ratio 10 X-X C/N Ratio 20
t:,-D. C!N Ratio 30

"-
~ E
1§ 2.0 1§ 2.0-

..~ "'
~ ""' J.O-

4 5 6
Time of cultivation (days)
Time of cultivation (days)
FIG. 4. Effects of Corn Steep Liquor on Yeas
FIG. 3b. Effects of CfN Ratio on Yeast Growth.
Growth.
Hydrocarbon concentration-5.0%
0 - 0 C/N Ratio 10 x-x C!N Ratio 20 0 - 0 control x - x 0.0001% added
D.-D. C!N Ratio 30 D.-D. 0.001% added D-0 0.01% added
C!N ratio is 26.3

tained 100 ml of cultural media was adde

corn steep liquor at concentration of O.Ol.9i
0.001% and 0.0001%, respectively.
The results of this experiment are show
in Fig. 4.
This shows that the addition of corn stee
liquor is very effective on shortening the tim
of lag phase, and that 0.01% addition an
0.001% addition have almost equal effect o
4 6
yeast growth.
Time of cultivation (days)
Assimilation of individual pure hydrocarbon b
FIG. 3c. Effects of CjN Ratio on Yeast Growth. Y-3 strain
Hydrocarbon concentration-7.5% One hundred ml of sterile mineral salt sol1
0-Q C/N Ratio 10 X - X C/N Ratio 20
D.-D. C/N Ratio 30 tions, in 500 ml shaker flasks, containing 5.0:
 Studies on Utilization of Hydrocarbon s by Yeasts. Part I 1007

of pure hydrocarbons, 0.17% of urea and

0.01% of corn steep liquor were inoculated
with 2 ml of seed culture.
The medium of seed culture contained a
mixture of pure hydrocarbons to be tested,
mineral salts solution and urea.
Pure alkanes tested were n-pentane, n-
hexane, n-heptane, n-octane, n-decane, n-
undecane, n-dodecane and n-tridecane.
The results are shown in Fig. 5. This
shows that aliphatic series lower than c9 are
not or very slightly assimilated. FIG. 6. The Yeast Y -3 Strain Grown on Malt
H ydrocarbons of C 10 to C 13 are well as- Agar ( X 1000).
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similated.
After one day a dry pellicle is formed, but
no sediment is observed. After 3 days sedi-
-;c 18 ment is formed.
e 16 Growth on malt agar; After 3 days at 30oC,
g
14
"' cells are oval to cylindrical, (3-5) x (7-15),u,
3 12
single, in pairs or in short chains. After 3
10
-~c 8 days at 30°C the streak culture is white, dull,
"
'"0 6 raised. (Fig. 6)
-;
-~
Slide culture; Pseudomycelium formation.
c.
0
2
Sporulation (Plaster of Paris blocks method);
The spores are round, 2-4 per ascus, with an
Time of cultivation (days) oil-drop inside.
FIG. 5. Growth Curves of Y -3 Strain in Media Fermentation; Very slight or absent.
Containing Various H ydrocarbons. Sugar assimilation; Glucose + Maltose
@--@ nonane
0---D decane
!::.-!::. undecane
0 ---0 dodecane
x- x tridecane Galactose +
No growth was observed when employed hydrocarbons Lactose - Saccharose +
lower than octane.
Raffinose + Arabinose -
It was observed that, after the stationary Rhamnose - Xylose +
phase, cells of Y-3 strain tended to lyse when Assimilation of potassium nitrate; Absent
employed odd-number hydrocarbon as carbon Splitting of arbutin; Positive
source. This is interesting observation but Gelatin stab; No liquefaction
the reason is not obvious. Ethanol as sole source of carbon; Weak
growth
Taxonomic studies on Y-3 strain
Heat resistance; No growth over 50°C
Diagnostic tests of Y -3 strain were carried
Optimal temperature; 2SOC to 30°C. Growth
Qut and the results showed that this strain
scanty at 3rC and no growth at 40°C.
was similar to Pichia sp. described in "The
Growth pH; from 3.0 to 8.0
Yeasts-A Taxonomic Study- " by Ladder
and Kreger-van Rij. SUMMARY
The description of the strain is as follows; 1) As nitrogen source, urea is more effec-
Growth in malt extract; After 3 days at tive than ammonium nitrate.
30°C, cells are oval to cylindrical (3-5) x 2) The C/N ratio 30 is most suitable within
{6-13),u, single, in pairs or in short chains. the limits of this experiment.
 1008 Kei ARIMA, Shigeo 0GINO, Keiji YANO and Gakuzo TAMURA

