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Kinetic analysis of bioethanol production from by

sugar fermentable fermentation derivativeed offrom


sugarcane byproductsby-products
Bianca Yadira Prez-Sariana1*, Aln Rogelio Gmez-Rincn2, Sergio SaldaaTrinidad2, Peggy Elizabeth Alvarez-Gutirrez2, Sebastian P. J. 1, Carlos Alberto
Guerrero-Fajardo3

Instituto de Energas Renovables-UNAM, Temixco, Morelos, 62580, Mxico

Universidad Politcnica de Chiapas, Tuxtla Gutirrez, Chiapas. 29010, Mxico

Departamento de Qumica, Universidad Nacional de Colombia, Bogot 11001,

Colombia.

*These authors contributed equally to this work

Corresponding author

Email addresses:
BYPS: bipes@ier.unam.mx, biyapesa@gmail.com,
ARGR: tool_alan@hotmail.com
SST: ssaldana@upchipas.edu.mx
PEAG: peggy.alvarez@hotmail.com
SPJ: sjp@ier.unam.mx
CAGF: caguerrerofa@unal.edu.co

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Abstract
Background

Bioethanol is considered as an alternative energy source that is renewable and


environmentally friendly, it can be produced from agricultural raw materials with high
sugar content. It can be obtained from common crops such as sugarcane (Saccharum
officinarum). Microorganisms such as yeast can produce ethanol through anaerobic
fermentation by conversion of glucose in bioethanol and carbon dioxide.
Results

Experimental design 2k factorial with central points was used with the substrate and
cell concentration as a variable with three levels for each parameter. This design was
performed to establish the formulation of media and fermentation conditions.
Our results show the kinetics of fermentation from sugarcane juice and sugarcane
molasses as substrate. BiomassCell concentration, bioethanol production, sugar
substrate consumption were analysed, the physical-chemical variables such as pH,
temperature were monitored. The model predicted that the maximum production of
bioethanol was using sugarcane juice to cell concentration of 1.2e+7 cfu/mL of yeast,
sugar concentration of 120.79 g/L of sugar, producing 40 g/L of bioethanol with a
yield of sugar consumption to bioethanol of 37.97%. Under these conditions,
experimental bioethanol production was 44.41 g/L and 38.35% of yield.
Conclusions

The evaluation of sugars fermentation Fermentation evaluation of sugars from


sugarcane juice and sugarcane molasses, allowed to do statistical analysis and
optimize the bioetanol production.

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Background
An alternative to fuel demand is the sustainable use of biomass conversion for energy
(bioenergy). Bioenergy has the potential to become an important part of sustainable
energy systems, contributing to reducing reduce emissions of greenhouse gases,
promoting sustainable development and may could gradually replace fossil fuels [1].
Ethanol derived from biomass is one of modern forms of bioenergy, it has the
potential to be a sustainable transport fuel and a fuel oxygenate that it can replace
gasoline [2].
There are several methods for transforming biomass into energy, the most widely used
is dare the thermochemical and biological processes [4]. The thermochemical heat is
usedused as an energy source of by biomass conversion and can be of three types:
combustion, pyrolysis and gasification.
Biological methods are based on the use of various types of microorganisms such as
bacteria, molds and yeasts, which degrade the molecules to simpler compounds of
high energy density, the best known is the alcoholic fermentation. In the literature
there are several types of microorganisms that produce biofuels, such as biodiesel, by
Rhodococcus opacus [5], E. Coli [6], Cyanobacteria and Microalgae among others.
The biohydrogen can be produced by Cyanobacteria and Microalgae [7] and mixed
acid organisms [8]. The biogas can also be produced by Cyanobacteria and
Microalgae [7]. Isobutanol may be produced by E. Coli [9] and others. And
bioethanol can be produced by Cyanobacteria and Microalgae [7], Clostridium [10],
Pichia stipitis [11], Laminaria digitata [12], Ceriporiopsis subvermispora [13],
Geobacillus [14], Saccharomyces cerevisiae, among others.
Saccharomyces cerevisiae is a microorganism producer of bioethanolAmong the
producers of bioethanol microorganism is the Saccharomyces cerevisiae, it is the most

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commonly used for fermentation, and like many organisms metabolizes glucose via
Embden-Meyerhof [15].
The aim of this study was to evaluate the fermentation of sugars to produce bioethanol
from sugarcane juice and sugarcane molasses, to do statistical analysis and to
optimize the system variables [3].

