Nihon Biseibutsu Seitai Gakkaiho (Bulletin of Japanese Society of Microbial Ecology) Vol 4, No. 2, 59-65, 1989 Inhibitory Effects of Volatile Amines Emitted by Bacterial Culture on the Growth of Fungi 1. The Department of Home Economics, Kyoritsu Women's Junior College, Hitotsubashi 2-chome 2-1, Chiyoda-ku, Tokyo 101, Japan. 2. Faculty of Education, Akita University, Tegatagakuen-cho 1-1, Akita 010, Japan. Abstract: It was previously reported in the results of the double Petri-dish assay that the growth of Aspergillus niger inoculated in a small dish was inhibited in a large Petri dish in which Serratia marcescens had been previously grown Volatile antifungal substances emitted by Serratia marcescens were collected and identified using the gas chromatographic-mass spectrometer Volatile substance was absorbed to hydrochloric acid, and then dried by lyophilization. The residue was dissolved with a NaOH solution, and was analyzed with 28% Pennwalt 223+4% KOH column. Ammonia, aminoethane and trimethylamine were identified by mass spectra of electric ionization and of chemical ionization with methane. The gases of these amines repressed the growth of Aspergillus niger. Key words: Volatile antifungal substances, bacterial colony, gas chromatographic-mass spectrometry, ammonia, aminoethane, trimethylamine Introduction Volatile substances produced by microorganisms have been studied on soil atmospheres (Banwart and Bremner, 1974), rumen fluids (Salsbury and Merricks, 1975), spoilage meats (Lee et al., 1979), cultures of pathogenic bacteria (Huysmans and Spicer, 1985; Zechman et al., 1986), and cultures of Clostridium (Brooks et al., 1970; Rimbault et al., 1986). However, the effects of these volatile products on other microorganisms have been little known. An inhibitory effect of volatile sulfur compounds against the germination of fungi (Kadota and Ishida, 1972) was reported, an action of volatile substances as a microbial phytontid (Tokin and Kamiyama, 1980) was described, and a suggestion that amines produced by Streptococcus lactis may act as a protective substance (Golovnya et al., 1969) was made. In the previous paper, the authors reported the inhibitory effects of microorganisms against other microorganisms using a newly designed method, i.e., the double Petri-dish assay method (Tanaka and Shoji, 1989). When a smaller Petri dish inoculated with a fungus was placed in a larger dish in which bacterial species had been previously grown, fungal growth was strongly repressed. This observation could be regarded as an effect of gaseous substances emitted by a bacterial culture in a larger dish, because the previously cultured bacterial culture and the fungal strain in the smaller dish could not communicate each other through their culture media. The present paper provides an evdience of the volatile antifungal substance emitted by bacteria using gas chromatograph-mass spectrometric (GC-MS) technique. Materials and Methods 1. The microorganisms, culture media, conditions of cultivation and reagents. Among the microbial strains used in the previ-
60 TANAKA and SHOJI ous paper (Tanaka and Shoji, 1989), Serratia marcescens IAM 12142 which showed the highest fungal growth-repressing activity was selected as a producer of the antifungal substances and Aspergillus niger obtained from the Applied Microbiology Laboratory of the Tokyo University of Agriculture was selected as a test microorganism in the experiments using the double Petri-dish assay. S. marcescens was cultured on Nutrient Agar in a Petri dish or Nutrient Broth in a shaking flask, at 30C. A. niger was cultured on Potato Dextrose Agar at 24C. When the two strains were cultured simultaneously in the double Petri-dish assay, they were incubated at 24C as described previously (Tanaka and Shoji, 1989). Potato Dextrose Agar was obtained from Eiken Kagaku Co., Ltd., meat extract and peptone from Kyokuto Kagaku Co., Ltd., and ammonium chloride, aminoethane hydrochloride and trimethylamine hydrochloride from Wako Pure Chemical Industries, Ltd. Other reagents used were all guaranteed grade. 2. Measurement of the activity of the antifungal substances (double Petri-dish assay). Spores of A. niger were inoculated on the center of the Potato Dextrose Agar plate in a small Petri dish (3cm ID). The dish was placed in a large Petri dish (9cm ID) into which 15ml of a sample of antifungal substances. The set of dishes was covered, sealed with Lab Sealing Tape (Scotch Co. Ltd.) and incubated. Measurement of the antifungal activities was performed by observing the growth of A. niger macroscopically, using another A. niger-inoculated small dish which had been placed in the large (9cm ID) dish containing distilled water or Nutrient Broth didn't culture S. marcescens instead of the sample, as a control. 