these samples were then filtered, plated, incu-
bated, and counted.

Long-Term Growth of Microorganisms in Fuel
Phase. To determine whether microorganisms
can live and multiply in jet fuel without the
presence of a discrete water phase, several gal-
lons of untreated jet fuel were chilled in a cold
room (3°C) for 3 days and filtered (Whatman
#1 filter paper) into a dry container. This sys-
tem contained such organisms as were indigenous
to the fuel and only that amount of water which
could be maintained in solution at 83°C; no sepa-
rate, discrete water phase was present when the
system was brought to room temperature. Sam-
ples were removed for counting immediately
after filtration and at intervals up to 63 days.
Incubation temperature was 23-25°C for this 63-
day period. .

Inhibition of Microbial Growth in Fuel-Water
Systems by Biobor-JF. To study the rate and
extent of microbial inhibition by Biobor-JF in
a fuel-water system, four laboratory “tanks” were
prepared using 2-liter graduated cylinders stop-
pered with cotton. 1950 ml of untreated com-
mercial jet fuel were placed in each cylinder.
Two bacterial and 2 fungal isolates were grown
in separate broth cultures, and 10 ml of each
culture were withdrawn, mixed together, and
washed twice with Bushnell-Haas salts solution
followed by centrifugation. The final mixed
suspension was added to 1 liter of the sterile salts
solution and a 50 ml aliquot of this suspension
was added to each tank. Tanks 1 and 2 were
designated as controls; tanks 3 and 4 were treated
by adding Biobor-JF to the fuel layer to a con-
centration of 270 ppm by weight (equivalent to
20 ppm elemental boron). After initial mixing,
samples were removed for counting at intervals
up to 88 days. Two hours after a uniform agi-
tation of 4 inversions (to simulate normal refuel-
ing turbulence), samples were taken with a sterile
probe from the aqueous layer and from the fuel
layer (4 inches below the fuel surface).

Inhibition of Microbial Growth on Solid Nu-
trient Media by Biobor-JF. Eight bacterial and
5 fungal isolates, selected from a total of 19 ap-
parently discrete types isolated from fuel (see
Appendix A)?, were grown in separate broth

1 Although the taxonomic characterization of the fuel isolates
was beyond the scope of this study, the more obvious physical
and cultural characteristics noted during the tests are shown
in Appendix A.

cultures; these were uniformly inoculated onto
the surfaces of individual petri dishes of solid
media using a sterile glass rod spreader. Bac-
teria and fungi were grown on nutrient agar and
Sabouraud Dextrose Agar respectively, using a
single isolate per dish. Whatman #2 filter paper
was cut into 6-mm discs using a paper punch;
the sterilized discs were dipped into Biobor-JF
and applied to the surface of the inoculated
plates. Plates were inspected for a zone of in-
hibition after a 48-hour incubation at 34°C.

In a second experiment, Biobor-JF was added
directly to nutrient agar and Sabouraud Dextrose
Agar to a final concentration of 400 ppm; it was
added after steam sterilization but before the
agar had solidified. Petri dishes were poured
with the treated agars and inoculated as pre-
viously described.

A third experiment was designed to determine
whether the microorganisms could utilize Biobor-
JF as a sole source of carbon. After sterilization
of a solid medium prepared by adding 15 g/liter
of agar to Bushnell-Haas salts solution, Biobor-
JF was added in concentrations of 0, 4,000, and
10,000 ppm. Four different bacterial isolates
were inoculated onto each of the 3 media.

Inhibition of Microbial Growth in Liquid Nu-
trient Media by Biobor-JF. Flasks containing
5 ml of sterile nutrient broth (beef extract 0.3%,
peptone 0.5%), in which 0, 200, 500, 1,000, 5,000,
or 10,000 ppm of Biobor-JF was incorporated,
were separately inoculated with 1-drop suspen-
sions of broth cultures of individual bacterial
isolates. These flasks were cotton-stoppered and
incubated with constant shaking at room tem-
perature (26°C) for 22 hours. Optical density
of the bacterial suspension was determined at
600 mp using a Coleman Junior spectrophoto-
meter with nutrient broth as the blank.

