REVISTA DE METALURGIA,
43 (3)
228-236, 2007
ISSN: 0034-8570
MAYO-JUNIO,
Biochemical analysis of the Hormoconis resinae
fungal mycelium in the corrosion of aeronautical
aluminium alloys*
R. Araya**, C. Bobadilla**, R. Vera** and B.M. Rosales***
Abstract
Biochemical analyses of the Hormoconis resinae fungal mycelium would explain behaviour
differences of corrosive and non-corrosive strains on Al and its aeronautical alloys. In previous works its aggressiveness had been studied through SEM-EDX surface analysis, electrochemical techniques and immersion testing. In this paper separation of the proteins of the
mycelium produced by a non-corrosive strain and its culture along three generations was performed. Cultures were prepared in batch in the presence and absence of pure Al and AA 2024,
AA 7005 and AA 7075 alloys. The mycelia grown throughout the three generations increasingly recovered usual characteristics at the third replication, included their corrosiveness on
Al and its alloys previously shown by all our strains. Amongst the bio-molecule fractions
isolated and analysed during this preliminary study only the proteins revealed changes with
the generation grown. When this fungal strain was cultured in the presence of alloy metal
sheets electrophoresis of the protean fraction was correlative with the distinct mycelia behaviour observed, including corrosiveness on Al and its alloys.
Keywords
Hormoconis resinae. MIC. Aeronautic. Aluminium alloys. Biochemical analyses.
Análisis bioquímico del micelio del hongo Hormoconis resinae en la
corrosión de aleaciones aeronáuticas de aluminio
Resumen
Las diferencias entre el comportamiento corrosivo y no corrosivo de una cepa del hongo
Hormoconis resinae sobre aluminio y sus aleaciones aeronáuticas se explicarían a través
de análisis bioquímicos del micelio. En trabajos previos, el comportamiento corrosivo se
estudió mediante análisis de superficie SEM-EDX, técnicas electroquímicas y ensayos de inmersión. En este trabajo, se llevó a cabo la separación de proteínas del micelio producido
por una cepa que perdió su corrosividad y su cultivo a través de tres generaciones. Cultivos
en batch, en presencia y ausencia de aluminio y sus aleaciones AA 2024, AA 7005 y AA
7075, a través de tres generaciones del micelio crecido, fueron recuperando sus características, incluida su habitual corrosividad, en la tercera replicación. De las fracciones de biomoléculas separadas y analizadas durante este estudio preliminar, sólo, las fracciones proteicas
revelaron cambios de una a otra generación. Cuando esta cepa del hongo se cultivó en
presencia de probetas de los metales, las modificaciones en la electroforesis de las respectivas fracciones proteicas fueron correlativas del comportamiento del micelio frente a la corrosión del aluminio y sus aleaciones.
Palabras clave
Hormoconis resinae. MIC. Aeronáutica. Aleaciones de aluminio. Análisis bioquímico.
* Trabajo recibido el día 21 de diciembre de 2005 y aceptado en su forma final el día 6 de marzo de 2007.
** Laboratorio de Corrosión, Instituto de Química, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2950, Valparaíso, Chile.
*** CIDEPINT, Av. 52 s/n entre 121 y 122, B1900AYB, La Plata, Argentina.
228
BIOCHEMICAL ANALYSIS OF THE HORMOCONIS RESINAE FUNGAL MYCELIUM IN THE CORROSION OF AERONAUTICAL ALUMINIUM ALLOYS
ANÁLISIS BIOQUÍMICO DEL MICELIO DEL HONGO HORMOCONIS RESINAE EN LA CORROSIÓN DE ALEACIONES AERONÁUTICAS DE ALUMINIO
1. INTRODUCTION
Several studies in the international literature have reported the effect of Hormoconis resinae in the corrosion of Al and its alloys. The mechanisms proposed to
explain the microbiologically influenced corrosion (MIC)
found have extended in the last decades to analysis of
in-service failures associated to different aluminium
alloys in aircraft integral fuel tanks[1-7]
Widespread techniques for these studies have been
optical microscopy and scanning electron microscopy
(SEM), together with several electrochemical techniques[7-15]. EDX (energy dispersive X-ray analysis) showed novel information on the existence of preferential
sites of attack and provided earliest evidence on possible biological factors determining the location of such sites[10-16]. First stages of corrosion showed evidence of
enhanced susceptibility of certain metal areas to localised attack, determined by alloy chemical heterogeneities
with respect to that of the alloy matrix, designed “secondary phases”[11-16]. Such results would suggest that
bio-molecules with different functional groups could be
needed to produce such localised up-take attacks.
