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`APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1979, p. 24-28
`0099-2240/79/07-0024/05$02.00/0
`
`Vol. 38, No. 1
`
`Toxic Effects of Some Alcohol and Ethylene Glycol
`Derivatives on Cladosporium resinae
`K. H. LEE* AND H. A. WONG
`Department of Biochemistry, University of Singapore, Singapore 3
`
`Received for publication 20 April 1979
`
`Eleven commercially available alcohol and ethylene glycol derivatives were
`tested for their toxicity toward a problem organism in jet fuel, Cladosporium
`resinae. In the presence of glucose, 20% (vol/vol) ethylene glycol monomethyl
`ether prevented spore germination and mycelial growth, and 10% (vol/vol) 2-
`ethoxybutanol, 10% 2-isopropoxyethanol, 10% 3-methoxybutanol, 5% 2-butyloxy-
`ethanol, 5% ethylene glycol dibutyl ether, and 5% diethylene glycol monobutyl
`ether were found to have similar effects. In a biphasic kerosene-water system, 3-
`methoxybutanol, 2-butyloxyethanol, and diethylene glycol monobutyl ether were
`again found to be more toxic than ethylene glycol monomethyl ether. Consider-
`able potassium efflux, protein leakage, and inhibition of endogenous respiration
`were observed in the presence of the more toxic compounds. 2-Butyloxyethanol
`also caused loss of sterols from cells.
`
`Microbiological contamination of jet aircraft
`fuel systems has been a major concern of airline
`operators, particularly in the tropics. The micro-
`organisms usually form a thick slimy sludge
`which is fily attached to the floor and sides of
`the integral fuel tanks whenever moisture is
`present to produce a fuel-water interface. The
`filamentous fungus Cladosporium resinae has
`been shown to be the predominent species pres-
`ent in the biological sludge (3, 4, 10) and is
`mainly responsible for the extensive corrosion
`frequently found in fuel tanks of both commer-
`cial and military jet aircraft (9).
`Previous work from this laboratory has shown
`that C. resinae can utilize C9 to C18 n-alkanes
`for growth, whereas the shorter-chain n-alkanes,
`particularly n-hexane and n-heptane, do not
`support growth of this organism and inhibit its
`growth on glucose (14). Similar effects were also
`observed when shorter-chain fatty acids and al-
`cohols (C6 to C12) were incubated with C. resinae
`monomethyl
`Ethylene
`glycol
`ether
`(13).
`(EGME), a short-chain glycol used primarily as
`an anti-icing additive in military jet aircraft, has
`been found to be toxic to C. resinae (5-7). The
`present paper reports work done on the action
`of some derivatives of ethylene glycol and other
`alcohols on C. resinae.
`
`MATERIALS AND METHODS
`Test organisms and conditions. Isolate 35A, iso-
`lated from Australian soil and identified as C. resinae
`forma avellaneum by D. G. Parbery, School of Agri-
`culture, University of Melbourne, was used in all ex-
`periments unless otherwise stated. The cultures were
`
`maintained on Bushnell-Haas glucose agar slants (2).
`Strains ATCC 22711 and ATCC 22712 were obtained
`from J. J. Cooney, Department of Biology, University
`of Dayton, Dayton, Ohio.
`All liquid culture experiments were carried out in
`triplicate in standard 250-nil Erlenmeyer flasks. For
`the preparation of liquid inoculum, the organism was
`grown on Bushnell-Haas glucose agar slants (2) for 1
`to 2 weeks at 30°C. A spore suspension practically free
`of mycelium as observed under a microscope was
`obtained by gently shaking the spores off an agar slant
`with 10 to 15 ml of sterile distilled water or basal salts
`medium. For each experiment, samples of the spore
`suspension were counted with a hemacytometer and
`diluted to give a concentration of 6 x 107 spores per
`ml. A known volume of the continuously agitated
`spore suspension was then pipetted into each flask.
