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~ BmmRT _DILLE, M.D.
`CHIEF, CIVIL AEROMEDICAL
`INSTITUTE
`
`~AM>
`P. v. SIEGEL, M.D.
`FEDERAL Am SuRGEON
`
`August 1967
`
`FEDERAL AVIATION ADMINISTRATION
`Office of Aviation Medicine
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2112 - 1/12
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`Qualified requesters may obtain Aviation Medical Reports from Defense Documentation
`Center. The general public may purchase from Clearinghouse for Federal Scientific
`and Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151
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`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 (1-13). Since 1956,
`when fuel system malfunctions in the B-47 air(cid:173)
`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(cid:173)
`patible with the fuel system components.
`Of particular interest recently has been the po(cid:173)
`tential usefulness of several boron-containing
`organic compounds which exhibit microbiocidal
`properties2
`• These compounds present a
`3
`10
`14
`15
`•
`•
`•
`•
`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(cid:173)
`erties of the additive under simulated field con(cid:173)
`ditions. Concurrent tests were conducted by the
`Aircraft Services Base, Federal Aviation Ad(cid:173)
`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(cid:173)
`uation consisted of the following :
`
`a. Comparative evaluation o£ seven nutrient
`media to determine the one most suitable for
`culturing the mixed population o£ microorgan(cid:173)
`isms found in fuel.
`
`b. Evaluation of a membrane filter procedure
`for measuring microbial concentration in both
`fuel and aqueous samples.
`c. Viability of microorganisms in turbine fuel.
`d. Kinetics of microbial mass transfer in fuel(cid:173)
`water systems.
`e. Effect of Biobor-JF on microbial growth in
`fuel-water systems.
`f. Effect of Biobor-JF on microbial growth in
`liquid and solid nutrient media.
`
`II. 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(cid:173)
`cedure described in the Society for Industrial
`Microbiology publication, "Proposed Procedures
`for Microbiological Examination of Fuels"16•
`The measured fuel samples were filtered by suc(cid:173)
`tion through membrane filters (Millipore mem(cid:173)
`brane, type HA, 47-mm dia., pore size 0.45p.)
`supported in sterile, stainless steel filter funnels
`(Gelman Instrument Co.) of approximately 1
`liter capacity. Each filter was washed with ap(cid:173)
`proximately 300 ml of sterile 0.1% aqueous Tri(cid:173)
`ton X-1001 to remove the residual kerosene from
`the membrane, followed by 100 ml rinse with
`0.01 M phosphate buffer, pH 6.8. The mem(cid:173)
`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(cid:173)
`phate buffer, pH 6.8, and filtered through mem(cid:173)
`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(cid:173)
`duced by Rohm and Haas Coonpany, Philadelphia, Pennsylvania.
`
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`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(cid:173)
`oratory}, .Double Strength Nutrient Agar, Tryp(cid:173)
`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(cid:173)
`tion Agar (Difco Manual #9, p. 245}. Repli(cid:173)
`cate samples were withdrawn from a constantly(cid:173)
`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, Sabol!raud Dextrose Agar supported fun(cid:173)
`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 niethod of counting,
`the following el:periments 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(cid:173)
`licate samples were prepared by transferring 1.0
`ml samples from each constantly-stirred micro(cid:173)
`bial suspension to the sterile funnel-filter as(cid:173)
`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(cid:173)
`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(cid:173)
`ism, allowing the broth to settle, then carefully
`decanting the fuel. Samples of 100 and 500 ml
`volumes were removed from
`the constantly(cid:173)
`stirred fuel suspension and transferred to the
`
`filter-funnel assembly, then washed with deter(cid:173)
`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(cid:173)
`bated at 34°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(cid:173)
`cubation to allow the nutrient to diffuse to the
`entrapped cells, a detergent rinse is necessary.
`To d~termine the effect of this detergent on the
`growth of the bacteria, aliquots from suspensions
`of known species (both gram-positive and gram(cid:173)
`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(cid:173)
`cubation on nutrient agar at 34°C.
