Japan. J. Microbiol.

Vol. 13 (1), 87-93, 1969

Rapid Gas Chromatographic Analysis of

Microbial Volatile Metabolites1

Masanori YOSHIOKA, Miyoshi KITAMURA, and Zenzo TAMURA

Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo,
and Department of Pediatrics, School of Medicine,
Tokyo Medical and Dental University, Tokyo

(Received for publication, September 20, 1968)

ABSTRACT

A direct injection of various culture supernatants into a column of Porapak Q was

investigated as a rapid and simple technique for gas chromatographic analysis of volatile
metabolites produced by microorganisms. The usual metabolites such as methanol,
ethanol, formic acid, 2-propanol (or acetone), 1-propanol, acetic acid, diacetyl, 1-butanol,
propionic acid, acetoin, butyric acid, 2,3-butanediol and lactic acid were detected with-
out any pretreatment. It was demonstrated that most strains of Bifidobacterium bifidum
(Lactobacillus bifidus) produced both acetic acid and lactic acid which contrasts with re-
sults from Escherichia coli and other Lactobacilli.

In microbiological experiments it is im-
portant to definitively identify organisms.
The identification steps, however, are often
time-consuming and laborious, and rapid
and simple methods are needed. Several
methods have been reported for differentiat-
ing bacteria by gas chromatography, by
which several compounds in small amounts
could be detected quantitatively and simul-
taneously. Oyama 18] applying bacterial
pyrolysates to a gas chromatograph, at-
tempted to detect the presence of bacteria
or substances of biological origin on Mars.
In the same manner, Reiner [10] used a
pyrolysis-gas-liquid chromatography for the
identification of bacterial strains. Abel et
1A part of this work was presented at the 18th
Meeting of Societas Paediatrica Japonica of North-
ern Part of Japan, Tokyo, October, 1967 and at the
88th Annual Meeting of Pharmaceutical Society of
Japan, Tokyo, April, 1968.
al. [1] and Yamakawa et al. [12] showed gas
chromatographic characteristic patterns of
cellular fatty acids and carbohydrates in
bacterial genera and species.
Alternatively, culture extracts were ana-
lyzed by gas chromatography from the point
of view that many bacterial metabolites are
volatile and useful for bacterial classifica-
tion [2, 4, 7, 9]. However, the extraction or
evaporation of metabolites from culture
supernatants is rather troublesome and un-
satisfactory.
The present paper deals with a rapid and
simple method for gas chromatographic
analysis of microbial volatile metabolites,
the direct injection of culture supernatants
into a column of Porapak Q, which was
recently developed for water analysis [5].
MATERIALS AND METHODS
Culture. A medium essentially that of

87
88 M. YOSHIOKA, M. KITAMURA AND Z. TAMURA

Yoshioka [13], containing 3.2 g of polypep- After two or three transfers, 0.1 ml of or-
tone (Daigo Nutritive Chemicals Ltd., Osa- ganism suspension was inoculated into 2 ml
ka), 8.0 g of casamino acids (Difco), 0.4 g of the medium and cultured at 37 C under
of cysteine, 4.8 g of yeast extract (Difco), the conditions shown in Table 2.
10 g of lactose (or glucose), and 3.2 g of Organisms were removed by centrifuga-
sodium chloride per liter of water, buffered tion at 1,300 x g for 15 minutes, and the
to pH 6.8, was used. The medium was supernatant recovered and stored at 4C
autoclaved at 115 C for 15 minutes. until used as the sample for gas chromato-
Each organism was cultured in the medi- graphy.
um at 37 C for 1 or 2 days aerobically or Gas chromatography. A Shimadzu Gas
anaerobically in an atmosphere containing Chromatograph Model GC-IB equipped
9 volumes of N2 and 1 volume of CO.. with hydrogen flame ionization detector

Fig. 1. Chromatograms of authentic compounds (9.1x108 mole in 5 Jul of the medium).

Black area shows the profile of medium blank. a, methanol; b, ethanol; c, formic
acid; d, 2-propanol; e, 1-propanol; f, acetic acid; g, diacetyl; h; 1-butanol;
propionic acid; j, acetoin; k, butyric acid; 1, 2, 3-butanediol; m, lactic acid. In the
lower chart, the range of vertical axe is expanded four times through retention time of
zero to three minutes. The arrow indicated the change of the range. For details of pro-
cedure, see text.
 GAS CHROMATOGRAPHY OF METABOLITES 89

was used. A glass tube (1.8 m X 4 mm

was packed with Porapak Q. The tempera-
ture of the column and sample chamber
was 210 C. The temperature of the detector
was 220 C. The flow rate of carrier gas, N2,
was 58 ml/min. The sensitivity was 100 and
the range 0.4 V. Five pl of the sample was
injected into the instrument.

