Eng. Life Sci. 2010, 10, No. 5, 439–445
Carlos Rolz
Roberto de León
Biochemical Engineering
Center, Research Institute,
Universidad del Valle de
Guatemala, Guatemala
439
Research Article
Converting developing and mature sugarcane
carbohydrates into ethanol
Experiments were performed employing cane particles obtained from sugarcane at
different growth stages until maturation measuring the amount of ethanol
produced and the carbohydrate consumption in order to estimate the sugarcane
growth stage where both parameters were optimized. Two non-flowering
commercial cane varieties NA56 and PR752002 were cultivated and samples taken
at different time intervals. Two Saccharomyces cerevisae strains were also compared
in the trials. Sucrose was poorly consumed in young cane, which was an unexpected result. Fructose on the other hand was the hexose that remained in the
medium at the end of the fermentations specially when using mature sugarcane.
There was an increasing trend in ethanol production as a function of days after
planting (DAP) as expected; however, a plateau was reached after 225 DAP and the
maximum value obtained was between 300 and 325 DAP. When these figures were
compared with the corresponding DAP used for sugar production, only 25 days
less were needed in the field for maximum ethanol production. On the other
hand, it was clear from the data that cane harvesting for ethanol production
should not be done after the recommended DAP for commercial sugar production. If this is done, the excess fructose present will not be completely utilized by
yeast. Finally, it was observed that the yeast with more affinity for sugarcane fibers
showed better ethanol yields in all samples tested.
Keywords: Carbohydrates / Ethanol / Extraction-fermentation / Saccharomyces cerevisae /
Sugarcane
Received: February 18, 2010; revised: July 14, 2010; accepted: July 23, 2010
DOI: 10.1002/elsc.201000030
1
Introduction
Energy required for economic development has come mainly
from fossil fuels extracted from deposits that took million of
years to form. Uncertainty about future supplies and environmental concerns related to the increase in atmospheric
carbon dioxide, and the corresponding climate change consequences, has motivated an interest in biofuels as these are
renewable and carbon neutral [1, 2]. Biofuels must meet two
important criteria. First, they should be obtained employing
processes with lower green-house-gas emissions than those
used with fossil fuels; second, their production should have
minor consequences for food security [3, 4]. Life cycle analysis
of ethanol production based in sugarcane and molasses as raw
materials has shown a positive energy balance and savings on
Correspondence: Carlos Rolz ([email protected]), Biochemical
Engineering Center, Research Institute, Universidad del Valle de
Guatemala, Guatemala 01015, Guatemala
Abbreviations: CBS, Centraalbureau voor Schimmelculture; DAP, days
after planting
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
green-house-gas emissions [5–15]. The concern about the fuel
versus food debate has been put in a favorable perspective that
does not require further unnecessary discussions [16, 17].
Recent unfavorable opinions have been expressed about
carbon accounting results in life cycle analysis that have not
considered direct and indirect effects of shifts in land-use
[18, 19]. However, these also have been set aside and considered irrelevant for the case of ethanol production from
sugarcane and molasses [17]. In fact, a huge trade between
southern and northern countries of fuel ethanol is becoming a
feasible future scenario based on certified production [17, 20,
21]. Brazil flexible ethanol program has been successful and
should be employed as model to follow [22–26].
Countries employing sugarcane for sugar production use
molasses in annexed plants as raw material for ethanol
production. However, there are also in operation in Brazil
autonomous installations where ethanol is produced directly
from harvested cane. Contrary to the bioethanol demand, the
sugar market is saturated, and the sudden price instabilities
and oscillations are caused by economic factors and short-term
shortages that occur at random due to natural disasters like
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440
hurricanes and drought periods that affect sugar plantations
worldwide.
Guatemala is producing ethanol from molasses in five
annexed plants; all of them export the product as no legislation
exists to make mandatory the use of the different bio-ethanolgasoline mixtures that are suitable for this purpose. Gasoline
consumption and sugarcane production in Guatemala is
around 1.7 109 L and 20 106 tons, respectively. Assuming
2.6 tons of molasses are available for each 100 tons of cane
processed, 50% of total sugars in the molasses, and 0.43
ethanol fermentation yield, the amount of ethanol obtained by
employing all the molasses would be 141.7 million liters. This
is not enough to meet the demand for a 10% mixture with
gasoline. Hence, direct utilization of sugarcane, or other
alternative crops, will be required to supply the rest, if this
scenario happens in the future. Sugarcane is usually harvested
according to parameters fixed by the sugar industry, which rest
on the premise of maximum sucrose content. As yeast is
capable of fermenting not only sucrose but also hexoses into
ethanol, the proper development stage to harvest sugarcane
directed only for ethanol production might be different than
the one for sugar production.
2
Materials and methods
2.1
Sugarcane and yeast strains
Two non-flowering cane varieties NA56 and PR752002 were
cultivated in ten rows, 10 m long and 1.5 m between rows, at
UVG-Proesur campus, Santa Lucia Cotzumalguapa, Escuintla,
situated in the Pacific lowlands at 300 m above sea level. To the
soil of each row, 32 kg of compost with 30% moisture content,
produced from a mixture of filter press mud (70%) and cane
bagasse (30%), were added initially as organic fertilizer. Two
Saccharomyces cerevisae strains were employed CBS 400 and
CBS 459 (Centraalbureau voor Schimmelcultures (CBS),
Utrecht) as explained in the text.
2.2
Eng. Life Sci. 2010, 10, No. 5, 439–445
C. Rolz and R. de León
pilot mill (Trapp TR-200). Both pulverized materials were
similar in physical appearance and represented a combined
sample of all internodes used. There were only two pulverized
samples for each DAP, one for each cane variety.
2.3
Ethanol production
Yeast was grown in a flask containing 30 g/L of Sabouraud
broth plus 1% sucrose for 48 h at 271C in a rotary shaker
(Incubator Shaker, Model 25, New Brunswick Scientific). The
contents were centrifuged at 101C and 4000 rpm (Eppendorf
Table-top Refrigerated Centrifuge Model 5804R). The yeast
biomass was once washed with distilled water. Then it was
suspended in distilled water to a known volume. A small
aliquot was used to obtain the yeast dry weight, by drying to
constant weight at 651C (Fisher Scientific Isotemp Incubator).
The rest was used to inoculate a known amount of pulverized
cane placed in a flask. The mean value of yeast added to each
flask was 63.8725.7 g of dry yeast per kg of dry cane particles.
At each DAP one sample of each cane variety was run in
duplicate for the two yeasts employed. As shown in Fig. 1 the
proportions used assured that the cane particles were initially
immersed in water, facilitating the extraction and the subsequent fermentation, or Ex-Ferm technique. The water-solid
ratio mean was 9.4571.60 mL per g of dry cane. The flasks
were kept for 72 h at 271C, a period of time three times longer
that the one previously found for complete sugar utilization
employing larger cane particles [27]. The longer time was
decided as it was suspected that in early cane sucrose, fructose
and glucose were not in a free state inside the sugarcane cells,
not easily extracted and not available to the yeast enzymes and
transport systems. The contents were filtered under vacuum
employing Whatman 1004-110 filter paper. During filtration
the solids were washed with distilled water. The liquid was
diluted to 250 mL. An aliquot was centrifuged at 101C and
4000 rpm and used to quantify ethanol by gas chromatography
and residuals sugars by high pressure liquid chromatography,
as explained below. The washed solids were discarded.
Cane sampling, handling and size reduction
Samples were taken at 98, 140, 168, 196, 222, 278, 307 and 335
days after planting (DAP). The samples consisted of three to
five stalks, chosen from different rows, cut at ground level. The
stalks were hand stripped of adhering top leaves and leaf
sheaths, weighed individually and their length recorded. The
stalks were sent immediately to UVG-Central campus at
Guatemala City, where they were kept at room temperature for
24 h and then processed. The second, third and fourth cane
internodes for each cane variety, counting from the bottom of
each stalk, were cut from the stalk manually employing a small
hand saw. The cut internodes from each stalk were mixed
together. With the first three cane samples, due to the stalk
relative fragility, the internodes were cut manually with a knife
into smaller circular pieces, which then were cut in half. These
were further pulverized employing a laboratory high-speed
cutting mill (IKA Works A11). For the rest of the samples the
initial internodes were fed directly to a hammer and impact
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Four of the eight flasks employed at 168 DAP at the
start of the fermentation. There were two flasks for each of the
two cane varieties and the two yeasts employed.
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Eng. Life Sci. 2010, 10, No. 5, 439–445
2.4
Converting sugarcane carbohydrates
Analytical methods
Cane moisture was determined by drying the pulverized
material at 651C until constant weight. Cane samples used for
initial sugar determination were prepared as follows.
Approximately 50 g of fresh pulverized material were mixed
with 250 mL of water in a beaker and brought to boiling
temperature, kept for 30 min and then left to cool. The
contents were filtered under vacuum employing Whatman
1004-110 filter paper. Individual sugars in the filtrate were
determined with an Agilent 1100 high pressure liquid chromatograph, an Agilent 1200 refractive index detector, a Zorbax
NH2, 25 cm long, 4.6 mm internal diameter column, employing acetonitrile in water (70–30), as the solvent phase. Ethanol
was quantified employing an Agilent gas chromatograph, with
an HP-Plot/Q, 30 m long, 32 mm internal diameter column.
Table 2. Physical changes data for the PR752002 cane variety
during growth and development expressed as the mean and
standard deviation of the cut stalks.a)
DAP
98
140
168
196
222
278
307
2.5
335
Statistical assessments
The cane stalk length, weight and water content data and the
ethanol production data were both analyzed by a two-way
analysis of variance as a function of DAP and cane variety
employing Statas Version 9.
3
Results and discussion
3.1
Physical and chemical changes during sugarcane
growth
The physical changes found during cane development and
growth are shown in Tables 1 and 2. The variation associated
with the mean for the three parameters measured, that is stalk
length, weight and water content, indicated that not all stalks
cut in any sample were similar in development and represented
Table 1. Physical changes data for the NA56 cane variety during
growth and development expressed as the mean and standard
deviation of the cut stalks.a)
DAP
98
140
168
196
222
278
307
335
a)
Stalk length (m)
Stalk weight (kg)
Water content (%)
0.8870.10
(0.11)
1.6870.18
(0.11)
2.3270.17
(0.07)
2.3070.39
(0.17)
2.4470.22
(0.09)
2.8670.12
(0.04)
2.8970.67
(0.23)
2.6270.38
(0.15)
0.4170.12
(0.29)
0.8770.15
(0.17)
1.6670.24
(0.14)
1.7670.15
(0.09)
1.7970.45
(0.25)
2.1470.16
(0.07)
2.0870.59
(0.28)
1.9070.44
(0.23)
87.5070.71
(0.0081)
78.4070.49
(0.0063)
79.0671.42
(0.0180)
77.2371.18
(0.0153)
73.2071.14
(0.0156)
69.5971.04
(0.0149)
68.8570.22
(0.0032)
69.5070.71
(0.0102)
The coefficient of variation is in parenthesis.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
441
a)
Stalk length (m)
Stalk weight (kg)
Water content (%)
0.7970.20
(0.25)
1.4670.31
(0.21)
1.9970.12
(0.06)
2.1270.15
(0.07)
2.3370.30
(0.13)
2.5570.32
(0.13)
2.4670.68
(0.28)
2.8170.16
(0.06)
0.4270.25
(0.60)
0.7870.15
(0.19)
1.0370.09
(0.09)
1.3170.20
(0.15)
1.5570.20
(0.13)
1.9670.37
(0.19)
2.1870.11
(0.05)
1.7970.40
(0.22)
87.1170.16
(0.0018)
79.3170.23
(0.0029)
79.0970.54
(0.0068)
72.5870.56
(0.0077)
72.9371.89
(0.0259)
69.1870.23
(0.0033)
67.4070.89
(0.0132)
66.4170.05
(0.0008)
The coefficient of variation is in parenthesis.
the natural variation found during sampling and in the field.