`In order to estimate the set(cid:173)
`Settling Rate.
`tling rate of bacteria placed in suspension by
`tank turbulence, a laboratory tank was prepared
`using a 2-liter, stoppered, graduated glass cylin(cid:173)
`der. Each such tank was inoculated with 100
`ml of Bushnell-Haas salts solution17 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(cid:173)
`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(cid:173)
`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(cid:173)
`water interface and from the water layer itself;
`
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`these samples were then filtered, plated, incu(cid:173)
`bated, and counted.
`Long-Term Growth of Miaroorganisms in Fuel
`Phase. To determine wh~ther microorganisms
`can live and multiply in jet fuel without the
`presence of a discrete water phase, sever:al gal(cid:173)
`lons of untreated jet fuel were chilled in a cold
`room (3°0) for 3 days and filtered (Whatman
`#1 filter paper) into a dry container. This sys(cid:173)
`tem contained such organisms as were indigenous
`to the fuel and only that amount of water which
`could be maintained in solution at 3°0; no sepa(cid:173)
`rate, discrete water phase was present when the
`system was brought to room temperature. Sam(cid:173)
`ples were removed for counting immediately
`after filtration and at intervals up to 63 days.
`Incubation temperature was 23-25°0 for this 63-
`day period.
`Inhibition of Miarobial 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(cid:173)
`pered with cotton. 1950 ml of untreated com(cid:173)
`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(cid:173)
`centration of 270 ppm by weight (equivalent to
`20 ppm elemental boron). After initial mixing,
`samples were removed for counting at intervals
`up to 33 days. Two hours after a uniform agi(cid:173)
`tation of 4 inversions (to simulate normal refuel(cid:173)
`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 Miarobial Growth on Solid Nu(cid:173)
`trient Media by Biobor-JF. Eight bacterial and
`5 fungal isolates, seleeted from a total of 19 ap(cid:173)
`parently discrete types isolated from fuel (see
`Appendix A)t, 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(cid:173)
`teria and fungi were grown on nutrient agar and
`Sabouraud Dextrose Agar respectively, using a
`single isolate per dish. Wha'tman #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(cid:173)
`hibition after a 48-hour incubation at 34°0.
`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(cid:173)
`viously described.
`A third experiment was designed to determine
`whether the microorganisms could utilize Biobor(cid:173)
`JF as a sole source of carbon. After sterilization
`of a solid medium prepared by adding 15 gjliter
`of agar to Bushnell-Haas salts solution, Biobor(cid:173)
`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 llliarobial Growth in Liquid Nu(cid:173)
`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(cid:173)
`sions of broth cultures of individual bacterial
`isolates. These flasks were cotton-stoppered and
`incubated with constant shaking at room tem(cid:173)
`perature (26°0} for 22 hours. Optical density
`of the bacterial suspension was determined at
`600 mp. using a Coleman Junior spectrophoto(cid:173)
`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 wa8 incorporated (after steri(cid:173)
`lization) were inoculated with 1-drop suspen(cid:173)
`sions of broth cultures of single fungal isolates
`from jet fuel. The flasks were incubated a:t 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
`
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`filter with the mycelium on it was transferred
`to an aluminum weighing dish and dried "in
`vacuo" over anhydrous CaS04 for 7 days (to
`constant weight) , weighed, and the growth de(cid:173)
`termined as mg o£ mycelium per flask.
`III. Results and Discussion.
`The membrane filtration technique gave rea(cid:173)
`sonably adequate precision as indicated by the
`data depicted in Table I; therefore, this tech(cid:173)
`nique was used to determine microbial concen(cid:173)
`tration of fuel samples.
`
`TABLE I. Precision of the Membrane Filtration Method
`
`Organism Sample
`Type
`Type 1
`
`No. of
`Repli-
`cates
`
`Sample
`Size, ml
`
`Colony Coefficient
`Count of Varia-
`±S.D.
