Biochemical Engineering Journal 119 (2017) 4251

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Effect of temperature on sugarcane ethanol fermentation: Kinetic

modeling and validation under very-high-gravity fermentation
conditions
Elmer Ccopa Rivera a,b, , Celina K. Yamakawa a , Marcelo B.W. Saad a , Daniel I.P. Atala c ,
Wesley B. Ambrosio c , Antonio Bonomi a,b , Jonas Nolasco Junior a , Carlos E.V. Rossell a
a
Laboratrio Nacional de Cincia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Caixa Postal 6192, CEP
13083-970, Campinas, So Paulo, Brazil
b
School of Chemical Engineering, State University of Campinas, P.O. Box 6066, 13081-970, Campinas, So Paulo, Brazil
c
British PetroleumBiofuels, CEP 04516-000 So Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In this work, a mechanistic model is developed to simulate the effect of temperature on Saccharomyces
Received 19 July 2016 cerevisiae growth and ethanol production of batch fermentations. A wide temperature range is used
Received in revised form 2 December 2016 to estimate the temperature-dependent kinetic parameters of the reaction kinetics. Because multi-
Accepted 4 December 2016
parameter estimation problems are complex, an optimization-based procedure is used to determine the
Available online 6 December 2016
optimum parameter values. The calculated reaction rates are used to construct a mechanistic fed-batch
model. Experimental data from several cycles of very-high-gravity (VHG) ethanol fermentation from
Keywords:
sugarcane are used to validate the model. Acceptable predictions are achieved in terms of the residual
Mathematical modeling
Parameter estimation
standard deviation (RSD). In addition, a suitable fermentation temperature prole, nutrient supplemen-
VHG ethanol fermentation tation and micro-aeration during cell treatment are essential factors to obtain a yield of up to 90%, with
Temperature a productivity of 10.2 g/L h and an ethanol concentration of 120 g/L.
Sugarcane 2016 Elsevier B.V. All rights reserved.

1. Introduction
Currently, ethanol fuel from sugarcane is used on a large scale as
hydrous ethanol in vehicles powered by ethanol or gasoline, also
referred to as ex-fuel vehicles. Additionally, this fuel is used as
an anhydrous ethanol blend with gasoline. The ethanol industry
in Brazil processes approximately 630 million tons of sugarcane to
produce 35.4 million tons of sugar, 28.5 million m3 of ethanol, and
32,300 GWh of electric power annually [1] and employs approxi-
mately 4.5 million people [2]. As a result, this industry continues to
present challenges regarding the increasing concern about environ-
mental impacts, primarily climate change effects and the reduced
dependence on fossil fuel resources.
The ethanol industry predominantly uses fed-batch fermenta-
tion with cell recycling; 7080% of distilleries utilize this mode of
operation to produce ethanol [3]. In this conguration, 99.5% of the
cells are reused in sequential fermentation (intensive recycling).
The high cell density inside the bioreactors contributes to reducing
Corresponding author at: School of Chemical Engineering, State University of
Campinas, P.O. Box 6066, 13081-970, Campinas, So Paulo, Brazil.
E-mail address: elmeralbertocr@gmail.com (E. Ccopa Rivera).
the fermentation time to 611 h and to increasing the ethanol yield
to 9092%. The nal ethanol concentration varies between 8 and
11 GL [4]. However, this current fermentation technology is lim-
ited in processing substrates at a concentration of up to 200 g/L TSAI
(total sugars as invert in which the original sucrose is equivalent
to 0.95 of the reducing sugars formed (glucose and fructose) and
glucose and fructose originally present), representing the poten-
tial of obtaining an ethanol concentration of at least 11 GL. The
increased amount of sugars and ethanol in conventional fermenta-
tion causes a decrease in the cell maintenance rate, thus reducing
the percentage of cell viability and stuck fermentation [5].
One method to improve the current ethanol fermentation prof-
itability is through process intensication. Very-high-gravity (VHG)
technology is one type of improvement process aimed at obtaining
high ethanol concentrations of up to 15 GL from moderately high
sugar concentrations (>250 g/L) [6]. Furthermore, VHG technology
enables reduction of the process water requirements, thus reduc-
ing the related distillation operational cost, vinasse generation and
its treatment cost, resulting in signicant energy savings [7].
Over the course of VHG fermentation, a cell encounters signi-
cant stress induced by osmotic pressure, which leads to variations
in the fermentation kinetics. Furthermore, the level of ethanol in the
late stage of the process depends strongly on the nutritional condi-

