Professional Documents
Culture Documents
Colombia.
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
-1-
Abstract
Background
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
-2-
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].
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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
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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
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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
-8-
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
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
- 15 -
- 16 -
- 17 -
- 18 -
Figure 6 - Response surface graphs and contour showing effect cell, sugar and
bioethanol concentration; from with SJ (a), SM (b).
- 19 -
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
- 20 -
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.
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
- 21 -
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%.
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
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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