3) The addition of corn steep liquor can

shorten the time of lag phase .
••• 4) Y-3 strain cannot assimilate the hydro-
•• I • carbons of aliphatic series lower than c9 .
1.0
•
• ••
5) Percentages of conversion from hydro-
e
~
carbon (calculated from optical density by
• •
§ • • •• • Fig. 7) into cell substance (dried cells) are as
'-
~
.::::
•
:/•.• follows;
n-decane 56.4% n-undecane 43.6%
·;;;"'

Q
il:
...'"' ./· n-dodecane 43.4% n-tridecane 47.5%

Acknowledgement. We are grateful! to Mr.

./. H. Wada of the Research Department, Phar-

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maceutical Division of Sumitomo Chemical

~
Co., Ltd. for the taxonomic studies.

Optical density (at 650 mp)

FIG. 7. The Relationship between Optical Density
and Dry Weight of Yeast Cell.
 [Agr. Bioi. Chern., Vol. 29, No. 11, p. 1009~1015, 1965]

Studies on Utilization of Hydrocarbons by Yeasts

Part II. Diterminal Oxidation of Alkanes by Yeasts

By Shigeo OGINO, * Keiji Y ANO, Gakuzo TAMURA

and Kei ARIMA

Department of Agricultural Chemistry, Faculty of

Agriculture, The University of Tokyo
Received June 8, 1965
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We found that the yeast Y-3 strain reported in the previous paperll has a diterminal
oxidation system of hydrocarbon.
This yeast capable of growing in mineral-salts solution with hydrocarbons as sole
source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-
undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic
acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was
also isolated from n-dccane cultures.
Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating
it with resting cells of this yeast.
1, 11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric
acid by the same treatment as described above.

