APPLID MicRomUoLoGY, Apr. 1975, p. 560-561 Copyright @ 1975 American Society for Microbiology
Vol. 29, No. 4 Printed in U.SA.
Production of Hydrogen Cyanide by Pseudomonas fluorescens L. R. FREEMAN,' P. ANGELINI, G. J. SILVERMAN,* ND C. MERRWT, JR. U.S. Army Natick Laboratories, Natick, Massachusetts 01760
Received for publication 14 January 1975
Two Pseudomonas fluorescens isolates were found to produce hydrogen cyanide when cultured on either Trypticase soy agar supplemented with 0.5% yeast extract or on irradiation-sterilized chicken.
In studying the volatiles produced by micro- were then placed in loosely enclosed flasks. organisms isolated from spoiled chicken it was After incubation at 10 C for 5 days, the volatiles noted that strains of Pseudomonas fluorescens produced by each culture were collected using produced hydrogen cyanide when grown on the high-vacuum, low-temperature distillation either sterile chicken or on Trypticase soy agar techniques of Merritt et al. (4). Water and (BBL) supplemented with 0.5% yeast extract. carbon dioxide fractions were separated from Michaels and Corpe (5) failed to detect the the condensate and the remaining "center" presence of hydrogen cyanide from cultures of fraction, consisting of compounds volatile at P. fluorescens although they did find that -80 C and nonvolatile at -140 C, was analyzed hydrogen cyanide was produced by Chromobac- by combined temperature programmed gas-liqterium violacium, Pseudomonas chlororaphis, uid chromatography and mass spectrometry and Pseudomonas aureofaciens. They were una- by the methods of Merritt et al. (3). ble to detect hydrogen cyanide in cultures of A 50-foot (ca. 15 m) support-coated open Pseudomonas aeruginosa, Serratia marcescens, tubular column (Perkin-Elmer) having an inBacillus subtilis, Staphylococcus aureus, and side diameter of 0.02 inch (ca. 1.15 cm) and a Escherichia coli. Sneath (8) had previously stationary phase of 1,2,3-tris(hydroxymethyl)was identified hydrogen cyanide in cultures of aminomethane(2 - cyanoethoxy)propane Chromobacterium violacium. Other gram-nega- used. The injection temperature was 150 C, and tive, motile, and pigmented bacteria are re- that of the interface was 200 C. The temperaported to produce hydrogen cyanide (1, 2, 6), ture was programmed at 6 C per min from -65 and these isolates, although designated as C to 125 C, and the linear flow rate of the carrier Pseudomonas sp. or Bacillus pyocyaneus, re- gas was 30 cm/s. The presence of hydrogen cyanide was verisemble Pseudomonas aeruginosa. Reports of the synthesis of hydrogen cyanide by fungi ante- fied from its mass spectrum. The gas-liquid dates that of bacteria (7). chromatography retention volume of hydrogen One reason why hydrogen cyanide was identi- cyanide in the sample was also identical with fied in the volatiles of P. fluorescens in this that of a known sample of hydrogen cyanide study but not by Michaels and Corpe (5) may when identical conditions for the gas-liquid have been due to differences in analytical tech- chromatography parameters were employed. Whether or not hydrogen cyanide is of taxoniques. Whereas they detected hydrogen cyanide calorimetrically, in this study the volatiles nomic value will depend upon the examination were concentrated and selectively fractionated of additional strains. It appears, though, that prior to identification by the more sensitive gas the capability of synthesizing hydrogen cyanide chromatography-mass spectrometry technique. may be limited since we did not find it present In this study the isolates were obtained from in the volatiles of a closely related taxon, spoiled chicken breast muscle held at 10 C for 5 Pseudomonas putida, in the other pseudomodays under aerobic conditions. Each culture nad isolates, or in isolates belonging to the was then inoculated onto both Trypticase soy genera Flavobacterium, Cytophaga, Moraxella, agar supplemented with 0.5% yeast extract and and Acinetobacter. None of the other volatile to chicken sterilized with 1.5 Mrad of gamma compounds identified in this study-dimethyl radiation at -30 C. The inoculated samples disulfide, methanol, ethanol, dimethyl sulfide, ethyl acetate, heptadiene, methyl propionate, I Present address: Brigham Young University, Provo, Utah and methyl thioacetate- characterized either
P. fluorescens or P. putida.
84602.
