J. Gen. Apple. Microbiol.

Vol. 10, No. 4, 1964.

ASSIMILATION OF HYDROCARBONS BY YEASTS

I. PRELIMINARY SCREENING

KAZUO KOMAGATA, TAKASHI NAKASE and NOBORU KATSUYA

Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan
Received August 20, 1964

Since the suggestion of DAVIS and UPDEGRAFF (1) stating the possibility
of producing foodstuffs from hydrocarbons by microorganisms, considerably
interesting reports concerning the hydrocarbon-utilizing microorganisms
have appeared in the literature (2, 3). Hydrocarbon-utilizing microorganisms
are found in several taxa, but the main members are limited to bacteria
and a few yeasts. TAUSSON(4) first reported the assimilation of hydro-
carbons by several kinds of yeasts, such as Debar yomyces, Endomyces,
Hansenula, Torulopsis and Monilia. Recent investigation has shown the
assimilation of hydrocarbons by Torulopsis colliculosa (5, 6), Candida
tropicalis (5, 6, 7), Candida lipolytica (5, 6), some Saccharomyces (8), and
certain Trichosporon (9). In this paper we describe the results of screening
hydrocarbon-utilizers from a large variety of yeasts which were freshly
isolated from various sources or obtained from several type culture collec-
tions. The detailed taxonomic studies of such yeasts will be discussed in
a following paper.

MATERIALS AND METHODS

Microorganisms. All yeasts used in this study were newly isolated or

obtained from type culture collections, including Institute for Fermenta-
tion, Osaka (IFO), Institute of Applied Microbiology, University of Tokyo
(TAM), Research Institute of Fermentation, Yamanashi University (RIFY),
and Laboratory of Fermentation, University of Tokyo (ATU). These
comprised of 498 strains belonging to 26 genera. Of 498 strains, 4 were
isolated by a kerosene enrichment culture, 306 from various sources including
fruits (66 strains), vegetables (52 strains), adhesives of neutralizing tanks
in the monosodium glutamate manufacturing process (34 strains of yeasts,
30 strains of yeast-like fungi), def atted soy bean flakes obtained from
manufacturing factory of soy bean protein (86 strains), and miscellaneous
sources (38 strains) using yeast extract-malt extract agar plates (Y. M. agar,
Difco.); the remaining 188 strains were supplied from type culture
collections.
Isolation of hydrocarbon-utilizing yeasts by kerosene enrichment culture.
Several hydrocarbon-utilizing yeasts were isolated by a kerosene enrichment
culture. The kerosene medium used for isolation was composed of kerosene,

313
314 KOMAG ATA, NAKASE and KATSUYA VOL. 10

20 ml; NH4C1, 5 g; Na2HPO4, 3.3 g; KH2PO4, 1.7 g; MgSO4' 7H2O, 0.5 g; NaCI,
0.5 g; thiamine hydrochloride, 0.1 mg; p-amino benzoic acid, 0.1 mg;
pyridoxine hydrochloride, 0.1 mg; pyridoxal, 0.02 mg; calcium pantothenate,
0.1 mg; nicotinic acid, 0.1 mg; biotin, 0.001 mg; folic acid, 0.001 mg;
riboflavin, 0.2 mg; and distilled water, 1000 ml; pH being adjusted to 7.0.
Small amounts of soil, sewage, or the other materials were added to the
above medium, and incubated with shaking for a week at 30°. After
several subcultures, hydrocarbon-utilizing yeasts were plated on kerosene
agar plates, and then transferred to kerosene agar slants. For the isolation
from air, kerosene agar plates were directly exposed to air. The kerosene
employed was a guaranteed brand, Japan Industrial Standard K-2203 grade
No. 1.
Screening tests of hydrocarbon-utilizing ,feasts. Hydrocarbon-utilizing
yeasts were preliminarily screened by growth tests on kerosene agar slants
for a week at 30°. The strains showing positive or doubtful results were
retested by using liquid medium. The cells grown on kerosene agar slants
were cultured in 500 ml-shaking flasks containing 50 ml of kerosene medium
with shaking at 30°. After a week, the ability to grow was detected by
an increase of the turbidity of the culture broth.
Utilization of pure hydrocarbons. A typical strain of each species
was examined to check its ability to utilize pure hydrocarbons. A medium
containing 20 ml of pure hydrocarbon per 1000 ml in place of kerosene in the
kerosene medium was used, and the procedures of cultivation were the same
as those described above. After a week, the ability of utilization was de-
tected by an increase of turbidity, and the cell number of the culture broth
was counted with a THOMA's hemacytometer. Hydrocarbons tested were n-
pentane, i-pentane, cyclopentane, n-hexane, i-hexane, n-heptane, n-octane, i-
octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tetradecane and n-
hexadecane. In the case of volatile hydrocarbons, such as n-pentane, i-pentane
and cyclopentane, rubber plugs were used instead of cotton plugs.

