Vol. 33, No. 4
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 977-979
Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
Ethylene Production by Soil Microorganisms P. J. CONSIDINE,'* N. FLYNN, AND J. W. PATCHING Department of Microbiology, University College, Galway, Ireland
Received for publication 11 August 1976
Ethylene-producing strains of Penicillium cyclopium and P. crustosum were isolated from soil. These isolates produced ethylene on a variety of carbon growth substrates including phenolic acids. The quantities of ethylene produced on the various substrates varied, and the substrate-ethylene production pattern for P. cyclopium strains differed significantly from that ofP. crustosum strains.
Ethylene has been identified as a component of soil atmospheres and, under certain conditions, has been shown to reach a concentration sufficiently high to influence plant growth and development (10-12, 14). The ethylene is apparently of microbial origin (10), but conflicting claims have been made to the relative contributions of various groups of microorganisms in the process of soil ethylene formation. Several species of ethylene-producing soil bacteria (9), fungi (5, 8), and yeasts (8) have been isolated. It has been suggested (8, 9) that methionine is the precursor of ethylene in both fungal and bacterial isolates. Previous work (4) has shown that phenolic acids, which are present in soil (13), promote ethylene production by the soil isolate Penicillium cyclopium. This note reports on attempts at isolating ethylene-producing microorganisms from soil by employing various types of isolation media. Air-dried deciduous forest soil (10 g) was blended with sterile water (90 ml) for 1 min. Agar media in petri dishes were inoculated with 0.1-ml portions of 10-fold dilutions of the suspension and incubated at 22°C. After 7 days the petri dish lids were replaced with modified petri dish bases. The modification consisted of inserting and sealing in each base a rubber serum cap (Suba-Seal, Freeman & Co., Barnsley, Yorkshire, England), which facilitated sampling of the gas atmosphere above the cultures. Each petri dish was then sealed with adhesive tape. Gas samples were removed daily and analyzed for ethylene. Colonies on plates that contained >1.0 ,1u of ethylene per liter were subcultured on the same medium used for their isolation and were then tested for the ability to produce ethylene in pure culture. All media contained the following (grams per liter): NH4NO3, 2.0; KH2PO4, 0.5; K2HPO4, 0.5; ' Present address: Department of Dairy and Food Microbiology, University College, Cork, Ireland.
MgSO4 4H20, 0.4; KCl, 0.5; CaCo3, 0.01; FeSO4, 7H20, 0.01; MnCl4H20, 0.01; NaMoO4-2H20, 0.01; ZnSO -7H2O, 0.01; Oxoid agar no. 1, 15.0. All other medium components were dissolved in 0.1 M phosphate buffer, adjusted to pH 7, filter sterilized, and added to autoclaved (121°C, 15 min) mineral salts-agar. Three isolation media were used: (i) soil extract medium, which contained 20% (vol/vol) of an extract prepared by autoclaving (121°C, 15 min) equal volumes of soil and water, centrifuging (3,000 x g, 15 min), and filtering the supernatant with Whatman no. 1 filter paper; (ii) G-M medium, which contained glucose (13.3 g/liter) and methionine (5.0 g/liter); and (iii) V-Y medium, which contained vanillic acid (13.3 g) and yeast extract (3.3 g/liter). Other media contained single carbon sources added to give final concentrations of 10 g/liter. Cultivation of isolates was carried out on solid slope media (10 ml) in Universal culture bottles plugged with cotton wool. Prior to gas analysis, the cotton wool plugs were replaced by alcohol-sterilized Suba-Seals. Gas samples were analyzed for ethylene by using a Pye series 104 chromatogram fitted with a glass column (1.5 m by 4 mm), packed with Porapak N (80 to 100 mesh), and with a flame ionization detector. The oven temperature was maintained at 100°C. Ethylene levels of 0.05 ,l/liter were detectable with this system. Significant levels (>1 ,ul/liter) of ethylene were detected within 10 days of sealing the plates containing the organisms growing on V-Y or G-M medium, but not until after 25 days for organisms growing on soil extract medium. Although the three isolation media used supported growth of bacteria, yeasts, and fungi, all of the isolates that produced ethylene in pure culture were found to belong to the genus Penicillium. Nine ethylene-producing isolates were obtained: seven on V-Y medium, one on G-M medium, and one on soil extract medium. The isolates were identified by the Common977