http://dx.doi.org/10.1016/j.bej.2016.12.002
1369-703X/ 2016 Elsevier B.V. All rights reserved.
 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251
tions for cell maintenance [8]. Another common source of kinetic
uctuation could be related to the variability of feedstock quality,
which is caused by the quality of molasses (related to the level of
exhaustion and sultation in the sugar production) and the quality
of sugarcane juice, which may vary during the sugarcane season,
depending on the variety, harvest period, climate conditions and
juice extraction procedure [9].
Rossell et al. [10] recently developed a novel continuous VHG
ethanol fermentation by enhancing the engineering design and
operation process. Ethanol fermentation in multistage bioreactors
with a well-dened temperature prole was considered in this
technology. Higher temperatures were considered in the initial
stages to maximize conversion, and lower temperatures were con-
sidered in the later stages to minimize the inhibition and cellular
damage from the high ethanol concentration. In addition, a dis-
tributed carbon source feeding was considered in the rst and
second stages to avoid inhibition by high sugar concentrations.
The process also included intracellular detoxication through a sec-
ond centrifugation followed by a cell reactivation stage to promote
membrane recovery and enzymatic restoration. The primary goal of
this technology was to maintain active and viable cells for an entire
harvest season, which is a critical condition to provide operating
stability and a high rate of sugar conversion to ethanol.
The primary challenge in the VHG fermentation process is to
determine the effect of temperature as well as high substrate and
ethanol concentrations on cell growth kinetics, which clearly affect
the performance of the fermentation process. Although different
empirical studies were developed for improving VHG processes,
they lack a systematic model-based approach. A realistic kinetic
model is required for the design, optimization and control of the
process.
Temperature is a crucial operating parameter due to its inu-
ence on the conversion of sugars to ethanol, which is an exothermic
reaction, i.e., heat is released. In the case of conventional fermen-
tation, a temperature control is required, often in the range of
3235 C [4]. For VHG fermentation, a quantitative understanding
of the effect of temperature on substrate consumption and ethanol
production rates must be investigated to dene the most suitable
operating conditions using the denition of optimum temperature
proles. Previous work [11] has addressed the effect of temperature
on ethanol production from sugarcane by S. cerevisiae, resulting
in the formulation of a mechanistic model, which includes terms
for the cell, substrate and ethanol inhibitions. The results reported
that the maximum specic growth rate increases as the temper-
ature increases. After approximately 37 C, the maximum specic
growth rate begins to decrease. The maximum ethanol and cell con-
centrations, i.e., the concentrations at which cell growth ceases, are
inversely related to the fermentation temperature. This model was
validated during batch fermentation under conventional fermen-
tation conditions.
This study investigated the effect of temperature on ethanol
production from sugarcane. For this purpose, a mechanistic
fermentation model was developed, with kinetic parameters deter-
mined using a model-based optimization algorithm. The model was
validated through experiments, including fed-batch fermentation
with cell recycling, operating under VHG conditions. The proposed
methodology drives the systematic development of an industrially
reliable mathematical model for VHG fermentation.
2. Material and methods
2.1. Microorganism
This study used an unclassied Saccharomyces cerevisiae strain
cultivated in the Bioprocess Development Laboratory at CTBE,
43