560
VOL. 29, 1975
561
NOTES
We express thanks to M. L. Bazinet and C. DiPietro for technical assistance. This research was supported by the U.S. Army Research Office, task order 74-386.
4. Merritt, C., Jr., S. R. Bresnick, M. L. Bazinet, J. T. Walsh, and P. Angelini. 1959. Determination of volatile components of foodstuffs. Techniques and their application to studies of irradiated beef. J. Agric. Food Chem.
LITERATURE CITED 1. Clawson, B. J., and C. C. Young. 1913. Preliminary report on production of hydrocyanic acid by bacteria. J. Biol. Chem. 15:419-422. 2. Lorck, H. 1948. Production of hydrocyanic acid by bacteria. Physiol. Plant. 1:142-146. 3. Merritt, C., Jr., P. Angelini, M. L. Bazinet, and D. J. McAdoo. 1966. Irradiation damage in lipids, p. 225-240. In R F. Gould (ed.), Advances in chemistry series. American Chemical Society Publications, Washington, D.C.
5. Michaels, R., and W. A. Corpe. 1965. Cyanide formation by Chromobacterium violaceum. J. Bacteriol. 89: 106-111. 6. Patty, F. A. 1921. The production of hydrocyanic acid by Bacillu pyocyaneous. J. Infect. Dis. 29:73-77. 7. Robbins, W. J., A. Rolnick, and F. Kavanagh. 1950. Production of HCN by cultures of basidiomycete. Mycologia 42:161-166. 8. Sneath, P. H. A. 1953. Fatal infection by Chromobacter-
1:784-787.
ium violaceum. Lancet il:276.
Vol. 29, No. 4 Printed in U.SA.
Production of Hydrogen Cyanide by Pseudomonas fluorescens L. R. FREEMAN,' P. ANGELINI, G. J. SILVERMAN,* ND C. MERRWT, JR. U.S. Army Natick Laboratories, Natick, Massachusetts 01760
Received for publication 14 January 1975
Two Pseudomonas fluorescens isolates were found to produce hydrogen cyanide when cultured on either Trypticase soy agar supplemented with 0.5% yeast extract or on irradiation-sterilized chicken.
In studying the volatiles produced by micro- were then placed in loosely enclosed flasks. organisms isolated from spoiled chicken it was After incubation at 10 C for 5 days, the volatiles noted that strains of Pseudomonas fluorescens produced by each culture were collected using produced hydrogen cyanide when grown on the high-vacuum, low-temperature distillation either sterile chicken or on Trypticase soy agar techniques of Merritt et al. (4). Water and (BBL) supplemented with 0.5% yeast extract. carbon dioxide fractions were separated from Michaels and Corpe (5) failed to detect the the condensate and the remaining "center" presence of hydrogen cyanide from cultures of fraction, consisting of compounds volatile at P. fluorescens although they did find that -80 C and nonvolatile at -140 C, was analyzed hydrogen cyanide was produced by Chromobac- by combined temperature programmed gas-liqterium violacium, Pseudomonas chlororaphis, uid chromatography and mass spectrometry and Pseudomonas aureofaciens. They were una- by the methods of Merritt et al. (3). ble to detect hydrogen cyanide in cultures of A 50-foot (ca. 15 m) support-coated open Pseudomonas aeruginosa, Serratia marcescens, tubular column (Perkin-Elmer) having an inBacillus subtilis, Staphylococcus aureus, and side diameter of 0.02 inch (ca. 1.15 cm) and a Escherichia coli. Sneath (8) had previously stationary phase of 1,2,3-tris(hydroxymethyl)was identified hydrogen cyanide in cultures of aminomethane(2 - cyanoethoxy)propane Chromobacterium violacium. Other gram-nega- used. The injection temperature was 150 C, and tive, motile, and pigmented bacteria are re- that of the interface was 200 C. The