The stages of massive corrosion disguise these initially selective attacks, but complementary techniques
were applied to investigate the possible existence of a
biological driving force responsible for the phenomenon. To this purpose measurements of several metalion uptake yielded results of great interest to further advance in the hypotheses proposed as a basis for the present work. Similar orders of magnitude of such metal
up-take for Fe, Zn, Al, Cu, Mg, and Na ions by the
Hormoconis resinae fungus[17 and 18] were found to those exposed for bacteria in the numerous works on the
subject in the revision work by Brown and Lester, 1979
[19]. This similarity suggests an interesting common pattern of behaviour for several microbial species in the
presence of varied essential metal ions (oligoelements).
Should these interactions depend on a biological driving force, the MIC phenomenon could be clarified by
knowing the effect of the presence or absence of essential metal ions on the biochemistry of microbial species. A valuable innovative tool would thus be explored for the design of MIC resistant alloys alternative to the
extended use of biocides as considered the only available solution.
Such knowledge could drastically improve the established treatment of this problem, namely the use of
biocides. These substances, of generally low specificity,
besides inactivating corrosive microorganisms, should
also affect other species, contributing to break the balance of the ecosystems they circulate alongside industrial
waste, including different drinking water sources.
Consideration of oligoelements as essential nutrients
is not only associated with the need of up-taking cerREV. METAL. MADRID,
tain metal ions by microbial species but also, albeit unexpected, when its growth is conditioned to the presence
of a piece of the respective metal. A first report of such
phenomenon was briefly mentioned long time ago in
a paper devoted to biocides evaluation[20] without further
analysis of any possible explanation. It describes the
difficulty to grow in laboratory microbial contaminants
of sea-water used as ballast in water displaced ship-fuel
tanks. However, the subject was not recognised as a valuable tool in the international literature until 1987 [10]
with the description of a MIC problem at a steel-works
plant. Failure occurrence was limited to a Sn-based babbitt antifriction coating of cold-steel roller mills, while
Pb-based homologous antifriction coatings under the
same whole set of operational conditions in the plant
was not susceptible to MIC. These results suggest that the
attack should not only be conditioned by the presence
of microorganisms, nor by other chemicals available in
the environment. It was found that it is also necessary
that some of the alloy components be an “oligoelement”
for the microbial contaminant/s. In fact the microbial
growth of the isolated bacteria, Pseudomonas maltophilia, showed growth stimulation in the presence of Sn+2
salts while Pb ions did not promote its amount of
growth.
Therefore, the aim of this work is to propose a “susceptibility” criterion allowing to detect “oligoelements”
involved in an alloy formulation towards a given microbe. With this purpose biochemical analysis of the
Hormoconis resinae fungal mycelium was approached,
in the presence and in absence of Al and three of its
aeronautical alloys having evidenced in the past very
intense susceptiblility[7-18].
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. Microorganisms and culture media
The strain applied to this study was one of various isolates from integral fuel tanks of military Argentine aircraft involved in a long duration study. After a four year storage period it showed unusual growth characteristics as loss of its dark coloration, lag phase duration
increase, less amount and slower mycelium production,
softer texture and lack of corrosiveness on aeronautical
Al alloys.
To establish the possible cause for these variations
this fungal strain[18] was grown as static batch cultures in
a modified (m) [6] Bushnell-Haas (B-H) medium[21] (Table
I) diluted to 1:10 solution with distilled water according
to the conditions followed in previous works[6, 8, 9 and 1118]. This diluted medium was used to support the fungal
growth and to simulate the drainage water usually found
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
229
R. ARAYA, C. BOBADILLA, R. VERA AND B.M. ROSALES
Table I. Bushnell and Haas Modified Nutrient Solution
Tabla I. Solución nutriente Bushnell y Haas Modificada
Compound
Weigth
MgSO4 7 H2O
KH2PO4
CaCl2
NH4NO3
Sol. 60% FeCl3
Distilled H2O
0.2 g
1.0 g
0.02 g
1.0 g
2 drops
1l
Concentrated solution pH = 5.4.
as sediment in integral fuel tanks of in service aircraft. Its
initial pH = 5.4 was adjusted to 6.5 and Jet A 1 fuel was
used as the only carbon source. In military aviation this
fungus appears since the 60´s as the most frequently reported corrosive microbial contaminant.