For fungal inhibition studies, separate flasks
containing 75 ml of sterile Sabouraud Dextrose
broth in which 0, 200, 500, 1,000, 5,000, or 10,000
ppm of Biobor-JF was incorporated (after steri-
lization) were inoculated with 1-drop suspen-
sions of broth cultures of single fungal isolates
from jet fuel. The flasks were incubated at room
temperature (26°C) for 5 days with intermittent
shaking. The medium was removed by filtering
the entire flask contents through a membrane
filter (Millipore, Type RA, pore dia. 1.20p, filter
diameter 47 mm) ; the mycelium was then washed
with 20 ml of 10% formalin. The pre-weighed





Taste V. Survival of Microorganisms in Fuel Phase

Time (days) Bacteria/liter Fungi/liter
0 18 1
9 20 1
17 10 2
24 7 1
63 8 2

Since absolutely anhydrous jet fuel is imprac-
tical to maintain in field refueling operations,
growth or survival of microorganisms under these
conditions was not studied.

In fuel-water systems, Biobor-JF appears to
inhibit fungi very effectively. Table VI indi-
cates that this biocidal action occurs within the
first four days of exposure.

There was no appreciable bacterial inhibition
in the water layer under the conditions of con-
stant Biobor concentration and the fuel/water
ratio existing in these laboratory experiments.

Taste VI. Biobor-JF Inhibition In Fuel-Water Systems’
Fuel Layer Water Layer

Tank Number acteria/  Fungi/  Bacteria/ = Fungi/ml

liter liter ml

Zero Time®

1 (control) 10 30 4.0 X10° 12 X10

2 (control) 120 30 «4.0 X10° 11 X10

3 (Biobor) 40 30 4.0 X10° 18 X10°

4 (Biobor) 20 40 3.0 X10° 11 X10°

4 Days

1 (control) 0 9800 828X100! 20 X10

2 (control) 20 7,200 61 X10° 10 X10

3 (Biobor) 10 0 31 X10° 0

4 (Biobor) 40 0 3.4 X10" 0

20 Days

1 (control) 2,200 2,700 48 X10° 74 X10

2 (control) 2,500 2,500, 2.0 X 10° 9.5 X10°

8 (Biobor) 20 0 80 X10 0

4 (Biobor) 20 0 9.0 X10 0

83 Days

1 (control) 1,510 2,600 3.28 X 10° 2.0 XK 10°

2 (control) 1,600 2,900 «757K 10° «1.27 X10"

3 (Biobor) 20 0 6.0 X10° 0

4 (Biobor) 20 0 4.0 X10° 0

YEach tank contained 1950 ml of jet fuel and 50 ml of Bushnell-
Haas salts solution. Tanks 3 and 4 contained Biobor-JF in
the fuel fraction at a concentration of 270 ppm by weight;
this concentration corresponds to 20 ppm of elemental boron.

°%Zero time—samples were removed just prior to the addition
of Biobor-JF to Tanks No, 3 and 4.

After the 33rd day, accurate sampling of the
water phase became impossible due to the for-
mation of fungal mats in the control tanks. De-
spite the lack of fungal counts in the treated
tanks, two small fungal colonies (white) ap-

peared in the water layer of tank #8 and grew
slowly to approximately.1 cm in diameter.

The complete absence of viable fungi in the
treated laboratory tanks on the 4th day is in-
teresting. Both phases remained free of viable
fungi for the remainder of the experimental
period although the fuel-water ratio was only
39:1.

With this fuel-water ratio, the quantity of
Biobor-JF present would give a concentration
in the water layer of approximately 10,000 ppm
at equilibrium. The greater fuel-water ratio
existing in an aircraft fuel tank would permit a
much higher Biobor-JF concentration to build
up in the static water phase.

Inhibition by Biobor-JF was less marked when
the organisms were incubated on a solid medium,
Uniformly-inoculated petri dishes of nutrient
agar showed no zone of inhibition around a
Biobor-JF-saturated paper disc after 48-hours’
growth with the organisms tested. Good growth
of all 8 bacterial isolates tested was obtained on
nutrient agar containing 400 ppm of Biobor-JF;
8 of 5 fungal isolates showed profuse growth on
Sabouraud Dextrose Agar at 72 hours with the
same Biobor-JF concentration.

Of the four bacterial isolates tested on solid
medium incorporating 4,000 ppm Biobor-JF as
the sole source of carbon, all exhibited growth
after 3 days at 34°C. On the 10,000 ppm
Biobor-JF medium, three of the four isolates
showed a slow, steady and eventually profuse
growth after one month at room temperature
(26°C). Of these three isolates which grew on
10,000 ppm, however, two were also capable of
growing on a solid medium containing only in-
organic salts; therefore, they must be capable
of using atmospheric CO, as a carbon source.
The remaining isolate of the three capable of
growth on 10,000 ppm could not grow on the
salts medium; therefore, it must have been utiliz-
ing the Biobor-JF as a source of carbon. None
of the fungal isolates was capable of growth in
the presence of even the lowest concentration of
Biobor-JF when no other carbon source was
present.