provided by the Faculty of Food Engineering/State University of
Campinas and originally obtained from the Santa Adlia sugarcane
mill. The stock culture was maintained in YPD (10 g/L yeast extract,
20 g/L peptone and 20 g/L dextrose) at 80 C with 30% (v/v) glyc-
erol.
2.2. Batch fermentations
A stock culture was activated in liquid YPD medium at 33 C
under agitation (250 rpm) for 24 h on an Innova 44 orbital shaker
(New Brunswick, NJ, USA). Then, a sample was streaked onto
YPD agar plates, incubated at 33 C for 48 h, and stored at 5 C.
The inoculum for all fermentations was prepared by transfer-
ring three loops from agar plates to a new semi-synthetic liquid
medium. The medium consisted of 2.30 g/L urea, 6.60 g/L K2 SO4 ,
3.0 g/L KH2 PO4 , 0.50 g/L MgSO4 7H2 O, 1.0 g/L CaCl2 2H2 0, 5.0 g/L
yeast extract, 25.44 mg/L trace elements (as detailed in Basso [12]),
3.0 ppm thiamine and 80.0 g/L TSAI. Then, the cells were incu-
bated at 33 C under agitation (250 rpm) for 12 h on the Innova 44
orbital shaker. Commercial crystal sugar was used as the source
of TSAI. After the inoculation period was complete, the cells were
recovered using a Beckman Avanti J-26 XP centrifuge (JLA-16.250
rotor, 5509 g, 10 C, 15 min). The cells were diluted with sterilized
potable water, and this suspension was inoculated in a 7.5 L Bioo
115 bioreactor (New Brunswick, NJ, USA). The initial cell concen-
tration was approximately 1 g/L. The propagation was conducted
in fed-batch mode at 33 C and aerated with the carbon source
limited to 18 g TSAI/L to minimize ethanol production through the
Crabtree effect [13]. The agitation and airow were controlled by
dissolved oxygen (DO) control in cascade mode, with the dissolved
O2 concentration maintained above 25% air saturation. The propa-
gation medium was the same as that used for the inoculum, with
180 g TSAI/L. After all sugars were consumed, to recover cells, the
fermented medium was centrifuged using a Beckman Avanti J-26
XP centrifuge (JLA-9.1000 rotor, 5509 g, 10 C, 15 min). The cells
were re-suspended with sterilized potable water and refrigerated
at 5 C until inoculation for further fermentations. The fermentation
medium for the cell propagation was similar to the semi-synthetic
medium described above. The salt and nutrient requirements were
varied according to the initial cell density. All batch fermentations
were performed in a 3 L Bioo 115 bioreactor (New Brunswick, NJ,
USA) under agitation at 200 rpm with a working volume of 2 L. The
experiments were performed using the initial cell and substrate
concentrations and at the temperatures shown in Table 1.
2.3. VHG fed-batch fermentation with cell recycling
The inoculum and cell propagation were conducted accord-
ing to the aforementioned batch fermentation. The inoculum
medium contained 5.0 g/L urea, 1.1 g/L (NH4 )2 HPO4 (DAP), 1.0 g/ L
MgSO4 7H2 O, 5.0 ppm ZnSO4 7H2 O, 3.0 ppm thiamine, 5.0 g/L yeast
extract and 80.0 g/L TSAI. The propagation medium contained
5.0 g/L urea, 1.1 g/L DAP, 1.0 g/L MgSO4 7H2 O, 5.0 ppm ZnSO4 7H2 O
and 150.0 g/L TSAI. The TSAI source consisted of 79% (w/w) from
sugarcane juice and 21% (w/w) from sugarcane molasses. The
expected nal concentration was 400 g TSAI/L. Physicochemical
treatment, referred to as clarication, was performed to reduce the
salt concentration, which inuences the fermentation performance
due to the elevated osmotic pressure. Substrate clarication was
performed with the addition of 85% of 0.4 mL/L phosphoric acid and
lime at a concentration of 8 Be until pH 6.4 was reached, followed
by heating at 95 C and the addition of 4 ppm non-ionic polymer
[14]. Several minutes of rest were required for ake formation and
decantation. These conditions allowed the drag of soluble impuri-
ties. Thus, a claried substrate with high quality, minimal turbidity
and low calcium concentration was produced. After clarication
44 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251

Table 1
Temperature and initial conditions of the batch experiments.

Temperature ( C) Initial condition (IC) Fermentation performance

Cell (g/L) Substrate (g/L) Ethanol (g/L) Productivity (g/L h) Yield (%)

24 (IC-1) 0.88 23.33 0.0 0.77 78.55

24 (IC-2) 17.17 291.10 1.47 4.96 91.11
27 5.13 161.44 0.82 5.97 87.31
30 (IC-1) 5.38 154.93 0.77 7.29 96.70
30 (IC-2) 4.93 161.11 0.81 7.09 90.81
30 (IC-3) 18.05 277.65 8.22 6.39 91.28
33 4.88 164.80 0.68 8.40 90.15
36 5.25 164.36 1.33 6.63 78.91

Fig. 1. VHG ethanol fermentation owchart.

was complete, 2-L Erlenmeyer asks containing the claried sub-
strate required for each fermentation were sterilized at 121 C for
30 min. The VHG ethanol fermentation was based on the patent
WO2014078924A1 of CNPEM/CTBE [10]. A schematic representa-
tion of the process is shown in Fig. 1. Fed-batch fermentations
were performed sequentially with cell recycling under six differ-
ent conditions. Under each condition, ve fermentation cycles were
performed. All stages shown in Fig. 1 are performed in each cycle.
Under the rst condition, referred to as the reference condition,
the fermentations were performed in the 3 L Bioo 115 bioreac-
tor (New Brunswick, NJ, USA), with a working volume of 2 L at
200 rpm and temperature controlled over the range of 2834 C.
The oxidation-reduction potential (ORP), pH, capacitance and DO
were monitored. A capacitance system (Aber Instruments, UK) was
correlated with viable cell concentrations. The fed-batch started
with an initial cell suspension volume of 0.9 L at a concentration
of approximately 80 g/L (dry basis), followed by substrate feeding
for 5 h. The temperature was set according to the ethanol con-
centration. The temperature started at 34 C and ended at 28 C
until all sugar was consumed, producing an ethanol concentra-
tion of approximately 120 g/L. The fermentation time for each cycle
was 11.5 h. A micro-aeration rate of 0.2 vvm was required when
the ethanol concentration reached 108 g/L. After fermentation was
complete, the cells were recovered using a Beckman Avanti J-26
XP centrifuge (JLA-9.1000 rotor, 13261 g, 20 C, 20 min). The cells
were re-suspended with 0.9 L of sterilized potable water, and then,
they were transferred back to the bioreactor vessel. Acid treatment
was performed on the cells by adding sulfuric acid (2 M) through
a silicon septum until a pH of 2.5 was reached. The acid treat-
ment conditions were 30 C, 0.2 LPM of air (positive pressure to
avoid contamination) and 600 rpm for 30 min. This cell suspension
was centrifuged as previously described. The cells were then re-
suspended with cooled reactivation medium that contained carbon,
nitrogen and phosphate sources. This cell suspension was trans-
ferred back to the bioreactor vessel for the cellular reactivation
stage. After this stage was complete, a new fed-batch fermentation
was performed. In the reactivation medium, sugarcane substrate
and DAP were used to supply carbon, phosphorus and nitrogen
for membrane restoration and cellular activity recovery. The cell
concentration ranged from 80 to 100 g/L (dry basis) in a medium
with 100 g/L TSAI and 3.1 g/L DAP. The operating parameters of the
bioreactor with a working volume of 0.9 L were 600 rpm, 33 C, and
2 L/min of air, which produced an environment with an ORP over
the range of +50100 mV. The residence time was 60 min.
To determine the robustness, stability and reproducibility of
the VHG ethanol fermentation described above, changes to cer-
tain key parameters, such as the DAP concentration, temperature
during fermentation, micro-aeration rate and acid treatment, were
investigated. Table 2 summarizes the conditions studied.
2.4. Analytical methods
All sucrose, glucose, fructose, and ethanol measurements from
batch fermentations were performed via high-performance liq-
 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251
Table 2
Process conditions for VHG fed-batch ethanol fermentation.
Condition Parameter assessed
1 Reference
2 DAP
3 Fermentation temperature
4 Fermentation temperature and
micro-aeration
5 Fermentation temperature and
micro-aeration
6 Acid treatment
Fig. 2. Pareto chart of the effects for the concentration of: A) cell, B) substrate and
C) ethanol (at a 95% signicance level).
uid chromatography (HPLC). Details are provided elsewhere [15].
Measurements of the same components in the VHG fed-batch fer-
mentations were also taken via HPLC. However, HPLC was applied
only for the rst and last samples during fermentation. Mea-
surements of the remaining samples, the feeding substrate and
the fermented medium concentration were conducted using mid-
infrared (MIR) spectroscopy. The technique aimed to use a rapid and
accurate method for analyzing VHG ethanol fermentation samples
instead of using HPLC. The cellular viability and budding were ana-
lyzed by staining the cells with methylene blue [16] and counted
using a Neubauer chamber in an optical microscope (Eclipse CI-S;
Nikon, Japan) for samples at the end of the fermentations. The dry
cell concentration was determined gravimetrically after centrifug-
ing, washing two times with Milli-Q water and drying at 80 C until
a constant weight was obtained in an analytical balance.
45
Description of conditions
Acid treatment: sulfuric acid
Reactivation: 100 g/L TSAI, 3.1 g/L DAP, micro-aeration rate 0.2 vvm,
33 C, 60 min
Fermentation: Temperature beginning at 34 C and ending at 28 C.
Temperature of 28 C and micro-aeration rate of 0.2 vvm were set
when the ethanol concentration reached 108 g/L
DAP concentration is increased to 5.6 g/L
Beginning at 34 C and ending at 30 C. This last temperature was set
when the ethanol concentration reached 108 g/L
Beginning at 34 C and ending at 30 C. The micro-aeration rate is
reduced by half when ethanol concentration reached 108 g/L
Returned to the same condition as that used in 3
Nitric acid instead of sulfuric acid
2.5. MIR analytical procedures
Calibration curves were developed for ethanol and sugars using
mid-infrared spectra and HPLC as primary reference analyses.
VHG fermentation samples were analyzed in a platinum dia-
mond attenuated total reectance (ATR platinum T Diamond 1
Re #263C3643) single reection cell, mounted in a mid-infrared
Bruker Alpha Fourier transform infrared (FT-IR) spectrometer
(Bruker Optics Inc., USA). Previously, a number of samples were
used to calibrate the spectrometer using HPLC as the primary
analyses. The ATR-MIR spectra were recorded using OPUS soft-
ware provided by Bruker Optics. The spectrum for each sample
was obtained by taking the average of 16 scans (resolution of
4 cm1 , between 5000 and 550 cm1 ), with a scanner velocity of
7.5 kHz (background of 16 scans), and using 100 L samples at 40 C
without cells (centrifuged sample). Air was used as the reference
background spectra, and the ATR diamond surface was cleaned with
ethanol (95% v/v) before each sample was analyzed to minimize or
avoid contamination between samples. A calibration model (mul-
tivariate regression) was applied using a partial least squares (PLS)
regression, with full cross validation of spectroscopic data and the
corresponding concentration values from the HPLC. The PLS cali-
bration requires the choice of suitable frequency ranges, optimum
data preprocessing, and an optimum number of factors. Thus, the
OPUS Quant Analyzer was used. This software employed an opti-
mization tool to select among eleven mathematical methods for
preprocessing the spectral data (e.g., vector normalization and sec-
ond derivative). In addition, the software evaluated the optimal
spectrum region to construct the calibration curve. After this anal-
ysis was complete, one method was selected for calibration study,
and the optimum number of terms in the PLS calibration models
was indicated by the lowest number of factors (PLS terms or rank),
yielding the minimum value of the root mean square error of cross
validation (RMSECV). The outliers were removed based on F-values
obtained from the OPUS software calculator. Statistics calculated
for the calibrations included the coefcient of determination in
cross validation (R2 ), the RMSECV, the bias (systematic averaged
deviation between the true and predicted values), the number of
PLS factors (rank), and the residual prediction deviation (RPD). Glu-
cose and fructose were analyzed using only one calibration curve
by reducing sugars (glucose plus fructose). The statistical results for
the PLS calibrations developed using the VHG fermentation sam-
ples are shown in Table 3. The obtained calibration curves can be
used to quantitatively determine these fermentation compounds
in VHG fermentation samples with high accuracy (R2 > 99, RPD > 5,
RMSECV (%) < 5). RPD values greater than 5 are considered appro-
priate for quality control and routine analysis [17].
46 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251

Table 3
Descriptive statistics of calibration for the measurement of VHG fermentation samples analyzed using attenuated total reectance mid-infrared spectroscopy.