The fungal non-corrosive strain had been stored at 75 oC temperature in the rich agar-malt medium during
various years, and their corrosive behaviour, as well as
other more obvious characteristics being recovered by
successive replications[15]. The strain became re-adapted to grow in diluted mB-H medium and Jet A1 turbofuel as only carbon source at its third generation evidencing all its original characteristics, amongst them its
corrosiveness.
2.1.2. Obtaining the homogenate
By re-suspension of a weighed biomass bulk of mycelium in a citrate-phosphate buffer, pH=5.0, the homogenate was obtained using physical methods of cellular rupture as ultrasound and maceration for its liquation.
2.1.3. Purification and characterisation of
biologic macromolecules
An extraction of proteins present in the crude homogenate of the fungus was carried out using chromatographic methods and electrophoresis in polyacrylamide
gels to establish the differences in protein composition
of the various cellular extracts grown with and without
Al-based alloys. Analogous preliminary characterisation
work on the polysaccharide, lipidic and nucleic acid
fractions separated from the grown mycelia did not seem to be influenced by the presence of metallic surfaces
on the physiological response to the corrosive behaviour of this fungus on Al and its aeronautical alloys.
Both lipids and phospholipids were isolated from
the crude homogenate for purification and characterisation through chromatographic methods in thin and
230
gaseous layer. Carbohydrates were characterised using
gaseous chromatography and mass spectrometry. As
none of these latter bio-molecules revealed neither alteration amongst culture generation nor any effect of the
presence of metals in the culture media, the biochemical further analyses were limited to the protean fraction.
The proteins present in the homogenate were precipitated by saline fractioning (with ammonium sulphate) and the fractions obtained subsequently dialysed
against a buffer and afterwards concentrated. Separation
of the precipitated and dialysed proteins was performed by chromatographic methods: a) molecular exclusion with Sephadex G-25, for isolation by molecular size and b) ionic interchange with DEAE-Sepharose, to
separate proteins by batch[22]. The proteins thus separated were submitted to electrophoresis in polyacrylamide gels-SDS [23] to estimate molecular weight by comparing their electrophoretic mobility with already known
standards.
2.1.4. Protein extraction
The treatment performed to obtain the protein homogenate was the following: the non-corrosive strain of
the fungus was incubated at 34 °C in static batch during one month, after which the mycelium was extracted from the culture medium and washed with pH 7.0
phosphate citrate buffer. Following overnight digestion
at 3 ºC, (digestion medium contained 0.5 % TWIN, 3.5 %
SDS, 3 % glycerol, 1.0 mM EDTA, 1.5 mM MgCl2 and
5.0 mM PMSF) the homogenate was centrifuged, the
overfloating separated from the pellet, and proteins were determined by quantification in the crude extract performed by the Lowry et al method[24], using the Folin
reactive and the Bradford method[25], based on adsorption of the Coomassie G-250 blue dye. In both methods
the standard used was bovine serum albuminae (BSA).
Proteins characterisation present in the crude homogenate of the fungus estimated molecular weight by
comparing their electrophoretic mobility. Thus differences in protein composition of the various cellular extracts grown with and without Al-base alloys was performed using electrophoresis in polyacrylamide gels (PAGESDS) at 10 %, dyed with coomassie blue and 30 μg protein was loaded into each gel track. The standard of
molecular weights used contained Bovine sero albuminae (BSA) 66 kD; Egg albuminae 45 kD and Carbonic
anhydrase of bovine erythrocytes 29 kD.
2.2. Metal samples
Al and AA2024, AA7005 and AA7075 alloys (Table II)
of 10 x 10 x 5 mm, polished with granullometry No 400
and 600 SiC papers were used for immersion in the microbial cultures, after degreasing with acetone.
