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`
`REVISTA ARGENTINA DE
`
`MICROBIOLOGIA
`
`VOLUMEN 30 N2 3 • JULIO- SETIEMBRE DE 1998
`
`INDICE
`
`* Bacterias heterotrofas y hongos en un sistema de manejo de combustible de
`aviacion y su relacion con el ensuciamiento del combustible.
`
`M. D. Ferrari, E. Neirotti, C. Albornoz
`
`105
`
`Produccion de antisueros fungicos especfficos en conejos.
`
`D. Perrotta, W. Vivot, W. Lee, C. Rivas, M. Yabo, L. Rodero, C. Canteros, G. Davel
`
`115
`
`* lnfluencia del pH sabre Ia toxicidad y Ia sobrevivencia de celulas totales y espo(cid:173)
`ras de Bacillus thuringiensis.
`
`S.C. Dias, M.A. Sagardoy
`
`Amilisis comparative de los patrones isoenzimaticos de ~-1 ,4 endoglucanasa en
`especies del genera Saccobolus {Ascobolaceae-Pezizales).
`
`A. M. Ramos, F. Forchiassin
`
`Factores que influyen en el desenquistamiento in vitro de Cryptosporidium sp.
`
`B. C. Pezzani, E. Bautista, A. Cordoba, M. M. De Luca, J. A. Basualdo
`
`INFORMES BREVES
`
`* Dificultad diagnostica en dos nifios hijos de madres seropositivas para el virus
`de Ia inmunodeficiencia humana tipo 1 (HIV-1).
`
`D. Liberatore, P. Olivari, M. Gomez Carrillo, M. Martinez, L. Garcfa, C. Rodriguez, L.
`Martinez Peralta, 0. Libonatti, M. M. Avila
`
`Presencia de micoplasmas en cultivos celulares en laboratories de Ia ciudad de
`Cordoba, Argentina.
`
`A. C. Cumino, P. Cordoba, T. M. Zapata
`
`122
`
`130
`
`138
`
`143
`
`147
`
`*En Ingles
`
`This materia I was <llpie<l
`atth,e NLM an<l maybe
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`

`
`REVISTA ARGENTINA DE
`
`MICROBIOLOGIA
`
`VOLUMEN 30 N2 3 • JULIO- SETIEMBRE DE 1998
`
`INDEX
`
`*Occurrence of heterotrophic bacteria and fungi in an aviation fuel handling system
`and its relationship with fuel fouling.
`
`M. D. Ferrari, E. Neirotti, C. Albornoz
`
`Specific fungi antisera production in rabbits.
`
`D. Perrotta, W. Vivot, W. Lee, C. Rivas, M. Yabo, L. Rodero, C. Canteros, G. Dave!
`
`*Influence of pH on the toxicity and survival of total cells and spores of Bacillus
`thuringiensis.
`
`S. C. Dias, M. A. Sagardoy
`
`Comparative analysis of isoenzyme patterns of ~-1.4 endoglucanase in species of
`the Saccobolus genus (Ascobolaceae-Pezizales).
`
`A. M. Ramos, F. Forchiassin
`
`Factors affecting the in vitro excystation of Cryptosporidium sp.
`
`B. C. Pezzani, E. Bautista, A. Cordoba, M. M. De Luca, J. A. Basualdo
`
`BRIEF REPORTS
`
`*Troublesome diagnosis in two children born to HIV-1 infected mothers.
`D. Liberatore, P. Olivari, M. Gomez Carrillo, M. Martinez, L. Garcfa, C. Rodriguez, L.
`Martinez Peralta, 0. Libonatti, M. M. Avila
`
`Mycoplasma presence in cell cultures in laboratories of Cordoba, Argentina.
`A. C. Cumino, P. Cordoba, T. M. Zapata
`
`105
`
`115
`
`122
`
`130
`
`138
`
`143
`
`147
`
`* In English
`
`This material \'¥3S'Cop:ied
`atth,e N LM and m.ay bi2
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`

`
`Revista Argentina de Microbiologfa (1 998) 30: 105-114- ISSN 0235-7541
`
`(Occurrence of heterotrophic bacteria and fungi in an
`aviation fuel handling system and its relationship with
`fuel fouling.)
