Polyurethane Foam. 1.9 cm, to fit snugly into 250-mni wide-mouthed jars. The dimension of the specimens for the growth curve

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1 APPLIE MIcRomoLwy, Dec. 168, p Vol. 16, No. 12 Copyright 168 American Society for Microbiology Printed in U.S.A. Effects of Jet-Fuel Microbial Isolates on a Polyurethane Foam H. G. HEDRICK AND M. G. CRUM Applied Science Laboratories, General Dynamics, Fort Worth Division, Fort Worth, Texas Received for publication 25 September 168 Jet-fuel microbial isolates were studied for effects on a polyurethane foam material that has been proposed as a baffling material for use in aircraft fuel tanks. Evidence was found that a polyesterurethane foam gave increased cell counts and oxygen uptake with a bacterial isolate, and extensive matting with fragmentation and decreases in tensile strength of the foam with a fungal isolate. The polyurethane foam was affected by activity of the jet-fuel microbial isolates to an extent that would cause serious microbiological problems in the fuel tanks ofjet aircraft. The application of an open-celled polyurethane foam as a baffling material to minimize sloshing and projectile impact has been proposed recently for jet aircraft fuel tanks. The addition of such a material to the fuel system of the aircraft raised several questions, such as the effects of the material on growth of certain microbial isolates and the effects of the microbial isolates on the physical integrity of the foam. Since we have previously investigated the effects of jet-fuel microbial isolates on organic aircraft fuel-tank coatings (2, 5), it was of interest to determine whether the polyurethane foam would serve as a substrate for jet-fuel microbial isolates. Edmonds and Cooney (3) reported that a polyesterurethane foam did not affect the rate or total amount of growth of a bacterial isolate, but it did enhance the growth of a fungal isolate in a fuelmineral salts system by functioning as a matrix for mycelial attachment. Our interest was extended to the effects of the foam on microbial growth for long exposure periods, and to the effects of the microbial isolates on certain physical properties of the foam. The purpose of this paper is to present the results of experiments undertaken to determine the effects of selected jet fuel-utilizing microbial isolates on a polyurethane foam. MATERIALS AND METHODS The polyurethane foam specimens were cut from a block sample of orange polyesterurethane foam (Scott Paper Co., Foam Division, Chester, Pa.). The specimens of the used foam were cut from samples taken from the fuel tanks of an F-105 jet aircraft and sent from Wright-Patterson Air Force Base, Ohio. The specimens used in the 0-day tensile strength experiment were cut to a dimension of 10.2 by 5.1 by cm, to fit snugly into 250-mni wide-mouthed jars. The dimension of the specimens for the growth curve experiments was 10.2 by 7.6 by 2.5 cm, to fit snugly into 450-ml wide-mouthed jars. The specimens for the Warburg determinations were cut into small pieces approximately 3 mm in length. The microorganisms included fuel isolates selected from a collection of bacteria and fungi known to grow in a medium with jet fuel as the sole carbon source (4). The bacterial isolate was Pseudomonas aeruginosa (GD-FW B-10) and the fungus was Cladosporium resinae (GD-FW F-8). The bacterial, or fungal, inoculum for each experiment was prepared by washing the cells, or spores and mycelium, grown in a fuelmineral salts medium (1), with physiological saline before use. The mixed inoculum was made by adding equal portions of the separate washings to a composite. One milliliter of inoculum was added to each specimen jar. The methods for determining the effects of the presence of new and used foam on the growth of the [bacterial isolate included plate counts and Warburg manometry. The plate counts were run on Tryptone Glucose Extract (TGE) Agar (Difco) in duplicate setups by the serial-dilution technique at intervals over a 60-day incubation period. The foam specimens were sterilized in the jars for 1 hr in an ethylene oxide Cryotherm autoclave (American Sterilizer Co., Erie, Pa.). The filtered fuel, autoclaved distilled water, and mineral salts solution were added at a 3:1 fuel-towater ratio. The inoculated jars were incubated under static conditions at 30 C with agitation at the counting periods. The Warburg determinations were conducted as described earlier (2) on 20-mg samples of the foam. The methods used in determining the effects of the microbial isolates on certain physical properties of the foam included visual observations for structural changes and analysis for changes in tensile strength. The specimens and the media were prepared as described above for the plate-count experiments. In the test setups containing fuel plus water or mineral salts,