Ethanol Glucose + Fructose Sucrose

Range (g/ L) 0129.7 0108.2 077.0

Spectra 487 70 355
Preprocessing Multiplicative scattering correction Vector normalization (SNV) No spectral data preprocessing
Frequency regions (cm1 ) 1636.2976.9 1739.51488.7; 1174.6854.5 1476.0860.6
R2 99.30 99.74 99.58
RMSECV (%) 2.64 1.28 0.863
Bias 0.000469 0.0279 0.00368
RPD 12 19.5 15.5
PLS terms 7 8 10

R2 : coefcient of determination in cross validation; RMSECV: root mean square error of cross validation; RPD: standard deviation/standard error of cross validation; PLS
terms: optimal number of terms used to develop the PLS models.

2.6. Determination of the fermentation performance parameters
The fermentative yield was calculated using Eq. (1), which is
based on the by-products method initially proposed by Finguerut
[18]:
Yield =
100
Aacetic
1.0032 + 0.54
P
(X + G + Sresidual ) + 0.504 P
+ 0.525
where P is the ethanol produced, X is the cell produced, G
is the glycerol produced, Sresidual is the TSAI residual, Aacetic is the
acetic acid produced and Alactic is the lactic acid produced.
The denition of yield based on the by-products method was
chosen because it is less sensitive to variations in the specic
weight of the fermented medium (which requires an accurate mea-
surement) compared with the conventional method. This method,
currently used in ethanol plants in Brazil, consists of measuring the
formation of the main by-products (cells, acetic acid, lactic acid and
CO2 ) as well as unconverted sugars related to ethanol formation.
The stoichiometric relationship among the acids and CO2 was based
on empirical data from the ethanol plants.
The volumetric productivity was calculated according to Eq. (2):
P nance by producing ethanol. Maintenance is the energy required
Productivity =
V.t
where P represents the ethanol produced in g, V is the nal volume
of the fermented medium in L and t is the total fermentation time
in h.
3. Modeling and parameter estimation
First, the temperature-dependent kinetic model for ethanol
fermentation was developed with experimental data from batch
experiments. The model was expanded to simulate fed-batch
ethanol fermentation operated under VHG conditions and vali-
dated against experimental observations.
3.1. Kinetic and theoretical aspects of batch fermentation
A mechanistic model that describes the kinetics of the concen-
trations of cells X (g/L), substrate S (g/L), and ethanol P (g/L),
in terms of three ordinary differential equations, i.e., Eqs. (3)(5),
was applied to the ethanol fermentation process [11]:
dX
= rx
dt
dS
= rs
dt
dP
= rp
dt
where rx , rs and rp represent the cell growth, substrate consumption
and ethanol production reaction rates, respectively (g/(L h)).
During VHG ethanol fermentation, the cell is exposed to stress
barriers, including osmotic stress due to high substrate concen-
tration, ethanol stress at the end of fermentation and high cell
densities inside the bioreactor. To simulate these phenomena in
terms of the physiological state of the cell, substrate saturation
Alactic
P
and inhibition terms were considered in the cell growth rate, rx ,
(1) in Eq. (6) (rst and second terms, respectively). The cell responses
to ethanol inhibition and inhibition due to high cell concentrations
were also incorporated into the model (third and fourth terms in
Eq. (6), respectively).
S P
 n  X
m
rx = max exp(Ki S) 1 1 X (6)
Ks + S Pmax Xmax
In the above equation, max denotes the maximum specic growth
rate (h1 ), Ks denotes the substrate saturation constant (g/L), Ki
denotes the substrate inhibition parameter (g/L), Xmax denotes the
cell concentration when cell growth ceases (g/L), Pmax denotes the
ethanol concentration when cell growth ceases (g/L) and m and
n denote parameters related to cellular and product inhibitions,
respectively.
Cells meet their energy requirements for growth and mainte-
(2) to support cell function, other than that required for cell synthe-
sis. For instance, repairing active cell material involves a protein
synthesis system, among others. The ethanol production rate, rp ,
is consistent with the growth and non-growth associated product
model proposed by Luedeking and Piret [19] and is represented in
Eq. (7):
rp = Yp/x rx + mp X (7)
where Yp/x denotes the LuedekingPiret growth-associated
constant (g/g) and mp denotes the LuedekingPiret non-growth-
associated constant (g/(g h)).
Similar to the ethanol production rate, the LuedekingPiret
equation was proposed for the substrate consumption rate, rs , as
shown in Eq. (8), by considering that the substrate consumed is
not only related to cell growth but is also used for other processes
requiring energy:
rs = rx /Yx + ms X (8)
where Yx and ms are the cell yield (g/g) and maintenance parameter
(3) (g/(g h)), respectively.
The kinetic model is formed using Eqs. (3)(8), which contain
eleven kinetic parameters. The model should include or be able to
(4)
estimate these parameters for estimating rx , rs and rp .
The reaction rates of the cell are affected by different factors,
(5)
and one of the most important of these is temperature.
 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251 47