Growth inhibition produced by various con-
centrations of Biobor-JF was more apparent in
broth cultures. These results are shown in Table
vil.



filter with the mycelium on it was transferred
to an aluminum weighing dish and dried “in
vacuo” over anhydrous CaS0, for 7 days (to
constant weight), weighed, and the growth de-
termined as mg of mycelium per flask.

III. Results and Discussion.

The membrane filtration technique gave rea-
sonably adequate precision as indicated by the
data depicted in Table I; therefore, this tech-
nique was used to determine microbial concen-
tration of fuel samples.

Tasre I. Precision of the Membrane Filtration Method

Organism Sample No. of Sample Colony Ooeficient
Type* Type Repli- Size,ml Count of Varia-
cates +8.D. tion, %
B Aqueous
Suspension 20 10 24045 5
B ” 20 10 19+2 1
B ” 10 10 148+7 5
F ” 6 10 3842 5
F ” 4 10 842417 5
B Fuel
Suspension 20 500.0 = 108+9 9
B ” 10 100.0 1742 12

*B= Bacteria, F=Fungi

The results shown in Table II illustrate that
Triton X-100 either has no significant effect on
microbial growth, or, if it does, a rinse with 100
ml of phosphate buffer is adequate for complete
removal of any residual detergent; therefore, a
100 ml rinse was selected as standard procedure.
Data in Table III illustrate that the settling
rate of microorganisms in jet fuel is a very sig-
nificant factor in determining a time for sampling
fuel from the aircraft. Microbial suspension
caused by refueling, flight, or other tank turbu-
lence can seriously affect field tests for fuel con-
tamination. Table III shows the number of
bacteria present 4 inches below the surface of a
laboratory tank up to 4 hours after agitation.

Taste II. Effect of Detergent’ Rinse on Bacterial
Growth Rate

No. of Organisms per Cultured Plate!
Volume Buffer

Rinse (ml) E. colt B. subtilis
0 129 32
25 133 81
100 136 32
500 140 28

*Each figure represents the mean of three individual
plate counts.

Taste III. Concentration of Bacteria Four Inches
Below Surface of Fuel at Various Time Intervals
After Suspension

Time After Shaking (min) Bacteria/25 mt

0 1,000

5 1,000

10 1,000

20 1,000

30 636

100 431

240 400

That microorganisms can distribute themselves
throughout the fuel under static conditions is
illustrated in Table IV. After 8 days’ incuba-
tion, considerable numbers of bacteria, which
were originally introduced into the water layer
only, were noted up to 14 inches above the fuel-
water interface; therefore, the organisms should
have no particular difficulty in contaminating
any new water pockets introduced by refueling
or condensation.

Taste IV, Vertical Distribution of Microorganisms in
Fuel-Water Systems After Standing for Hight Days

Distance Above
Fuel-Water

Tank No. Interface (inches) Bacteria /liter

Fungi/liter

2 14 20
7 320
1 20
3 14 70
8 9270
2 8790
— 0,25" 2.7x10°
4 4 120
7 20
1 1820

* Sampling of Water Layer below interface.

» Fungi were not counted separately in tank #3; counts
shown represent the total number of colonies which
grew on nutrient agar.

While DeGray and Killian’ and others have
established qualitatively the ability of micro-
organisms to remain viable in essentially “dry”
fuel, we were interested in determining whether
a population increase might occur under these
conditions. The results of this experiment are
shown in Table V. Neither bacteria nor fungi
multiplied appreciably without the presence of a
discrete water phase; however, considerable num-
bers of both remained viable for the period of
the experiment.


Preliminary studies established a Biobor-JF
distribution coefficient of approximately 440
where:

K (distribution coefficient) = Concentration of additive in water
"Concentration of additive in fuel
or: K = Weight of additive in water
Weight of additive in fuel
provided the fuel and water volumes
are equal.

The equation for calculating the concentration
of additive in the water layer which is achieved
by addition of a known amount of additive to a
defined system is as follows:

Kew.

C (watery VidtKev.

where K=distribution coefficient
W,=total weight of Biobor-JF in system
V:= volume of fuel
Vw= volume of water

Computations based on these formulae indicate
that the Biobor-JF concentration in the water
layer of an aircraft fuel tank, under field condi-
tions, would be expected to exceed the biocidal
level of 5,000-10,000 ppm when the fuel is re-
peatedly treated at the 270 ppm maximum level
recommended by SOHIO.