2 ...J VOL. 16, 168 JET-FUEL MICROBIAL ISOLATES 1827 the fuel-to-water ratio was 1:1 in order to establish for the tensile-strength testing. Foam specimens were an interface at the mid-region of the specimens. When set up for each medium with a set of uninoculated the fuel, water, or salts medium was used, the total controls. All of the specimen setups were incubated at volume was 150 ml. The prepared inocula of the 30 C under static conditions. A set of specimens was microbial isolates were added to the duplicate test removed at 30-day intervals for the gross observations setups. The duplicates provided four test specimens and the tensile-strength testing. The changes in tensile strength were determined on an Instron tester at a load rate of 5 inches (12.7 cm) per min. 10: l RESULTS AND DISCUSSION Changes in the growth response of the jet fuel- 10FS+ - utilizing P. aeruginosa isolate to the presence of a Now Fom new and a used polyesterurethane foam in fuel- FSk mineral salts and fuel-distilled water media are />~/Useshown Ls in Fig. 1. A high level of viable cells was 107 *Xt i f5 FS - maintained in the fuel-mineral salts medium with \/NoFoim both the new and the used foam after 60 days of l\\.f \/ incubation. The level of viable organisms in the presence of used foam was initially lower than 106 with the new foam; however, the two levels were about equal after 36 days, suggesting that the different samples of the foam were not contributing differently to the growth of the bacterial iso- 105 O late in the fuel-mineral salts medium. A decline in the number of viable cells began to show in the + control fuel-mineral salts medium without the z) 01 Ifoam, but the viable count began to increase to- E 10o, ward the end of the 60-day period. The level of c io8 viable cels and the rate of growth of the bacterial isolate in the presence of the new foam in the fuel- O mineral salts medium were similar to those re- 43?ported by Edmonds and Cooney (3). These re- (5 10 Asults suggest that the bacterial isolate was not 5 \ FOW* utilizing the foam material in place of the nu- No/Nmki trients supplied by the fuel-mineral salts medium l' *\ / A more significant difference in growth re- lo, t FDW sponse of this isolate was found in the fuel-dis- *.: / No Foam tilled water medium. The level of viable cells was I>1\F. / + greater in the presence of the new foam than in FW+ used that of the used foam. This finding suggested that evidently been leached out of the sample of used foam. These data extend the experiments of Edmonds and Cooney (3) by indicating that more than one test medium should be employed for,/; *~/...' usedthe new foam was supplying nutrients that had 104 ~ i..n/ detecting growth-supporting properties of orgamc substrates. The Warburg experiments showed more oxygen uptake by the bacterial isolate in the new foam than in that used over a 75-hr incubation period O00 (Fig. 2). In the mineral salts medium plus foam, 100 I I higher oxygen uptake was found in the used foam The addition of foam to the fuel-mineral salts Incubation Time - days medium gave an increase in oxygen uptake. The FIG. 1. Growth response of Pseudomonas aeruginosa comparative amount and rate of oxygen uptake in the presence of new and used foam. (FS, fuel + were greater by the bacterial isolate with the polymineral salts medium; FD W, fuel + distilled water urethane foam than with the polyurethane fuelmedium). tank coatings as reported by Crum et al. (2).

