Rapid Gas Chromatographic Analysis of Microbial Volatile Metabolites1

Transcription

1 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, diacety1, 1-butanol, propionic acid, acetoin, butyric acid, 2,3-butanediol and lactic acid were detected without any pretreatment. It was demonstrated that most strains of Bifidobacterium bifidum (Lactobacillus bifidus) produced both acetic acid and lactic acid which contrasts with results from Escherichia coli and other Lactobacilli. In microbiological experiments it is important 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 differentiating bacteria by gas chromatography, by which several compounds in small amounts could be detected quantitatively and simultaneously. Oyama [8] applying bacterial pyrolysates to a gas chromatograph, attempted 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 Northern Part of Japan, Tokyo, October, 1967 and at the 88th Annual Meeting of Pharmaceutical Society of Japan, Tokyo, April, 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 analyzed by gas chromatography from the point of view that many bacterial metabolites are volatile and useful for bacterial classification [2, 4, 7, 9]. However, the extraction or evaporation of metabolites from culture supernatants is rather troublesome and unsatisfactory. 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

2 88 M. YOSHIOKA, M. KITAMURA AND Z. TAMURA Yoshioka [13], containing 3.2 g of polypeptone (Daigo Nutritive Chemicals Ltd., Osaka), 8.0 g of casamino acids (Difco), 0.4 g of cysteine, 4.8 g of yeast extract (Difco), 10 g of lactose (or glucose), and 3.2 g of sodium chloride per liter of water, buffered to ph 6.8, was used. The medium was autoclaved at 115 C for 15 minutes. Each organism was cultured in the medium at 37 C for 1 or 2 days aerobically or anaerobically in an atmosphere containing 9 volumes of N2 and 1 volume of CO2. After two or three transfers, 0.1 ml of organism suspension was inoculated into 2 ml of the medium and cultured at 37 C under the conditions shown in Table 2. Organisms were removed by centrifugation at 1,300 ~g for 15 minutes, and the supernatant recovered and stored at 4 C until used as the sample for gas chromatography. Gas chromatography. A Shimadzu Gas Chromatograph Model GC-IB equipped with hydrogen flame ionization detector Fig. 1. Chromatograms of authentic compounds (9.1 ~10-8 mole in 5ƒÊl 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; i, 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 procedure, see text.

3 GAS CHROMATOGRAPHY OF METABOLITES 89 was used. A glass tube (1.8m ~4mm i.d.) was packed with Porapak Q. The temperature 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 Đl of the sample was injected into the instrument. RESULTS In Fig. 1, the first peak of the medium was attributed to water, the second and the third to unknown compounds, though the retention times of the two peaks corresponded to those of acetic acid and propicnic acid. Retention times and peak heights of authentic compounds are given in Table 1. The peak heights of these authentic compounds in the medium were not changed by incubation at 37 C for 2 days or addition of ammonia. Retention time of acetone was the same as that of 2-propanol. All compounds except formic acid and lactic acid were semiquantitatively analysed, since their peak heights were proportional to the concentrations as shown by Table 1. Retention time and peak height of authentic compounds obtained from the upper chart of Fig, 1. Fig. 2. Calibration curve of formic acid (- -) and acetic acid (- ~-). Five Đl of the medium containing each amount of acid was injected. The peak height of medium blank was subtracted from that of acid. Chromatographic condition: the same as in the lower chart of Fig. 1, for formic acid; the same as in the upper chart for acetic acid. acetic acid in Fig. 2. But 2.5 ~10-8 mole of formic acid was well detected like the other compounds, and hence the ordinal quantity of metabolites in the medium could be analysed. The data on the culture supernatants of the organisms is summarized in Table 2 and Fig. 3. The products of Escherichia coli 2 JAM 1016 did not depend on the culture condition but on the carbon source, while Saccharomyces carlsbergensis Hansen IAM 4727 formed only ethanol aerobically and ethanol and lactic acid anaerobically. It was taxonomically interesting that diacetyl was produced by Aerobacter aerogenes, and acetic acid by Klebsiella pneumoniae, 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- * Values minus the peak height of medium blank. matographic profiles. Insignificant peaks at the retention time of 2.6 minutes in the

4 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. 1.