978
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Effect of carbon source on the production of ethylene by P. cyclopium and P. crustosum strainsa Ethylene production (,l1/liter per 24 h)
Penicillium crustosum sras
Penicillium cyclopium strains
Growth substrate A
B
C
D
E
strains
F
1
2
3
22 21 30 32 29 287 27 270 203 Glucose 6 7 5 5 10 112 192 158 7 G-M 211 152 218 8 6 301 249 8 219 Acetate NDb 65 70 57 65 0 0 60 0 Citrate 110 108 8 11 15 99 127 115 163 2-Ketoglutarate 137 126 129 6 10 115 10 148 129 Malic acid ND 94 100 52 82 104 0 0 0 Succinate 74 90 87 17 22 114 27 88 75 Glutamic acid 24 15 15 31 21 20 11 16 15 Vanillic acid 87 168 74 138 306 125 88 178 75 p-Hydroxybenzoic acid 36 44 20 10 54 17 31 13 9 Protocatechuic acid 30 14 37 100 124 78 130 12 29 Ferulic acid 126 326 360 355 101 207 151 129 V-Y 135 288 158 140 317 182 68 1,152 3,091 3,883 Sabaroud dextrose a The highest levels of ethylene produced on the various media are recorded. Uninoculated media did not produce detectable levels of ethylene. b ND, Not determined.
wealth Mycological Institute (Kew, Surrey, England). Three of the ethylene producers isolated on vanillic acid-yeast extract medium were identified as strains of P. crustosum. The other isolates were identified as strains of P. cyclopium. The nine isolates were grown on media containing various carbon sources. The quantities of ethylene produced by the various strains depended on the carbon source (Table 1). P. cyclopium strains produced significantly higher quantities of ethylene when grown on media containing acetate or tricarboxylic acid cycle intermediates than on media containing glucose even though growth on the latter substrate was substantially greater. A completely opposite pattern was observed for P. crustosum strains. Little or no ethylene was produced on media containing acetate or tricarboxylic acid cycle intermediates, although growth on these substrates was similar to that obtained with P. cyclopium strains. Glucose supported good ethylene production. This suggests that ethylene production occurs via different pathways in P. cyclopium and P. crustosum. Alternatively, different regulatory mechanisms exist in both organisms. The results indicate that ethylene biosynthesis in P. cyclopium strains is similar to that for P. digitatum (3). The results also support previous findings (3, 4, 6, 7) that methionine, which is an immediate precursor of ethylene in plant tissue (1) and a postulated precursor in Mucor hiemalis (2) and bacteria (9), is not an immediate precursor of ethylene in Penicillium
spp.
Significant increases in ethylene production were obtained with all isolates cultivated in media containing complex supplements such as yeast extract or mycological peptone (a component of Sabaroud dextrose medium). These large increases cannot be accounted for by the relatively smaller increases in mycelial mass. It was found that all of the phenolic acids tested supported growth and ethylene production in strains of P. cyclopium and P. crustosum. This supports our previous suggestion (4) that phenolic acid-utilizing organisms may contribute to the total production of ethylene in soil, particularly in soils containing high levels of degradable phenolic polymers. LITERATURE CITED 1. Abeles, F. B. 1973. Physiology and biochemistry of ethylene production, p. 30-57. In F. B. Abeles (ed.), Ethylene in plant biology. Academic Press Inc., London. 2. Bird, C. W., and J. M. Lynch. 1974. Formation of hydrocarbons by microorganisms. Chem. Soc. Rev. 3:309-328. 3. Chou, T. W., and S. F. Yang. 1973. The biogenesis of ethylene in Penicillium digitatum. Arch. Biochem. Biophys. 157:73-82. 4. Considine, P. J., and J. W. Patching. 1975. Ethylene production by microorganisms grown on phenolic acids. Ann. Appl. Biol. 81:115-119. 5. Dasilva, E. J., E. Henricksson, and L. E. Henriksson. 1974. Ethylene production by fungi. Plant Sci. Lett. 2:63-66. 6. Jacobsen, D. W., and C. H. Wang. 1968. The biogenesis of ethylene in Penicillium digitatum. Plant Physiol. 43:1959-1966. 7. Ketring, D. L., R. E. Young, and J. B. Biale. 1968. Effects of mono fluoroacetate on Penicillium digita-
VOL. 33, 1977
8. 9. 10. 11.
