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Enzyme and Microbial Technology 40 (2007) 187194

Rapid communication
Stimulation of novel thermostable extracellular lipolytic
enzyme in cultures of Thermus sp.
Alberto Domnguez
a
, Pablo Fuci nos
b
, M. Luisa R ua
b
, Lorenzo Pastrana
b
,
Mara A. Longo
a
, M. Angeles Sanrom an
a,
a
Department of Chemical Engineering, University of Vigo, Lagoas-Marcosende, 36310 Vigo, Spain
b
Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain
Received 29 September 2005; received in revised form 5 September 2006; accepted 12 September 2006
Abstract
Selected organisms from the genus Thermus (T. aquaticus YT1, T. thermophilus HB8 and HB27) offer new opportunities for biocatalysis and
biotransformations as a result of the extreme stability of their enzymes. In order to favour the secretion of extracellular lipolytic enzymes the effect
of temperature and carbon source has been studied. All strains were able to grow within a wide temperature range (from 60 to 80

C) with an
optimum value of 70

C for extracellular lipolytic activity.


On the other hand, several sugars were used as carbon source in the culture medium at different concentrations (from 0.5 to 14 g dm
3
).
Supplementation of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose) was carried out in an attempt to increase biomass and
lipolytic enzyme production. The inuence of carbon source was variable, depending on the strain. Nevertheless, the addition of lowconcentrations
of mono- or disaccharides helped to improve the productivity of the process in most cases. The most signicant effects on extracellular lipolytic
activity were detected in T. thermophilus HB27 and T. aquaticus YT1 cultures, using sucrose and fructose as carbon source at an initial concentration
of 0.5 g dm
3
. Moreover, the effect of addition of lipids (olive oil, coconut oil and tributyrin) was tested. In T. thermophilus HB27 cultures, the
highest extracellular lipolytic activities were obtained when olive oil was added (70% higher than those obtained in control cultures).
2006 Elsevier Inc. All rights reserved.
Keywords: Thermostable lipolytic enzymes; Carbon source; Temperature; T. thermophilus HB8; T. thermophilus HB27; T. aquaticus YT1
1. Introduction
In recent years, there has been an increasing interest in the
study of enzymes from extremophiles, since they are not only
more thermostable but often more resistant to chemical agents
and extreme pHvalues than their mesophilic homologues [13].
Lipolytic enzymes catalyze a wide number of different reac-
tions, most of them of industrial application. In spite of their
potential interest, the available information about the production
of lipolytic enzymes by thermophilic microorganisms is scarce,
and it is mainly focused on the detection of this activity in some
bacteria isolated from hot springs, as well as the stability of the
enzymes at high temperatures [4,5].
The ability of T. thermophilus (HB8 and HB27) and T. aquati-
cus YT1 to produce signicant lipolytic activity when cultivated

Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.
E-mail address: sanroman@uvigo.es (M.A. Sanrom an).
in a complex medium has been demonstrated in our laboratory.
All enzymes were stable at 80

C over 30 min and showed a


remarkable activity on fatty acid esters with acyl chains longer
than 10 carbon atoms [6]. Two proteins with lipolytic activity
were identied in intra- and extracellular extracts from the three
Thermus strains cultures by zymogramanalysis, with molecular
weights of 34 and 62 kDa [7,8]. The culture time-course in a
basal medium has been described [8] and the inuence of some
variables (i.e. gas environment) in biomass and enzyme produc-
tion has been investigated, both in Erlenmeyer asks [8] and a
stirred tank bioreactor [9].
In previous papers [69], we have shown that enzyme produc-
tion was not fully associated to growth rate, although absolute
values of total lipolytic activity and biomass were positively
correlated. However, cell growth was relatively low, and lipoly-
tic activity appeared to be largely retained within the biomass.
Therefore, it would be interesting to nd culture conditions (i.e.
medium composition, pH, temperature, aeration), allowing to
improve growth and/or favour enzyme secretion.
0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2006.09.006
188 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
In this work, optimisation of lipolytic enzyme production by
T. thermophilus HB8, HB27 and T. aquaticus YT1 has been
attempted. The inuence of incubation temperature and the
effect of the type and concentration of carbon source in the cul-
ture medium have been studied.
2. Materials and methods
2.1. Microorganisms and culture conditions
T. thermophilus HB8, HB27 and T. aquaticus YT1 were kindly provided
by Dr. J. Berenguer (Universidad Aut onoma, Madrid, Spain).
Submerged cultures were carried out in 1000 cm
3
Erlenmeyer asks
with 200 cm
3
of medium (pH 7.5), prepared in distilled water and composed
of 8 g dm
3
casein peptone, 4 g dm
3
yeast extract and 3 g dm
3
NaCl.
Throughout this study, the basal medium was consecutively supplemented with
several concentrations (0.5, 1.5, 7 and 14 g dm
3
) of carbohydrates (glucose,
fructose, maltose and sucrose). Also, some experiments were carried out in
culture media supplemented with lipids, namely olive oil, coconut oil and
tributyrin (concentrations 1 and 10 g dm
3
). Culture inoculation and sample
preparation were realised according to Fuci nos et al. [8]. Experiments were
done in duplicate and samples were analysed in triplicate. The values in the
gures correspond to mean values with a standard deviation less than 15%.
2.2. Analytical methods
2.2.1. Cell growth determination
Cell concentration was monitored spectrophotometrically at 600 nm and the
obtained values were converted to g cell dry wt dm
3
using calibration curves
previously determined.
2.2.2. Lipolytic activity assay
It was determined using p-nitrophenyl laurate (p-NPL) as substrate following
the method described in Fuci nos et al. [7]. One activity unit was dened as the
amount of enzyme that produced 1 mol of p-nitrophenol per min under standard
assay conditions. The activities were expressed in Udm
3
.
2.2.3. Protein concentration
It was determinedaccordingtoLowryet al. [10], usingbovine serumalbumin
as standard.
2.2.4. Total carbohydrates concentration
It was determined by the phenol-sulphuric method of Dubois et al. [11],
using d-glucose as standard.
3. Results and discussion
In this work, some strategies to enhance lipolytic enzyme
production by three Thermus strains have been investigated. In
a previous paper [6], the selected strains were reported to show
remarkable activity on fatty acid esters with acyl chains longer
than 10 carbon atoms, and the activity towards these substrates
appeared to increase as substrate chain-length diminished.
Therefore, the denomination lipolytic enzyme/activity has
been utilised throughout this work, since incontrovertible attri-
bution of true lipase activity would require further investigation.
Moreover, an operational classication of enzyme activities
as intra- or extracellular has been utilised. Thus, activity
detected in the culture medium after biomass separation by
centrifugation was considered as extracellular, while that recov-
ered in solution after sonication of the buffer-resuspended cells
and elimination of cell debris was considered as intracellular.
Although the occurrence of cell-bound enzyme would be
possible and is not regarded here, this categorization is based on
usual industrial practice, and it is the most interesting from the
standpoint of industrial application of the described enzymes.
3.1. Effect of temperature on growth and lipolytic activity
The three studied strains were grown in shake asks,
within a wide range of temperature (from 60 to 80

C). The
cultures were stopped after 30 h of incubation, since previous
experiments indicated that, no signicant increases in lipolytic
enzyme activity were attained later in ask cultures. Besides,
cell lysis was mostly negligible at this time. Moreover, the
authors indicated that no kinetic typication of the enzymes as
primary metabolites was possible for any of the Thermus strains,
because of the lack of a good tting of the experimental lipolytic
activity production rates to the Luedecking & Piret model
[68].
Time-course of cultures and nal biomass, intra- and
extracellular lipolytic activity are shown in Fig. 1 and Table 1,
respectively. In the three studied strains, the increase in temper-
ature seemed to have a negative effect on biomass production:
nal cell growth at 60

C was almost two-fold higher than


that at 80

C. However, this effect was more remarkable in T.


thermophilus HB8 cultures, while T. thermophilus HB27 and
T. aquaticus YT1 only showed a signicant decrease in cell
growth at temperatures above 70

C. These results are close


to those reported by Oshima and Imahori [12], who found
that the optimal growth temperature for a locally isolated T.
thermophilus strain was 70

C. When biomass production


proles (Fig. 1) are considered, it can be seen that stationary
phase is approached rather rapidly at 80

C. Similar nal
cell growth levels are attained at 60 and 70

C, although a
longer lag phase is generally observed at 60

C, as it was
foreseeable.
More signicant differences between strains were observed
for lipolytic enzyme production. Maximum intracellular
activity was very similar in all T. aquaticus YT1 cultures, with
an average value of 66 Udm
3
, and production proles were
fairly close. On the other hand, the increase in temperature had
an acute effect on T. thermophilus HB8 and HB27 intracellular
enzyme levels. Final activities of 125 and 93 Udm
3
were
obtained, respectively, operating at 80

C, about two-fold higher


than those attained at 60

C, while intermediate values were


obtained at 70

C. Although the shape of the production patterns


was somewhat similar to those of cell growth for each inde-
pendent temperature, enzyme production cannot be considered
strictly growth associated. First, the highest activity levels were
found at 80

C, which was the least favourable condition for


biomass production. Secondly, at this temperature, attainment
of maximumenzyme levels was delayed with respect to those in
cell growth. Therefore, some additional feature must be involved
in the mechanismof lipolytic enzyme production at intracellular
level.
T. thermophilus strains have been reported to produce
chaperonins, a class of proteins implicated in the folding of
other proteins. Taguchi et al. [13] recovered and puried two
A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194 189
Fig. 1. Time course of cultures of Thermus strains, grown at different temperatures: () 60

C, () 70

C, and () 80

C.
intracellular chaperonins from late log phase T. thermophilus
HB8 cultures performed at 75

C, and demonstrated their ability


to promote refolding of several enzymes at high temperatures.
However, the effect of chaperonins on protein refolding is
dependent on the temperature: above 60

C spontaneous
refolding fails but if the native protein is sufciently stable, the
chaperonin induces productive refolding in an ATP-dependent
manner. At temperatures below 60

C (i.e. 50

C) spontaneous
refolding of the proteins occurs, and the chaperonin arrests this
spontaneous refolding in the absence of ATP.
It could be hypothesised that the occurrence of chaperonins
within T. thermophilus cells might contribute to the phenomena
described in this work, by favouring lipolytic enzyme folding
and therefore stability at high temperature. Since the mechanism
of chaperonin action seems to be dependent of temperature, this
could account to a certain extent for the differences observed in
intracellular lipolytic enzyme levels for T. thermophilus HB27
and HB8.
As for extracellular lipolytic activities, they were rather low
in comparison with the intracellular enzymes. In all cases, the
highest nal values were obtained when operating at 70

C. A
parabolic dependence of temperature was postulated for extra-
cellular lipolytic activity. Experimental results for nal activity
were tted to polynomic equations, and subsequently derivated
to obtain the theoretical optimum. According to this procedure,
optimal temperatures were found at 66.9, 70.6 and 70.5

C for
T. thermophilus HB8 and HB27 and T. aquaticus YT1, respec-
tively.
The lower extracellular enzyme activity at high tempera-
ture could in part be due to the secretion of specic proteases
during the last phase of the cultures. This fact was described
by Matsuzawa et al. [14] in previous studies carried out with
other Thermus strains. Also, temperature-dependent differences
in extracellular enzyme activity could be related to the occur-
rence of special enzyme secretion mechanisms in the studied
microorganisms, a hypothesis that has already been proposed
by the authors in a previous work [8] to explain the seemingly
independent dynamics of extracellular lipolytic activity and
intracellular enzyme and biomass production. Several species
of Thermus genus are able to form, under certain environmental
conditions, clusters of cells surrounded by a membrane formed
by the fusion of the individual external membranes of each cell.
These formations are usually known as rotund bodies [15]. It
might be assumed that proteins could be rstly secreted by indi-
vidual cells to this internal cavity and, from there poured into
the culture medium. Therefore, the integrity and permeability
of the external membrane of the rotund bodies might be impor-
tant to control the liberation of enzymes to the culture medium,
and these properties could be inuenced by the temperature.
Thus, the combined and opposed effects of increased release of
190 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
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enzymes to the culture medium and thermal or proteolytic deac-
tivation at high temperatures could account for the results found
in the present work.
Although maximum intracellular lipolytic activity was
obtained at 80

C in T. thermophilus HB8 cultures, the scarce


benecial effect in the other studied strains and the consequent
increase in production costs does not recommend operating at
this temperature. Besides, one of the most interesting aspects
in this eld is the enhancement of enzyme secretion, which in
this case appears to be favoured at 70

C (especially for T. ther-


mophilus HB27). Therefore, all subsequent experiments were
carried out employing 70

C as culture temperature.
3.2. Effect of the type and concentration of carbon source
A high degree of nutritional diversity has been detected in
Thermus strains (i.e. T. thermophilus HB8, T. aquaticus YT1) by
Degryse et al. [16], Alfredsson et al. [17] and Santos et al. [18].
These strains grewwell on different carbon sources, although the
presence of glucose, galactose, fructose, sucrose or maltose in
the culture mediumseemed to have a particularly positive effect.
However, the above-mentioned studies were carried out by
means of agar plate cultures, and only provided qualitative
results. Besides, they focused on the investigation of cell
growth, and no information was given on the production of
potentially interesting enzymes. In the present work T. ther-
mophilus HB8, HB27 and T. aquaticus YT1 have been grown
in submerged cultures, using a complex medium supplemented
with several concentrations (from 0.5 to 14 g dm
3
) of mono-
and disaccharides (glucose, fructose, maltose and sucrose). The
end-culture values of biomass, intra- and extracellular lipolytic
activity (after 30 h of incubation) for the three strains are
compared in Figs. 24, respectively. The data are expressed as
percentages referred to the results obtained in the basal medium
(with no additional carbon source) at 70

C, shown in Table 1.
The effect of carbon source appeared to be somewhat
strain-dependent and therefore difcult to generalise. However,
some common patterns could be established for the tested
strains. As it can be observed in Fig. 2, supplementation of
the basal medium with low concentrations (up to 1.5 g dm
3
)
of mono- or disaccharides led, in most cases, to a moderate
increase (2050%) in biomass production. Higher carbon
source concentrations did not result in further improvements in
cell growth, with the exception of T. thermophilus HB8 cultures,
in which biomass was increased two-fold and 2.5-fold when
the basal medium was supplemented with sucrose (7 g dm
3
)
or maltose (14 g dm
3
). Also, it is noteworthy that cell growth
was signicantly diminished when high levels of glucose were
added to the medium, in all the studied strains.
When intracellular lipolytic activity production was con-
sidered (Fig. 3), the general behaviour of the microorganisms
was similar to that described for cell growth. However, the
comparison of both sets of data indicated that intracellular
enzyme and biomass productions were not fully associated. The
addition of low concentrations (0.51.5 g dm
3
) of mono- and
disaccharides generally resulted in a noticeable amelioration
in enzyme production, the most promising results having been
A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194 191
Fig. 2. Inuence of carbon source on biomass production by Thermus strains,
after 30 h of culture time. Carbon source: () glucose, () fructose, () maltose,
and () sucrose (100%biomass production: T. thermophilus HB8, 0.75 g dm
3
;
T. thermophilus HB27, 1.04 g dm
3
; T. aquaticus YT1, 1.18 g dm
3
).
obtained with disaccharides, and for T. thermophilus HB8.
Carbon source supplementation above this level does not
seem to be advisable, since intracellular lipolytic activities
remained mostly unaltered, or decreased. Only T. thermophilus
HB8 and HB27 showed any amelioration in intracellular
enzyme levels at high carbon source concentrations, with
three-fold and 1.6-fold rises in activity (referred to control) in
the presence of sucrose (14 g dm
3
) and maltose (7 g dm
3
),
respectively.
Finally, extracellular lipolytic enzyme concentrations were
assessed. No signicant levels of extracellular activity were
detected for T. thermophilus HB8, in any case. As for the other
two strains (Fig. 4), the general effect of carbon source supple-
mentation was similar to that previously described for biomass
Fig. 3. Inuence of carbon source on intracellular lipolytic activity production
by Thermus strains, after 30 h of culture time. Carbon source: () glucose, ()
fructose, () maltose, and () sucrose (100% lipolytic activity: T. thermophilus
HB8, 72.80 Udm
3
; T. thermophilus HB27, 81.79 Udm
3
; T. aquaticus YT1,
68.70 Udm
3
).
and intracellular enzyme. A slight enhancement in extracellular
enzyme production was obtained when low concentrations of
the different carbon sources were added. The best results were
obtained with sucrose for T. thermophilus HB27 and fructose
for T. aquaticus YT1 (both sugars at 0.5 g dm
3
).
Total carbohydrates and protein consumption were evalu-
ated in all the cultures. The assessed nutrients were not totally
depleted in any case, and most consumption occurred during
the early stages of the cultures. Low nutrients consumption
in Thermus strains has already been mentioned in previous
works [6,19,20]. No relevant information could be inferred from
these data, concerning the dynamics of cell growth and lipolytic
enzyme production on different carbon sources.
192 A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194
Fig. 4. Inuence of carbon source on extracellular lipolytic activity production
by T. thermophilus HB27 and T. aquaticus YT1, after 30 h of culture time.
Carbon source: () glucose, () fructose, () maltose, and () sucrose (100%
lipolytic activity: T. thermophilus HB8, 4.50 Udm
3
; T. thermophilus HB27,
34.40 Udm
3
; T. aquaticus YT1, 27.25 Udm
3
).
To our knowledge there are few reports about the produc-
tion of thermostable lipases or esterases from thermophilic
microorganisms, and none on how it can be affected by the
choice and concentration of carbon source. However, the inu-
ence of this factor on lipolytic enzyme secretion by mesophilic
microorganisms has been investigated. The results are very dif-
ferent depending on the microorganism and the experimental
conditions employed. Generally, monosaccharides have been
reported to favour lipase production by mesophilic microor-
ganisms. So, Benjamin and Pandey [21] found that glucose
and fructose enhanced lipase activity in submerged cultures of
Candida rugosa. Dalmau et al. [22] proposed a mixture of com-
pounds as optimum carbon source for lipase production by the
same microorganism. Polysaccharides (i.e. starch) and glycerol
have been described as poor carbon sources for lipase production
by C. rugosa and Yarrowia lipolytica [2123], although they led
to good results in the case of Penicillium citrinum and Rhizopus
delemar [24,25]. Costa et al. [26] concluded that the ability of the
yeast Issatchenkia orientalis to secrete lipolytic activity in sub-
merged culture was improved by using monosaccharides (glu-
cose, fructose), although di- and polysaccharides (sucrose, lac-
tose, maltose, starch) as well as glycerol were not recommended.
The results found in the present work seemto indicate that the
presence of certain carbon sources in the culture medium might
have aninuence oncell growthandlipolytic enzyme production
by Thermus strains. However, the observed effects appeared to
be somehowless dramatic than those reported in some instances
for mesophilic microorganisms. This could be related to the
extreme environments in which thermophilic microorganisms
are usually found, generally characterised by lownutrients avail-
ability. Therefore, the strains are naturally conditioned to survive
in oligotrophic environments, and inhibition by excess of sub-