`
`M. D. FERRARI*, E. NEIROTTI, C. ALBORNOZ
`{§!r:tro de Investigaciones Tecnol6gicas, Administraci6n Nacional d~ Combustibles, Alcohol y Portland (ANCAP), Pando,
`Canelone5:YJ 1000 (ji~guay/
`·correspondencia. FAX:+ 598-2-292 2o:!'t'!+ 598-2-292 2rrli3. E-mailerrari@fing.edu.uyJ
`
`SUMMARY
`
`( Clean, dry and contaminant-free fuel is necessary for safe and economical aircraft operation.
`Microbial growth in aviation fuel handling systems can alter the quality of the product. This paper
`reports the occurrence of heterotrophic bacteria and fungi in a handling system of jet A-1 aviation
`turbine fuel. A total of 350 samples were collected during 1990-1996. The aerobic microorganisms
`in fuel samples were mainly fungi, 85% of samples containing $ 100 cfu/1 (range 0 (< 1 cfu/1) to
`2000 cfu/1). The predominant fungi were Cladosporium and Aspergillus. Water was observed mainly
`in samples extracted from the drainage pipes of two tanks used frequently as intermediate storage
`tanks. The aerobic heterotrophic microorganisms found in water samples were mostly bacteria,
`counts varying from 100 to 8.8x1 07 cfu/ml, with 85% of samples containing 104 - 107 cfu/ml. There
`was a preponderance of Pseudomonas spp. Bacterial contaminants belonging to the genus
`Flavobacterium and Aeromonas were also identified. Sulphate reducing bacteria were detected in
`80% of water samples. It was not possible to assign a maximum microbial contamination level above
`which maintenance is required and it is suggested that analysis of successive samples from the
`same site are necessary for this purpose. Microbial sludges produced in the laboratory and collected
`from a contaminated tank bottom were analysed chemically. The data are presented and discussed.
`Samples collected from the supply pipes of tanks and refueller trucks during the period surveyed
`always met the standard specifications./
`
`Key words: microbial contaminants, heterotrophic bacteria, fungi, aviation fuel.
`
`RESUMEN
`
`Bacterias heter6trofas y hongos en un sistema de manejo de combustible de aviaci6n y su
`relaci6n con el ensuciamiento del combustible. El funcionamiento seguro y econ6mico de las
`aeronaves exige un combustible esencialmente limpio, seco y sin contaminantes. El crecimiento
`microbiano en los sistemas de manejo de combustible de aviaci6n puede alterar el mantenimiento
`de su calidad. Este articulo informa sobre Ia presencia de bacterias heterotr6ficas y de hongos en
`un sistema de producci6n, almacenamiento y distribuci6n de combustible de aviaci6n jet A-1. Se
`analizaron 350 muestras durante 1990-1996. Los microorganismos aerobics presentes en las
`muestras de combustible fueron principalmente hongos. Los niveles fungicos variaron entre 0 (< 1
`ufc/1) y 2000 ufc/1, pero el 85% die ron $ 100 ufc/1. Las colonias fungicas predominantes pertene(cid:173)
`cfan a los generos Cladosporium y Aspergillus. Se observ6 agua basicamente en muestras extraf(cid:173)
`das de las purgas de dos tanques usados frecuentemente como tanques de almacenamiento in(cid:173)
`termedio. Los microorganismos heter6trofos aerobics encontrados en agua fueron bacterias. Los
`nivqles bacterianos variaron entre 100 y 8.8x10 7 ufc/ml y el 85% estuvieron en el range 104 - 107
`ufc/ml. Hubo preponderancia de Pseudomonas spp. Tambien se identificaron contaminantes
`bacterianos del genera Flavobacterium y Aeromonas. Se detectaron bacterias reductoras del sulfate
`en el 80% de las muestras de agua. No fue posible asignar valores numericos maximos por enci(cid:173)
`ma de los cuales el sistema requiere mantenimiento. El analisis de muestras sucesivas de un mis-
`
`This mate ri 31 was .;:o-p+ed
`at th<e NLM and may be
`Soubject. US Copyright LavF:s
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`

`
`106
`
`Revista Argentina de Microbiologia (1998} 30: 105-114
`
`---~-
`
`mo punto de muestreo se requiere para ese fin. Se efectu6 Ia caracterizaci6n qufmica de lodos
`microbianos producidos en el laboratorio y recogidos del Iondo contaminado de un tanque. Se pre(cid:173)
`sentan y discuten los resultados analfticos obtenidos. En el perfodo relevado todas las muestras
`extrafdas de los cafios de expedici6n de los tanques y de los camiones tanque reunieron siempre
`las especificaciones estandares.
