Tuatara: Volume 19, Issue 1, November 1971

A Comparison of the Growth of New Zealand Soil, Fuel and Air Isolates of the ‘Kerosene Fungus’ Cladosporium Resinae (Lindau) De Vries in Aviation Turbine and Lighting Kerosene

A Comparison of the Growth of New Zealand Soil, Fuel and Air Isolates of the ‘Kerosene Fungus’ Cladosporium Resinae (Lindau) De Vries in Aviation Turbine and Lighting Kerosene

Introduction

The First Report in the open scientific literature of the occurrence of Cladosporium resinae (Lindau) de Vries in hydrocarbon fuels appears to be that of Hendey in 1964. Hazzard (1963) positively identified C. resinae in aviation kerosene in Australia in 1961 but his results are contained in a technical report which has not been widely circulated. In the same year Prince (1961) reported finding a Cladosporium sp. in jet fuel in the United States of America and his photographs indicate that it was probably C. resinae. Contamination of aviation fuels with C. resinae has subsequently been shown to be widespread. The fungus has been isolated from aviation fuels, aircraft tanks or filters in Australia (Hazzard, 1963), Brazil (Gutheil, 1966), Denmark, England, India, Japan, Nigeria, New Zealand, Syria (Anon., 1968) and U.S.A. (Engel and Swatek, 1966).

Parbery (1967) first reported consistently isolating C. resinae from Australian soils in 1967. He was later (1969) able to show that this fungus is widespread in soils in Australia, Europe and Britain. The results of a soil survey in New Zealand support his findings (Sheridan, Steel and Knox. 1971).

Four forms of C. resinae are known to exist (De Vries, 1955) but only one, f. avellaneum, has apparently been recovered from kerosene-type fuels (Hazzard, 1963; Hendey, 1964). C. resinae f. resinae has arisen in culture from f. avellaneum (Hendey, 1964) and been isolated occasionally from soil (Parbery, 1968). An albino, f. albidum, has also arisen in culture from f. avellaneum (Parbery, 1968; Sheridan, Steel and Knox, 1971). The only form isolated from kerosene fuels and soils in New Zealand is C. resinae f. avellaneum (Sheridan, Steel and Knox, 1971). The other two forms have been found here as saltants in culture — the fourth form (f. sterile) has not been seen by us (Sheridan — unpublished). All of the above forms (except f. sterile) have been tested in our laboratory for ability to grow in kerosene. Parbery's (1968) isolates of f. resinae from soils are reported by him as not growing in kerosene. As far as we are aware no one has tested isolates of C. resinae from the air for their ability to grow in kerosene.

This paper compares the growth of New Zealand soil, fuel and air isolates of the ‘kerosene fungus’ C. resinae in aviation turbine and lighting kerosene.

Figure 1. Growth of soil isolates of C. resinae f. avellaneum in kerosene after six weeks. Left: Aviation turbine kerosene. Right: lighting kerosene. Centre: Uninoculated control.

Figure 1. Growth of soil isolates of C. resinae f. avellaneum in kerosene after six weeks. Left: Aviation turbine kerosene. Right: lighting kerosene. Centre: Uninoculated control.

Materials and Methods

Isolations from Soils, Fuels and Air

Isolations were made from soil by the creosoted matchstick method (Sheridan, Steel and Knox, 1971).

Isolations were made from aviation fuels by passing 250ml. of the fuel through a sterile millipore membrane filter, pore size 0.45 micron, in a manner similar to that employed by Hazzard and Kuster (1962). The filter membrane was then placed inside a sterile Petri dish, V-8 juice agar at 50° C. poured gently over its surface, and the plate incubated at 25° C. for five days. A number of filters carrying C. resinae and a number of isolates of the fungus on agar media were kindly sent to us by Mr. W. Johnston of B.P. New Zealand Ltd.

Isolations were made from the air using the methods described by Sheridan and Nelson (1971).