`tion,%
`
`B Aqueous
`Suspension
`
`B
`B
`F
`F
`B
`
`B
`
`Fuel
`Suspension
`
`20
`20
`10
`6
`4
`
`20
`10
`
`1.0
`1.0
`1.0
`1.0
`1.0
`
`240±5
`19±2
`148±7
`38±2
`342±17
`
`500.0
`100.0
`
`103±9
`17±2
`
`5
`11
`5
`5
`5
`
`9
`12
`
`1B =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(cid:173)
`nificant factor in determining a time for sampling
`fuel from the aircraft. Microbial suspension
`caused by refueling, flight, or other tank turbu(cid:173)
`lence can seriously affect field tests for fuel con(cid:173)
`tamination. Table III shows the number of
`bacteria present 4 inches below the surface of a
`laboratory tank up to 4 hours after agitation.
`TABLE II. Effect of Detergent Rinse on Bacterial
`Growth Rate
`
`Volume Buffer
`Rinse (ml)
`0
`25
`100
`500
`
`No. of Organisms per Cultured Plate'
`
`E. coli
`129
`133
`136
`140
`
`B. subtilis
`32
`31
`32
`28
`
`1 Each figure represents the mean of three individual
`plate counts.
`
`TABLE III. Concentration of Bacteria Four Inches
`Below Surface of Fuel at Various Time Intervals
`After Suspension
`
`Time After Shaking (min)
`
`Bacteriaj85 ml
`
`0
`5
`10
`20
`30
`100
`240
`
`1,000
`1,000
`1,000
`1,000
`636
`431
`400
`
`That microorganisms can distribute themselves
`throughout the fuel under static conditions is
`illustrated in Table IV. After 8 days' incuba(cid:173)
`tion, considerable numbers of bacteria, which
`were originally introduced into the water layer
`only, were noted up to 14 inches above the fuel(cid:173)
`water interface; therefore, the organisms should
`have no particular difficulty in contaminating
`any new water pockets introduced by refueling
`or condensation.
`
`TABLE IV. Vertical Distribution of Microorganisms in
`Fuel-Water Systems After Standing for Eight Days
`
`Tank No.
`2
`
`3
`
`4
`
`Distance Above
`Fuel-Water
`Interface (inches) Bacteriajliter
`14
`20
`320
`7
`1
`20
`14
`70
`8
`9270
`2
`3790
`-0.25"
`2.7x1010
`14
`120
`20
`7
`1
`1320
`
`Fungi/liter
`20
`0
`0
`b
`b --
`b
`
`b
`
`0
`0
`0
`
`• Sampling of Water Layer below interface.
`b Fungi were not counted separately in tank #3; counts
`shown represent the total number of colonies which
`grew on nutrient agar.
`
`While DeGray and Killian7 and others have
`established qualitatively the ability of micro(cid:173)
`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(cid:173)
`bers of both remained viable for the period of
`the experiment.
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`TABLE v. Survival of Microorganisms in Fuel Phase
`
`Time (days)
`0
`9
`17
`24
`63
`
`Bacteria/liter
`18
`20
`tO
`7
`8
`
`Fungi/liter
`1
`1
`2
`t
`2
`
`Since absolutely anhydrous jet fuel is imprac(cid:173)
`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(cid:173)
`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(cid:173)
`stant Biobor concentration and the fueljwater
`ratio existing in these laboratory experiments.