Microbial oxidation of aliphatic hydrocabon
is believed to occur by a monoterminal oxida-
tion.2-5)
Recently, A. S. Kester and J. W. Foster 6 )
showed that hydrocarbons were oxidized by
a diterminal oxidation mechanism in coryne-
bacterial organisms besides a monotcrminal
mechanism.
The present paper deals with diterminal
oxidation of hydrocarbons by the yeast Y -3
strain.
MATERIASL AND METHODS
Organism and culture methods
All the experiments were performed with the yeast,
• Present address; Research Department, Pharmaceutical
division of Sumitomo Chemical Co., Ltd. Kasugade·cho,
Konohana-ku Osaka.
1) K. Arima, S. Ogino, K. Yano and G. Tamura, This
Journal, 29, 1004 (1965).
2) J. E. Stewart, R. E. Kallio, D.P. Stevenson, A. C. Jones
and D. 0. Schissler, f. Bacteriol., 78, 441 (1959).
3) E. R. Leadbetter and J. W. Foster, Nature, 184, 1428.
4) M. H. Proctor, Biochim. Biophys. Acta, 42, 559 (1960).
5) M. T. Heydeman, ibid., 42, 557 (1960).
6) A, S. Kester and J. W. Foster, f. Bacteriol., 85,859 (1963).
Y -3 strain, tentatively assigned to Pichia sp. in the
previous paper. 1 >
The composition of mineral salts solution used in
this work was disodium phosphate 12 aq. 2.5 g,
monopotassium phosphate 3.5 g, magnesium sulfate
7 aq. 1.0 g, sodium chloride 0.5 g, urea 0.85 g, corn
steep liquor 0.1 ml and tap water II.
The hydrocarbons used were n-undecane of 95%
purity and n-decane of 98% purity (Tokyo Chemical
Industry Co., Ltd.)
Production of various acids in amounts suitable for
isolation was carried out in 5 I shaker flask contain-
ing 1.51 of mineral salts solution and 37.5 g of in-
dividual hydrocarbon as the sole source of carbon,
so the CjN ratio was 30.
From two to five flasks were employed in each
experiment. The mineral salts solution was sterilized
for 15 min. at 120°C, and hydrocarbon was added
just before inoculation. The flasks were shaken on
the rotary shaker at 30°C.
Extraction of acids
The kieselguhr was added to the broth and filtered
under reduced pressure. The filtrate was acidified
to pH 2. 0 with H2S04 and extracted two to three
 1010 Shigeo 0GINO, Keiji Y ANO, Gakuzo TAMURA and Kei ARIMA
times in a separatory funnel with equivalent volumes
of ether.
The combined ether extracts were shaken wit.h
0.5N-NaOH and separated in a funnel. The ether
layer was washed by shaking with distilled water.
The alkaline solution and washed liquor were com-
bined and, after the acidification, were extracted
with ether again.
The ether layer was dried by anhydrous sodium
sulfate.
The dried ether extract was concentrated to dry-
ness and crude acid was obtained at the yield of 30
mgjl. broth.
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Thin-layer chromatography
For time course of acid formation 50 ml of broth
were taken out at intervals of adequate time and
extracted as described above. The crude acids mix-
tures were dissolved into 0.5 ml of ethanol. Twenty
,ul of ethanolic acids solution were spotted on kiesel-
gel G plate.
Two different solvent systems were used, in the
ascending technique; solvent A, benzene-dioxane-acetic
acid (90: 25: 4), solvent B, benzene-ether-acetic acid
(9: 2: 1).
The plate was dried in an oven at l05°C for 1 hr,
and acid spots were developed by spraying with
bromocresol green indicator solution.
Partition column chromatography
For the isolation of various acids, partition column
chromatographic technique was used. Crude acids
mixtures obtained as described above were dissolved
in chloroform and adsorbed on silicic acid column.
Elution was carried out by chloroform followed by
chloroform-ethanol mixtures (97% chloroform).
Dioic acids were eluted in the order of chain
length.
Crude acids were recrystallized from chloroform
and n-hexane mixtures.
Gas chromatography
Gas-liquid chromatography was carried out as fol-
lowing conditions.
chromatograph Shimadzu Model GC-1B
column dimension 150x0.4cm
solid support
chromosorb W (60/80 mesh)
stationary phase DEGS-H 3P0 4 (1-1)
temperature l96°C
carrier gas nitrogen at 30 mljmin
detector flame ionization detector type
HFD-1
Chemical method
Identification of each pure acid was made by co-
chromatography on thin-layer, by molecular weight
determination (Rast or titration), by elementary ana-
lysis and by comparison of the infrared absoption
spectrum of the isolated acid with that of an authen-
tic compound.
The mixture of acids was identified by thin-layer
chromatography and gas chromatography.
Azelaic acid, pimelic acid, glutaric acid and sebacic
acid were obtained from Tokyo Chemical Industry
Co., Ltd.
1,11-Undecane dioic acid was synthesized by
Willgerodt reaction. 7l
Procedures of resting cell experiments
Yeast cells were collected at the log phase by
centrifugation.
Cells were washed four times with the mineral
salts solution without the nitrogen source and were
suspended in the same salts solution.
The reaction mixtures were prepared as follows:
to a Monod test tube were added 7 ml of mineral
salts solution with no nitrogen source, 1 ml of
0.01 M-dioic acid solution (neutralized to pH 7.0
by 1 N-NaOH) and 2 ml of cell suspension, the cell
concentration of which was five times as much as the
concentraticm in broth at maximum growth in optical
density.
The mixtures were incubated overnight at 30°C on
Monod shaker
RESULTS
Acid formation
Acid production during growth of Y -3 strain
on n-undecane was tested. The results are
shown in Fig. 1.
This shows that about five spots are deve-
loped, and that the acid which appears at
early stage and has the largest RF value dis-
appears at 140 hr cultivation.
The amounts of the two acids which begin
to appear at 30 hr after inoculation are
gradually increasing with the passage of culti-
vation time.
These three acids were isolated from n·
undecane culture and identified. But the
7) D. B. Pattison and M. Carmack, ]. Am. Chem. Soc., 68,
2034 (1946).
 Studies on Utilization of Hydrocarbons by Yeasts. Part II. lOll

by the same method.