In conclusion, Biobor-JF appears to be an
effective fungal inhibitor for jet fuels when used
at the recommended concentrations. Continued
use could eliminate fungi from any new water
pockets formed in the fuel tanks from condensa-
tion or from cooling of water-saturated fuel.
Our experiments indicate that Biobor-JF is a
less effective anti-bacterial agent in fuel-water
systems. Whether or not resistant strains of
bacteria or fungi would develop and prove to be
a problem in practical applications cannot be
predicted from existing data. Further field
studies, under conditions more favorable to
microbial growth, are indicated.



AM 67-21

EVALUATION OF A-BIOCIDAL TURBINE-FUEL ADDITIVE

Charles R. Crane, Ph.D.
Donald C. Sanders, M.S.

Approved by Released by
J. Rosert Dit, M.D. P. V. Simcrn, M.D.
Crier, Crviz ArromMEpicaL Feprra, Ar Surcron
Institute

August 1967

FEDERAL AVIATION ADMINISTRATION
Office of Aviation Medicine



p

n

2

mp

p

REFERENCES

Dowauvr, T. B., “Microbiological Fuel Contamina-
tion and Corrosion,” Lockheed Field Service Digest,
Vol. 7, No. 5, March-April 1961, pp. 3-13.

. Scovitte, W. H. and Gron, G. M., “A Fuel Soluble

Additive for Sterilization of Turbine Fuel Systems,”
Standard Oil Company (Ohio), Report for presen-
tation to the Aviation Technical Service Committee
of the American Petroleum Institute, Montreal, May
1964, pp. 1-39.

Cocxey, R. R., Honor, H. M., Iverson, W. P., and
Witxes, C. E., “Microbial Contamination of Air
Force Petroleum Products,” Air Force Aero Propul-
sion Laboratory, Wright-Patterson Air Force Base,
Ohio, Technical Documentary Report’ No. RTD-TDR-
63-4119, January 1964, pp. 1-37.

Rogers, J. D., Krynirsxy, J. A., and CHuRcHIrL,
A. V., “Jet Fuel Contamination: Water, Surfactants,
Dirt and Microbes,” Society of Automotive Engi-
neers Publication 5830, New York, October 1962,
pp. 281-292.

Leonarp, J. M., “Fuel Fungi,” Naval Research Re-
view, 1960, pp. 16-18.

Prince, A. E., “Microbiological Sludge in Jet Air-
craft Fuel,” Developments in Industrial Microbiology,
Vol. 2, 1961, pp. 197-208.

DeGray, R. J, and Kiran, L, N., “Life in Essen-
tially Nonaqueous Hydrocarbons,” Developments in
Industrial Microbiology, Vol. 8, 1962, pp. 296-303.

. Baxanauskas, S., “Bacterial Activity in JP-4 Fuel,”

Materials Laboratory, Wright-Patterson Air Force
Base, Ohio, WADC Technical Report No. 58-82,
March 1958, pp. 1-15, AD 151084.

Wuson, J. G, “Microbial Growth in Fuels and
Fuel Systems,” Journal of the American Association

for Contamination Control, Vol. 2, No. 1, 1963, pp.
11-12,

DeGray, R. J. and Kriuan, L. N., “Bacterial Slime
and Corrosion in Petroleum Product Storage,” Indus-
trial and Engineering Chemistry, Vol. 52, No. 12,
1980, pp. 744—76A.

Hi, E, C., “Biological Problems of Fuel Storage,”
Chemistry in Britain, Vol. 1, No. 5, 1965, p. 190.
Lonpon, S, A., FINEFRocK, V. H., and Kruxran, L. N.,
“Microbial Activity in Air Force Jet Fuel Systems,”
Aerospace Medical Research Laboratories, Wright-
Patterson Air Force Base, Ohio, Report No. AMRL-
TR-65-80, April 1965, pp. 1-25.

Nunn, D., “Microbiological Investigations,” A. F.
Aero Propulsion Laboratory, Wright-Patterson Air
Force Base, Ohio, Report No. RTD-TDR-63-4118,
Part II, February 1965, pp. 1-40.

Wricut, R. H. and Hostetter, H. F., “Microbiologi-
cal Diesel Fuel Contamination,” Society of Automo-
tive Engineers Publication 651B, New York, January
1963, pp. 1-12.