`Chemicals. The ethylene glycol derivatives and
`other alcohols were obtained from Fluka, Basel, Swit-
`zerland, and E. Merck AG, Darmstadt, West Germany.
`Commercial jet fuel (Jet A-1) was kindly supplied by
`Shell Co., Singapore. D-[U-14C]glucose was purchased
`from Radiochemical Centre, Amersham, England. All
`other compounds were analytical grade or the best
`grade available and were from Merck or Sigma Chem-
`ical Co., St. Louis, Mo.
`Abbreviations. The names of the various com-
`pounds are abbreviated as follows: ethylene glycol
`monomethyl ether or 2-methoxyethanol, EGME; 2-
`ethoxyethanol, 2-EE; 2-isopropoxyethanol, 2-IE; 3-
`methoxy-l-butanol, 3-MB; 1-methoxy-2-propanol, 1-
`MP; 2-butyloxyethanol, 2-BE; diethylene glycol mon-
`omethyl ether, DEGMME; diethylene glycol dimethyl
`ether, DEGDME; diethylene glycol monoethyl ether,
`DEGMEE; diethylene glycol diethyl ether, DEGDEE;
`diethylene glycol monobutyl ether, DEGMBE; and
`ethylene glycol dibutyl ether, EGDBE.
`Effect of compounds on spore germination and
`mycelial growth of C. re8inae in glucose medium.
`
`24
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`VOL. 38, 1979
`
`EFFECTS OF ETHYLENE GLYCOLS ON C. RESINAE
`
`25
`
`A 0.5-ml amount of the spore suspension in water was
`added to 40 ml of a mixture of Bushnell-Haas mineral
`salts medium containing 1% glucose and appropriate
`volumes of the substances being tested. The final
`concentrations of each of the substances tested ranged
`from 1 to 20% (vol/vol). The flasks were incubated
`statically at 300C for 21 days. To test for effects on
`spore germination, flasks containing 40 ml of mineral
`salts medium with 1% glucose and a test compound
`were inoculated with 0.5 ml of spore suspension. Ger-
`mination was considered inhibited if white mycelial
`colonies were not observed after 21 days of incubation
`at 300C. To test for inhibitory effect on mycelial
`growth, 1-ml samples of the filter-sterilized test com-
`pounds were added aseptically to flasks containing
`mineral salts medium, 1% glucose, and germinating
`spores in the form of tiny white mycelial colonies.
`Incubation was carried out at 300C, and evidence for
`increase in mycelial mass was observed for 20 days.
`Flasks were incubated for an additional 21 days if no
`growth was observed.
`Effects of compounds on C. resinae in a bi-
`phasic system containing Jet A-1 fuel and min-
`eral salts solution. The ratio of fuel to water found
`in the fuel tanks of airplanes is usually between 1,000:
`5 and 1,000:1. The experiments were therefore carried
`out using one of these ratios. A 0.1-ml amount of a
`spore suspension in Bushnell-Haas salts medium was
`added to 100 ml of Jet A-1 fuel in a 250-ml Erlenmeyer
`flask. The test substance was added so that its final
`concentration ranged from 0.05 to 0.2% (vol/vol) with
`respect to the fuel.
`Effect of antifungal compounds on potassium
`efflux and protein leakage. The procedure for these
`experiments has been reported previously (15). Potas-
`sium was analyzed by flame photometry, and protein
`was analyzed by the method of Lowry et al. (8).
`Extraction of sterols by the test compounds. A
`cell suspension in mineral salts medium (4 ml, 1 to 2
`mg of cells per ml) was gently shaken with 1 ml of test
`compound at 30°C for 5 h in a 25-ml flask. Also, cells
`were incubated without the addition of any test com-
`pound. At the end of the incubation period, the cells
`were harvested by vacuum filtration over preweighed
`Whatman glass fiber filter paper. The filtrate was
`shaken vigorously with 5 ml of chloroform-methanol
`(2:1, vol/vol) in a 50-ml screw-capped tube for 15 min
`at room temperature. The organic phase was sepa-
`rated, dried over anhydrous Na2SO4, and analyzed for
`sterols by the colorimetric method of Searcy and
`Bergquist (12). The reagent volumes were reduced
`proportionately to enable the assay of 10 to 50 jig of
`sterols. Ergosterol was used as the standard.