Stalk length and weight showed a larger variation than water
content as they were metrics obtained for each stalk. The
results for water content had a lower variation as it was done
on composite samples. Stalk length and weight increased with
DAP and water content decreased from 87 to below 70% for
both cane varieties. A two-way analysis of variance indicated
that the significant increase in weight with DAP found was
independent of the cane variety, and their interaction was
negligible. However for the stalk length increase the response
of the two cane varieties was significantly different
(p 5 0.0354), at any DAP value. Finally the decrease in water
content also was significantly different (p 5 0.0017) for the two
cane varieties, but in this case the interaction with DAP was
also significant (p 5 0.0066). In the literature differences in
stalk weight, length and water content among cultivars has
been reported in studies where comparison among cultivars
was the main objective. For example, Legendre and Burner
[28] found differences in stalk weight and dry matter biomass
between several cultivars and hybrids. Pammenter and Allison
[29] found significant differences in dry matter accumulation,
but not in stalk length increment for two cultivars.
The sugar accumulation and the amount of individual
sugars found during cane development and growth in the
combined samples at each DAP are shown in Figs. 2 and 3.
Total sugars increased and there was a significant sucrose
accumulation with maturation. On the other hand, glucose
and fructose concentrations, rich in young cane, practically
disappeared with DAP. However in the 335 DAP sample,
sucrose had decreased and both reducing sugars had increased.
Sucrose is photosynthesized in the leaf from CO2 and transported to the stalk, where it is either used to support growth
and plant respiration or is stored. Sucrose accumulation is a
complex metabolic process that involves transport between
different plant cell compartments, is the net result of
enzymatic synthesis and breakdown, and starts during stem
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442
Eng. Life Sci. 2010, 10, No. 5, 439–445
C. Rolz and R. de León
Figure 2. Sugar accumulation and individual sugars distribution
in samples of sugarcane variety NA56 at different DAP.
Figure 3. Sugar accumulation and individual sugars distribution
in samples of sugarcane variety PR752002 at different DAP.
elongation. However sucrose is constantly in a dynamic state,
subject to a complex cycle of breakdown and re-synthesis
[30–35]. Significant differences in sucrose content among
cultivars are commonly observed and are the main reason
behind breeding programs [36]. The decrease in sucrose
content in the last mature cane sample and the corresponding
increase in reducing sugars has been noted and reported in the
literature, although the specific DAP when this happens varies
according to the cane cultivar [37, 38] and could be considered
the onset of plant senescence. Overall the results obtained for
the physical and chemical changes just mentioned were in
concordance with the known concepts of sugarcane plant
physiology and growth [28, 39–41].
Figure 4. Ethanol production in liters per ton of fresh cane at
different DAP values for the two cane varieties and the two
yeasts employed. The error bars are standard deviations.
commercial cane varieties and S. cerevisaes strains. A plateau
was reached after 255 DAP and the maximum value between
300 and 325 DAP. The days necessary for the maximum may
vary depending on different growth periods. A two-way
analysis of variance indicated that the increase in ethanol
production with DAP found was dependent on the yeast strain
(p 5 0.0040) but not in the cane variety (p 5 0.3241). However
the three binary interactions between DAP, the cane variety
and the yeast strain were significant. During filtration of the
flask contents it was observed that the yeast CBS 422 showed
an attachment to the cane fibers during fermentation, as the
liquid fraction obtained was practically transparent. This was
not observed with yeast CBS 459 as the liquid fraction
obtained was very turbid. The physical attachment of ethanolic
yeasts to lignocellulosic fibers has been observed before and
recently it has been proposed as a method of yeast entrapment
[42]. In Fig. 4 it is easily seen that for both cane varieties the
curves for the strong attached yeast were always above the yeast
that remained mainly in suspension. The simultaneous sugar
extraction and fermentation, or Ex-Ferm technique [27]
functioned well in all pulverized cane samples. For all DAP
samples the mean value of Y (ethanol yield) was 0.3770.15 for
the NA56 cane variety and 0.3470.13 for the PR752002,
taking into account the values obtained for both yeast strains.
The maximum ethanol production figure at 307 DAP of 80 L
per ton of fresh cane by the yeast CBS 422 employing the
sugars present in the NA56 cane variety as substrate is
comparable to Brazilian average figures [21].