3. Preparation of a GC-MS analysis sample Cells of S. marcescens were inoculated with a bent glass rod on the surface of a Nutrient Agar plate medium prepared in a large Petri dish (25cm ID). A small Petri dish (3cm ID) containing a filter paper wetted with 0.2ml of 2N-HC1 solution was placed in the large dish. Then the large dish was covered and sealed with Lab Sealing Tape and incubated. HCl solution in the filter paper that absorbed gaseous substances was transfered by washing with a small quantity of distilled water into a mini-vial (1ml-capacity) and lyophilized. The lyophilized sample was used for a GC-MS analysis after being dissolved into 0.3ml of 1N- NaOH solution which was injected into the minivial with a syringe through a Teflon septum of the vial's cap. 4. GC-MS analysis A GC-MS analysis was performed with JMS- DX300 (Japan Electron Optics Laboratory (JEOL) Co. Ltd., Tokyo, Japan) which was connected with a data processor, JMA-DA5000 (JEOL Co. Ltd.). Gas chromatographic separation was performed with a glass column (3m long, 3mm ID) packed with 28% Pennwalt 223+4% KOH (80 100 meshes; Alltech Associates Inc., Illinois, US) (Gruger, 1972; Dunn et al., 1976). The temperature and the flow rate of the helium gas were 120C isothermally and 15mlmin-1, respectively. A mass spectrometer operated in both the electron-impact ionization mode and the chemical ionization mode (methane). Component identities obtained by electron-impact ionization were comfirmed by computer matching with compounds in the National Bureau of Standards/National Institutes of Health/Environmental Protection Agency (NBS/NIH/EPA) library data. Results S. marcescence was inoculated in the Nutrient Broth in a shaking flask and incubated with shaking for 2 days. The cultured broth (ph8.6) showed the following properties in the double Petri-dish assay: (1) it inhibited strongly the growth of A. niger as culture of S. marcescence grown on the surface of the Nutrient Agar medium; (2) its inhibitory activity against the
Inhibitory effect of volatile amines on fungi 61 growth of A. niger did not change when the broth was heated gently for 20min at 100C in boiling water; (3) its activity disappeared when the broth was heated with a burner and was boiled for 20 min; (4) the activity disappeared when the broth was acidified with 1N-HC1; (5) its activity did not change when the broth was alkalized with 1N- NaOH; (6) its activity in the broth which was once acidified with 1N-HC1 was recovered when alkalized with an excess amount of IN-NaOH; (7) a cell-free filtrate of the broth obtained through a membrane filter (0.45pm) showed almost the same extent of activity as that of the broth itself; and (8) the activity disappeared when the broth was mixed with Dowex 50W-2 (H-form) (data not shown). These results indicated that the fungal growthinhibitory substances were produced extracellularly in water-soluble forms having volatile and basic natures. In addition, the inhibitory effect against fungal growth decreased, when S. marcescence was cultured on the Nutrient Agar plate medium in a Petri dish (9cm ID) with two small Petri dishes (3cm ID) one of which contained agar medium inoculated with A. niger and the other a filter paper wetted with 1ml of SN-H2SO4 solution (data not shown). These results revealed that the fungal growthinhibiting volatile substances produced by S. marcescence can be collected with an acid solution. The analysis of GC-MS was performed with volatile substance produced by S. marcescence and collected with HCI solution. Gas chromatography of a gaseous sample of the alkalized liophilized specimen exhibited four peaks eluted between 1.0 and 3.5min (Fig. 1). The first peak was identified as air from its retention time of 1.0 min. The second peak was identified as ammonia from the retention time of 1.5min and electronimpact mass spectrum (molecular mass M=17) of an authentic compound (Figs. 2a and 3a). Generally, electron-impact ionization of low molecular primary amine yields a small number of fragment ions (mainly m/e 30) and a low intensity molecular peak. Third and fourth peaks eluted at 2.7 and 3.1min were identified as trimethylamine (molecular mass M=59) (Figs. 2b and 3b) and aminoethane (molecular mass M=45)(Figs. 2c and 3c), respectively, from a profile of fragment inos (Fig. 2), the results of computer matching in the NBS/NIH/EPA library data (Fig. 3), and a profile of chemical ionization mass spectrum (characteristic peaks M+1=60 and 46; Figs. 4a and 4b). Inhibition effects of these three substances against fungal growth were examined as follows. Each 10ml solution of ammonium chloride, trimethylamine hydrochloride or aminoethane Fig. 1. Chromatogram of gaseous substance produced by S. marcescens. The organism was grown in a large dish in a double Petri-dish assay. Gaseous product was collected in HC1 solution with a filter paper in a small dish. Column used was 28%Pennwalt 223+4%KOH column (3m long, 3mm ID).