3 1828 HEDRICK AND CRUM APPL. MICROBIOL. o 0 25 so Incubation Time -hours FiG. 2. Oxygen utilization by Pseudomonas aeruginosa in the presence of new and used foam. (FS, fuel + mineral salts medium; F, fuel medium; S, mineral salts medium). Total fluid volume of reaction flasks: 1.0 ml of saline-washed cells, 1.0 ml of JP-4 fuel, 1.0 ml of mineral salts solution (or 2.0 ml when fuel not used). These data suggest that the foam may be more subject to effects of the bacterial isolates than are the polyurethane fuel-tank coatings. Observations on representative foam specimens taken from the five types of media after 0 days of exposure to the microbial isolates are shown in Fig. 3. No changes were present in the gross structure of the foam specimens exposed to the bacterial isolate, P. aeruginosa. The specimens exposed to the fungal isolate, C. resinae, had heavy mycelial matting in the fuel-water and fuelmineral salts media. In some of these setups, the reticulate structure of the foam was completely plugged with mycelium and the structure was fragmenting (as in the fuel plus salts column in Fig: 3). This type of microbial effect would become a serious detriment in an aircraft fuel-tank environment. The fungal isolate appears to cause more visual destruction and impairment of the polyfoam than does the bacterial isolate in the exposure conditions used in these experiments. These observations substantiate the report by Edmonds and Cooney (3) that the foam served as a matrix for mycelial attachment. The average tensile-strength changes of replicate specimens of the foam exposed in the five fluid media to the microbial isolates are presented in Table 1. The largest change in tensile strength over the controls was found in the fuel-mineral salts medium with C. resinae and with the mixed microbial inoculum. The fungal isolate also showed some reduction in tensile strength in the water and mineral salts media. The changes in the tensile strength of the foam caused by the bacterial isolate were not as great as those caused by the fungus. A composite of the trends of the tensile-strength changes in the foam exposed to the microbial isolates is presented in Fig. 4. These results indicate that the polyesterurethane foam was affected by the action of the jetfuel microbial isolates as evidenced by an increased bacterial cell count obtained in the pres-

4 VoL. 16, JET-FUEL MICROBIAL ISOLATES 182 e.. I. CONTROL FUEL WATER 5ALT$ FUEL+ FiG. 3. Representative foam specimens after exposure to microbial activity for 0 days. WATER TABLE 1. Tensile-strength changes (psi) in polyurethane foam specimens Fluid media and incubation time (days) FUEL +.5ALT5 Microbial isolates Fuel Water Salts" Fuel + water Fuel + salts Cladosporium resinae Pseudomonas aeruginosa Mixedb Cont role abushnell-haas mineral salts medium. bc. resinae plus P. aeruginosa.,cno organisms.

5 1830 HEDRICK AND CRUM APPL. MICROBIOL. 40 1' rii& oe! -o Control 0 i30 2d 101-.gO (4-~ ~ ~... ~Mixed Bhria i... _. ~caol X _. Mixed - Fungus AWZ-pl7F R o* Conol mm*becteria 05L,-.J. - ",sc%%... A i *i- %%, ' Fungus -- d.ft."unsmix \*\\\\- * Funguss- Changes in tensile strength of the foam ex- different Incjubation Time days - FIG. 4. posed to three types of microbial systems in fluid media. ence of the foam, an increased oxygen uptake by the bacterial isolate, production of extensive fungal matting in the reticular matrix of the foam, degradation of the foam structure by the fungal isolate with fragmentation, and changes in the tensile strength of the physical structure of the foam. The addition of the foam to aircraft fuel tanks as a baffling material would contribute to increased activity of jet fuel-utilizing microbial isolates. ACKNowLEDGMENmS This work was conducted under the General Dynamics Corp. IRAD program. We acknowledge the receipt of the used foam from J* A. Ryan, Wright-Patterson Air Force Base, Ohio, and the assistance of the Engineering Test Laboratory, General Dynamics, in the tensile-strength tests. LITERATURE CITED 1. Bushnell, L. D., and H. F. Haas The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41: Crum, M. G., R. J. Reynolds, and H. G. Hedrick Microbial penetration and utilization of organic aircraft fuel-tank coatings. Appl. Microbiol. 15: Edmonds, P., and J. J. Cooney Microbial growth in a fuel-water system containing polyesterurethane foam. Appl. Microbiol. 16: Hedrick, H. G., R. J. Reynolds, and M. G. Crum Identification and viability of microorganisms from jet fuel samples. Develop. Ind. Microbiol. : Reynolds, R. J., M. G. Crum, and H. G. Hedrick Studies on the microbiological degradation of aircraft fuel tank coatings. Develop. Ind. Microbiol. 8: Umbreit, W. W., R. H. Burris, and J. F. Stauffer Manometric techniques, 4th ed., p Burgess Publishing Co., Minneapolis.