Results and discussion


Growth kinetic of the yeast S. cerevisiae Y2034.

Microbial growth explains because increase or decline of bioethanol production, in


other words: carbohydrates consumption is associated with specific growth rate and
bioethanol production rate. In a submerged culture of microorganisms, it can be
differentiated at least four phases: lag phase or adjustment, exponential phase,
stationary phase and death phase [16].
In exponential phase, number differential of cells with respect to time, is equal to
specific growth rate () for the number of cells, in stationary phase the number of
cells with respect to time is equal to zero and in death phase there is a reduction of
viable cell, this reduction is represented for k [17].
Growth kinetic of yeast S. cerevisiae with 50 and 150 g/L initial sugar from sugarcane
juice (SJ) is displayed in Figure 1. In graph a can be observed lag phase from 0 to 6h,
exponential phase lasted 12 h (6 to 18 h), where was 0.30 h-1 (), 0.281 h-1 () and
0.280 h-1 (). Death phase was comprised between 18 and 24 h where k was -0.080,
-0.034 and -0.018 for 1e+7, 2e+7 and 3e+7 cfu/mL initials cell concentration. Graph
b, shows lag phase from 0 to 6 h, exponential phase was 12 h (6 to 18 h), where was
0.404 h-1 (), 0.321 h-1 () and 0.332 h-1 (). Death phase was 18 to 24 h where k
was -0.050, -0.004 and -0.012 for 1e+7, 2e+7 and 3e+7 cfu/mL initials cell
concentration, respectively.

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Growth kinetic of yeast S. cerevisiae with 200 g/L initial sugar from SJ is showed in
Figure 2. It can be observed lag phase from 0 to 6 h, exponential phase lasted 12 h (6
to 18 h), where was 0.248 h-1, 0.248 h-1 and 0.224 h-1 for 1e+7, 2e+7, 3e+7 cfu/mL
initials cell concentration, respectively. Neither death phase was observed nor it could
see stationary phase due to long time intervals.
Comparing Figure 1 to Figure 2 was observed that in the graph a, the exponential
phase was higher than in the graph b, although in Figure 2 there was no death phase,
but it was obtained maximum number of cells, approximately 1.1e+10, 1.4e+10,
1.6e+10 to 1e+7, 2e+7 and 3e+7 cfu/mL initials cell concentration. This was because
of there was highest sugar concentration therefore fermentation would take longer.
Growth kinetic of yeast S. cerevisiae with 50 and 150 g/L initial sugar from sugarcane
molasses (SM) is displayed in Figure 3. In graph a can be observed that lag phase
from 0 to 6 h, exponential phase lasted 12 h (6 to 18 h), where was 0.260 h-1 (),
0.246 h-1 () and 0.239 h-1 (). Death phase was 18 and 24 h where k was -0.013,
-0.012 and -0.170 for initial cell concentrations 1e+7, 2e+7 and 3e+7 cfu/mL.
Graph b in Figure 3 was observed lag phase from 0 to 6 h, exponential phase was 12 h
(6 to 18 h) where was 0.340 (), 0.333 () and 0.337 (). Death phase was 18 to
24 h where k was -0.085, -0.023 and -0.012 for initial cell concentrations of 1e+7,
2e+7 and 3e+7 cfu/mL, respectively.
Growth kinetic of yeast S. cereviasie with 200 g/L initial sugar from SM is showed in
Figure 4. It can be observed that the adaptation phase was in a time interval from 0 to
6 h, exponential phase lasted 12 h (6 to 18 h) where was 0.007, 0.008 and 0 to 1e+7,
2e+7 and 3e+7 cfu/mL initial cells concentration, respectively. The death phase was
not observed.