REV. METAL. MADRID,
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
BIOCHEMICAL ANALYSIS OF THE HORMOCONIS RESINAE FUNGAL MYCELIUM IN THE CORROSION OF AERONAUTICAL ALUMINIUM ALLOYS
ANÁLISIS BIOQUÍMICO DEL MICELIO DEL HONGO HORMOCONIS RESINAE EN LA CORROSIÓN DE ALEACIONES AERONÁUTICAS DE ALUMINIO
Table II. Chemical Composition of the Al Alloys (w/w%)
Tabla II. Composición química de las aleaciones de Aluminio (% m/m)
Alloy
Zn
Cu
Mn
Si
Mg
Fe
Cr
Ti
Al
7005
7075
2024
Pure Al
4.7
5.2
0.12
—
0.05
1.6
4.5
—
0.45
0.09
0.53
—
0.10
0.23
0.14
—
1.2
2.6
1.5
—
0.18
0.24
0.29
—
0.16
0.13
—
—
0.03
0.02
0.02
—
Rest
Rest
Rest
99.9
2.3. Methods
2.3.1. Drop test
SEM-EDX was used to evaluate the alloys’ MIC increased
susceptibility with the successive fungal generation cultures of the strain, initially non-evidencing corrosive behaviour. This susceptibility was followed through the
drop test. It consists on putting in a Petri dish with sterile mB-H solution a metal sample polished up to 0.25
mm diamond paste. With all sterilised instruments and
in a laminar flow cabinet, a piece of the metal sterilised
in alcohol, dried by quick passage on a flame, was inoculated with a small piece of mycelium carefully cut
with a tweezers. Placed the culture at the interphase of
the mB-H nutrient solution and jet A1 fuel each Petri
dish was covered and metal seeded with the microbe
was maintained at 34 oC [12, 13, 17 and 17] up to 10 days.
Referred to proteins present in the homogenate harvested from the different cultures of third generation,
on the contrary, the results allowed to establish differences in the composition of the cellular extracts grown
in the presence and absence of Al-base alloys as compared with the first generation culture (Fig. 3 and 4).
Then, the protein profiles of the newly grown mycelium showed differences in the protein composition depending on the different alloys presence or absence.
In tracks 1, 2 and 3, two bands may be observed
coinciding in all three samples, one of which also appears in track 4. In track 5, two diffuse bands should also
be similar to those found in 1, 2, 3 and 4 tracks. In track
2.3.2. SEM – EDX analysis
After 10 day’s incubation the metal samples were retired
from the cultures and allowed to dry on filter paper in
the culture oven. Then EDX on attacked secondary phases was performed. After samples sputtering SEM observation were performed and micrographs of the microbe/metal aspect registered.
3. RESULTS
3.1. Biochemical analysis
A non-corrosive strain of the fungus Hormoconis resinae
showed during first replication that neither the mycelia
caused any corrosion on the tested pure Al nor on its
three alloys. The result of protean profile of this first
culture of non-corrosive strain grown in the presence
of Al and the three Al alloys coincided with the control
(culture grown in absence of the metals) in associated
bands to the molecular weights 66 and 31 kDa, as can
be seen in figures 1 and 2. An exception can be seen
for AA 7075 alloy, which only presents one of these
bands.
REV. METAL. MADRID,
Figure 1. Protein electrophoresis of the non-corrosive strain
of the Hormoconis resinae mycelium, in presence and absence of AA 2024 and AA 7005, stained with Coomassie dye, first
generation.
Figura 1. Electroforesis de proteínas de la cepa no corrosiva
del micelio Hormoconis resinae, en presencia y ausencia de AA
2024 y AA 7005, teñida con tinción de Coomassie, primera
generación.
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
231
R. ARAYA, C. BOBADILLA, R. VERA AND B.M. ROSALES
Figure 2. Protein electrophoresis of the non-corrosive strain
of the Hormoconis Resinae mycelium, in presence and absence of pure Al and AA 7005, stained with silver dye, first generation.
Figura 2. Electroforesis de proteínas de la cepa no corrosiva del
micelio de Hormoconis resinae, en presencia y ausencia de Al puro y AA 7005, teñida con tinción plata, primera generación.
4, an obvious difference with respect to the other tracks
is observed. In this case three well defined bands, the
first one of which coincides with the band of the standard molecular weight corresponding to 66 kDa. The
second band coincides with the second band observed
in 1 to 3 track samples and the third band does not coincide with any other sample bands.
In figure 4 the protean bands expressed by Hormoconis resinae have been summarized for the first and
third generations with their respective molecular weight.
The results indicate that all the first generation present
a band at 66 kDa, disappearing in the third generation
samples, except when Hormoconis resinae is cultured in
the presence of the AA 7075 alloy. Certainly, the cultures grown at the third generation produced a lower
amount of protean bands. The tendency is that the molecular weight values of the small proteins in the first
generation is higher in the third generation, except for
the exhibited in the presence of AA 7075. Changes observed in the molecular weight bands of Hormoconis
resinae cultured in the presence de AA 2024 y AA 7005
are similar amongst them, but higher to those of the first
generation, diverging from this tendency when
Hormoconis resinae is cultivated in the presence of AA
7075. At the third generation in front to the AA 7075
alloys it expresses new proteins of lower molecular
232
Figure 3. Protein electrophoresis of the Hormoconis resinae
mycelium, in presence and absence of pure Al and AA 7005,
stained with silver dye, third generation (corrosiveness recovered).