tum metabolism and on ethylene biosynthesis. Plant Cell Physiol. 9:617-631. Lynch, J. M. 1972. Identification of substrate and isolation of microorganisms responsible for ethylene production in the soil. Nature (London) 240:45-46. Primrose, S. B. 1976. Ethylene-forming bacteria from soil and water. J. Gen. Microbiol. 97:343-346. Smith, K. A., and S. W. F. Restall. 1971. The occurrence of ethylene in anaerobic soil. J. Soil. Sci. 22:430-443. Smith, K. A., and P. D. Robertson. 1971. Effect of
NOTES
979
ethylene on root extension of cereals. Nature (London) 234:148-149. 12. Smith, K. A., and R. S. Russell. 1969. Occurrence of ethylene and its significance in anaerobic soil. Nature (London) 222:769-771. 13. Whitehead, D. C. 1964. Identification of p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids in soil. Nature (London) 202:417-418. 14. Yoshida, T., and T. Suzuki. 1975. Formation and degradation of ethylene in submerged rice soils. Soil Sci. Plant (Tokyo) Nutr. 21:129-135.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 977-979
Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
Ethylene Production by Soil Microorganisms P. J. CONSIDINE,'* N. FLYNN, AND J. W. PATCHING Department of Microbiology, University College, Galway, Ireland
Received for publication 11 August 1976
Ethylene-producing strains of Penicillium cyclopium and P. crustosum were isolated from soil. These isolates produced ethylene on a variety of carbon growth substrates including phenolic acids. The quantities of ethylene produced on the various substrates varied, and the substrate-ethylene production pattern for P. cyclopium strains differed significantly from that ofP. crustosum strains.
Ethylene has been identified as a component of soil atmospheres and, under certain conditions, has been shown to reach a concentration sufficiently high to influence plant growth and development (10-12, 14). The ethylene is apparently of microbial origin (10), but conflicting claims have been made to the relative contributions of various groups of microorganisms in the process of soil ethylene formation. Several species of ethylene-producing soil bacteria (9), fungi (5, 8), and yeasts (8) have been isolated. It has been suggested (8, 9) that methionine is the precursor of ethylene in both fungal and bacterial isolates. Previous work (4) has shown that phenolic acids, which are present in soil (13), promote ethylene production by the soil isolate Penicillium cyclopium. This note reports on attempts at isolating ethylene-producing microorganisms from soil by employing various types of isolation media. Air-dried deciduous forest soil (10 g) was blended with sterile water (90 ml) for 1 min. Agar media in petri dishes were inoculated with 0.1-ml portions of 10-fold dilutions of the suspension and incubated at 22°C. After 7 days the petri dish lids were replaced with modified petri dish bases. The modification consisted of inserting and sealing in each base a rubber serum cap (Suba-Seal, Freeman & Co., Barnsley, Yorkshire, England), which facilitated sampling of the gas atmosphere above the cultures. Each petri dish was then sealed with adhesive tape. Gas samples were removed daily and analyzed for ethylene. Colonies on plates that contained >1.0 ,1u of ethylene per liter were subcultured on the same medium used for their isolation and were then tested for the ability to produce ethylene in pure culture. All media contained the following (grams per liter): NH4NO3, 2.0; KH2PO4, 0.5; K2HPO4, 0.5; ' Present address: Department of Dairy and Food Microbiology, University College, Cork, Ireland.