`
`Palabras claves: contaminantes microbianos, bacterias heter6trofas, hongos, combustible de avia(cid:173)
`ci6n.
`
`INTRODUCTION
`
`Clean, dry contaminant-free fuel is needed for
`safe, economical aircraft operation (1 ,9, 19). Micro(cid:173)
`bial growth in aviation fuel handling systems can
`alter the quality of the product and requires spe(cid:173)
`cial maintenance procedures. Such systems usu(cid:173)
`ally have five components: water, mineral salts, air,
`fuel and hydrocarbon degrading microorganisms.
`Water remaining in the fuel tank bottom, specially
`in small crevices and seams, allows the prolifera(cid:173)
`tion of microorganisms which grow in biofilms at
`the water-oil interface and on the water-metal in(cid:173)
`terfaces (tank surfaces, floating suctions and pipe
`wall) (8, 1 0). The biofilm consists of microbial cells
`embedded in an organic polymer matrix of micro(cid:173)
`bial origin (extracelullar polysaccharide substance,
`EPS) (1 0). Water, organic and inorganic nutrients
`are retained and concentrated in the biofilm (1 0).
`The material accumulated at the fuel-water inter(cid:173)
`face is composed of rust, dirt, sand, clay, fuel oxi(cid:173)
`dation products (such as gums), fuel biodegrada(cid:173)
`tion products, EPS, and microbial biomass, gen(cid:173)
`erally surrounded by a fuel-water emulsion
`(1 0, 15, 19). Microorganisms can form oil-in-water
`emulsions, either by direct surface action, or by
`extracellular biosurfactant production, as a means
`of increasing the accessibility of hydrocarbons for
`biodegradation (10). The characterization of fuel
`contaminants is important in order to identify the
`main cause of deterioration (8, 12, 15, 19,22).
`The microorganism most often implicated in the
`biodeterioration of aviation fuels is the fungus
`Hormoconis resinae (synonym Cladosporium
`resinae), the imperfect form of Amorphoteca
`resinae (1 0, 16,19-21 ). The fungal biomass has
`sufficient mechanical strength and volume to block
`filters and drains and once attached to tank floors
`
`and walls is difficult to dislodge by cleaning (9).
`Other genera of fungi (Aspergillus, Fusarium,
`Penicillum, Trichosporium), yeasts (Candida) and
`bacteria (Arthrobacter, Bacillus, Micrococcus,
`Pseudomonas, Serratia, Streptomyces) also grow
`on hydrocarbon fuels (4, 13-15,19,21 ,22) and some
`have been identified in aviation fuels. If aerobic
`microorganisms consume all the oxygen available
`and sulphates are present in the water, the con(cid:173)
`ditions will be favourable for the growth of
`anaerobic sulphate reducing bacteria (9, 19). Hy(cid:173)
`drogen sulphide produced by these bacteria can
`be a cause of metal corrosion, jet fuel failing the
`copper corrosion test, and black, unpleasant
`smelling drainage waters (9).