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Comparing Figure 3 to Figure 4 shows that in the graph a, exponential phase was
higher than the graph b, although in phase Figure 4 there was no death phase, but a
maximum number of cells, approximately 4.2e+10 (), 4.5e+10 () and 5.3e+10
() to 1e+7, 2e+7 and 3e+7 cfu/mL initial cells concentration respectively. This was
because of there was highest sugar concentration therefore fermentation would take
longer.
Consumption kinetics of sugars

Sugar consumption from SJ


In Figure 1 sugar consumption with 50 g/L initial sugar was 47.68 g/L, 47.72 g/L and
47.68 g/L; with 125 g/L initial sugar was 110.51 g/L, 110.48 g/L and 110.30 g/L, and
with 200 g/L initial sugar (Figure 2) was 183.21 g/L, 183.99 g/L and 183.53 g/L to
1e+7, 2e+7 and 3e+7 cfu/mL cell initial concentration, respectively.
Sugar consumption from SM
Figures 3 sugar consumption with 50 g/L initial sugar was 46.67 g/L, 46.63 g/L and
46.67 g/L; with 125 g/L was 112.53 g/L, 112.47 g/L and 112.13 g/L, and 200 g/L
initial sugar (Figure 4) was 184.54 g/L, 183.47 g/L and 184.51 g/L to 1e+7, 2e+7 and
3e+7 cfu/mL initial, respectively. As we can observed sugar consumption was a
higher than 89%.
Effect of sugar concentration on the rate of net growth in the yeast S.
cerevisiae Y2034

In the reactions no catalyzed the product formation by microorganisms depends


linearly on concentration of added substrate. While that catalyzed reactions by
microorganisms, usually a hyperbolic dependence of rate is obtained relative to
substrate concentration, distinguishing itself three different parts of curve. In first part,
at low substrate concentrations, rate is proportional to substrate concentration, such
that if substrate concentration is doubled, the velocity is doubled (first order reaction).

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The second part of curve at intermediate substrate concentrations, the rate increases is
less than first part with increasing concentration, starting around the half of the
maximum speed. Last part of the curve, at high substrate concentrations, the, rate is
independent of substrate concentration (zero order reaction), and so the rate achieved
is close to the maximum [17].
Figure 5, graph a showed the effect of substrate sugar concentration to SJ on net
growth rate of the yeast, which was 0.251 h-1, 0.260 h-1 with 1e+7 cfu/mL, 0.254 h-1,
0.275 h-1 with 2e+7 cfu/mL and 0.251 h-1, 0.295 h-1 with 3e+7 cfu/mL from 50 g/L to
125 g/L of sugar concentration. But from 125 to 200 g/L of sugar ubstrate
concentration, growth rate remains constant.
Figure 5, graph b showed the effect of the concentration of substratesugar
concentration to SM on net growth rate of the yeast. The bBehaviorsbehaviours of
these curves showed that from 50 g/L to 125 g/L the growth rate for were 0.250 h-1,
0.265 h-1 to 1e+7; 0.283 h-1, 0.317 h-1 to 2e+7 cfu/mL; 0.281 h-1, 0.308 h-1 to 3e+7
cfu/mL. But from 125 to 200 g/L growth rate remains constant, which means that at a
concentration greater than 125 g/L, rate was independent of the sugar concentration.
Summary of all results is shown in Table ??????
Statistical analysis

The statistical significance of the corresponding model equation was checked by F


test analysis of variance (ANOVA, Table 2 and 3).
The adequacy of the models was expressed by the coefficient of determination R2,
which proved to be 1 and 0.9998 for the production of bioethanol from SJ and SM,
respectively. These values indicate 100% of the variability of response in the
production of bioethanol from SJ and 99.98% of the variability of response in SM.