MgSO4 4H20, 0.4; KCl, 0.5; CaCo3, 0.01; FeSO4, 7H20, 0.01; MnCl4H20, 0.01; NaMoO4-2H20, 0.01; ZnSO -7H2O, 0.01; Oxoid agar no. 1, 15.0. All other medium components were dissolved in 0.1 M phosphate buffer, adjusted to pH 7, filter sterilized, and added to autoclaved (121°C, 15 min) mineral salts-agar. Three isolation media were used: (i) soil extract medium, which contained 20% (vol/vol) of an extract prepared by autoclaving (121°C, 15 min) equal volumes of soil and water, centrifuging (3,000 x g, 15 min), and filtering the supernatant with Whatman no. 1 filter paper; (ii) G-M medium, which contained glucose (13.3 g/liter) and methionine (5.0 g/liter); and (iii) V-Y medium, which contained vanillic acid (13.3 g) and yeast extract (3.3 g/liter). Other media contained single carbon sources added to give final concentrations of 10 g/liter. Cultivation of isolates was carried out on solid slope media (10 ml) in Universal culture bottles plugged with cotton wool. Prior to gas analysis, the cotton wool plugs were replaced by alcohol-sterilized Suba-Seals. Gas samples were analyzed for ethylene by using a Pye series 104 chromatogram fitted with a glass column (1.5 m by 4 mm), packed with Porapak N (80 to 100 mesh), and with a flame ionization detector. The oven temperature was maintained at 100°C. Ethylene levels of 0.05 ,l/liter were detectable with this system. Significant levels (>1 ,ul/liter) of ethylene were detected within 10 days of sealing the plates containing the organisms growing on V-Y or G-M medium, but not until after 25 days for organisms growing on soil extract medium. Although the three isolation media used supported growth of bacteria, yeasts, and fungi, all of the isolates that produced ethylene in pure culture were found to belong to the genus Penicillium. Nine ethylene-producing isolates were obtained: seven on V-Y medium, one on G-M medium, and one on soil extract medium. The isolates were identified by the Common977
978
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Effect of carbon source on the production of ethylene by P. cyclopium and P. crustosum strainsa Ethylene production (,l1/liter per 24 h)
Penicillium crustosum sras
Penicillium cyclopium strains
Growth substrate A
B
C
D
E
strains
F
1
2
3
22 21 30 32 29 287 27 270 203 Glucose 6 7 5 5 10 112 192 158 7 G-M 211 152 218 8 6 301 249 8 219 Acetate NDb 65 70 57 65 0 0 60 0 Citrate 110 108 8 11 15 99 127 115 163 2-Ketoglutarate 137 126 129 6 10 115 10 148 129 Malic acid ND 94 100 52 82 104 0 0 0 Succinate 74 90 87 17 22 114 27 88 75 Glutamic acid 24 15 15 31 21 20 11 16 15 Vanillic acid 87 168 74 138 306 125 88 178 75 p-Hydroxybenzoic acid 36 44 20 10 54 17 31 13 9 Protocatechuic acid 30 14 37 100 124 78 130 12 29 Ferulic acid 126 326 360 355 101 207 151 129 V-Y 135 288 158 140 317 182 68 1,152 3,091 3,883 Sabaroud dextrose a The highest levels of ethylene produced on the various media are recorded. Uninoculated media did not produce detectable levels of ethylene. b ND, Not determined.