All isolates were grown on V-8 juice agar (Sheridan, Steel and Knox, 1971) until required for testing on kerosene.

Harvesting Growths

The mycelial mat produced by the fungus was harvested after six weeks by carefully removing it with a bent nichrome wire. In general the complete growth could be removed in this way, thus obviating the necessity for filtration. The growth was then pressed gently between a few thicknesses of blotting paper to remove excess kerosene, placed in a pre-weighed aluminium cup and dried to constant weight at 80° C. in a hot-air oven. Constant weight was achieved in two days in the case of growths from aviation turbine kerosene. Growths from lighting kerosene required somewhat longer.

Results

The fungus grew at the interface between the mineral salts medium and kerosene (Fig. 1) producing a dark brown fungal mat of mycelium in the case of f. avellaneum and a white fungal mat in the case of f. albidum (Fig. 2). After six weeks when the growths were harvested, black sclerotial-type bodies were present in some cases adhering firmly to the side of the bottle. These were possibly immature ascocarps. The fungus produced asexual spores within the kerosene.

Figure 2. Growth of C. resinae in aviation turbine kerosene after six weeks. Left: Soil isolates) f avellaneum Right: Air isolates) Centre: Soil isolates — f. albidum

Figure 2. Growth of C. resinae in aviation turbine kerosene after six weeks.
Left: Soil isolates) f avellaneum
Right: Air isolates)
Centre: Soil isolates — f. albidum

All isolates tested were able to grow on both aviation turbine and lighting kerosene. Table 1 shows the results of three separate tests where the growth of soil, fuel and air isolates of C. resinae was compared under similar conditions. The figures in the table refer to dry weight of growth harvested from two bottles. Before harvesting the fungus in the first test, V-8 juice agar plates were inoculated with each isolate. The resulting growth was used to inoculate kerosene in the second test. The process was repeated for the third test (note that in the third test 8oz. bottles were used containing 100ml. of mineral salts medium and 25ml. kerosene). Thus the requirements of ‘Koch's Postulates’ were satisfied.

Figure 3. Dry weight of soil, fuel and air isolates of C. Resinae after six weeks’ growth in aviation turbine kerosene.

Figure 3. Dry weight of soil, fuel and air isolates of C. Resinae after six weeks’ growth in aviation turbine kerosene.

In Third Test used 8oz. medicine flats with 100ml. B/H + 25ml. kerosene.

isolates was greater in lighting kerosene than in aviation turbine kerosene. The opposite was true of the air isolates. However, in the second test (see Table 1) the amount of growth produced by air isolates in aviation turbine and lighting kerosene was very similar. Visual observation from the time of inoculation indicated that the growth of all isolates was initially slower in lighting kerosene. By the third or fourth week it had caught up and overtaken that in aviation turbine kerosene.

The indications from the above results are that soil and air isolates of C. resinae grow better and faster in kerosene than do fuel isolates. Because of the small number of isolates involved it was decided to include a greater number of soil and fuel isolates in another experiment. Results are shown in Fig. 5. The greatest amount of growth was produced by a soil isolate; some fuel isolates, although producing visible growth, did not produce enough for harvesting. It is obvious from Fig. 5 that there is a wide range of ability of isolates of the fungus to grow in aviation turbine kerosene.

Some isolates of f. avellaneum produced a reddish brown pigmentation of the mineral salt medium. The nature of this pigment is being investigated.

C. resinae f. resinae has been shown to grow in aviation turbine kerosene but has not yet been tested in lighting kerosene. The white form, f. albidum, was recovered from two bottles containing aviation turbine kerosene which had been inoculated with the fuel isolates of f. avellaneum (C94, C95) but it was not tested further.