5 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 Dentistry, Tokyo Medical and Dental University, Tokyo. g. Kindly provided by Biofermin Pharmaceutical Co.,Ltd., Tokyo. h. 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 ƒêg of B12 to 2 ml of the medium. k. Not assigned (Rt 2.6 min). Œ. Cultured in the brain heart infusion. m. Kindly provided by Dr. H. Fujita, National Cancer Center, Tokyo. n. 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 L, casei, L. plantarum and L. delbrueckii were not assigned. As expected, Propionibacterium fermented lactose to propionic acid and glucose to propionic acid and acetic acid. C, liquefaciens P-15 grew well in the medium, but no volatile compounds were found. In the same medium the other strains of Corynebacterium and Actinomyces would not grow. It is often said that Bifidobacterium bifidum is morphologically similar to Corynebacterium and Actinomyces. However 47 of 51 strains of B. bifidum [14] uniformly produced acetic acid and lactic acid. Such fermentativity was differed from that of E. coli. Moreover, it was interesting that some strains produced a slight amount of ethanol. DISCUSSION In contrast with the columns usually used, the Porapak Q column makes possible the injection of culture supernatant without pretreatment, as well as the analysis of many samples in a short time. Of course, nonvolatile ingredients in the medium such as amino acids and lactose accumulated at the entrance of the column and discolored it; but this difficulty was obviated by re-

6 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.

7 GAS CHROMATOGRAPHY OF METABOLITES 93 newing the entrance part every thirty injections. Rogosa [11] and Cecchini [3] also analysed bacterial metabolites with this type of column, but they were unable to analyse certain important metabolites generally produced 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 metabolites that normally would be destroyed or abandoned in chemical or biochemical pretreatment, 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. bifidum, E. coli, lactobacilli and A. aerogenes. 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, without the appearance of ghost peaks which normally render the use of these media impossible. 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 Department of Pediatrics, School of Medicine, Tokyo Medical and Dental University) and Dr. Shigetake Yoshioka (Department of Pediatrics, School of Medicine, 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. REFERENCES [1] Abel, K., DeSchmertzing, H., and Peterson, J. I Classification of microorganisms by analysis of chemical composition. I. Feasibility of utilizing gas chromatography. J. Bacteriol. 85: [2] Bawdon, R. E., and Bassette, R Differentiation of Escherichia coli and Aerobactor aerogenes by gas liquid chromatography. J. Dairy Sci. 49: [3] Cecchini, G. L., and O'brien, R. T Detection of Escherichia coli by gas chromatography. J. Bacterial. 95: [4] Henis, Y., Gould, J., and Alexander, M Detection and identification of bacteria by gas chromatography. Appl. Microbiol. 14: [5] Hollis, O. L., and Hayes, W. V Water analysis by gas chromatography using porous polymer columns. J. Gas Chromatog. 4: [6] Kitamura, M Analysis of intestinal flora by gas chromatography. Acta Paed. Japon. 72: (in Japanese) [7] O'Brien, R. T Differentiation of bacteria by gas chromatographic analysis of products of glucose catabolism. Food Technol. 21: [8] Oyama, V. I Use of gas chromatography for the detection of life on Mars. Nature 200: [9] Packett, L. V., and McCune Determination of steam-volatile organic acids in fermentation media by gas-liquid chromatography. Appl. Microbial. 13: [10] Reiner, E Identification of Bacterial strains by pyrolysis-gas liquid chromatography. Nature 206: [11] Rogosa, M., and Love, L. L Direct quantitative gas chromatographic separation of C2-CB fatty acid, methanal, and ethyl alcohol in aqueous microbial fermentation media. Appl. Microbiol. 16: [12] Yamakawa, T., and Ueta, N Gas chromatographic studies of microbial components. Carbohydrate and fatty acid constitution of Neisseria. Japan. J. Exp. Med. 34: [13] Yoshioka, S On the bifidus factor. Acta Paed. Japan. (Oversea Ed.) 6: [14] Yoshioka, M., Yoshioka, S., Tamura, Z., and Ohta, K Growth responses of Bifidobacterium, bifidum to coenzyme A, its precursors and carrot extract. Japan. J. Microbiol. 12: