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Applied and Environmental Microbiology, August 1999, p. 3594-3598, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transport and Utilization of Hexoses and Pentoses
in the Halotolerant Yeast Debaryomyces hansenii
Alexandra
Nobre,
Cândida
Lucas,* and
Cecília
Leão
Departamento de Biologia, Centro de
Ciências do Ambiente, Universidade do Minho, 4709 Braga
Codex, Portugal
Received 13 January 1999/Accepted 20 May 1999
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ABSTRACT |
Debaryomyces hansenii is a yeast species that is known
for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was
investigated with respect to the utilization of pentoses and hexoses in
mixtures and as single carbon sources. Growth parameters were
calculated for batch aerobic cultures containing pentoses, hexoses, and
mixtures of both types of sugars. Growth on pentoses was slower than
growth on hexoses, but the values obtained for biomass yields were very
similar with the two types of sugars. Furthermore, when mixtures of two
sugars were used, a preference for one carbon source did not inhibit
consumption of the other. Glucose and xylose were transported by cells
grown on glucose via a specific low-affinity facilitated diffusion
system. Cells derepressed by growth on xylose had two distinct
high-affinity transport systems for glucose and xylose. The sensitivity
of labeled glucose and xylose transport to dissipation of the
transmembrane proton gradient by the protonophore carbonyl cyanide
m-chlorophenylhydrazone allowed us to consider these
transport systems as proton symports, although the cells displayed
sugar-associated proton uptake exclusively in the presence of NaCl or
KCl. When the Vmax values of transport systems
for glucose and xylose were compared with glucose- and xylose-specific
consumption rates during growth on either sugar, it appeared that
transport did not limit the growth rate.
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INTRODUCTION |
Debaryomyces hansenii is
a xylose-utilizing yeast that exhibits an industrially interesting
xylitol/ethanol production ratio (17, 20). Xylose
fermentation and subsequent ethanol production have been studied in
several other yeast species, including Pichia stipitis,
Candida shehatae, and Pachysolen tannophilus, but
the ethanol productivity of these organisms compares very poorly with that of Saccharomyces cerevisiae when glucose-based
substrates are used (6, 18, 22, 26, 27). This is due to
several factors, including the low resistance to ethanol stress of the yeasts (25). Optimization of biomass and xylitol production from xylose by yeasts has been studied by using batch and continuous cultures (3, 20). However, industrial substrates, such as the hemicellulose hydrolysates obtained from several sources, are
usually mixtures of hexoses and pentoses. Upon hydrolysis, hemicellulose yields D-xylose as the major component, as
well as other sugars, including D-glucose,
D-mannose, D-galactose, and
L-arabinose in variable combinations depending on the
source of the raw material. Utilization of mixed substrates by yeasts has so far received little attention; the only exceptions have been
studies of ethanol production from glucose-xylose mixtures by P. tannophilus (9) and consumption of xylose in complex sugar mixtures by several strains of S. cerevisiae
(26). On the other hand, D. hansenii, which is an
osmotolerant yeast, may be a very attractive microorganism for polyol
production (1). The purpose of the present work was to
evaluate growth parameters in mixed sugar cultures and to elucidate the
underlying transport systems for the sugars and the corresponding
regulation in D. hansenii INETI CL18.
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MATERIALS AND METHODS |
Microorganism and media.
D. hansenii INETI CL18, which
was obtained from the Instituto Nacional de Engenharia e Tecnologia
Industrial, Portugal, was originally isolated from sugarcane. It was
maintained at 4°C in solid medium containing 2% (wt/vol) glucose,
2% (wt/vol) peptone, and 1% (wt/vol) yeast extract. Cells were
cultivated in liquid mineral medium by using 0.5% (wt/vol) ammonium
sulfate as the nitrogen source (23) and different carbon
sources (D-glucose, D-galactose,
D-mannose, D-xylose, and
L-arabinose), as indicated below.
Culture conditions.