RESULTS

In Fig. 1, the first peak of the medium Fig. 2. Calibration curve of formic acid
was attributed to water, the second and the and acetic acid (-x-). Five ul of the medium
containing each amount of acid was injected.
third to unknown compounds, though the
The peak height of medium blank was sub-
retention times of the two peaks corre-
tracted from that of acid. Chromatographic
sponded to those of acetic acid and pro- condition : the same as in the lower chart of
pionic acid. Retention times and peak Fig. 1, for formic acid ; the same as in the
heights of authentic compounds are given upper chart for acetic acid.
in Table 1. The peak heights of these acetic acid in Fig. 2. But 2.5x 10-8 mole of
authentic compounds in the medium were formic acid was well detected like the other
not changed by incubation at 37 C for 2 compounds, and hence the ordinal quantity
days or addition of ammonia. Retention of metabolites in the medium could be
time of acetone was the same as that of analysed.
2-propanol. The data on the culture supernatants of
All compounds except formic acid and the organisms is summarized in Table 2
lactic acid were semiquantitatively ana- and Fig. 3. The products of Escherichia coli
lysed, since their peak heights were propor- 2 IAINI 1016 did not depend on the culture
tional to the concentrations as shown by condition but on the carbon source, while
Saccharomyces carlsbergensis Hansen JAM
Table 1. Retention time and peak height of
authentic compounds obtained from 4727 formed only ethanol aerobically and
the upper chart of Fig, 1. ethanol and lactic acid anaerobically.
It was taxonomically interesting that di-
acetyl was produced by Aerobacter aero-
genes, and acetic acid by Klebsiella pneumo-
niae, since both organisms belong to the
Enterobacteriaceae as does E. coli, and often
have been confused with each other.
In addition to lactic acid, small amounts
of acetic acid and/or propionic acid were
detected in the culture supernatants of some
strains of Lactobacilli. Ethanol was also
formed by L. helveticus and L. fermenti.
Therefore, hetero- and homo-fermentativity
was easily discriminated between gas chro-
matographic profiles. Insignificant peaks at
the retention time of 2.6 minutes in the
90 M. YOSHIOKA, M. KITAMURA AND Z. TAMURA

Table 2. Chromatographic peak heights of culture supernatants.

Coromatographic condition as in the lower chart of Fig. I.
 GAS CHROMATOGRAPHY OF METABOLITES 91

Table 2. (Continued)

a. Minus the peak height of medium blank.

b. A; aerobic, N; anaerobic condition.
c. L; lactose, G; glucose.
d. Kindly provided by Dr. H. Taniguchi, Department of Bacteriology, School of Medicine, Tokyo
Medical and Dental University, Tokyo.
e. Kidly provided by Dr. G. Tamura, Laboratory of Fermentation and Microbiology, Department
of Agricultural Chemistry, Tokyo.
f. Kindly provided by Dr. K. Takamori, Department of Oral Microbacteriology, School of Den-
tistry, Tokyo Medical and Dental University, Tokyo.
g. Kindly provided by Biofermin Pharmaceutical Co., Ltd., Tokyo.
Ii. Kindly provided by Dr. S. Yoshioka, Department of Pediatrics, School of Medicine, Tokyo
Medical and Dental University, Tokyo.
i. Assigned to diacetyl (Rt 4.6 min).
j. Added 0.2 pg of B12 to 2 ml of the medium.
k. Not assigned (Rt 2.6 min).
1. Cultured in the brain heart infusion.
in. Kindly provided by Dr. H. Fujita, National Cancer Center, Tokyo.
a. Kindly provided by I. Takazoe, Department of Biology, Tokyo Dental College, Tokyo.
o. Added 0.1 ml of skimmed cow's milk to 2 ml of the medium.

chromatograms of culture supernatants of duced acetic acid and lactic acid. Such fer-
L. casei, L. plantarum and L. delbrueckii mentativity was differed from that of E. coli.
were not assigned. Moreover, it was interesting that some
As expected, Propionibacterium fer- strains produced a slight amount of ethanol.
mented lactose to propionic acid and glu-
DISCUSSION
cose to propionic acid and acetic acid.
C. liqucfaciens P-15 grew well in the medi- In contrast with the columns usually
um, but no volatile compounds were found. used, the Porapak Q column makes possible
In the same medium the other strains of the injection of culture supernatant without
Corynebactcrium and Actinomyces would pretreatment, as well as the analysis of
not grow. many samples in a short time. Of course,
It is often said that Bifido bacterium bifi- nonvolatile ingredients in the medium such
dum is morphologically similar to Coryne- as amino acids and lactose accumulated at
bacterium and Actinomyces. However 47 of the entrance of the column and discolored
51 strains of B. bifidum [14] uniformly pro- it; but this difficulty was obviated by re-
92 M. YOSHIOKA, M. KITAMURA AND Z. TAMURA

Fig. 3. Representative chromatograms of culture supernatants. Black area shows the profile
of medium blank. Chromatographed as shown in the lower chart of Fig. 1.
 GAS CHROMATOGRAPHY
newing the entrance part every thirty injec-
tions. Rogosa [11] and Cecchini [3] also ana-
lysed bacterial metabolites with this type of
column, but they were unable to analyse
certain important metabolites generally pro-
duced by bacteria, such as lactic acid. Our
method was superior in that we were able
to detect lactic acid and more compounds.
It should also be possible to detect metabo-
lites that normally would be destroyed or
abandoned in chemical or biochemical pre-
treatment, since the culture is used directly.
The medium used in this experiment was
easily prepared and suitable not only for
B. bifidum but for many other as well. It
was also useful for the identification of
B. bifiditin, E. coli, lactobacilli and A. aero-
genes. Kitamura [6] used this method to
identify the bacterial flora of the feces of
newborn infants.
This method also permitted the direct
injection and analysis of natural culture
media, such as Brain Heart Infusion, with-
out the appearance of ghost peaks which
normally render the use of these media im-
possible. We suggest that the method has
practical applications in industry, such as
in the analysis of culture supernatants in
breweries and for the identification of
pathogenic bacteria in clinical medicine.
ACKNOWLEDGEMENT
We thank Prof. Keizo Ohta (Director of Depart-
ment of Pediatrics, School of Medicine , Tokyo Medi-
cal and Dental University) and Dr . Shigetake
Yoshioka (Department of Pediatrics, School of Medi-
cine, Tokyo Medical and Dental University) and
Dr. Keijiro Samajima (Faculty of Pharmaceutical
Sciences, University of Tokyo) for relevant advice .
We are also grateful to Miss Yukiko Saito for
valuable technical assistance.
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