62 TANAKA and SHOJI Fig. 2. Electron-impact ionization mass spectra of volatile amines produced by S. marrcescens. (a): ammonia (molecular mass M=17) (b): trimethylamine (molecular mass M=59) (c): aminoethane (molecular mass M=45) hydrochloride (10u-100mM, respectively) was transferred into a Petri dish (9cm ID), and a small Petri dish (3cm ID) containing the Potato Dextrose Agar plate medium on which A. niger was inoculated was placed in the large dish. Immediately after adding a portion of 1N-NaOH (final concentration, 0.1N) to the amine solution, the large dish was covered, closed up with Lab Sealing Tape and then incubated at 24C. The fungal growth was compared with a control experiment in which the amine solution in the large dish was replaced with distilled water. After 4 days of the culturing, the fungal growth was not observed when over 0.03mmol of ammonium chloride or aminoethane or over 0.01mmol of trimethylamine was added to the dish. However, A. niger in the small dish of the control grew all over the medium and formed spores (Table).
Inhibitory effect of volatile amines on fungi 63 (a) (b) (c) Fig. 3. Electron-impact ionization mass spectra of volatile amines in NBS/NIH/EPA (a): ammonia; (b): trimethylamine; (c): aminoethane. library data base. Discussion The gaseous active substances produced by S. marcescence were identified as ammonia, aminoethane and trimethylamine based on the data on their retention times, electron-impact ionization and chemical ionization mass spectra. In addition, the authentic samples of these chemicals strongly inhibited the growth of A.niger in gaseous forms. There is a possibility that some other unknown active substances exist, because the collection method of the antifungal substances has not been examined in detail. However, the main components of the gaseous antifungal substances produced by the bacterial culture seem to be these three volatile amines. Alcohols, aldehydes, amines, esters, ketones and sulfur compounds have been reported as gaseous or volatile substances emitted from natural soils (Banwart and Bremner, 1974; Vlasenko et al., 1978), and nutrient-supplemented soils (Francis et al., 1975). The cultures of pathogenic bacteria (Huysmans and Spicer, 1985; Zechman et al., 1986), Clostridium (Brooks et al., 1969; Pons et al., 1985) and Streptococcus lactis (Golovnya et al., 1969) emitted these substances. However, their effects against other microorganisms were not described in these papers. Some volatile sulfur compunds produced by microorganisms have been reported
64 TANAKA and SHOJI Fig. 4. Chemical ionization mass spectra of volatile amines produced by S. marcescens. (a): trimethylamine (molecular mass M+1=60) (b): aminoethane (molecular mass M+1=46) Table. Inhibitory effect of volatile amines against the growth of A. niger in Petri dish under gaseous condition. Each amine (ammonium chloride, aminoethane hydrochloride or trimethylamine hydrochloride) transfered into large Petri-dish (9cm ID) under alkalin condition. A small Petri-dish (3cm ID) in which A. niger inoculated on Potato Dextrose Agar placed in the large dish. Growth of fungi was observed after 4 days incubation. (-): Fungi didn't grow. (++++++): Fungi grew in Petri dish (3cm ID).
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