RESULTS

The results of screening tests of hydrocarbon-utilizing yeasts are shown

in Table 1. Of 498 strains of yeasts tested, 56 strains utilized kerosene as
a sole source of carbon. Almost all the strains utilizing kerosene belonged
to the genus Candida and included 10 species: C. parapsilosis (26 strains),
C. intermedia (9 strains), C. lipolgtica (8 strains), C. tropicalis (5 strains),
C. cloacae (1 strain), C. maltosa (1 strain), C. guillierraondii (1 strain),
C. albicans (1 strain), C. rugosa (1 strain), and C. krusei (1 strain). The
remaining yeasts were 1 strain of Hansenul a anomaly and 1 strain of
Rhodotorula rubra. The new species of Candida cloacae and Candida maltosa
were identified by the authors, and detailed taxonomical characteristics will
be described in a following paper. Almost all the yeasts mentioned above
1964 Assimilation of Hydrocarbons by yeasts 315

Table I. Results of screening.

Number of strains
316 KOMAGATA, NAKASE and KATSUYA VOL. 10

Table 1. (continued)
1964 Assimilation of Hydrocarbons by yeasts 317

Table 1. (continued)
318 KOMAGATA,NAKASE and KATSUYA VOL. 10Y

Table 1. (continued)

Table 2. Kerosene-utilizing yeasts and their sources of isolation.

Freshly isolated Stock cultures

1964 Assimilation of Hydrocarbons by yeasts 319

demonstrated good growth in kerosene medium, except C. krusei, H. anomaly

and R. rubra which exhibited rather poor growth. The most abundant
growth in kerosene medium was shown by 3 strains of C. tropicalis and
1 strain of C. cloacae which were isolated from air and mud respectively
by a kerosene enrichment culture. These kerosene-utilizing yeasts were
found also in the isolates from vegetables, butter, etc., and in stock
cultures maintained in type culture collections. The number of strains
and species of hydrocarbon-utilizing yeasts and sources of isolation are
shown in Table 2. Pure hydrocarbons of C9-C16, such as n-nonane, n-decane,
n-undecane, n-dodecane, n-tetradecane, and n-hexadecane were readily
attacked by kerosene-utilizing yeasts. As shown in Table 3, the cells over

Table 3. Utilization of pure hydrocarbons.

109 per ml were harvested in the culture broths containing hydrocarbons,

such . as n-nonane, n-decane, n-undecane, n-dodecane, n-tetradecane,
n-hexadecane, and kerosene. n-Octane was utilized only by C. parapsilosis
Y-2-10, but not effectively. R, rubra Y-19-5 assimilated n-decane slightly
but failed to utilize other hydrocarbons. C. krusei IFO1063 and H. anomaly
320 KOMAGATA,
NAKASEand KATSUYA VOL. 10

ATU Will-5 could not assimilate any of these pure hydrocarbons though
weak but apparent growth was observed in kerosene medium.