Batch cultures were grown with a
liquid/air ratio of 1:5 and were incubated at 30°C with orbital
shaking at 160 rpm (Certomat HK; B. Braun, Melsungen, Germany). Growth
was monitored by measuring the optical density (absorbance at 640 nm)
with a spectrophotometer (Spectronic 21; Bausch & Lomb) and by
determining dry weight. The experiments were performed by taking 10-ml
samples which were filtered through ME 25/41 ST mixed ester membranes
(Schleicher and Schuell, Dassel, Germany) and then washed with an
identical volume of distilled water and dried at 80°C overnight. The
specific growth rates (µmax) during the exponential phase
of growth were determined either by measuring the optical density or by
determining the dry weight. Yield coefficients
(YX/S) were based on dry weights and substrate
concentrations in the stationary phase. Specific consumption rates for
glucose or xylose were calculated by determining µmax/YX/S.
Estimating sugar concentrations in growth media.
Sugar
concentrations in growth media were determined by high-performance
liquid chromatography. The system used included a model 307 pump
(Gilson, Villiers le Bel, France) and a model 132 RI detector (Gilson).
Compounds were separated by using a Merck Polyspher OA KC column
(catalog no. 51270) at 50°C; 1 mM sulfuric acid at a flow rate of 0.5 ml min
1 was the eluent. Quantification was performed by
the internal standard method.
Measuring initial uptake rates.
Cells were harvested in the
exponential phase of growth (optical density at 640 nm, 0.6 to 0.7) by
centrifugation with a model 4K10 centrifuge (B. Braun, Osterod Harz,
Germany), washed twice with 200 ml of ice-cold distilled water (5 min
at 12,200 × g each time), and suspended at a final
concentration of 20 to 25 mg (dry weight) ml1 in ice-cold
distilled water. To estimate the initial rates of uptake of labeled
glucose and xylose at pH 5.0, we used a previously described method
(14) and aqueous solutions of [U-14C]glucose
and [U-14C]xylose having specific activities of 8.5 and
7.4 MBq mmol1, respectively (3% ethanolic solutions;
Amersham, Buckinghamshire, England). The concentration of the final
cell suspension was 8 to 10 mg (dry weight) ml1. The
sampling times used were 0, 5, and/or 10 s; each experiment was
repeated three times (the linearity of uptake was maintained for up to
20 s). Kinetic constants were estimated from Eadie-Hostee plots
and were confirmed by a computer nonlinear regression analysis performed with GraphPad PRISM (GraphPad Software, Inc.). No quenching effects were observed in uptake experiments, even in the presence of
high concentrations of NaCl.
The method used to estimate the initial rates of proton uptake after
glucose or xylose was added in the absence of NaCl or in the presence
of several NaCl concentrations was the method described previously
(10). All of the experiments were performed at 30°C.
The effects of other sugars on uptake of glucose or xylose
(
14) were determined by using each sugar at concentrations
of
200 and 20 mM to study inhibition of the low-affinity and
high-affinity
uptake systems, respectively. The effect of ethanol on
sugar transport
was determined, and the exponential inhibition constant
(
ki) (
24,
25) and MIC of ethanol
(C
min) (
24,
25) were also determined.
To do
this, cells were incubated for 2 min in the presence of
different
concentrations of ethanol (5 to 15%, vol/vol), after
which uptake was
assayed. The same method was used to determine
the effect of the
protonophore carbonyl cyanide
m-chlorophenylhydrazone
(CCCP)
(concentration in the assay mixture, 50 µM) on sugar transport.
The
effect of starvation was investigated by incubating the cells
in
mineral medium without a carbon source at 30°C for different
periods
of time. Samples were centrifuged, washed twice in ice-cold
distilled
water, and assayed as described above. A cycloheximide
MIC of 200 µg
ml
1 was used to prevent protein synthesis during
starvation in
controls.
Reproducibility of the results.
All of the experiments were
repeated at least three times with independent batches of cells. The
standard deviations are presented below.
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RESULTS |
Growth in batch culture.