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The lower the coefficient of variation (CV) (0.12% for bioethanol production from SJ
and 0.62% to SM), the greater the accuracy and reliability of the experiments carried
out. Chance probability for models less than 0,00501 was also noted that models were
highly significant and no significant chance of lack of fit for models indicate that the
experimental data obtained are in good agreement with the model. TIf the value of
testing the lack of fit for the model was significant (P-value < 0.05) to X1, X2, X1X2,
X22 to SJ and X2 to SM.
Optimization of production of bioethanol

Figure 6 and 7 showed that 3D graphics for response surface plotted the regression
equation. Using the response surface plots, the interaction between two variables and
their optimal level are easy to understand and locate. Graphs a and b show interaction
between cell concentration and sugar concentration respect bioethanol production and
yield.
Yields of sugar consumptionversion to bioethanol are shown in Figure 7, results
showed that optimum level (Table 4) was observed near the initial value of biomass
concentration and near the central value of sugar concentration. Myers and
Montgomery describe a multiple response method called desirability [18]. The
method makes use of an objective function D; it reflects the desirable ranges for each
response (di). The desirable range is from zero to one (from least to most desirable,
respectively). This work was obtained 0.952 of desirability for SJ, and 0.943 for SM.
Culture in bioreactor

The optimum condition using sugarcane juice with 1.2e+7 cfu/mL initial cell
concentration and 120.79 g/L initial sugar concentration was evaluated using a
bioreactor, experimental bioethanol production was 44.41 g/L with a conversion yield
of sugar consumption to bioethanol of 38.35% (Figure 8), in this process the total

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consumption of sugars was 115.79 g/L. It may be noted that the yeast metabolized
95%.

Conclusions
It is Eestablished a fermentation processes that allowed the construction of the growth
kinetics of Saccharomyces Cerevisiae Y2034 and their effect with sugar concentration
to find the optimal conditions for bioethanol production.
The result of statistical analysis we found that the best theoretical condition for
bioethanol, was using sugarcane juice with 1.2e+7 cfu/mL initial cell concentration,
120.79 g/L initial sugar concentration, 40 g/L of bioethanol production. Under these
conditions bioethanol production was 44.41 g/L and 38.35% of conversion yield.
The oOptimization was an alternative to improve bioethanol production in sugarcane
juice.
Through the growth kinetics of the yeast Saccharomyces cerevisiae Y2034, it was
determined that the best substrate for cells production was sugarcane molasses.

Methods
Substrate

The sugarcane juice (SJ) was extracted from a local extractor, while the sugarcane
molasses (SM) was obtained from local sugar milltalent in the state. SJ had total
soluble solids concentration of 27 Bx and SM had 83 Bx. Both substrates were used
as basis for the preparation of the fermentation medium, and these were supplemented
with salts: 0.02 g/L magnesium sulfatesulphate (MgSO47H2O), 0.2 g/L ammonium
phosphate ((NH4)2SO4) and 2 g/L yeast extract [19].
Microorganism

The yeast Saccharomyces cerevisiae Y2034 was kindly donated the ceparium
Biotechnology Laboratory of Universidad Politcnica de Chiapas. The strain was

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maintained in YPD agar, (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose,
and 2% w/v agar) kept slants at 4C. YPD liquid is used as inoculum shaken at 150
rpm, 30 C during 24 h.
Fermentation process

Fermentation process was performed in triplicate with the two substrates in 250 mL
flasks with constant agitation at 180 rpm, 5 pH [20] and 30 C temperature [21]. The
final volume used for the fermentations was 150 mL being stirred for 24 h.
For validation experiments with optimum conditions was performed with a Bioreactor
Applikon using 2.5 L of SJ in a 3 L bioreactor (Applikon, Foster City, CA) equipped
with two six-blade Rushton turbines. The pH was monitored using an autocleavable
electrode (Applikon) and controlled at 5 0.48 by a Bioconsole ADI
1035/Biocontroller 1010 (Applikon). The experiments were performed at 30 C and
stirred at 180 rpm.
Analytic methods