`
`TABLE VI. Biobor-n' Inhibition In Fuel-Water Systems'
`
`Water Layer
`Fuel Layer
`Tank Number Bacteria/
`Fungi/ Bacteria/
`Fungi/ml
`liter
`liter
`ml
`
`Zero Time'
`t (control)
`2 (control)
`3 (Biobor)
`4 (Biobor)
`4 Days
`t (control)
`2 (control)
`3 (Biobor)
`4 (Biobor)
`20 Days
`t (control)
`2 (control)
`3 (Biobor)
`4 (Biobor)
`33 Days
`t (control)
`2 (control)
`3 (Biobor)
`4 (Biobor)
`
`to
`t20
`40
`20
`
`0
`20
`tO
`40
`
`2,200
`2,500
`20
`20
`
`t,5t0
`t,600
`20
`20
`
`30
`30
`30
`40
`
`9,800
`7,200
`0
`0
`
`2,700
`2,500
`0
`0
`
`2,600
`2,900
`0
`0
`
`4.0 xto•
`4.0 xto•
`4.0 x to•
`3.0 xto•
`
`3.23X to•
`6.1 x1o•
`3.t xto•
`3.4 xto•
`
`4.8 X108
`2.0 X tO"
`8.0 X tO"
`9.0 X tO"
`
`3.28 X tO"
`7.57 X tO"
`6.0 X tO"
`4.0 XtO"
`
`t.2 XtO'
`t,t X tO'
`1.3 X tO'
`t.t XtO'
`
`2.0 X tO'
`t.O X10'
`0
`0
`
`7.4 X tO'
`9.5 X tO'
`0
`0
`
`2.0 xto•
`t.27X to•
`0
`0
`
`1Each tank contained 1950 ml of jet fuel and 50 ml of Bushnell(cid:173)
`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.
`2Zero 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(cid:173)
`mation of fungal mats in the control tanks. De(cid:173)
`spite the lack of fungal counts in the treated
`tanks, two small fungal colonies (white) ap-
`
`peared in the water layer of tank #3 and grew
`slowly to approximately 1 em in diameter.
`
`The complete absence of viable fungi in the
`treated laboratory tanks on the 4th day is in(cid:173)
`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 eqnilibrium. 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;
`3 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°0. 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°0). Of these three isolates which grew on
`10,000 ppm, however, two were also capable of
`growing on a solid medium containing only in(cid:173)
`organic salts; therefore, they must be capable
`of using atmospheric C02 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(cid:173)
`ing the Biobor-JF as a source of carbon. None
`of the fnngal 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(cid:173)
`centrations of Biobor-JF was more apparent in
`broth cultures. These results are shown in Table
`VII.
`
`5
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2112 - 7/12
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`TABLE VII. Effect of Biobor-JF on Bacterial Growth in Liquid Culture
`
`Biobor-JF
`Concentration
`(ppm)
`0
`200
`500
`1,000
`5,000
`10,000
`
`Known Species'
`E. coU A. aerogenes B. subti!is S. marcescens
`0..64
`0.57
`0.48
`0.86
`0.68
`0.62
`0.77
`0.39
`0.66
`0.57
`0.77
`0.39
`0.63
`0.10
`0.71
`0.53
`0.10
`0.04
`0.00
`0.00
`0.03
`0.00'
`0.00
`0.00
`
`Optical Density at 600 mp.
`Fuel Isolates
`#11
`#13
`#14
`0.21
`0.08
`0.40
`0.06
`0.17
`0.40
`0.051 0.15
`o:37
`0.03' 0.13
`0.12
`0.00
`0.04
`0.00
`0.02
`0.00
`0.00
`
`#10
`0.42
`0.38
`0.17
`0.05
`0.00
`0.00
`
`#16
`0.45
`0.43
`0.40
`0.34
`0.00
`0.00
`
`#17
`0.44
`0.41
`0.37
`0.28
`0.01
`0.00
`
`#19
`0.66
`0.72
`0.65
`0.62
`0.00
`0.00
`
`'Fuel. isolate #11 showed a slight granularity in the broth culture at the 500 and 1,000 ppm concentrations.
`'A. aerogenes showed a slight growth on the second day at the 10,000 ppm concentration.
`3Pure cultures of Escherichia coU, Aerobacter aerogenes, Bacillus subti!is, 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(cid:173)
`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(cid:173)
`tween 1,000 and 5,000 ppm, E. coli and Fuel
`Isolate # 13 showed significant growth at the
`highest concentration used, 10,000 ppm. A. aero(cid:173)
`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.