D. hansenii was grown on
pentoses or hexoses as single carbon and energy sources, and growth
parameters (µmax, YX/S,
µmax/YX/S) were calculated (Table
1). Growth on glucose and growth on
mannose resulted in similar growth rates, and growth on galactose
resulted in a slightly lower value. Growth on xylose or arabinose was
slower than growth on hexoses. In spite of the differences, the final biomass yields for all of the sugars assayed were similar.
Growth on mixtures of two sugars (1% [wt/vol] each) was investigated
by using all of the possible combinations of the hexoses
and pentoses
mentioned above. Representative results obtained
with mixtures of two
hexoses, mixtures of two pentoses, and mixtures
of one hexose and one
pentose are presented in Table
1. The biomass
yields obtained with
hexose and hexose-pentose mixtures were slightly
lower than the yields
obtained with the same amount of sugar alone.
Diauxic growth with
similar growth parameters was observed when
glucose was mixed with
either mannose or galactose, and the glucose
was consumed first. With
all of the other mixtures, consumption
of the two sugars was
nondiauxic; the beginning of consumption
of the second substrate
generally followed a lag phase that was
longer than the lag phase for
the first substrate, after which
consumption of the two sugars
proceeded simultaneously. For example,
in the case of the
glucose-xylose mixture, xylose consumption
began only when the glucose
concentration was less than 20% of
the original concentration.
However, the same µ
max was observed
in both phases of
growth (Table
1). The experiments were repeated
with a lower
concentration (0.1%, wt/vol) of each sugar, but still
no distinct
µ
max value was determined during the second growth
phase.
Similar results were obtained for all of the other mixtures
examined.
Utilization of hexoses was preferred to utilization
of pentoses, in the
following order: glucose, mannose, galactose,
xylose,
arabinose.
With hexose-arabinose or pentose-arabinose mixtures, no consumption of
arabinose was observed unless the medium pH was readjusted
from pH 2.2 to 2.5 to pH 5.5 (initial pH of the growth medium)
after the first
carbon source was consumed. The influence of the
initial pH on
consumption of arabinose as a single carbon source
was examined at pH
values between pH 1.8 and 7.3 in approximately
0.5-pH unit increments.
The µ
max varied, and the optimum µ
max was
observed when the initial pH was 5.2. At initial pH values
below 2.6 no
growth was
detected.
Glucose and xylose transport on glucose-grown cells.
Uptake of
glucose (Fig. 1) and uptake of xylose
(data not shown) by cells of D. hansenii growing on glucose
and collected in the mid-exponential phase exhibited Michaelis-Menten
kinetics. Both transport systems had low affinities for their
substrates; the Km for glucose was approximately
eight times lower than the Km for xylose (Table
2), whereas the Vmax values for the two sugar
transport systems were very similar. Xylose inhibited glucose uptake
competitively (Fig. 2), with a
Ki of 175 mM. Galactose, arabinose, mannose, and
2-deoxyglucose were also tested as potential inhibitors of glucose
transport but had no effect.
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FIG. 1.
Eadie-Hofstee plot and direct plot (inset) of initial
rates of uptake of labeled glucose in glucose-grown cells ( ) and
xylose-grown cells ( ). d.wt., dry weight.
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FIG. 2.
Inhibition of low-affinity glucose transport in
glucose-grown cells by xylose. Symbols:  , no xylose; , 300 mM
xylose;  , 400 mM xylose;  , 500 mM xylose. (Inset) Effect of
xylose concentration on Km for glucose. d.wt.,
dry weight.
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The initial glucose uptake rates were approximately constant and did
not exhibit any definite variation at external pH values
ranging from
3.0 to 7.0, the
Vmax value being 8.86 ± 1.31 mmol
h
1 g (dry weight)
1. Furthermore,
when uptake was measured in the presence of the
protonophore CCCP, the
Vmax values were about the same as the
Vmax values in the absence of CCCP (data
not shown). Ethanol inhibited
the initial rates of uptake of glucose
and xylose in a noncompetitive
way.