`The negative effects of microbial growth are: 1)
`clogging of filters and drainage pipes with micro(cid:173)
`bial slime, 2) failure in water separators due to
`emulsion formation, 3) pitting and corrosion in
`floating suction, steel and aluminum tank walls, 4)
`malfunction of fuel gauges due to biofilm produced
`by microorganisms that colonise probes, and 5)
`fuel failing to comply with standard specifications
`(9, 12,19,21 ). In addition, microbial growth can take
`place in the filter separator producing brown spots
`and viscous deposits, increasing the pressure
`drop, and finally blocking the equipment (9}.
`The most practical means of controlling micro(cid:173)
`bial growth is to keep the fuel free of water. Prop(cid:173)
`erly designed and operated equipment for fuel
`storage and distribution, coupled with good house(cid:173)
`keeping, will limit the availability of water to the
`microorganisms. The maintenance of minimal
`water in the system is currently attained by the use
`of storage tanks with a slop towards a sump and
`equipped with floating suction, by daily draining of
`water, settling of the fuel before expediting, and
`the use of water separators (8,9, 15, 19). Biocides
`
`-~ ;
`
`- - ·b
`
`.sao
`
`This material was copied
`at the NLM and may be
`-o;hjWs"""~~-;-j.
`
`J &Ll&t
`
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`
`"7-~-------
`
`Heterotrophic bacteria and fungi in aviation fuel
`
`107
`
`as fuel additives are not included in the ASTM
`standard specifications (1) and they are not ap(cid:173)
`proved by the lATA for continuous use (9).
`The monitoring of microbial growth in fuel han(cid:173)
`dling systems is difficult because the water-oil in(cid:173)
`terface is not usually accessible for sampling.
`There are neither standards for microbiological
`quality of fuels nor any general consensus on the
`microbiological methods to be used. The level of
`microorganisms in samples of fuel and tank drain(cid:173)
`age waters will have different implications depend(cid:173)
`ing on the climate, the season, the analytical meth(cid:173)
`odology followed, and the fuel handling system
`considered. Documented data about the occur(cid:173)
`rence of microorganisms in such systems is scare
`in the literature. The majority of the literature about
`occurrence of microorganisms usually deals with
`aqueous systems (11 ,23).
`the occurence of
`This paper
`reports
`heterotrophic bacteria and fungi in a storage and
`distribution system of jet A-1 aviation turbine fuel.
`The level of microbial contaminants in 350 sam(cid:173)
`ples collected during 1990-1 996 was evaluated.
`Chemical characterization of microbial sludges
`produced in the laboratory and collected from the
`field was performed. The detection of potential
`problems related to microbial growth in fuel tanks,
`using data obtained from microbial counts in fuel
`samples, is also discussed.
`
`MATERIALS AND METHODS
`
`Samples
`
`Samples were collected respectively in a petro(cid:173)
`leum refinery and its aviation fuel distribution plant
`located in an international airport. Four storage
`tanks containing jet A-1 aviation fuel were sam(cid:173)
`pled in the refinery. Tanks had floating suction and
`were 3000 to 5000 m3 in size. In the distribution
`plant, samples were taken from seven storage
`tanks and four refueller trucks (tank-type vehicles).
`Tanks, which were 90 m3 in size, were vertical,
`completely lined internally and equipped with float(cid:173)
`ing suction. Fuel was normally transferred from
`tanks to trucks through a common, single pipeline
`provided with filters. Samples were wtihdrawn
`before and after filtering. Truck tanks were also
`equipped with filters. Samples (1-1 aliquots) were
`
`obtained from suction, drainage and supply pipes.
`Before sampling, the valves were opened and liq(cid:173)
`uid was allowed to run for some seconds. Bottom
`samples (1 I) were obtained through the tank roofs
`using a samplig bottle (2). In some instances,
`deposits in tank bottom crevices were removed
`with sterile spatulas or forceps. All samples were
`collected in sterile Pyrex glass bottles (serie 1395,
`Corning, USA), provided with hydrocarbon-re(cid:173)
`sistant caps, and were kept at room temperature
`during transportation to the laboratory. Analyses
`were performed within 7 h (water samples) or 24
`h (fuel samples) of sampling.