Figura 3. Electroforesis de proteínas del micelio de Hormoconis
resinae, en presencia y ausencia de aluminio puro y AA 7005,
teñida con tinción plata, tercera generación ( corrosividad recuperada).
weight, in the order of 25 kDa and it maintains two
bands (65 y 42 kDa), while bands of high molecular
weight (84 y 78 kDa) disappear respect to the first generation.
For the third generation, the fungus grown in the
absence of the alloys (control) presents two different
bands (85 y 34 kDa) from those expressed by the same generation of the strain growing in the presence of
the alloys the arrows point at the above described differences, as can be seen in figure 3 once it recovered its
corrosive behaviour.
3.2. SEM – EDX analysis
EDX on table III shows the chemical composition of
the main attacked second phase of the alloys, which reach for AA7005 to the highest Zn content (14.07 %) and
which is much higher than the that in the matrix, as
shown in table II (4.7 %). In table III the EDX corresponding to second phases of the AA 7075 evidenced
areas also enriched in Zn, Mg and Fe.
After carrying out replication cultures up to a third generation, the strain of the fungus recovered the corrosive behaviour, the attack becoming very intense on the
REV. METAL. MADRID,
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
BIOCHEMICAL ANALYSIS OF THE HORMOCONIS RESINAE FUNGAL MYCELIUM IN THE CORROSION OF AERONAUTICAL ALUMINIUM ALLOYS
ANÁLISIS BIOQUÍMICO DEL MICELIO DEL HONGO HORMOCONIS RESINAE EN LA CORROSIÓN DE ALEACIONES AERONÁUTICAS DE ALUMINIO
Table III. EDX on second phases of the alloys W (%)
100
Al puro
95
Tabla III. EDX de las segundas fases de las aleaciones W(%)
90
Control
AA 7075
85
AA 2024
Control
80
AA 7005 Al puro
75
AA 7005
Control AA 2024
70
AA 7075
65
Patrón de PM
60
AA 2024
EDX on a
second
phase
AA 7005
EDX on a
second
phase
AA 7075
EDX on
matrix
x 2500
AA 7075
EDX on a
second
phase x 5000
60.40
2.48
9.83
27.30
—
—
85.94
—
—
—
14.07
—
88.66
—
—
3.04
8.31
—
78.73
—
1.24
4.92
11.33
3.77
55
50
Al
Mn
Fe
Cu
Zn
Mg
45
40
35
30
25
20
-1
0
1
2
Primera generación
Tinción Comassie
3
4
5
Primera generación
Tinción Plata
6
7
8
9
10
11
12
Tercera generación / Tinción Plata
Figure 4. Molecular weight of proteins in the crude homogenate of the fungus Hormoconis resinae in the presence of
aeronautical aluminium alloys.
Figura 4. Pesos moleculares de proteínas de los homogenados crudos del hongo Hormoconis resinae en presencia de
aleaciones aeronáuticas de aluminio.
alloy secondary phases and following polygonal ways as
in works for the previous corrosive behaviour [9, 11 and
17]. SEM aspect of the respective metal samples can be seen in figure 5 to allow comparison to the previous aspects under corrosive cultures[11 and 17].
Drop tests shows that the fungal mycelium produced only a soft attack on the alloys surface, pure Al was
not affected and crystallographic attack nucleated preferentially on second phases.
4. DISCUSSION
SEM micrographs after the drop test on metal samples in
figures 6a-6c reveal a very slight attack corresponding to
non-corrosive behaviour produced by the mycelium
was restricted to surface Al2O3 film damage and slight
secondary phase dissolution below hyphae traces.
The displacement of hyphae is not polygonal, joining secondary phases, as it had been observed when
corrosive behaviour occurred[17] Polygonal trace through
second phases evidence a surface chemotactic or attraction effect on the mycelium, due to their higher local
chemical content in certain elements throughout the
matrix. This increased content in “oligoelements” respect to the matrix content would trigger the change in
direction once fully up-taken the necessary element/s
at a given second phase. Then the mycelium grows following a straight line up to the nearer second phase,
up to 50 mm distances, to up-take again the same oligoelements[17].