Vmax
decreased exponentially with the ethanol concentration,
which is
consistent with the equation obtained for other mediated
transport
systems (
24,
25). On the basis of the results of
these
experiments, a
ki value for ethanol of 0.6 M
1 was estimated, the ethanol C
min being
approximately
zero.
Glucose and xylose transport on xylose-grown cells.
We also
measured transport of glucose and xylose in cells of D. hansenii growing exponentially on xylose. With these cells, the
Eadie-Hofstee plots of the initial rates of glucose uptake (Fig. 1) and
xylose uptake (data not shown) were biphasic. The lower-affinity
component had kinetic parameters similar to the parameters obtained for
the low-affinity glucose-xylose uptake observed with glucose-grown
cells (Table 2). In addition to the low-affinity component, a higher-affinity system for glucose seemed to
operate in xylose-grown cells. Similar results were obtained for xylose
transport. The kinetic parameters estimated for these systems are shown
in Table 2. The Km and
Vmax values for the higher-affinity transport of
glucose were different from the values for xylose uptake. Mannose
competitively inhibited high-affinity glucose transport
(Ki, 0.38 mM), whereas galactose, xylose, and arabinose did not. On the other hand, xylose uptake was not inhibited by any of these sugars (data not shown).
The
Km values for the high-affinity glucose and
xylose transport systems were not affected by the extracellular pH (the
pH
was varied from 3.0 to 7.0), while the
Vmax
for either glucose
or xylose uptake decreased slightly at pH values
below 5.0 (data
not shown). Both the glucose transport system and the
xylose transport
system were strongly inhibited by the protonophore
CCCP (the
Vmax values decreased 82 and 67%,
respectively). Both glucose uptake
and xylose uptake were inhibited by
ethanol in a noncompetitive
way. As observed with glucose-grown cells,
the
Vmax values decreased
exponentially with the
ethanol concentration; in these experiments
the
ki values for ethanol were 0.98 and 0.80 M
1 for glucose transport and xylose transport,
respectively, and
the ethanol C
min values were 860 mM and
almost zero for glucose
transport and xylose transport,
respectively.
Regulation of glucose and xylose transport systems.
Carbon
source starvation of glucose-grown cells in mineral medium for 2 h
resulted in a gradual increase in the activity of the high-affinity
transport system for glucose (Fig. 3),
which was prevented by the presence of cycloheximide. Transfer of
glucose-grown cells to mineral medium containing 2% xylose resulted
(within 10 min) in the formation of both the high-affinity transport
system for glucose and the high-affinity transport system for
xylose (Fig. 3), which was again prevented by cycloheximide.
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FIG. 3.
(A) Effect of starving glucose-grown cells in mineral
medium without a carbon source on the formation of the high-affinity
transport system for glucose. Symbols: and ,
[U-14C]glucose;  , [U-14C]xylose. (B)
Appearance of the high-affinity transport systems for glucose and
xylose: Vmax values for
[U-14C]glucose ( and ) and
[U-14C]xylose ( and ) after glucose-grown cells
were transferred to medium containing 2% xylose. Open symbols, cell
suspensions incubated in the presence of cycloheximide; solid symbols,
cell suspensions incubated in the absence of cycloheximide. d.wt., dry
weight.
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H+ movements associated with sugar uptake.
In many
cases, when the mechanism of sugar transport in yeasts is an
H+ symport, transient alkalinization of an aqueous cell
suspension occurs during initial uptake of the substrate
(13). In cells of D. hansenii grown on any of the
hexoses or pentoses mentioned above as a single carbon source, addition
of glucose, mannose, galactose, xylose, or arabinose did not result in
extracellular alkalinization. However, in xylose-grown cells incubated
for 2 min in the presence of 1 M NaCl or 1 M KCl (but not in the
presence of 1 M LiCl, 1 M MgCl2, or 1 M CaCl2),
addition of glucose, mannose, galactose, or xylose elicited
alkalinization. The initial proton uptake rates followed saturation
kinetics, and the corresponding Km and
Vmax values for glucose and xylose, calculated
from Eadie-Hofstee plots, are presented in Table
3. The Km values
obtained were identical to the corresponding Km
values estimated with labeled sugars, but the
Vmax values were considerably lower than the
values shown in Table 2. Labeled glucose and xylose uptake parameters were reestimated by incubating cell suspensions in buffer containing 1 M NaCl (Table 3). The Km values obtained in this
way did not differ from the Km values determined
in the absence of NaCl (Table 2), but the Vmax
values decreased, reaching levels close to those for proton uptake.