The sugars concentration was measured with for Millers method [22]. The count of
viable cells was determinated with Neubauers Chamber adapted to an optical
microscope; the trypan blue was used as dye of viable cells [23]. To bioethanol
concentration were sampled in the aqueous phase, centrifuged at 5000 rpm for 5 min
at 5 C, supernatant was changed to a new tube [24] and the precipitate was discarded.
Samples were analysed by gas chromatography Agilent Technologies 6850 with data
acquisition system with software A.02.01 Agilent Cerity.
For validation experiments with optimum conditions, YSI 2700 SELECT equipment
Biochemistry analyzer was used in manual mode and a membrane (enzyme oxidase
alcohol immobilized) to ethanol 2786 with software included, the equipment was
calibrated with a standard solution of ethanol 2 g/L. Culture samples of 1 mL were

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taken every 3 h and centrifuged at 5000 rpm for 5 min at 5C. The supernatant was
filtered through a 0.22 m filter (Millipore, Bedford, MA, USA).
Experimental design

Experimental design 2k factorial with central points was used to optimizeThe


experimental design with two input variables; initial cell concentration, initial sugar
concentration, and two output variables; bioethanol concentration and yield of sugar
consumption to bioethanol yield conversion of sugar to bioethanol from SJ and SM,
each variable with three levels (Design Expert 7.0.0 software). The experimentals
designed and is shown in Table 5.

Competing interests
The authors declare that they have no competing interest.

Authors' contributions
BYPS performed the experiments, analyzed the results and prepared of the
manuscript. ARGR carried out validation of optimum conditions. SST participated in
the experimental design and fermentation process. PEAG participated in the
microorganism culture and substrate characterization. SJP coordinated the study and
revised the manuscript. CAGF helped to structure and draft the manuscript. All
authors read and approved the final manuscript.

Acknowledgements
This work was supported by Science and Technology Council 100212 to the project
PAPIIT 103410. We would like to thank M.B Jos Raunel Tinoco of the Instituto de
Biotecnologa, and Dr. Alejandro Tllez Jurado of the Universidad Politcnica de
Pachuca, for the crucial technical support during this work.

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Figures

Figure 1 - Growth kinetic of yeast S. cerevisiae Y2034 and consumption sugar


kinetic from SJ with 50 g/L (a) and 150 g/L (b) ( 1e+7, 2e+7 and 3e+7
cfu/mL).

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Figure 2 - Growth kinetic of yeast S. cerevisiae Y2034 and consumption sugar


kinetic from SJ with 200 g/L ( 1e+7, 2e+7 and 3e+7 cfu/mL).

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Figure 3 - Growth kinetic of yeast S. cerevisiae Y2034 and consumption sugar


kinetic from SM with 50 g/L (a) and 150 g/L (b) ( 1e+7, 2e+7 and 3e+7
cfu/mL).

Figure 4 - Growth kinetic of yeast S. cerevisiae Y2034 and consumption sugar


kinetic from SM with 200 g/L ( 1e+7, 2e+7 and 3e+7 cfu/mL).

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Figure 5 - Effect sugar concentration on rate net growth in yeast S. cerevisiae


Y2034, SJ (a) SM (b) ( 1e+7, 2e+7 and 3e+7 cfu/mL).

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Figure 6 - Response surface graphs and contour showing effect cell, sugar and
bioethanol concentration; from with SJ (a), SM (b).

Figure 7 - Response surface graphs and contour showing effect cell


concentration, sugar concentration and yield; from with SJ (a), SM (b).

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Figure 8 - Growth kinetic bioethanol production () and consumption sugar


kinetic () from optimum conditions.

Tables
Table 1. Summary results.

SJ

Factor 1

Factor 2

(cfu/mL)

(g/L)

3.0e+07

50

0.254

3.0e+07

125

0.251

3.0e+07

200

0.271

2.0e+07

50

0.251

(h-1)

2.0e+07

125

0.295

2.0e+07

200

0.297

1.0e+07

50

0.262

1.0e+07

125

0.302

dt
(h)
2.71
9
2.55
1
2.55

Ks

SM

(h-1)

(h-1)
0.281

4.715

0.279

0.317

8
2.75
2
2.54
9
2.37

0.308

0.283
11.50
8

0.309

0.317
0.324

0
2.64

0.290

4
2.29

12.06

9
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0.326

0.335

dt
(h)
2.19
0
2.18
8
2.01

m
Ks

(h-1)

8.795

0.330

0
2.19
0
2.17
0
2.07

10.44
3

0.342

0
2.56
0
2.65

12.84

0.365

1.0e+07

200

0.303

2.28

0.340

2.45

1
0
Factor 1: cell concentration, Factor 2: sugar concentration, : specific growth rate, dt:
doubling time, Ks: saturation constant, m: maximum growth rate.