wealth Mycological Institute (Kew, Surrey, England). Three of the ethylene producers isolated on vanillic acid-yeast extract medium were identified as strains of P. crustosum. The other isolates were identified as strains of P. cyclopium. The nine isolates were grown on media containing various carbon sources. The quantities of ethylene produced by the various strains depended on the carbon source (Table 1). P. cyclopium strains produced significantly higher quantities of ethylene when grown on media containing acetate or tricarboxylic acid cycle intermediates than on media containing glucose even though growth on the latter substrate was substantially greater. A completely opposite pattern was observed for P. crustosum strains. Little or no ethylene was produced on media containing acetate or tricarboxylic acid cycle intermediates, although growth on these substrates was similar to that obtained with P. cyclopium strains. Glucose supported good ethylene production. This suggests that ethylene production occurs via different pathways in P. cyclopium and P. crustosum. Alternatively, different regulatory mechanisms exist in both organisms. The results indicate that ethylene biosynthesis in P. cyclopium strains is similar to that for P. digitatum (3). The results also support previous findings (3, 4, 6, 7) that methionine, which is an immediate precursor of ethylene in plant tissue (1) and a postulated precursor in Mucor hiemalis (2) and bacteria (9), is not an immediate precursor of ethylene in Penicillium
spp.
Significant increases in ethylene production were obtained with all isolates cultivated in media containing complex supplements such as yeast extract or mycological peptone (a component of Sabaroud dextrose medium). These large increases cannot be accounted for by the relatively smaller increases in mycelial mass. It was found that all of the phenolic acids tested supported growth and ethylene production in strains of P. cyclopium and P. crustosum. This supports our previous suggestion (4) that phenolic acid-utilizing organisms may contribute to the total production of ethylene in soil, particularly in soils containing high levels of degradable phenolic polymers. LITERATURE CITED 1. Abeles, F. B. 1973. Physiology and biochemistry of ethylene production, p. 30-57. In F. B. Abeles (ed.), Ethylene in plant biology. Academic Press Inc., London. 2. Bird, C. W., and J. M. Lynch. 1974. Formation of hydrocarbons by microorganisms. Chem. Soc. Rev. 3:309-328. 3. Chou, T. W., and S. F. Yang. 1973. The biogenesis of ethylene in Penicillium digitatum. Arch. Biochem. Biophys. 157:73-82. 4. Considine, P. J., and J. W. Patching. 1975. Ethylene production by microorganisms grown on phenolic acids. Ann. Appl. Biol. 81:115-119. 5. Dasilva, E. J., E. Henricksson, and L. E. Henriksson. 1974. Ethylene production by fungi. Plant Sci. Lett. 2:63-66. 6. Jacobsen, D. W., and C. H. Wang. 1968. The biogenesis of ethylene in Penicillium digitatum. Plant Physiol. 43:1959-1966. 7. Ketring, D. L., R. E. Young, and J. B. Biale. 1968. Effects of mono fluoroacetate on Penicillium digita-
VOL. 33, 1977
8. 9. 10. 11.
tum metabolism and on ethylene biosynthesis. Plant Cell Physiol. 9:617-631. Lynch, J. M. 1972. Identification of substrate and isolation of microorganisms responsible for ethylene production in the soil. Nature (London) 240:45-46. Primrose, S. B. 1976. Ethylene-forming bacteria from soil and water. J. Gen. Microbiol. 97:343-346. Smith, K. A., and S. W. F. Restall. 1971. The occurrence of ethylene in anaerobic soil. J. Soil. Sci. 22:430-443. Smith, K. A., and P. D. Robertson. 1971. Effect of
NOTES
979
ethylene on root extension of cereals. Nature (London) 234:148-149. 12. Smith, K. A., and R. S. Russell. 1969. Occurrence of ethylene and its significance in anaerobic soil. Nature (London) 222:769-771. 13. Whitehead, D. C. 1964. Identification of p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids in soil. Nature (London) 202:417-418. 14. Yoshida, T., and T. Suzuki. 1975. Formation and degradation of ethylene in submerged rice soils. Soil Sci. Plant (Tokyo) Nutr. 21:129-135.