REV. METAL. MADRID,
In spite of their biochemical characteristics as oligoelements, they did not seem to exert any attraction effect
on the displacement direction of the non-corrosive strain
mycelia. Their attack is too slight to be the result of any
up-take process by the first generation culture of this
non-corrosive strain.
With the overfloating samples, a SDS-PAGE polyacrylamide electrophoresis was performed, as shown in figure 3. Therefore it can be inferred that the fungus
grown separately in the presence of AA2024, AA7005
and pure Al presents the same band type, and the fungus grown in the presence of AA7075 presents two different bands from the other alloys, one of them coincides with the control sample of Hormoconis resinae. In
the presence of this alloy the fungus of the 3rd generation maintains two bands (66 and 44 kDa) amongst those expressed by the fungus of the 1st generation.
Moreover, it can be observed that when comparing the
bands expressed by the fungus in 1st and third generation, two bands of the higher molecular weight (78 and
y 84 kDa) dissapear and a new protein of lower molecular weight (25 kDa), is expressed. From this point of
view and comparing to the proteins expression exhibited by the fungus of third generation in the presence
of pure Al it is evident that the fungal mycelium grown
in the presence of the AA 7075 alloy absolutely differs
from the others samples in its protean expression. This
fact could be explained analyzing the components of
each alloy given that is that alloy which presents the
highest mass % of those considered as “oligoelements”,
zinc, magnesium and iron. This fact could be interpreted as the absence of any physiological effect of either
presence or absence of the metal phase when the fungal strain had lost its capacity to produce its original corrosive behaviour, as shown during the first generation.
On the contrary, as it was previously demonstrated[15],
when corrosiveness of a given strain has recovered at
the third successive replication (or generation), the mycelium composition would express a different biochemi-
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
233
R. ARAYA, C. BOBADILLA, R. VERA AND B.M. ROSALES
Figure 5. SEM aspect of the Hormoconis resinae corrosive strain attack on Al alloys[17].
Figura 5. Aspecto MEB del ataque de la cepa corrosiva de Hormoconis resinae sobre aleaciones de Al [17] .
cal response depending on the presence or absence of
metallic Al and its three alloys.
This type of chemical differences could be considered an evidence of a biological driving force for microbial species ability of being or not corrosive to certain
metal or alloy. The susceptibility to MIC of a given alloy
would thus be modified on the basis of this biochemical knowledge for a corrosive microbial species present
in a given service condition.
At present there is no information at international
level to explain in biochemical terms fungi corrosiveness, nor the effect nutrients, included oligoelements,
able to modify that characteristic.
However, from the results exposed in this paper it
can be proposed that as well the lost as the recovered
corrosiveness were correlative from reversible abilities of
the fungus to adapt to different nutritional sources causing also simultaneous modification in the other referred characteristics. Both, the corrosiveness loss and recovery were associated to nutritional sources modification, from jet fuel as the only C source to malt agar
extract and vice versa. The strain only seems to have
modified its characteristics and behavior according the
nutrients available.
By analogy to the advance attained from measurements of metallic-uptake by the Hormoconis resinae
fungus[17 and 18] compared to results reported for different bacterium/metallic-ion systems[19], it was interesting to take a similar reference study, though with bacteria[26 and 27]. Zinkevich et al.[28] report that the presence
or absence of metallic iron in the cultures of two different sulphate-reducing bacteria (SRB) strains induced
exopolymer production of different chemical composi-
234
tion, which in this case it occurs in carbohydrate biomolecules. This culture regulation of the microbe physiological response to the metal presence at molecular level could be the answer also found for the fungus.
In the presence of toxic-ions as those of Hg, As, etc.[19]
increased extracellular polymeric substances (EPS) production stimulated their selective up-take decreasing concentration in the medium, to allow survival of responsible
bacterium during this “detoxification” processes. EPS production was reported as a physiological response provoked by high toxic ion concentrations, as Brown and Lester
reported through the many examples exposed about “metal up-take” by bacteria in their review paper, already in
1979. Such information would be useful as a basis to demonstrate microbial species selection for bio-extraction
of metal ions contaminating waters and soils.