Hence, one proton per glucose or xylose molecule was transported in the
presence of 1 M NaCl.
The minimum incubation period in the presence of 1 M NaCl for detection
of a lower
Vmax
assayed, 30 s, was still long enough
to determine the observed decrease in the
Vmax,
as shown in Fig.
4. The
Vmax of proton uptake
increased with increasing salt concentration
(Fig.
5). The 1:1
proton-sugar stoichiometry mentioned above was
valid only for salt
concentrations greater than 600 to 800 mM
(Fig.
5).
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FIG. 4.
Effects of incubation with 1 M NaCl on the
Vmax values of the high-affinity transport
systems for radiolabeled glucose () and xylose (). d.wt., dry
weight.
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FIG. 5.
Vmax values for
[U-14C]glucose () and [U-14C]xylose
() and proton uptake after glucose () and xylose () were added
as a function of NaCl concentration in suspensions of xylose-grown
cells. (Inset) Ratio between Vmax from proton
uptake and labeled glucose () or xylose () uptake as a function
of NaCl concentration in the assay mixture. d.wt., dry weight.
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No extracellular alkalinization elicited by glucose or xylose was
detected in glucose-grown cell suspensions incubated in
the absence and
in the presence of NaCl or
KCl.
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DISCUSSION |
Our data show that growth of D. hansenii on glucose or
mannose resulted in approximately the same µmax and the
same biomass yield. On the other hand, the growth rate on xylose or
arabinose was lower, whereas similar biomass yields were
obtained. With sugar mixtures, either diauxic or nondiauxic sugar
consumption was observed. The same type of nondiauxic growth of
P. tannophilus was observed when a glucose-xylose
mixture was used (9). In D. hansenii, pentose
metabolism and hexose metabolism proceed without any particular
drawbacks, in contrast to what has been described previously for other
yeasts (2, 18, 22, 26), since, disregarding the type of
consumption of each sugar mixture, the yields are rather similar. Our
data suggest that mixtures of hexoses and pentoses, such as those
present in hemicellulose hydrolysates, are probably completely consumed
by D. hansenii as long as pH of the medium can be maintained
close to 4 to 5. Hemicellulose extracts used industrially usually
undergo acid hydrolysis, but the pH of the solution is normally
neutralized with CaCO3.
When D. hansenii was grown on glucose, it exhibited a
low-affinity glucose transport system. The absence of simultaneous
proton uptake, the insensitivity of glucose uptake to CCCP and to
changes in the external pH, and the relatively low level of inhibition by ethanol led us to conclude that glucose uptake occurs by facilitated diffusion. Xylose competitively inhibited glucose transport and had a
Ki identical to its transport
Km, which suggests that xylose shares
glucose-facilitated diffusion with even lower affinity.
In contrast, D. hansenii cells induced by growth on xylose
were different. Radiolabeled glucose and xylose had uptake kinetic parameters whose affinity was much higher than the affinity in glucose-grown cells and did not act as mutual inhibitors, which indicated that these sugars are probably transported by different permeases. Both sugar transport systems in these cells were inhibited by the protonophore CCCP. The inhibition by ethanol was characterized by ki values higher than the values determined
for facilitated diffusion in glucose-grown cells but lower than the
previously published values for active transporters of the proton
symport type (21, 25). This finding is probably related to
the fact that in xylose-grown cells glucose and xylose are taken up by two transport systems that have different sensitivities for ethanol, the reason why the ki for ethanol has an
intermediate value. This is not a result that allows one to distinguish
easily between facilitated diffusion and active transport. However, it
reinforces the conclusion that permeases are involved in sugar uptake
and corroborates other data.