`Glucose uptake. Glucose uptake in the presence
`and absence of the test compounds was measured by
`a [14C]glucose uptake method described previously
`(15). The final concentration of the antifungal com-
`pounds tested was from 1 to 5% (vol/vol).
`Endogenous respiration. Cells were initially
`grown in Bushnell-Haas medium containing 1% glu-
`cose in 1-liter Fernbach flasks for 3 to 7 days. Har-
`vested cells were washed free of glucose and resus-
`pended in the salts medium to give a cell concentration
`of 5 to 10 mg (dry weight) per ml. A 50-ml sample of
`the cell suspension was incubated with 1 ml of D-
`
`['4C]glucose solution (5 ,uCi/ml, 0.125 ACi/lumol) at
`300C for 2 h. The labeled cells were harvested, washed
`thoroughly with distilled water (three times, 50 ml
`each) by vacuum filtration on Whatman glass fiber
`filter paper, and finally resuspended in the mineral
`salts medium to give a cell concentration of 2 to 3 mg
`(dry weight) per ml. Production of I'CO2 from the
`washed labeled cells was measured in the presence
`and absence of the various compounds. A 3-ml amount
`of the cell suspension was placed in a screw-capped
`bottle (30-ml capacity) equipped with a rubber seal
`through which materials could be injected into the
`bottle. A scintillation vial, which contained 1 ml of
`'4CO2-trapping agent (16) and was tightly capped with
`a rubber stopper, was connected to the cell chamber
`by a length of polythene tubing. At zero time, 1 ml of
`20% (vol/vol) test compound was injected into the cell
`chamber. After a 2-h incubation at 300C in a shaker
`bath, 1 ml of 40% trichloroacetic acid was injected into
`the cell suspension to stop the reaction. About 100 ml
`of nitrogen gas was pumped into the cell chamber to
`drive the gas produced by the cells into the vial. After
`standing for 4 h or longer in the shaker bath, the vial
`was removed, and 10 ml of scintillation fluid [4 g of
`2,5-diphenyloxazole and 0.2 g of 1,4-bis-2-(5-phenyl-
`oxazolyl)benzene in 1 liter of toluene] was added to it.
`Radioactivity was measured in a Packard Tri-Carb
`liquid scintillation spectrometer.
`
`RESULTS AND DISCUSSION
`The two most common biocides currently
`being used in jet aircraft are Biobor JF (a prod-
`uct of U.S. Borax and Chemical Corp.) and
`EGME. Both are commercially available. The
`former is a mixture of two boron compounds,
`2,2-oxybis-(4,4,6-trimethyl- 1,3,2-dioxaborinane)
`2,2-(1-methyltrimethylenedioxyl)-bis-(4-
`and
`methyl-1,3,2-dioxaborinane). It has been ob-
`served that Biobor JF at the recommended con-
`centration of 270 ul/liter of fuel gives rise to a
`deposit of boric acid after standing for some
`length of time, both in the laboratory and in fuel
`tanks (unpublished data). The effectiveness of
`EGME, an anti-icing agent, as a biocide is still
`doubtful, as shown by the occasional isolation of
`C. resinae from storage tanks containing fuel
`treated with 0.05 to 0.15% (vol/vol) EGME (7,
`11).
`Eleven commercially available aliphatic alco-
`hols, mostly related to EGME, were tested for
`antifungal activity in the present study. The
`effects of these compounds on the growth of C.