`
`Microbiological analysis
`
`Aerobic heterotrophic bacteria and fungi in water
`and sludge samples were enumerated by the
`spread plate method using saline solution as dilu(cid:173)
`ent (6). In fuel samples, microorganisms were
`quantified by the 0.45-~m membrane filter method
`and n-hexane sterilized by a 0.22-~m membrane
`filter was used as washing solvent (1 9). When
`samples were composed of water and fuel to(cid:173)
`gether, the phases were aseptically separated af(cid:173)
`ter decantating and were then analyzed. Total
`aerobic heterotrophic microorganisms (bacteria
`and fungi) were enumerated on Difco plate count
`agar (6). Fungal counts were performed using
`Difco malt agar with penicilin-G (60 mg/1) and
`Rose Bengal (5 mg/1). All plates were incubated
`at 30°C. Sulphate-reducing bacteria in water sam(cid:173)
`ples taken from drainage pipes were determined
`by the MPN method using lactate medium (3).The
`microbial colonies and the samples were exam(cid:173)
`ined microscopically (Nikon Microphot, phase con(cid:173)
`trast, brigth field, 20x ocular, 10x-100x objectives).
`Predominant microorganisms were isolated, tested
`for hydrocarbon utilization as sole carbon source,
`and identified at genus level. The hydrocarbon
`utilization test was performed in culture tubes us(cid:173)
`ing Bushneli-Hass mineral broth
`(5) with
`n-hexadecane (1% v/v) and resazurin (1 ppm) at
`27"C (7, 18). Classification of bacterial isolates was
`performed using methods outlined by Smibert and
`Krieg (17): cell morphology, Gram stain, motility,
`growth at 42°C, and biochemical tests (oxidase,
`catalase,
`pigment production,
`cellobiose,
`D-manitol, L-arabinose, salicin, starch hydrolysis,
`
`This material was co·p:ied
`atth,e N LM and may bi2
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`
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`ANACOR EX. 2110 - 9/16
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`

`
`108
`
`Revista Argentina de Microbiologfa (1998) 30: 1 05-114
`
`gelatin, and Tween 80). Additional biochemical
`data were obtained using the Oxi/Ferm Tube II
`multitest system (F. Hoffmann-La Roche & Co. Ltd.
`Diagnostica, Basle, Switzerland). Sludges, if
`present, were removed aseptically from the
`water-fuel interface, examined microscopically and
`cultured in the media previously described. The
`analytical procedures were carried out in a laminar
`flow cabinet (class II) using hydrocarbon-compat(cid:173)
`ible materials. Quality control checks of culture
`media, membrane filters and cabinet were rou(cid:173)
`tinely performed.
`
`Characterization of materials at the
`water-fuel interface
`
`Materials at the water-fuel interface were sepa(cid:173)
`rated by decantation in an Imhoff cone followed
`by centrifugation at 6000 g for 15 min. The
`air-dried residue was fractioned by solvent extrac(cid:173)
`tion under ultrasonic agitation using n-hexane, and
`a mixture of equal parts of toluene, acetone, and
`methanol (TAM) (15, 19,22). Quantitative data were
`obtained by drying at 60°C and weighing the in(cid:173)
`soluble residue after each extraction. n-Hexane
`solubles are primarily fuel. TAM soluble materials
`are chemical and biological degradation products,
`some components of microbial cells, and water.
`The insoluble residue is mainly composed of dirt,
`rust, EPS, and biomass (15, 19). This residue was
`studied by thermogravimetric analysis (TGA) us(cid:173)
`ing a Shimadzu analyzer, model DT-40. Mass loss
`was measured during heating at 1 ooc/min to
`65oac under nitrogen and then during burning at
`65oac in air (19). Volatiles at low temperature (be(cid:173)
`low 2ooac) are the remaining solvents and water,
`volatiles at high temperature (200 to 650°C) are
`the volatile organic matter, solids ignited at 650ac
`comprise EPS, biomass and other organic sub(cid:173)
`stances (19). Crystalline phases present in the fi(cid:173)
`nal ash obtained from TGA (oxides, rust, metals)
`were identified by X-ray powder diffractometry
`(Philips diffractometer, model 141 o, Co tube, au(cid:173)
`tomatic slit) assisted by the PC-PDF data retrieval
`program, version 2.13 (ICDD, Swarthmore, USA).