3.3
3.2
Sugar consumption
Ethanol production
In Fig. 4 the ethanol production data as a function of DAP are
shown for the two cane varieties and yeasts tested. The
observed increasing trend in production with DAP is what was
expected, as more carbohydrates were available to yeast due to
the cane stalk development. It is interesting to point out that
the trend shown by the four set of data is similar; hence it is
quite probable that the results are applicable to most
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Tables 3–5 present data on sucrose, glucose and fructose
consumption at the end of the fermentation as a function of DAP
and expressed as percentage of the initial content in the flask, for
the two cane varieties and the two yeasts tested. Sucrose was
completely consumed in all tests only in mature cane, that is, at
and after 278 DAP. Sucrose was poorly consumed in young cane,
as the data for the 98 DAP sample shows. Glucose was completely
consumed; with the exception of the sample at 168 DAP, where
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Eng. Life Sci. 2010, 10, No. 5, 439–445
Converting sugarcane carbohydrates
Table 3. Sucrose consumption during the extraction fermentation
trials expressed as percentage of the initial content in the flask.
DAP Sucrose
NA56
CBS 422
98
140
168
196
222
278
307
335
Sucrose
NA56
CBS 459
Sucrose
PR752002
CBS422
61.8571.05 63.0071.00 65.6671.68
99.4070.09 97.1170.02 99.4170.07
94.0870.60 85.9871.32 95.6870.03
98.5470.41 99.2370.14 99.4070.10
93.5970.15 89.5471.20 100.0070.00
100.0070.00 100.0070.00 100.0070.00
100.0070.00 100.0070.00 100.0070.00
100.0070.00 100.0070.00 100.0070.00
Sucrose
PR752002
CBS459
67.4372.18
99.1270.48
87.6571.35
99.5670.02
87.5270.58
100.0070.00
100.0070.00
100.0070.00
Table 4. Glucose consumption during the extraction fermentation
trials expressed as percentage of the initial content in the flask.
DAP Glucose
NA56
CBS 422
Glucose
NA56
CBS 459
98
140
168
196
222
278
307
335
100.0070.00
100.0070.00
87.5270.93
100.0070.00
100.0070.00
100.0070.00
100.0070.00
100.0070.00
100.0070.00
100.0070.00
89.4270.48
100.0070.00
100.0070.00
100.0070.00
100.0070.00
100.0070.00
Glucose
PR752002
CBS422
100.0070.00
100.0070.00
90.8570.41
100.0070.00
100.0070.00
95.3376.61
100.0070.00
100.0070.00
Glucose
PR752002
CBS459
100.0070.00
100.0070.00
89.1170.72
100.0070.00
100.0070.00
100.0070.00
100.0070.00
100.0070.00
Table 5. Fructose consumption during the extraction fermentation
trials expressed as percentage of the initial content in the flask.
DAP Fructose
NA56
CBS 422
Fructose
NA56
CBS 459
98
140
168
196
222
278
307
335
90.41713.56
69.7678.32
26.32710.49
88.37716.44
a)
100.0070.00
76.5470.18
28.4373.32
93.1479.70
a)
a)
66.01748.07 62.39753.19
a)
a)
a)
a)
Fructose
PR752002
CBS422
100.0070.00
76.6470.18
38.9774.07
95.3576.58
100.0070.00
54.45764.41
Fructose
PR752002
CBS459
100.0070.00
71.8875.48
35.4678.63
89.7671.42
a)
62.61752.87
a)
19.29741.16
a)
75.65734.43
Cases in which more fructose was analytically detected than the
original fructose quantity in the pulverized cane sample.
consumption values were close to 90%. The consumption of
fructose was erratic. It was totally consumed in young cane, in
which fructose and glucose relative concentrations were high.
However during stalk elongation or DAP values between 140 to
222 fructose consumption dramatically decreased. In mature
cane, more fructose was analytically detected at the end of the
fermentation than the fructose quantity contained in the initial
pulverized cane samples. The different experimental yeast utilization trends found for the three sugars need an explanation.
S. cerevisae strains show a preference for glucose over other
hexoses as a source of carbon and energy. Glucose uptake by the
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
443
cell is mediated by a family of hexose transporters, which also
transport fructose, although glucose is first depleted from the
fermentation medium. This discrepancy in uptake rates has been
shown to be not only strain dependent but also influenced by
external factors like ethanol and nitrogen concentrations and is
more pronounced at the end of any batch fermentation [43, 44].