temperaported to produce hydrogen cyanide (1, 2, 6), ture was programmed at 6 C per min from -65 and these isolates, although designated as C to 125 C, and the linear flow rate of the carrier Pseudomonas sp. or Bacillus pyocyaneus, re- gas was 30 cm/s. The presence of hydrogen cyanide was verisemble Pseudomonas aeruginosa. Reports of the synthesis of hydrogen cyanide by fungi ante- fied from its mass spectrum. The gas-liquid dates that of bacteria (7). chromatography retention volume of hydrogen One reason why hydrogen cyanide was identi- cyanide in the sample was also identical with fied in the volatiles of P. fluorescens in this that of a known sample of hydrogen cyanide study but not by Michaels and Corpe (5) may when identical conditions for the gas-liquid have been due to differences in analytical tech- chromatography parameters were employed. Whether or not hydrogen cyanide is of taxoniques. Whereas they detected hydrogen cyanide calorimetrically, in this study the volatiles nomic value will depend upon the examination were concentrated and selectively fractionated of additional strains. It appears, though, that prior to identification by the more sensitive gas the capability of synthesizing hydrogen cyanide chromatography-mass spectrometry technique. may be limited since we did not find it present In this study the isolates were obtained from in the volatiles of a closely related taxon, spoiled chicken breast muscle held at 10 C for 5 Pseudomonas putida, in the other pseudomodays under aerobic conditions. Each culture nad isolates, or in isolates belonging to the was then inoculated onto both Trypticase soy genera Flavobacterium, Cytophaga, Moraxella, agar supplemented with 0.5% yeast extract and and Acinetobacter. None of the other volatile to chicken sterilized with 1.5 Mrad of gamma compounds identified in this study-dimethyl radiation at -30 C. The inoculated samples disulfide, methanol, ethanol, dimethyl sulfide, ethyl acetate, heptadiene, methyl propionate, I Present address: Brigham Young University, Provo, Utah and methyl thioacetate- characterized either
P. fluorescens or P. putida.
84602.
560
VOL. 29, 1975
561
NOTES
We express thanks to M. L. Bazinet and C. DiPietro for technical assistance. This research was supported by the U.S. Army Research Office, task order 74-386.
4. Merritt, C., Jr., S. R. Bresnick, M. L. Bazinet, J. T. Walsh, and P. Angelini. 1959. Determination of volatile components of foodstuffs. Techniques and their application to studies of irradiated beef. J. Agric. Food Chem.
LITERATURE CITED 1. Clawson, B. J., and C. C. Young. 1913. Preliminary report on production of hydrocyanic acid by bacteria. J. Biol. Chem. 15:419-422. 2. Lorck, H. 1948. Production of hydrocyanic acid by bacteria. Physiol. Plant. 1:142-146. 3. Merritt, C., Jr., P. Angelini, M. L. Bazinet, and D. J. McAdoo. 1966. Irradiation damage in lipids, p. 225-240. In R F. Gould (ed.), Advances in chemistry series. American Chemical Society Publications, Washington, D.C.
5. Michaels, R., and W. A. Corpe. 1965. Cyanide formation by Chromobacterium violaceum. J. Bacteriol. 89: 106-111. 6. Patty, F. A. 1921. The production of hydrocyanic acid by Bacillu pyocyaneous. J. Infect. Dis. 29:73-77. 7. Robbins, W. J., A. Rolnick, and F. Kavanagh. 1950. Production of HCN by cultures of basidiomycete. Mycologia 42:161-166. 8. Sneath, P. H. A. 1953. Fatal infection by Chromobacter-
1:784-787.
ium violaceum. Lancet il:276.