`Microbial sludges were produced in the laboratory
`as a reference material. A mixture of hydrocarbon(cid:173)
`utilizing microorganisms isolated from jet A-1 fuel
`(two fungi and three bacteria) were inoculated into
`
`500-ml Pyrex glass bottles containing 250 ml of
`fuel and 50 ml of Bushneii-Hass mineral broth.
`Sterile controls were also performed. Bottles were
`kept at room temperature in the dark for six
`months and were agitated manually once a week.
`
`Statistical analysis
`
`The statistical software used was Sigmastat for
`Windows, version 1.01, (Jande! Scientific, San
`Rafael, CA, USA).
`
`RESULTS AND DISCUSSION
`
`Figure 1 illustrates the typical levels of fungi found
`in samples of fuel extracted from a tank at differ(cid:173)
`ent zones: suction, drains and bottom. Figure 2
`gives the numbers of aerobic heterotrophic bac-
`
`103
`
`A
`
`~ ;
`
`%
`
`'i
`
`" "
`" z
`
`% "' z
`z " "
`*
`* * ~ * * * *
`1 2 3 4 56 7 8 9101112131415161718192021222324
`
`Order of sample
`
`B
`
`::! 102
`2
`0
`·a,
`c 10 1
`:J
`ll..
`
`10°
`
`103
`
`::! 102
`il
`
`': :1~~11::91.:11111.
`I
`~
`,_ ·····''··'''-'
`
`1 2 3 4 56 7 8 9101112131415161718
`
`Order of sample
`
`103
`
`c
`
`...J 102
`
`10°
`
`I I I I~~~ I"!"-~~ ~
`
`1 2 3 4 5 6 7 8 9 10 1112 13 14
`
`Order of sample
`
`Figure 1. Fungal counts in samples of jet A-1 fuel ex(cid:173)
`tracted from a storage tank at different points: A) drain(cid:173)
`age pipes, B) suction pipe, and C) tank bottom. The as(cid:173)
`terisk (*) indicates fungal counts below 1 cfu/L.
`
`This materialwas<oj:fie-d
`at the N LM an-d may bi!
`~ubje<t US Copyright Laws
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2110 - 10/16
`
`

`
`Heterotrophic bacteria and fungi in aviation fuel
`
`109
`
`108
`
`108
`
`105
`
`_,
`~ 107
`u
`,;
`.fB
`0 ro
`..c
`u :;:
`0. _g
`e
`~ 104
`.c
`0
`:0 e 103
`
`Q)
`<(
`
`lll~m I
`
`II
`
`1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
`
`Order of sample
`Figure 2. Levels of aerobic heterotrophic bacteria in
`water samples collected from the drainage pipes of a jet
`A-1 tank used as an intermediate storage tank.
`
`teria in water samples obtained from the drainage
`pipes of the same tank. The microbial counts
`showed high variability and did not follow a nor(cid:173)
`mal distribution pattern according
`to
`the
`Kolmogorov & Smirnov normality test (P < 0.05).
`This was true for the total number of samples and
`for those obtained from a single source.
`The frequency of occurrence of fungi and total
`aerobic heterotrophic microorganisms in fuel, tak(cid:173)
`ing into account all the samples analyzed, is pre(cid:173)
`sented in Figure 3. The aerobic microorganisms
`in fuel samples were mainly fungi, the counts vary(cid:173)
`ing from 0 (< 1 cfu/1) to 2000 cfu/1. 50% of the
`samples contained :s; 10 cfu/1 and 85% :s; 100 cfu/
`I. Predominant fungal colonies belonged to the
`genera Cladosporium and Aspergillus and utilized
`hydrocarbon as a sole carbon source. It is known
`that such fungal spores can survive in fuel (19).