Cuurcnin, A. V. and LearHen, W. W., “Research
on Microbiological Sludge Inhibitors,” Directorate
of Materials and Processes, Wright-Patterson Air
Force Base, Ohio, Report No. ASD-TDR-62-368,
April 1982, pp. 1-71, AD 284183.

Mutter, B, M. (Editor), “Proposed Procedures for
Microbiological Examination of Fuels,” Committee
on Microbiological Deterioration of Fuels, Society
for Industrial Microbiology, Washington, D.C., Spe-
cial Publication No. 1, July 1963, pp. 1-35.
BusHNeELL, L. D, and Haas, H. F., “The Utilization
of Certain Hydrocarbons by Microorganisms,” Jour-
nal of Bacteriology, Vol. 41, 1941, pp. 653-672.

10.

11.

12.

14,

i.

16.

17.

OTHER PERTINENT REFERENCES NOT CITED

Guyyes, G. J. and Bennet, E. O., “Bacterial De-
terioration of Emulsion Oils, I. Relationship between
aerobes and sulfate-reducing bacteria in deteriora-
tion,” Applied Microbiology, Vol. 7, 1959, pp. 117-121.

Henerx, R. B., “Compilation of References on Micro-
Diological Contamination of Fuels,” Armed Services
Technical Information Agency, Arlington Hall Sta-
tion, Arlington, Virginia, November 1, 1961, pp.
16-21, AD 266 000.

10

3. Kies, D, E, and Leonarp, J. M., “Microbial In-
hibitors for Systems of Jet Fuel and Water,” Chem-
istry Division, U. Naval Research Laboratory,
Washington, D.C., NRL Report No. 5501, August
31, 1980, pp. 1-10, AD 244511.

4. Raysonp, R. L., “Microbial Oxidation of n-Parat-
finic Hydrocarbons,” Developments in Industrial
Microbiology, Vol. 2, 1931, pp. 23-32.

. SUMMER, W., “Microbially Induced Corrosion,” Cor-
rosion Technology, Vol. 7, No. 9, 1960, pp. 287-288.



Culture Number
1

2

3

o-B

11

12

13

14

16

17

18

19

APPENDIX A
CHARACTERISTICS OF FUEL ISOLATES

Characteristics

Fungus: light-brown growth on SDA}, colonies round and convex, no diffius-
able pigment produced.

Fungus: profuse white growth on SDA turning light-grey with age, brown
pigment diffuses into medium.

Fungus: long, white filamentous growth on surface, light-brown pigment
diffuses into medium in old cultures.

Fungus: rich brown coloration, profuse growth on SDA with yellowish-brown
pigment diffusing into medium.

Fungus: sparse, yellowish growth on SDA becoming greenish-brown with
age, yellow fruiting bodies appear in old cultures.

Fungus: profuse, white, silky growth on SDA, colonies exude an opaque
pigment into medium with age and surface growth becomes a greyish-pink
in color.

Fungus: profuse, light-brown growth on SDA, raised, slightly-convex colo-
nies with white edges, no diffusable pigment.

Fungus: raised, green colonies on SDA with white edges.

Bacterium : rods growing in singles, pairs and chains, round ends, length about
2-4X width, gram-positive, non-acid-fast, forms pellicle in NB*, colonies
circular, convex, entire, moist, cream-colored on NA.°

Bacterium: large rods with rounded ends, mostly single rods showing granu-
lar staining, gram-negative, forms pink pellicle in NB with some growth in
depth; round, convex, moist, pink colonies on NA.

Bacterium: rods, mostly long chains with much branching, gram-positive,
granular growth in NB, profuse white colonies with convoluted surfaces
on NA.

Bacterium: small rods, short, almost coccoid in shape, gram-negative, motile,
shows uniform, abundant growth throughout medium in NB; colonies on
WA are circular, raised, entire, white, moist, and translucent.

Bacterium : rods, rounded ends, length approximately 6X width, singles, pairs,
long chains, gram-positive, non-acid-fast, forms slight pellicle in NB with
heavy flocculent growth in depth. Forms irregular, raised, undulate,
opaque, white colonies with translucent centers on NA.

Bacterium: rods, singles, pairs and short chains, gram-positive, non-acid-fast,
produces slightly granular, white pellicle in NB. Colonies on NA are cir-
cular, convex, entire, ivory-colored and opaque.

Bacterium: rods with rounded ends, singles, pairs and chains, gram-positive,
non-acid-fast, forms white pellicle in NB, colony appearance same as cul-
ture #16.