`resinae 35A in 1% glucose-mineral salts medium
`are shown in Table 1. The toxicities of all the
`compounds tested were at least comparable to
`that of EGME. 2-EE, 2-IE, 3-MB, 2-BE, DEG-
`DEE, and DEGMBE were more toxic than
`EGME; 20% EGME prevented spore germina-
`tion and mycelial growth, whereas 10% 2-EE,
`10% 2-IE, 10% 3-MB, 5% 2-BE, 5% DEGMBE,
`and 5% EGDBE were found to have a similar
`
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`26
`
`Compound
`
`APPL. ENVIRON. MICROBIOL.
`
`Mycelial growth at the following concn:
`
`LEE AND WONG
`TABLE 1. Growth of C. resinae 35A in 1% glucose-mineral salts medium in the presence of various
`compoundsa
`Spore gerni-nation and mycelial growth at the
`following concn:
`10%
`5%
`1%
`EGME
`+
`+++
`++++
`2-EE
`-
`+
`+++
`2-IE
`-
`+
`+++
`3-MB
`-
`+
`+++
`++++
`1-MP
`+++
`+
`++++
`2-BE
`+++
`+++
`-
`-
`-
`+
`DEGDME
`+++
`++++
`-
`+
`+++
`++++
`DEGDEE
`++
`+
`-
`+
`+++
`+++
`+++
`-
`+++
`-
`+
`DEGMME
`++++
`-
`+
`++++
`+++
`+
`+++
`-
`DEGMEE
`++++
`-
`+
`++++
`+++
`-
`-
`+++-
`-
`-
`DEGMBE
`-
`+++
`EGDBE
`+++-
`-
`-
`-
`-
`+++
`-
`a ++++ Growth of C. resinae in controls without inhibitors; +++, ++, and + refer to the degree of growth
`relative to that of the controls. -, No visible growth after 42 days of incubation at 30°C.
`
`1%
`++++
`
`20%
`-
`-
`-
`-
`-
`
`-
`
`5%
`+++
`+
`+
`+
`+++
`
`10%
`+
`-
`-
`-
`+
`
`20%
`-
`-
`-
`-
`-
`
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`
`TABLE 2. Growth of C. resinae 35A in a static two-
`phase fuel-water (1,000:1) system containing various
`compoundsa
`Spore germination and mycelial
`growth after 30 days with the
`following concn (% of total vol):
`
`Compound
`
`0.2
`0.1
`0.05
`EGME
`+
`+++
`-
`-
`2-EE
`+++
`+
`-
`2-IE
`+
`+++
`-
`-
`3-MB
`++
`-
`-
`2-BE
`++
`-
`DEGDEE
`+
`++
`-
`-
`DEGMBE
`++
`-
`-
`EGDBE
`++
`a ++++, Growth of C. resinae in controls without
`inhibitors; +++, ++, and + refer to the degree of
`growth relative to that in the controls.
`
`effect. Similarly, the potencies of these six com-
`pounds, when evaluated under simulated field
`conditions by observing the growth of C. resinae
`35A in static two-phase fuel-water systems
`(1,000:1), were greater than that of EGME (Ta-
`ble 2). No growth was observed when EGME
`was used at 0.2%, whereas 3-MB, 2-BE, and
`DEGMBE were effective in controlling the
`growth of the fungus at 0.1%.
`Figures 1 and 2 show the uptake of D-[ U-
`"4C]glucose in the presence of various concentra-
`tions of the compounds tested. At a final con-
`centration of 1% 2-BE, glucose uptake was only
`25% that of the controls. For DEGMBE, the
`inhibition was about 25% at a 1% concentration
`and 40% at a 3% concentration. With 3% (final
`concentration) 2-BE and EGDBE glucose up-
`take by the cells was almost completely in-
`
`hibited. EGME at a 5% concentration, on the
`other hand, did not inhibit glucose uptake at all.