In fact in wine fermentations, yeasts are forced to use fructose in
conditions of nitrogen starvation and high ethanol concentrations, which induce microbial stress and promote what is known
in the trade as stuck fermentations [45]. On the other hand, it is
presently accepted that the first step in sucrose utilization by
ethanolic yeasts is its complete hydrolysis into glucose and fructose by an extracellular invertase. Invertase is encoded by a family
of SUC genes, which also generate an intracellular constitutive
invertase. This membrane-bound enzyme breaks down sucrose
into its hexose components during active sucrose transport
directly into the cell, which is another uptake mechanism present
in ethanolic yeasts [46, 47]. These premises provide an explanation to our experimental results, which have shown the complete
glucose consumption in all the cane samples, irrespective of the
initial glucose concentration, amount conformed by the sum of
the naturally present in the cane particles and that generated by
sucrose hydrolysis. Fructose on the other hand, the amount
originally in the cane particles and that produced during
fermentation by sucrose hydrolysis, accumulated in the medium
possible due to the impairment of the fructose transport system;
hence more fructose was analytically detected at the end of the
fermentation than the fructose quantity contained in the initial
pulverized cane samples.
The partial sucrose consumption found with the early cane
samples, however, needs another explanation. Sucrose is
present in all plants and plays a series of vital roles that
promote plant growth and development, like the allocation of
carbon sources in different cell compartments and inducing
hexose-based sugar signals that control synthesis [48, 49]. In
sugarcane, sugar accumulation during growth and development is highly correlated with the activities of various enzymes
that breakdown sucrose: neutral and soluble acid invertases,
and enzymes that synthesize sucrose, sucrose-phosphate
synthase and sucrose synthase [50]. Sucrose is usually mobilized within the cell by sucrose transporters [51, 52]. It is then
possible that sucrose in the early cane samples was not totally
available to the yeast fermenting action, as it was being
transported within the cell by the transporter molecules. In
mature cane however, sucrose had accumulated during growth
within the cell as a storage reserve and was totally available to
the yeast enzymes. This reasoning explains our results satisfactorily but no experimental proof is provided. It is proper to
point out that in actual practice only mature sugarcane will be
used for any biotechnological process so that the experimental
finding relating sucrose consumption in early sugarcane
previously discussed has no immediate practical application.
4
Concluding remarks
As far as we could detect, there were no previous reports in the
literature regarding ethanol production data directly from
sugarcane at different growth stages until maturation and
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444
C. Rolz and R. de León
senescence. The sample at 307 DAP gave the maximum
experimental ethanol yield per ton of fresh cane for the two
non-flowering cane varieties tested, although a plateau might
exist between 300 and 325 DAP. These cane varieties in
Guatemala are usually harvested around 350 DAP for
commercial sugar production. Hence a difference of approximately 25 days less in the field might be needed for maximum
ethanol production. On the other hand, it is clear from the
data that cane harvesting for ethanol production should not be
done after the recommended DAP for commercial sugar
production. If this is done, the excess fructose present will not
be completely utilized by yeast. The difference in harvest times
will not dramatically change current agricultural practices,
which include one planting and three to four ratoon crops in
close correlation with the start of the rainy season.
It was found that fructose was the hexose that remained in
the medium at the end of the fermentation. Hence it is a
critical parameter to minimize when selecting yeast varieties
for ethanol production from sugarcane. Finally it was found
that yeasts showing strong attachment to sugarcane fibers
showed better ethanol yields in all the DAP interval.
Acknowledgements
This work was partially financed by Project FONACYT 094-2006
of the Guatemalan National Science Council (CONACYT). The
authors appreciate the chromatographic analysis done by Maria
del Carmen Samayoa and Fabiola de Micheo and acknowledge
the technical help in preparing the samples and conducting the
experiments by Carlos Arias. The critical remarks and useful
suggestions of three anonymous reviewers are also appreciated.
Conflict of interest
The authors have declared no conflict of interest.
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