Bacterium : rods forming long chains, gram-positive, does not stain easily with
aqueous stains; colonies on NA are roughly circular, umbonate, with undu-
late edges and considerable yellowish growth below surface of medium ;
colony has hard, white surface becoming rough with age; small circular,
umbonate, white colonies grow on surface of NB but do not grow in depths.

Bacterium : short rods, singles, pairs, short chains; have rounded ends, motile,
gram-negative, forms slight white pellicle in NB with white clumps grow-
ing in depths; has white, convex, slightly rough colonies on NA.

> SDA—Sabouraud’s dextrose agar

*NB—Nutrient broth
*NA—Nutrient agar



Taste VII. Effect of Biobor-JF on Bacterial Growth in Liquid Culture

BiobordF Optical Density at 600 mp

Concentration Known Species? Fuel Teotates
(ppm) E. cob A. aerogenes B. subtitie S. marcescens| #10 #11 #13 #15 #16 H1T_—«H#:19
0 064 0.57 0.48 0.86 0.42 0.08 0.21 040 045 0.44 0.66
200 0.68 0.62 0.39 OTT 038 0.06 017 0.40 048 0.41 0.72
500 0.66 0.57 0.39 0.77 0.17 0.05' 0.15 037 040 037 0.65
1,000 0.63 0.53, 0.10 O71 0.05 0.08' 0.13 0.12 0.34 0.28 0.62
5,000 0.10 0.04 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.01 0,00
10,000 0.03 0.00? 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00

+Puel. isolate #11 showed a slight granularity in the broth culture at the 500 and 1,000 ppm concentrations.

24, aerogenes showed a slight growth on the second day at the 10,000 ppm concentration.

spure cultures of Hscherichia coli, Aerobacter aerogenes, Bacillus subtilis, and Serratia marcescens were generously supplied by
Dr. L. Vernon Scott and his staff, Department of Microbiology, University of Oklahoma Medical Center, Oklahoma City,

Oklahoma.

Bacteria exhibited considerable species varia-
tion in their ability to grow in the presence of
higher concentrations of the additive in liquid
media. Whereas most of the organisms tested
were completely inhibited at concentrations be-
tween 1,000 and 5,000 ppm, £. coli and Fuel
Isolate #13 showed significant growth at the
highest concentration used, 10,000 ppm. A. aero-
genes exhibited a similar resistance to inhibition
by Biobor-JF.

Generally, the fungi all respond to the same
concentration ranges of Biobor-JF; however,
resistant cultures were noted. The results of this
experiment are shown in Table VIII.

Taste VIII. Effect of Biobor-JF on Fungal Growth in
Liquid Culture’

Biobor-JF | Milligrams of Mycetium/75 ml Growth Medium

Concentration |Fuel Isolate No. 1 2 8 4 6
(ppm)

0 155 454 425 185 78

200 159 889 352 120 55

500 139 391 O 114 51

1,000 58 289 367 71 48

5,000 0 283 0 0 46

10000 0 7 0 0 16

All flasks were incubated for 5 days at 26°C with intermittent
agitation

Fungal isolates #2 and #6 were the least
inhibited by Biobor-JF, showing only 37.5% and
41% inhibition respectively at the 5,000 ppm
concentration. Isolate #6 grew to 19% of the
control weight at 10,000 ppm.

Field tests were conducted concurrently with
the laboratory testing in cooperation with the
Engineering Division, Aircraft Services Base
(Engineering Report on Jet Fuel Additive
Biobor-JF for Biocidal Effects, Report #66-25,
prepared by AC-884). Samples were collected
from symmetrically-located control and Biobor-
J¥F-treated tanks on a Convair 880 aircraft dur-

ing routine operation over a 3-month period.
Representative data from the aircraft fuel sam-
ples are shown in Table IX. These data proved
to be inconclusive, partially due to the cold-
weather conditions which inhibited growth even
in the control tanks. This lack of microbial
growth in the untreated fuel prevented significant
comparisons to be made with populations in the
Biobor-JF-treated tanks.

Taste IX. Microorganisms in Fuel from Convair 880
during Field Tests

Bacterta/Liter Fungi/IAter

Sampling | Control Biobor-Treated | Control Biobor-Treated
Date Tank Tank Tank Tank
127 6 4 10 4
2-3 8 20 114 10
2-12 486 6 954 2
217 58 38 70 “4
841 10 6 10 10
3-19 10 22 lt 0
8-24 382 41 84 0
427 2 22 16 0
5-6 26 3 12 0

iSamples on this date were collected immediately prior to
addition of the Biobor-JF (270 ppm).