Uptake of mannose also occurred via the glucose transport system, while
the xylose transport system did not transport any of the other
monosaccharides and thus was apparently specific for this sugar. Also,
in C. shehatae, facilitated diffusion and sugar proton
symports have distinct specificities for different pentoses and hexoses
(14).
The specific rate of consumption of glucose by D. hansenii
was lower than the glucose transport capacity (facilitated diffusion Vmax) (Tables 1 and 2), suggesting that glucose
transport does not limit growth on this sugar. For cells growing on
xylose, the specific rate of consumption of this sugar was considerably
higher than the Vmax of the high-affinity
transport system, indicating that glucose-xylose-facilitated diffusion
could also play an important role in sustaining growth on xylose.
Consistent with these conclusions is the finding that growth in
mixtures of glucose and xylose was nondiauxic; this means that as soon
as a low concentration of glucose in the growth medium is reached,
xylose may compete with glucose transport by the facilitated diffusion
system. This should enable induction (in the presence of glucose) of
xylose high-affinity transport, as well as the xylose
catabolism-specific enzymes (5, 12).
The concentrative monosaccharide transport systems usually have been
described as proton symports that are driven by the proton motive force
generated by the plasma membrane H+ ATPase (for example,
the H+-xylose symport described in E. coli
[11] and the H+-glucose and
H+-xylose symports in different yeasts [4, 7, 13,
14]). Surprisingly, in D. hansenii, no proton
uptake was detected when glucose or xylose was added to xylose-grown
cell suspensions. If we take into consideration (i) the fact that
D. hansenii is a halotolerant yeast (1, 19), (ii)
the fact that it has been postulated that a Na+-glycerol
symport occurs in this yeast (15), and (iii) the fact that
this yeast has been described as an organism that regulates K+ and Na+ intracellular contents as an even
interchange, substituting one ion for the other and generating ion
potential from high intracellular sodium contents (16, 19),
it is not unlikely that glucose and xylose high-affinity transport
systems are affected by a salt gradient over the plasma membrane.
Proton uptake was detected exclusively in the presence of salt, and at
salt concentrations above a certain level stoichiometry was determined.
The results favored recognition of the glucose and xylose high-affinity
transport systems as proton symports that may indirectly depend on the
presence of salt to determine sensible variations in the proton motive force, which can be critical for proton uptake detection.
Starvation resulted in gradual induction of the high-affinity
glucose-proton symport, whereas transfer of glucose-grown cells to
xylose-containing medium resulted in the gradual appearance of
high-affinity glucose- and xylose-proton symports. On the basis of
these results we concluded that the glucose-proton symport was subject
to glucose repression, while the xylose-proton symport requires
induction by the substrate. This type of transport regulation is
similar to what has been described previously for glucose and xylose
transport in C. shehatae (14) and P. stipitis (7), as well as for xylose transport in
Candida utilis (8). Furthermore, the results
obtained in the transport studies performed with D. hansenii
were consistent with the pattern observed for consumption of mixed
substrates. In conclusion, we stress that the results obtained in this
study support the hypothesis that D. hansenii is a good
candidate for biodegradation of hemicellulose hydrolysates and,
therefore, for further biochemical engineering in which the goal is
improving xylose consumption and xylitol production.
| |
ACKNOWLEDGMENTS |
This work was financed in part by European Union project
BIOTECH PL 95016. A. P. Nobre was the recipient of Ph.D.
grant PRAXIS XXI/BD/3488/94.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biologia, Universidade do Minho, Campus de Gualtar, 4709 Braga Codex, Portugal. Phone: 351-53-604313/11/10. Fax: 351-53-678980. E-mail: clucas{at}bio.uminho.pt.
| |
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Applied and Environmental Microbiology, August 1999, p. 3594-3598, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