The acid isolated from n-decane also agreed
with sebacic acid. IR-spectra of these acids
are shown in Fig. 2.
8000 This shows that the acids isolated from n-
undecane culture are 1,11-undecane dioic acid,
azelaic acid and pimelic acid, respectively.
D The acid isolated from n-decane cui ture is
the same as sebacic acid.
z"o 30
9
44 68 92 116 140 164 Degradation of 1,11-undecane dioic acid and
hrs hrs hrs hrs hrs hrs hrs hrs
azelaic acid by resting cells of Y-3 strain
FIG. 1. Chromatogram of Organic Acids Produced The acids obtained from resting cell incu-
by Y -3 Strain. bation mixture were extracted as described
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Solvent benzene/dioxane/acetic acid=!l0/25/4 above.

other two acids which began to appear at
140 hr cultivation were not isolated.
Identification of acids
Chemical properties of organic acids obtain-
ed from n-undecane and n-decane cultures
are shown in Table I.
These acids were isolated by column chro-
matography and recrystallized from adequate
solvents.
The acid which had largest RF value agree-
ed with 1,11-undecane dioic acid synthesized
by Willgerodt reaction by cochromatography
of thin-layer.
The second acid agreed with azelaic acid
and the third also agreed with pimelic acid
Identification of the extracts was carried
out by thin-layer chromatography and gas
chromatography.
In thin-layer chromatography two solvent
systems were used.
The results are shown in Fig. 3 and 4. This
shows that 1,11-undecane dioic acid is trans-
formed into azelaic acid, pimelic acid and
glutaric acid, and that azelaic acid is changed
into pimelic acid and glutaric acid.
These results are supported by gas chromato-
graphy as shown in Fig. 5 and 6.
DISCUSSION
The experiments reported here show that
hydrocarbon is oxidized by a diterminal oxida-
tion mechanism in yeast.

TABLE I. CHEMICAL PROPERTIES OF ORGANIC ACIDS

PRODUCED BY Y -3 STRAIN

Melting point Molecular Elementary

wt. analysis
~ ...-'-----... ...-'-----...
Authentic Theo- Theo-
Sample acid Mixed Found retical Found
retical
0 {C: 61.13 C: 61.06
1,11-Undecane dioic acid 111.0~111.5 112.0 111.0 220 216.3
H: 9.29 H: 9.32
{C: 57.10 C: 57.43
Azelaic acid 105.5~106.0 106.5 105.5 *195 188.2
H: 8.51 H: 8.57
{C: 52.39 C: 52.49
Pimelic acid 104.0~105.0 104.5 104.0 0
]62 160.2
H: 7.59 H: 7.55
{C: 59.29 C: 59.38
Sebacic acid !33.5~134.5 134.5 133.0 202.0
H: 8.89 H: 8.97
Rast Method; ' Titration
 1012 Shigeo 0GINO, Keiji YANO, Gakuzo TAMURA and Kei ARIMA

Wavenumber (cm-1)
FIG. 2a. Infrared Spectrum of Acid Produced from n-Undecane by Y -3 Strain.
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Solid line: authentic l,ll·undecane dioic acid

Broken line: microbial product (nujol)

4.000 3,200 2,400

Wavenumber (Zm-1)
FIG. 2b. Infrared Spectrum of Acid Produced from n-Undecane by Y-3 Strain.
Solid line: authentic azelaic acid
Broken line: microbial product (nuiol)

3,200 2,400 1,900

Wavenumber (cm- 1)
FIG. 2c. Infrared Spectrum of Acid Produced from n-Uudecane by Y -3 Strain.
Solid line: authentic pimelic acid
Broken line: microbial product (nujol)
 Studies on Utilization of Hydrocarbons by Yeasts. Part II. 1013