`Water was mainly observed in samples ex(cid:173)
`tracted from the drainage pipes of two tanks lo(cid:173)
`cated in the refinery, which are frequently used as
`intermediate storage tanks. The aerobic hetero(cid:173)
`trophic microorganisms found in water samples
`were mostly bacteria. Bacterial counts varied from
`1 00 to 8.8x1 07 cfu/ml. Figure 4 shows that 85%
`of samples contained 104 - 107 cfu/ml. Sulphate
`reducing bacteria were detected in 80% of the
`water samples. 50% of the positive samples gave
`values over 100 MPN/ml. Those collected from the
`supply lines of tanks and refueller trucks during
`the period surveyed always met the standard
`specifications.
`Predominant aerobic bacteria in fuel and wa(cid:173)
`ter samples were isolated. They grew successfully
`
`175
`
`150
`
`Q)
`
`(/) 125
`0 c
`[g 100
`:J
`0 u
`0 ....
`0
`(jj
`..c
`E
`:J z
`
`75
`
`50
`
`25
`
`0
`
`0·1
`
`3-4
`2-3
`1-2
`Fungi, log (cfull)
`
`4-5
`
`175
`
`150
`
`(/) 125
`Q) u
`c
`~ 100
`:J u
`u
`0
`0
`(jj
`..c
`E
`:J z
`
`50
`
`75
`
`25
`
`0
`
`0-1
`
`1-2 2-3
`
`3-4
`
`4-5
`
`Aerobic heterotrophic microorganisms, log (cfull)
`
`Figure 3. Frequency of occurrence of fungi and aerobic
`heterotrophic microorganisms in jet A-1 fuel samples.
`
`25
`
`20
`
`"' Q)
`0 c 15
`~ :;
`u
`u
`0
`0
`Q; 10
`..c
`E
`:> z
`
`0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9
`
`Aerobic heterotrophic bacteria, log (cfulmL)
`Figure 4. Frequency of occurrence of aerobic
`heterotrophic bacteria in water samples collected from
`drainage pipes of jet A-1 fuel storage tanks.
`
`This material W3S'Copi-ed
`at the NLM and may b<!
`Sou hje1.:t US Copyright Laws
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2110 - 11/16
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`

`
`11 a
`
`Revista Argentina de Microbiologfa (1998) 3a: 1 a5-114
`
`on culture medium containing hydrocarbon as sole
`carbon source. There was a preponderance of
`Pseudomonas spp. Bacterial contaminants be(cid:173)
`longing to the genus Flavobacterium and Aero(cid:173)
`monas were also identified.
`$ince the distribution of the data was not nor(cid:173)
`mal, it was analyzed by the Mann-Whitney rank
`sum and Kruskai-Wallis analysis of variance on
`ranks tests. There were no significant differences
`(P > 0.05) in the microbial counts between sam(cid:173)
`ples collected: a) from different sampling points
`within the same tank (drainage pipes, suction pipe,
`and bottom), b) from different storage tanks, c)
`from different refueller trucks, d) before and after
`filtering, and e) in different seasons.
`When the value of the fungal counts in a fuel
`sample was higher than 50 cfu/1, a new sample
`was taken from the same sampling point, usually
`within 15 days. The new sample generally gave
`low fungal counts. The profiles of fungal counts in
`successive fuel samples illustrate this behaviour.
`A typical profile is shown in Figure 1.
`During the period surveyed, a tank located in
`the distribution plant showed high values of fun(cid:173)
`gal counts (higher than 50 cfu/1) in six successive
`
`samples collected from the drainage pipes. These
`samples were taken over three months and the
`fungal count range was 54 to 540 cfu/1. Previously,
`the counts had fallen in the range 0 (<1 cfu/1) to
`40 cfu/1. The di