DISCUSSION

From the taxonomical point of view, it is of interest that almost all

the hydrocarbon-utilizing yeasts belong to the genus Candida, and that the
powerful utilizers are probably restricted to certain species of Candida.
However, the relationship between the hydrocarbon-utilizing yeasts and
their habitats is not so clear. Nearly all the hydrocarbon-utilizing yeasts,
including 26 strains of C. parapsilosis, 7 strains of C. lipolytica and 1
strain of C. maltosa, were isolated from adhesives of neutralizing tanks in
the monosodium glutamate manufacturing process. This finding is somewhat
peculiar, because such adhesives are not considered to be related to
hydrocarbons. From this same source, 30 strains of yeast-like fungi were
also isolated, but none of these exhibited growth in the medium containing
kerosene as a sole source of carbon. In comparison with newly isolated
yeasts, 5 strains of hydrocarbon-utilizing yeasts obtained from type culture
collections, C. lipolytica IFO 0746, C. rugosa IFO 0750, C, albicans IFO 1060,
C. tropicalis IFO 0006, and C. tropicalis RIFY WF-191, also exhibited good
growth in kerosene medium. It seems that yeasts would not lose completely
or partially their ability to assimilate kerosene during the long period of
cultivation. We could not discuss relationship between the kinds of hy-
drocarbons and assimilatory abilities by yeasts, because the hydrocarbons
tested were rather few. However, it is clear that n-paraffins containing
over 9 carbon atoms were easily attacked.

SUMMARY

The authors described the results of preliminary screenings of

hydrocarbon-utilizing yeasts of which 56 strains were obtained. Fifty four
strains belonging to the genus Candida: C. parapsilosis (26 strains),
C. intermedia (9 strains), C. lipolytica ($ strains), C. tropicalis (5 strains),.
C. cloacae (1 strain), C. maltosa (1 strain), C. guilliermondii (1 strain),
C. rugosa (1 strain), C. albicans (1 strain), and C. krusei (1 strain). The
remaining two strains were Hansenula anomala and Rhodotorula rubra.
All strains belonging to the genus Candida, except C. krusei, exhibited the
powerful kerosene-utilizing ability. Stock cultures obtained from type
culture collections also exhibited the strong kerosene-assimilatory ability as
well as these newly isolated strains. n-Paraffins containing more than 9
carbon atoms were easily attacked by hydrocarbon-utilizing yeasts.
The authors wish to thank Prof. K. Arima, Laboratory of Fermentation, University
of Tokyo, Prof. H. Iizuka, Institute of Applied Microbiology, University of Tokyo, Dr.
T. Hasegawa, Institute for Fermentation, Osaka, and Dr. S. Goto, Research Institute
of Fermentation, Yamanashi University, who kindly supplied the stock cultures.
1964 Assimilation of Hydrocarbons by yeasts 321

REFERENCES

(1) J. B. DAVIS and D. M. UPDEGRAFF: Bacteriol. Rev., 18, 215 (1954).

(2) G. W. FURS: Arch. Mikrobiol., 39, 375 (1961).
(3) J. W. FOSTER: Antonie van Leeuwenhoek., 28, 241 (1962).
(4) T. A. TAUSSON: Microbiology (U. S. S. R.), 8, 828 (1939), cited from Chem.
Abstr., 35, 3673 (1941).
(5) F. JUST, W. SCHNABEL and S. ULLMANN: Die Brauerei Wiss. Beilage 4, Heft
8, 57 (1951), cited from reference (2).
(6) F. JUST, W. SCHNABEL and S. ULLMANN: Die Brauerei Wiss. Beilage 4, Heft
9, 71 (1951), cited from reference (2).
(7) W. HOERBURGER: Forschungs Ber. Wirtsch.-u. Verkehrsministeriums Nordrhein-
Westfalen No. 131, 2211 (1955).
(8) R. G. HARRIS and R. J. STRAWINSKY: U. S. Patent 2,697,061 (1954).
(9) H. IIZUKA, S. SHIIO and N. SETO: Paper read at the Annual Meeting of Agr.
Chem. Soc. Japan, April, 1963.