Wavenumber (cm-1)
FIG. 2d. Infrared Spectrum of Acid Produced from n-Decane by Y -3 Strain.
Solid line: authentic sebacic acid
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Broken line: microbial product (nujol)

==
OQo=O 0
occo 0
0 Q c-:_; cJ o
0 0
0<:)00 0 0 C~) C) 0 0

cont 9D 9D liD liD 9D 7D SD SD 6D

.~g R.C. R.C. ~B . . . . .
SD
cont. 9D 9D liD liD 9D SD 6D SD
FIG. 3. Thin-layer Chromatogram from ~B R.C. R.C. ~B
SD
Resting Cell Experiments.
Solvent: benzene-dioxane-acetic acid (90: 25: 4) EIG. 4. Thin-layer Chromatogram from
Cont.: no substrate 11 D: l,ll·undecane dioic acid Resting Cell Experiments.
9D: azelaic acid 8D: suberic acid
7D: pimelic acid 6D: Adipic acid SD glutaric acid Solvent: benzene·ether·acetic acid (9: 2: 1)
R. C.: treated with resting cells. Cont.: no substrate llD: l,ll·Undecane dioic acid
9D: azelaic acid, 8D: suberic acid
7D: pimelic acid, 6D: adipic acid SD: glutaric acid
R. C.: treated with resting cells.
The dioic acids produced by this yeast are
metabolized by the resting cells in such a
way as other dioic acids the carbon skeletons from n-undecane culture besides 1,11-undecane
of which are shorter by two atoms than that dioic acid.
of the substrate, are produced. Dioic acids pathway reported here has not
We believe that this degradation is probably yet been demonstrated hitherto in yeast.
carried out by ~-oxidation mechanism. These observations show that diterminal
In bacteria, Kester and Foster 61 obtained oxidation of alkanes may be widely distribut-
decane and dodecane dioic acids etc., but ed in microorganisms.
shorter chain-length dioic acids than the sub- We could not isolate the monoic acids such
strate alkanes of their series were not demon- as undecanoic acid, decanoic acid and nona-
strated. noic acid etc. from either cultural filtrate or
We could isolate azelaic and pimelic acid cells.
 1014 Shigeo 0GINO, Keiji YANO, Gakuzo TAMURA and Kei ARIMA

Azelaic acid

j
Pimelic acid

l
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
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Min.
FIG. 5. Gas Chromatogram of Acids Extracted from Incubation Mixtures.
Azelaic acid was treated with resting cells.

Glutaric acid
I

Azelaic acid
Pimelic acid
j l
I
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Min.
FIG. 6. Gas chromatograms of Acids Extracted from Incubation Mixtures.
1.ll·undecane dioic acid was treated with resting cells.

This may merely mean that these acids do way, although we could not isolate them.
not accumulate in detectable amounts. There are at least two kinds of degradation
After hydrolysis of extracts from cells, the pathway of alkanes in microorganisms: monoic
acid mixtures, the carbon-chain length of acid pathway and dioic acid pathway.
which was longer than C 13 , were isolated. The scheme of these pathways is shown in
Anyhow, there may be a monoic acid path- Fig. 7.
 Studies on Utilization of Hydrocarbons by Yeasts. Part II. 1015

n-undecane "
,,,~"'

[HO-CHdCH2)g-CH20H] <--------
"'
[CH 3 -(CH2)g-CH 20H)

1
HOOC-(CHz)g-COOH < - - - - - - - - - CH 3-(CH2)g-COOH
1
1,11-undecane dioic acid undecanoic acid

1
HOOC-(CH2) 7-COOH <---------
1
CH 3-(CH2)7-COOH
azelaic acid nonanoic acid

1
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HOOC-(CH2)5-COOH <--------- CHa-(CH2)s-COOH

pimelic acid heptanoic acid

1
HOOC-(CH2)a-COOH <----------
1
CH 3 -(CH 2) 3-COOH
glutaric acid pentanoic acid
.), .),

1
dioic acid
1
monoic acid
pathway pathway
FIG. 7. Scheme of Degradation Pathway.