`The question as to the cause of the antifungal
`activity of these alcohol derivatives led to an
`investigation of their effects on the fungal cell
`membrane by studying potassium loss from the
`cell. It was found that 2-BE at a 20% concentra-
`tion caused almost complete loss of cellular po-
`tassium, whereas at the same concentration
`EGME resulted in the efflux of only 27% of the
`potassium. In fact, EGME, when compared with
`the other alcohols tested, caused the least
`amount of potassium efflux (Table 3). A greater
`loss of cellular soluble protein was also observed
`in the presence of alcohols other than EGME
`(Table 4). Thus, it appears that one of the effects
`of these alcohols is on the membrane of C.
`resinae since the amount of potassium efflux
`and loss of cellular soluble proteins would reflect
`in some measure the degree of damage to mem-
`brane function. EGME is therefore the least
`toxic of all of the compounds tested.
`The probable mechanism of action of these
`alcohols on cell membranes was then investi-
`gated by testing the incubating medium for the
`presence of membrane components, such as ste-
`rols. Except for 2-BE, no sterols were found in
`the medium after the cells were incubated with
`the alcohol derivatives at 20% (vol/vol) concen-
`trations for 5 h. About 6.0 ,ug of sterol (as ergo-
`sterol) per mg of cell dry weight was released by
`20% 2-BE. Thus, 2-BE appears to cause damage
`to the cell membrane by removing some of the
`integral components of the membrane. Thus,
`the effect of the other antifungal ethylene glycol
`derivatives on the membrane was probably to
`cause a disarrangement of membrane structure
`
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`VOL. 38, 1979
`
`EFFECTS OF ETHYLENE GLYCOLS ON C. RESINAE
`
`27
`
`Substance (20% final concn)
`
`TABLE 3. Effect of antifungal substances on
`potassium efflux of C. resinae 35A at 30°C
`K+ loss after a 5-h
`incubation (% of to-
`tal cellular K+)
`0.95
`27.33
`91.00
`58.20
`58.20
`45.79
`56.68
`
`None (control)
`EGME
`2-BE
`DEGDEE
`DEGMBE
`EGDBE
`3-MB
`
`Substance
`(20% final concn)
`
`TABLE 4. Effect of antifungal substances on protein
`leakage of C. resinae 35A at 30°C
`Protein leakage after
`a 5-h incubation (%
`of total soluble pro-
`tein)
`2.26
`8.51
`25.27
`25.75
`34.80
`28.60
`25.22
`
`None (control)
`EGME
`2-BE
`DEGDEE
`DEGMBE
`EGDBE
`3-MB
`
`a
`
`s 1w
`IL
`
`/
`
`TOE h mn.
`FIG. 1. Effect of test compounds on the uptake of
`D-f'4C]glucose. Values are expressed as counts per
`minute per milligram (dry weight) of cells. A 1% final
`concentration of each was used. Symbols: 0, control;
`*,2-BE; A, DEGMBE.
`
`200
`
`A.
`
`4-,
`
`l 2
`
`3
`
`7
`
`FIG. 2. Effect of test compounds on the uptake of
`D-['4Cjglucose. Values are expressed as counts per
`minute per milligram (dry weight) of cells. A 5% final
`concentration was used for EGME, and a 3,o' final
`concentration was used for the other compounds.
`Symbols: O, control; O, EGME; A, DEGMBE; *, 2-
`BE; V7, EGDBE.
`without causing the disintegration of the com-
`ponents of the membrane. This disorganization
`could therefore have resulted in the inhibition
`of the uptake of substrates, such as glucose.
`Furthermore, the endogenous respiration of
`
`TABLE 5. Effect of compounds on the endogenous
`respiration of three C. resinae strainsa
`Endogenous respiration (% of con-
`trols) in:
`ATCC
`22711
`
`35A
`
`ATCC
`22712
`
`Compound (5% final
`concn)
`
`EGME
`98
`97
`99
`2-BE
`30
`50
`53
`DEGMBE
`40
`59
`55
`DEGDEE
`50
`52
`54
`a Rate of 14C02 expiration was measured as counts
`per minute per milligram of cell dry weight after a 2-h
`incubation at 30°C.