20n 4-5-65, the aircraft was refueled with military JP-4 fuel
containing 0.10-0.15% of the Phillips PFA-5SMB deicing
additive which is also known to exhibit marked biocidal
effects. Refueling with JP-4 was continued during routine
operation for one week. On 4-13-65, all tanks were drained
and refilled with Conoco Jet-50 fuel; Biobor-JF was added
to the #3 tank to a concentration of 270 ppm. ‘Tests were
continued in the usual manner until termination of the
field project.

A comparison between the counting method
used in this study and the SOHIO test method,
(STM R-54-65-T), which utilizes an extraction
of the fuel sample with nutrient broth, indicated
that the methods gave comparable results pro-
vided the fuel contained no minute water droplets
clinging to the sides of the container. The pres-
ence of these water droplets, which was noted in
some field samples, would produce an erroneously

high count by the SOHIO method.


Media Selection. Media tested for use with
the membrane filter counting method included
Nutrient Agar (Fisher Scientific Co.), Standard
Plate Count Medium (Baltimore Biological Lab-
oratory), Double Strength Nutrient Agar, Tryp-
tone-Glucose Extract Agar (Difco Manual #9,
p. 57), Nutrient Broth enriched with 0.1% yeast
extract and 0.1% glucose, Sabouraud Dextrose
Agar (Fisher Scientific Co.) and Czapek Solu-
tion Agar (Difco Manual #9, p. 245). Repli-
cate samples were withdrawn from a constantly-
stirred suspension of mixed bacteria and fungi
which had been isolated from fuel. Each sample
was filtered and the filters incubated at 37°C on
poured plates of the above test media. Counts
of bacterial colonies present after 24, 48, and 72
hours of incubation on the first five media failed
to show any significant differences among the
various media in terms of the number of colonies
or rate of growth. Of the last two media, which
are designed primarily for the cultivation of
fungi, Sabouraud Dextrose Agar supported fun-
gal growth considerably better than the Czapek
Solution Agar. Nutrient Agar and Sabouraud
Dextrose Agar were therefore selected for the
cultivation of bacteria and fungi respectively.

Accuracy of Membrane Filter Method. To
establish the degree of precision to be expected
from the membrane filter method of counting,
the following experiments were conducted.

1. Aqueous Suspensions: Separate suspensions
of bacteria and fungi were prepared in phosphate
buffer (0.01 M, pH 6.8) by the direct addition
of a diluted broth culture of the organism. Rep-
licate samples were prepared by transferring 1.0
ml samples from each constantly-stirred micro-
bial suspension to the sterile funnel-filter as-
sembly which contained 100 ml of phosphate
buffer. The addition of the 1.0 ml sample to the
buffer provided a more uniform distribution of
the organisms over the filter surface. After ap-
plication of suction, an additional 100 ml of
sterile buffer was used to wash down the sides
of the funnel.

2. Fuel Suspensions: Separate suspensions of
bacteria and fungi in fuel were prepared by
shaking fuel with a broth culture of each organ-
ism, allowing the broth to settle, then carefully
decanting the fuel. Samples of 100 and 500 ml
volumes were removed from the constantly-
stirred fuel suspension and transferred to the

filter-funnel assembly, then washed with deter-
gent and buffer as previously described.

The washed filters from both aqueous and fuel
suspensions were then transferred to petri dishes
containing the appropriate medium and incu-
bated at 84°C. Colony counts were made using
a standard Quebec counter after 24 hours of
incubation for the bacterial plates and 48 hours
for the fungal plates. The results are shown in
Table I; the coefficient of variation ranged from
5% to 12%.

Detergent Effect. Since the jet fuel must be
removed from the membrane filter prior to in-
cubation to allow the nutrient to diffuse to the
entrapped cells, a detergent rinse is necessary.
To determine the effect of this detergent on the
growth of the bacteria, aliquots from suspensions
of known species (both gram-positive and gram-
negative) were transferred into approximately
10 ml of sterile, aqueous 0.1% Triton X-100
contained in a sterile filter-funnel unit. After
removal of the detergent solution by suction, the
filter was washed with 0, 25, 100, or 500 ml of
sterile phosphate buffer (0.01M, pH 6.8); the
500 ml rinse was added in five 100-ml portions
to assure complete removal of the detergent.
Colony counts were made after 26 hours of in-
cubation on nutrient agar at 34°C.