Discussion

All soil and air isolates and the majority of fuel isolates tested by us were able to grow in aviation turbine and lighting kerosene in the absence of any other source of carbon. Isolates varied considerably in the amount of growth produced after six weeks, indicating that many strains of the fungus exist, differing in their ability to grow in and utilise kerosene. All isolates grew well on V-8 juice agar without creosote, no difference being noted in the rate of growth. Further evidence for the existence of a number of strains of this fungus is obtained from the amount of reddish brown pigment produced by isolates and present in the aqueous phase when growing in kerosene. Some isolates produced an appreciable amount of this pigment, others none at all.

The amount of growth of soil and fuel isolates in general decreased on successive transfers through kerosene while that of air isolates increased. The reason for this is not known. Further work is necessary in relation to air isolates of the fungus because contamination of fuel by airborne spores is likely to occur.

Acknowledgments

The authors wish to thank Mr. W. Freitag and Mr. W. Johnston of B.P. New Zealand Ltd. for their help and interest in this work, particularly for supplying us with samples of kerosene. Financial assistance provided by the Victoria University of Wellington, Internal Research Committee, is gratefully acknowledged. Finally, our thanks are due to all those within the Botany Department of this university, and elsewhere, who have assisted us in so many ways.

References

Anon., 1968: Catalogue of the Culture Collection of the Commonwealth Mycological Institute, Kew.

De Vries., G. A., 1955: Cladosporium avellaneum de Vries, a synonym of ‘ Hormodendrum’ resinae Lindau. Antonie van Leeuwenhoek 21, 166-68.

Engel, W. B., and Swatek, F. E., 1966: Some ecological aspects of hydrocarbon contamination and associated corrosion in aircraft. Dev. Indust. Microbiol. 7, 354-66.

Gutheil, N. C., 1966; Ocorrência de Cladosporium resinae (Lindau) de Vries em querosene de aviaçao no Brasil. Comunicaçao prévia foi apreséntada ao 1° Simposie de Fermentacao, realizado pela Associaçao Brasileira de Quimica, em Sao Paulo, de 9 a 13 de Nov. de 1964.

Hazzard, G. F., 1963: Fungal growths in aviation fuel systems. Part 4. Fungi in Aviation Fuel Systems in Australia and in the Far East. Defence Standards Lab. (Australia) Rep. 252, 52 pp.

——, and Kuster, E. C., 1962: Fungal growths in aviation fuel systems. Part 2. Test methods. Defence Standards Lab. (Australia) Rep. 252, 14 pp.

Hendey, N. L., 1964: Some observations on Cladosporium resinae as a fuel contaminant and its possible role in the corrosion of aluminium alloy fuel tanks. Trans. Br. mycol. Soc. 47 (4), 467-75.

Parbery, D. G., 1967: Isolation of the kerosene fungus Cladosporium resinae from Australian soil. Trans. Br. mycol. Soc. 50 (4), 682-85.

——, 1968: The soil as a natural source of Cladosporium resinae. Biodet. of Materials., Elsevier Pub. Co., Lond., 371-80.

——, 1969: The natural occurrence of Cladosporium resinae. Trans. Br. mycol. Soc. 53 (1), 15-23.

Prince, A. E., 1961: Microbiological sludge in jet aircraft fuel. Dev. Indust. Microbiol. 2, 197-203.

Sheridan, J. E., 1971: The ‘kerosene fungus’ Amorphotheca resinae Parbery as a natural component of the airspora and the possible role of birds in dissemination. N.Z. Jl. Science (in press).

——, Steel, Jan, and Knox, M. D. E., 1971: The natural occurrence of the ‘kerosene fungus’ Amorphotheca resinae in New Zealand soil. N.Z. Jl. Science 14 (1), 147-60.

Sheridan, J. E., and Nelson, Jan, 1971: The selective isolation of the ‘kerosene fungus’ Cladosporium resinae from the air. Int. Biodetn. Bull. (in press).

* Fungal mat dried to constant weight in hot-air oven at 80° C.

** In First and Second Test used 16oz. medicine flats with 200ml. B/H + 50ml. kerosene.