`
`C. resinae was inhibited by some of these com-
`pounds (Table 5). In the three strains studied,
`the degree of inhibition ranged from 51% in the
`presence of DEGMBE for ATCC 22711 to 70%
`in the presence of 2-BE for isolate 35A. No
`inhibition was observed with the same concen-
`tration of EGME. The reason for the differences
`in the sensitivities to these alcohols observed
`among the different strains of C. resinae is not
`known. The action of these compounds in in-
`hibiting endogenous respiration might be attrib-
`uted to their effect on the intracellular mem-
`brane or their nonspecific blocking of enzymic
`sites or both (1).
`ACKNOWLEDGMENTS
`This work was supported by a grant from the Ministry of
`Science and Technology, Republic of Singapore.
`
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`28
`
`LEE AND WONG
`
`APPL. ENVIRON. MICROBIOL.
`
`We thank Boon Kheng Tan for excellent technical assist-
`ance.
`
`LITERATURE CITED
`1. Bell, G. H. 1971. The action of monocarboxylic acids on
`Candida tropicalis growing on hydrocarbon substrates.
`Antonie van Leeuwenhoek J. Microbiol. Serol. 37:385-
`400.
`2. Bushnell, L. D., and H. F. Haas. 1941. The utilization of
`certain hydrocarbons by microorganisms. J. Bacteriol.
`41:653-673.
`3. Edmonds, P., and J. J. Cooney. 1967. Identification of
`microorganisms isolated from jet fuel systems. Appl.
`Microbiol. 15:411-416.
`4. Hendey, N. I. 1964. Some observations on Cladosporium
`resinae as a fuel contaminant and its possible role in
`the corrosion of aluminum alloy fuel tanks. Trans. Br.
`Mycol. Soc. 47:467-475.
`5. Hendey, N. I., V. J. Bagdon, G. E. Ernst, D. E.
`Klemme, and J. M. Leonard. 1971. Biocidal proper-
`ties of an anti-icing additive. NRL Report no. 7337.
`Department of the Navy, Washington, D.C.
`6. Hitzman, D. 0. 1964. The control of bacterial and fungal
`growth in jet fuels by use of a fuel additive. Dev. Ind.
`Microbiol. 6:105-116.
`7. London, S. A., V. H. Finefrock, and L. N. Killian.
`
`1964. Microbial activity in air force jet fuel systems.
`Dev. Ind. Microbiol. 6:61-79.
`8. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J.
`Randall. 1951. Protein measurement with the Folin
`phenol reagent. J. Biol. Chem. 193:265-275.
`9. Parberry, D. G. 1971. Biological problems in jet aviation
`fuel and the biology of Amorphotheca resinae. Mater.
`Org. 3:161-208.
`10. Prince, A. E. 1961. Microbiological sludge in jet aircraft
`fuel. Dev. Ind. Microbiol. 2:197-203.
`11. Rogers, M. R., and A. M. Kaplan. 1965. A survey of
`microbiological contamination in a military fuel distri-
`bution system. Dev. Ind. Microbiol. 6:80-94.
`12. Searcy, R. L., and L. M. Bergquist. 1960. A new colour
`reaction for the quantitation of serum cholesterol. Clin.
`Chim. Acta 5:192-199.
`13. Teh, J. S. 1974. Toxicity of short-chain fatty acids and
`alcohols towards Cladosporium resinae. Appl. Micro-
`biol. 28:840-844.
`14. Teh, J. S., and K. H. Lee. 1973. Utilization of n-alkanes
`by Cladosporium resinae. Appl. Microbiol. 25:454-457.
`15. Teh, J. S., and K. H. Lee. 1974. Effects of n-alkanes on
`Cladosporium resinae. Can. J. Microbiol. 20:971-976.
`16. Woeller, F. H. 1961. Liquid scintillation counting of 14CO2
`with phenethylamine. Anal. Biochem. 2:508-511.
`
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