Settling Rate. In order to estimate the set-
tling rate of bacteria placed in suspension by
tank turbulence, a laboratory tank was prepared
using a 2-liter, stoppered, graduated glass cylin-
der. Each such tank was inoculated with 100
ml of Bushnell-Haas salts solution’” containing
2 ml of a suspension of 2 bacterial and 2 fungal
types previously isolated from fuel. 1900 ml of
fuel was layered over the 100 ml of salts solution.
After 8 days’ incubation at room temperature,
the cotton plug was replaced by a glass stopper
and the fuel-water mixture was shaken vigor-
ously. At various time intervals after shaking,
samples of 25 ml each were aseptically withdrawn
from exactly 4 inches below the fuel surface,
plated, and counted.

Distribution of Organisms in Fuel-Water Sys-
tem. To establish the distribution of bacteria
in fuel tanks under static conditions, 3 “tanks”
were prepared as described above and incubated
for 8-12 days at room temperature. Using a
long, sterile, tubular probe, fuel samples were
removed at various distances above the fuel-
water interface and from the water layer itself;


EVALUATION OF A BIOCIDAL TURBINE-FUEL-ADDITIVE

I. Introduction.

Microbial growth in kerosene-type fuels is
often associated with the fouling of fuel screens
and capacitance probes, as well as the corrosion
of aluminum-alloy fuel tanks (1-18). Since 1956,
when fuel system malfunctions in the B-47 air-
craft were traced to microbial sludge formation,
considerable effort has been expended to develop
a fuel additive which would retard the growth
of these organisms and, at the same time, be com-
patible with the fuel system components.

Of particular interest recently has been the po-
tential usefulness of several boron-containing
organic compounds which exhibit microbiocidal
properties?*?°1415, These compounds present a
unique solution to the problem of getting the
biocidal material into contact with the organisms
which proliferate in the water layer, since they
could be introduced as a fuel-biocide solution
and, due to their water solubility, would tend to
saturate any water pockets which exist in the
tank. Biobor-JF, developed by the Standard
Oil Company of Ohio, is such a product.

An evaluation of the effectiveness of Biobor-JF
as a biocidal jet fuel additive was initiated in a
two-phase program. One phase was a carefully
controlled laboratory study of the biocidal prop-
erties of the additive under simulated field con-
ditions. Concurrent tests were conducted by the
Aircraft Services Base, Federal Aviation Ad-
ministration, Oklahoma City, Oklahoma using
the fuel-additive mixture in an agency-owned
Convair 880 aircraft performing routine flight
operations. This report, however, is concerned
primarily with the laboratory studies.

The laboratory studies conducted in this eval-
uation consisted of the following :

a, Comparative evaluation of seven nutrient
media to determine the one most suitable for
culturing the mixed population of microorgan-
isms found in fuel.

b. Evaluation of a membrane filter procedure
for measuring microbial concentration in both
fuel and aqueous samples.

¢. Viability of microorganisms in turbine fuel.

d. Kinetics of microbial mass transfer in fuel-
water systems.

e. Effect of Biobor-JF on microbial growth in
fuel-water systems.

£. Effect of Biobor-JF on microbial growth in
liquid and solid nutrient media.

Il. Experimental.

Fuel. A commercial kerosene fuel, Conoco
Jet-50, was used in all laboratory and field
studies except as noted in Table IX.

Counting of Microorganisms

1. Fuel Samples: Microorganisms in jet fuel
were counted using a modification of the pro-
cedure described in the Society for Industrial
Microbiology publication, “Proposed Procedures
for Microbiological Examination of Fuels”.
The measured fuel samples were filtered by suc-
tion through membrane filters (Millipore mem-
brane, type HA, 47-mm dia., pore size 0.45)
supported in sterile, stainless steel filter funnels
(Gelman Instrument Co.) of approximately 1
liter capacity. Each filter was washed with ap-
proximately 300 ml of sterile 0.1% aqueous Tri-
ton X~100 to remove the residual kerosene from
the membrane, followed by 100 ml rinse with
0.01 M phosphate buffer, pH 6.8. The mem-
brane filter was transferred aseptically from the
filter assembly to a petri dish (Lab-Tek Plastics
Co., 60 x 20 mm) containing the appropriate
growth medium. The filter was placed on the
agar, face-up, covered, and incubated.

2. Water Samples: Microorganisms in water
samples were serially diluted with 0.01 M phos-
phate buffer, pH 6.8, and filtered through mem-
brane filters as described above except that they
did not receive the detergent wash.

1 Triton X-100 is an alkyl phenoxy polyethoxy ethanol pro-
duced by Rohm and Haas Company, Philadelphia, Pennsylvania.



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