Table 2. Variance analysis to for bioethanol production fromof sugarcane juice.

Source

Estimated

Sum of

Degree

Mean

coefficient

squares
1630.0631

freedom
5

F-value P-value
square
326.0126 133008.4 0.0001

Model
Intercept
X1
X2
X1X2
X12

41.4200
0.8300
16.2600
0.6700
0.2100

4.1168
1585.3502
1.8225
0.1228

1
1
1
1

4.1168
1585.350
1.8225
0.1228

1679.601
646799.9
743.5536
50.1015

0.0001
0.0001
0.0001
0.0002

X22

-3.5400

34.5942

34.5942

14113.94

0.0001

Residual
0.0172
7
0.0025
Lack of Fit
0.0172
3
0.0057
Pure Error
0.0000
4
0.0000
Corr. Total
1630.0
12
X1: cell concentration, X2: sugar concentration, F: Fisher test, P-value: probability
distribution value. The correlation coefficient (R2) was 1, adjusted correlation
coefficient was 0.99 and coefficient of variation was 0.12%.
Table 3. Variance analysis to for bioethanol production fromof sugarcane
molasses.

Source
Model
Intercept
X1
X2
X1X2
X12
X22
Residual
Lack of Fit
Pure Error
Corr. Total

Estimated

Sum of

Degree

coefficient

squares
1700.8963

freedom
5

0.0323
1699.8300
0.6006
0.4024
0.1635
2.1143
2.1143
0.0000
1703.010

1
1
1
1
1
7
3
4
12

37.0162
0.0733
16.8317
-0.3875
-0.3817
0.2433

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Mean

F-value
square
340.1793 1126.241

P-value

0.0323
1699.830
0.6006
0.4024
0.1635
0.3020
0.7048
0.0000

0.7533
0.0001
0.2013
0.2863
0.4859

0.1068
5627.675
1.9885
1.3324
0.5412
0.0000

0.0001

X1: cell concentration, X2: sugar concentration, F: Fisher test, P-value: probability
distribution value. The correlation coefficient (R2) was 0.99, adjusted correlation
coefficient was 0.98 and coefficient of variation was 0.62%.

Table 4. Optimum parameters.

Substrate

Factor 1

Factor 2

Response 1

Response 2

Desabirility
(cfu/mL) (g/L)
(g/L)
(%)
SJ
1.2e+7
120.79
40
37.97
0.95
SM
1.2e+7
140.13
40
32.12
0.94
Factor 1: cell concentration, Factor 2: sugar concentration, Response 1:
bioethanol production, Response 2: conversion yield

Table 5. Design layout

Tests

Factor 1

Factor 2

Response 1 (g/L)

Response 2

(cfu/mL)

(g/L)

SJ

SJ

SM

SM

1
3.0e+07
200
55.83
52.75
30.92
30.83
2
2.0e+07
125
41.43
36.94
37.81
33.74
3
1.0e+07
50
21.73
20.04
47.57
44.87
4
3.0e+07
50
22.03
20.29
48.24
45.44
5
2.0e+07
50
21.53
20.19
47.10
45.24
6
2.0e+07
125
41.43
36.94
37.81
33.74
7
3.0e+07
125
42.43
37.57
38.78
34.31
8
2.0e+07
125
41.43
36.94
37.81
33.74
9
2.0e+07
200
54.16
54.71
29.92
30.82
10
1.0e+07
200
52.83
54.050
29.31
30.44
11
2.0e+07
125
41.43
36.940
37.81
33.74
12
1.0e+07
125
40.76
36.08
37.19
33.06
13
2.0e+07
125
41.43
36.940
37.81
33.74
Factor 1: cell concentration, Factor 2: sugar concentration, Response 1: bioethanol
production, Response 2: yield

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