`
`ing routine operation over a 3-month period.
`Representative data from the aircraft fuel sam(cid:173)
`ples are shown in Table IX. These data proved
`to be inconclusive, partially due to the cold(cid:173)
`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.
`TABLE IX. Microorganisms in Fuel from Convair 880
`during Field Tests
`
`TABLE VIII. Effect of Biobor-JF on Fungal Growth in
`Liquid Culture'
`
`3 " 6
`
`Biobor-JF
`Mi!Ugrams of Myce!ium/15 m! Growth Medium
`Concentration Fuel Isolate No.
`1
`ll
`(ppm)
`155 454 425 135 78
`0
`159 389 352 120 55
`200
`139 391
`0 114 51
`500
`1,000
`58 289 367
`71 48
`5,000
`0 283
`0
`0 46
`10000
`0
`0 15
`7
`0
`1All 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 Additi:ve
`Biobor-JF for Biocidal Effects, Report #66-25,
`prepared 'by AC-884). Samples were collected
`from symmetrically-located control and Biobor(cid:173)
`,JF -treated tanks on a Convair 880 aircraft dur-
`
`6
`
`Fungi/ Liter
`Bacteria/ Liter
`Sampling Control Biobor-Treated Control Biobor-Treated
`Tank
`Date
`Tank
`Tank
`Tank
`1-271
`4
`4
`6
`10
`10
`2-3
`8
`20
`114
`2-12
`2
`486
`954
`6
`14
`2-17
`58
`38
`70
`10
`3-11
`6
`10
`10
`10
`22
`0
`3-19
`11
`3-24
`32
`41
`0
`84
`2
`0
`22
`4-27'
`15
`26
`0
`3
`12
`5-6
`'Samples on this date were collected immediately prior to
`addition of the Biobor-JF (270 ppm).
`'On 4-5-65, the aircraft was refueled with military JP-4 fuel
`containing 0.10-0.15% of the Phillips PFA-55MB 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(cid:173)
`vided the fuel contained no minute water droplets
`clinging to the sides of the container. The pres(cid:173)
`ence of these water droplets, which was noted in
`some field samples, would produce an erroneously
`high count by the SOHIO method.
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2112 - 8/12
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`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 :
`
`K•Wt
`C (water) = Vr + K • Vw
`where K =distribution coefficient
`Wt =total weight of Biobor-JF in system
`Vr=volume of fuel
`Vw=volume of water
`
`Computations based on these formulae indicate
`that the Biobor-JF concentration in the w11ter
`layer of an aircraft fuel tank, under field condi(cid:173)
`tions, would be expected to exceed the biocidal
`level of 5,000-10,000 ppm when the fuel is re(cid:173)
`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(cid:173)
`tion or from cooling of water-saturated fuel.
`Our experiments indicate that Biobor-JF is a
`less effective anti-bacterial agent in fuel-water
`·whether or not resistant strains of
`systems.
`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.
`
`7
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2112 - 9/12
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`CFAD v. Anacor, |PR20‘|5-01776 ANACOR EX. 21 ‘I2 -10/12
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2112 - 10/12
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`

`
`2
`
`3
`
`4
`
`6
`
`7
`
`9
`
`9-B
`10
`
`11
`
`12
`
`13
`
`14
`
`Culture Number
`1
`
`APPENDIX A
`CHARACTERISTICS OF FUEL ISOLATES
`0 haracteristics
`Fungus: light-brown growth on SDA1 , colonies round and convex, no diffius(cid:173)
`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(cid:173)
`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 2
`, colonies
`circular, convex, entire, moist, cream-colored on NA.3
`Bacterium: large rods with rounded ends, mostly single rods showing granu(cid:173)
`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
`NA 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(cid:173)
`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(cid:173)
`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(cid:173)
`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
`
`16
`
`1'7
`
`18
`
`19
`
`9