strates is often encountered [27].
Supplementation of the medium with low concentrations of
mono- anddisaccharides generallyresultedinimprovedbiomass
and lipolytic enzyme production. However, when high carbohy-
drate concentrations were used, no further improvements were
observed, and in some cases (i.e. glucose) strong decreases
in biomass and enzymatic activity were detected. This could
be attributed to the occurrence of Maillard reactions between
amino compounds and reducing sugars, promoted by the high
culture temperatures utilised and favoured by values of pH in
the medium between 7.5 (initial) and 8.5 (at the end of culture).
This kind of reaction is often found in cultures of thermophilic
strains and may result in products that can be inhibitory to
the microorganisms [27]. In the present case, this potentially
inhibitory effect appears to be most remarkable with monosac-
charides, and more precisely glucose. This could be due both
to the higher reactivity of this sugar towards Maillard reaction,
and a possible stronger toxic effect of the formed products. Sus-
ceptibility of disaccharides to undergo the reaction is smaller
(i.e. maltose) or nonexistent (i.e. sucrose, non-reducing sugar),
which agrees with the lesser adverse effects associated to their
presence in the culture medium.
With respect to the mechanismby which disaccharides might
be metabolised by the studied strains, the results seem to indi-
cate that they are not massively hydrolysed in the mediumbefore
being incorporated to the cells. Extracellular hydrolysis of both
disaccharides would imply the generation of glucose, which was
shown to exert a strong inhibitory effect on both biomass and
enzyme production, yet no drastic negative repercussions were
detected when high concentrations of maltose or sucrose were
considered. Therefore, either the disaccharides are hydrolysed
at approximately the uptake rate by the cells, thus maintain-
ing a concentration below the critical one for inhibition, or
they are directly up taken by the cells. The latter hypothesis
is supported by the recently ascertained genome sequence of T.
thermophilus, in which maltose/trehalose permease and ABC
transporter encoding genes were found [28]. Besides, the mal-
tose/trehalose ABC transporter was recently reported to also
recognize sucrose [29]. The genes encoding two -glucosidades,
a -glucosidase and a maltodextrin glucosidase were also found,
but there are no indications that these enzymes could be secreted
by the microorganisms to the culture medium.
Finally, the effect of the addition of lipidic compounds to the
culture mediumin biomass and lipolytic enzyme production has
been assessed in T. thermophilus HB27 cultures. This strain was
selected for this preliminary study, due to the higher enzyme
secretion levels detected in previous cultures, and the more
abundant information available on its lipolytic enzymes [7]. The
presence of lipids has been reported to be crucial for lipases and
esterases production by a number of microorganisms [30]. Thus,
A. Domnguez et al. / Enzyme and Microbial Technology 40 (2007) 187194 193
Fig. 5. Intra- and extracellular lipolytic activity production by T. thermophilus
HB27 cultures in presence of several lipidic compounds with different con-
centrations (1 and 10 g dm
3
): B, control; OO, olive oil; CO, coconut oil; TB,
tributyrin.
the basal medium was supplemented with different concentra-
tions (1 and 10 g dm
3
) of triglycerides (olive oil, coconut oil
and tributyrin). Intra- and extracellular enzyme activities after
30 h culture time are presented in Fig. 5. Tributyrin appeared
to have a strong inhibitory effect in the microorganism, while
cell growth did not undergo dramatic changes with the addition
of olive or coconut oil. On the other hand, the presence of olive
oil helped to increase lipolytic enzyme production levels, both
intra and extracellular up to 33% and 70%, respectively. As it
occurred with carbohydrates, the best results were obtained with
low concentrations of added carbon source. Therefore, it would
appear that the presence of lipids could trigger to some extent the
production of lipolytic enzymes. The occurrence of a biphasic
medium could lead to hydrophobic interactions that might alter
the permeability of the membranes surrounding the already
mentioned rotund bodies, therefore accounting for the observed
variations in enzyme secretion levels. However, further inves-
tigation is required on this topic before attempting any denite
conclusion.
4. Conclusions
According to the results obtained, it can be concluded that
the progressive increase in incubation temperature appeared to
have a negative effect on cell growth, and 70

C was selected
as an optimum for this variable, a compromise in terms of
biomass, intra- and extracellular enzyme production, and cost
optimisation.
Disaccharides (i.e. sucrose and maltose) have been estab-
lishedas goodcarbonsources allowingtoimprove growthand/or
favour enzyme secretion by these microorganisms, although the
most adequate concentration seems to depend on the strain.
On the other hand, the addition of high levels of glucose or
fructose to submerged cultures of Thermus sp. did not bring
about signicant ameliorations in lipolytic activity. Moreover,
the addition of some lipidic compounds (olive and coconut oils)
to the culture medium increased total lipolytic activity levels up
to two-fold with respect to the control culture. These promising
results encourage to realise more studies in which surfactants
or other lipidic compounds would be added to culture medium.
These compounds could help to increase the secretion of lipoly-
tic enzymes due to changes in cell wall permeability or surfactant
effects on cell bound enzyme.
Acknowledgements
This work was nanced by the Spanish Ministry of Science
and Technology and European FEDER(Project PPQ2001-3361)
and XUNTA de Galicia (Project PGIDT03PXIB30103PR).
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