On the contrary, it is likely that this ion up-take mechanism could differ from the one responsible for metallic corrosion. EPS secreted by corrosive species can
contribute to the corrosive process not only by facilitating irreversible cell attachment leading to metal surface colonization but also by binding metal ion species, as
demonstrated for SRB by Beech and Cheung in 1995
[29]. Also differences encountered in the EPSs chemical
composition secreted by SRB into the bulk solution and
within the biofilm[29] would support the fact that the EPS
composition could be strongly affected by the proximity to the metal surface.
MIC seems to depend from the microbial capacity
to produce EPSs able to colonise a given metal surface
through atom up-take and removal from the lattice. This
would also imply an oxidation step for ions irreversible
attachment. In our case, the analogous role of bacterial
REV. METAL. MADRID,
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
BIOCHEMICAL ANALYSIS OF THE HORMOCONIS RESINAE FUNGAL MYCELIUM IN THE CORROSION OF AERONAUTICAL ALUMINIUM ALLOYS
ANÁLISIS BIOQUÍMICO DEL MICELIO DEL HONGO HORMOCONIS RESINAE EN LA CORROSIÓN DE ALEACIONES AERONÁUTICAS DE ALUMINIO
Figure 6. SEM aspect of the non-corrosive strain of Hormoconis resinae on: a) AA 2024 alloy. b) AA 7005 alloy. c) AA 7075
alloy. d) Pure aluminium.
Figura 6. Aspecto MEB de la cepa no corrosiva de Hormoconis resinae sobre: a) La aleación AA 2024. b) La aleación AA 7005.
c) La aleación AA 7005. d) Aluminio puro.
EPS should be assigned to the fungal mycelium, whose
local adherence and corrosiveness to the metal surfaces have been reported to determine the attack localisation and intensity in service military aircraft[12, 13 and 17].
The mentioned difference in the protean profile of
the mycelia grown in the presence and absence of Al
and its alloys should also control distinct type and rate
of acid formation, not only through Jet A1 degradation
but also by autolysis of mycelia of different age cultures,
previously studied trough electrochemical and surface
REV. METAL. MADRID,
analysis techniques with very corrosive strains of
Hormoconis resinae[6-9 and 13-18]. The main results revealed in those papers are a great effect of the fungal culture on: a) the pitting potential decrease of the Al-base
alloys, b) the increasing current density of oxygen reduction and c) the pH decrease with incubation time
of the modified B-H diluted nutrient medium.
Other manifestation of the existence of a biochemical
response of the fungus to metal surface composition
could be noticed comparing the hyphae aspect shown in
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570
235
R. ARAYA, C. BOBADILLA, R. VERA AND B.M. ROSALES
figures 6a-6c. SEM micrographs of the mycelium growing on the alloys and on pure Al respectively, above
1,000 magnifications, reveal that pure Al did not stimulate fungal sporulation while the alloys promote it.
These results provide new evidence about the corrosion risk of military aircraft in service due to the high surface area to culture volume of Hormoconis resinae ratio
in heavily contaminated integral fuel tanks[12, 13 and 30].
5. CONCLUSIONS
A non-corrosive strain of the fungus Hormoconis resinae
showed during its first replication that the mycelia neither caused any corrosion on the pure Al nor on the AA
2024, AA 7075 and AA 7005 alloys.
In the present study only the protean biomolecules
of the mycelia manifested variations associated to the
corrosiveness as well as to the presence or absence of Al
alloys metal samples in the cultures.
At the third generation, coinciding with the recovery
of the lost corrosiveness, differences in the protein composition of the mycelia appeared when the fungal strain
was grown in presence or absence of the alloys. This
differences, non-detected for the first generation of the
same non-corrosive strain, indicate that when the corrosive behaviour was recovered the fungal mycelium
expressed some proteins in absence of alloys (control of
the third generation), not present when the fungus grew
in the presence of any alloy.
This evidence demonstrates a significant effect of the
metal surfaces on enhancing the corrosive behaviour of
the strain likely related to fungal metabolic needs.
This was also verified through morphological changes observed on the fungal hyphae depending on the
metallic substratum colonised.
REFERENCES
[1] D.G. PARBERY, Mater. Org. 6 (1971) 161-208
[2] D.G. PARBERY, Biodeterioration of metals, Applied
Science Publishers Ltd., England, 1972, pp. 65-74.
[3] P. MC KENZIE, A.S. AKBAR AND J.D. MILLER,
Institute of Petroleum (Technical paper), 1977, pp.
37-50.
[4] D. CABRAL, Int. Biodeterioration Bull. 16 (1980)
23-27.
[5] J.D. BU’LOCK, J.E. SMITH AND D.R. BERRY, Eds.,
Industrial Mycology, Pub. Edward Arnold (UK),
1975, pp. 33-57.
[6] B.M. ROSALES, S. CHICHIZOLA AND D. BALEANI,
Proc. 1st NACE Latin American Region Corros.
Cong. and 1st Venezuelan Corros. Cong., 2, paper
94054, 1994, pp. 1-11.
[7] B.M. ROSALES, E.S. AYLLÓN AND M.C. LEIRO,
New Methods for Corrosion Testing of Aluminium
236
View publication stats
Alloys, ASTM STP 1134, Agarwala, V. S., Ugiansky,
G. M. Eds. ASTM, Philadelphia, USA, 1992, pp. 5059.
[8] B.M. ROSALES AND E.R. DE SCHIAPPARELLI,
Mater. Perform. 19 (1980) 41-44.
[9] E.R. DE SCHIAPPARELLI AND B.M. ROSALES,
Mater. Perform. 19 (1980) 47-50.
[10] O.A. BISCIONE, E.S. AYLLÓN AND B. M.ROSALES,
Int. Biodeterioration 23 (1987) 159-166.
[11] E.S. AYLLÓN AND B.M. ROSALES, Corrosion NACE
44 (1988) 638-43.
[12] M. IANNUZZI, G. DUFFÓ AND B.M.ROSALES.
Proc. 15th Int. Corros. Cong., Paper 77, Granada,
Spain, 2002.
[13] M. IANNUZZI AND B.M. ROSALES. Materi. Perform. J. NACE (2003) 62-66.
[14] B.M. ROSALES, A.C. CABEZAS, S.E. CHICHIZOLA AND A.FERNÁNDEZ, 3 rd Latin American
Biodegradation & Biodeterioration Symp.,
Florianópolis, Brazil, paper 78, 1998.
[15]A.M. PALERMO, S. CHICHIZOLA AND B.M. ROSALES, Proc. Second Latin American Biodegradation
and Biodeterioration Symp., Gramado, Brasil, 1995.
[16] E.S. AYLLON, B.M. ROSALES, Corrosion NACE,
50, Nº 8 (1994) 571-575.
[17] B.M. ROSALES, Proc. 11th Int. Corrosion Cong.,
Florence, Italy, 4, 1990, p. 359-366.
[18] B.M. ROSALES, A. PUEBLA, D. CABRAL, Proc. XI
Int. Corros.. Cong., Houston, USA, 5B, 1993, pp.
3773-3785.
[19] M.I. BROWN AND J.N. LESTER, Water Res. 13
(1979) 817-837.
[20] D.E. KLEMME AND R. NEIHOF, Memorandum
Report 3212, USA, (1976) pp. 1-15.
[21] L.D. BUSHNELL AND H.F. HAAS, Bacteriology 41
(1941) 653-673.
[22] J.C. JANSON, L. RYDEN, In protein purification.
VCH Publishers, 1989, pp. 233-267.
[23] U.K. LAEMMLI, Nature 227 (1970) 680-685.
[24] O.H. LOWRY, N.J. ROSEBOROUGH, A.L. FALL,
R.J. RANDALL, J.Biol. Chem. 193 (1951) 265-275.
[25] M.M. BRADFORD, Anal. Biochem. 72 (1976) 248254.
[26] A. RAJASEKAR, S. MARYTHAMUTHU, N. MUTHUKUMAR, S. MOHANAN, P. SUBRAMANIAN AND
N. PALANISWAMY, Corros. Sci. 47 (2005) 257-271.
[27] D.J. HANSEN, D.J. TIGHE-FORD AND G.C. GEORGE, Int. Biodeterioration Bull. 17 (1981) 103112.
[28] V. ZINKEVICH, I. BOGDARINA, H. KANG, M.A.W.
HILL, R. TAPPER, AND I.B. BEECH, Int. Biodeterioration Biodegradation 37 (1996) 163-172.
[29] I.B. BEECH AND C.W.S. CHEUNG Int. Biodeterioration Biodegradation 35 (1995) 59-72.
[30] B. ROSALES AND E.R DE SCHIAPPARELLI, Int.
Biodeterioration Bull., 16 Summer (1980) 313 6 .
REV. METAL. MADRID,
43 (3), MAYO-JUNIO, 228-236, 2007, ISSN: 0034-8570