0.5 10.0
0.41 0.43
0.4 0.38 8.4
7.8

max (h-1)
7.2

Yp/x (g/g)
0.3 0.31 6.8 6.6
5.9
0.2 0.18 5.2 5.1
0.1 3.6
0 2.0
21 24 27 30 33 36 39 21 24 27 30 33 36 39
73 0.1
71 0.088 0.089
Xmax (g/L)

71.0

Yx (g/g)
70 69.4
0.076 0.075
68 68.1 0.064 0.066
67.1 0.059
67 0.052 0.054
66.3
65 0.04
21 24 27 30 33 36 39 21 24 27 30 33 36 39
122 0.0058
120 0.0052
Pmax (g/L)

118.6 0.0048

Ki (g/L)
117 116.6 0.0046 0.0046
0.0043
115 115.1 0.0039 0.0039
113.8
112 112.8 0.0033 0.0033
110 0.0027
21 24 27 30 33 36 39 21 24 27 30 33 36 39
Temperature (oC) Temperature (oC)

Fig. 3. Optimal values of the kinetic parameters at 24, 27, 30, 33 and 36 C (solid symbols) and their corresponding t using Eqs. (9) and (10) (solid curves).

(A) 18 (B) 18 (C) 16

14 14 13
Cell (g/L)
Cells (g/L)

Cells (g/L)
11 11 10
7 7 6
4 4 3
0 0 0
180 0 3 5 8 10 13 180 180 0 2 4 6 8 10
Time (h) Time (h)
Substrate (g/L)

Substrate (g/L)

144 144
Substrate (g/L)

144
108 108 108
72 72 72
36 36 36
0 0 0
80 0 3 5 8 10 13 85 90 0 2 4 6 8 10
Time (h) Time (h)
64 72
Ethanol (g/L)

68
Ethanol (g/L)

Ethanol (g/L)

48 51 54
32 34 36
16 17 18
0 0 0
0 3 5 8 10 13 0 2 4 7 9 11 0 2 4 6 8 10
Time (h) Time (h) Time (h)

Fig. 4. Experimental data from the batch fermentations (Cells (); Substrate () and Ethanol ()) compared with simulated concentration time curves (): A) 27 C, B) 30 C
and Initial Condition IC-2 in Table 1 and C) 33 C.

3.2. Modeling of the temperature inuence where T is the temperature ( C) at which the kinetic parameters
are evaluated, A and C are pre-exponential factors and B and D are
In this study, the temperature-dependent parameters are mod- exponential factors.
eled using Eqs. (9) and (10). As demonstrated in previous studies, A main feature of the proposed temperature-dependent kinetic
these equations have been effectively used to simulate the effects model is the behavior of X, S, and P when the temperature changes.
of temperature on cell growth in sugarcane juice, molasses [9] and Given that the some parameters are modeled as being tempera-
bagasse hydrolysates [20]: ture dependent, the kinetic rates change when the temperature
is varied. This approach enables the simulation of an adequate
temperature prole for improving the fermentation performance.
Parameter (T ) = A exp (B/T ) + C exp (D/T ) (9) For instance, in VHG fermentation, a gradual reduction in tem-
perature during fermentation is required, which alleviates the
inhibition effects while maintaining sufcient rates of cell division
Parameter (T ) = A exp (B/T ) (10) and ethanol production [21].
48 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251
3.3. Parameter estimation procedure
An accurate estimation of model parameters is a major issue in
the development of mechanistic models. Here, a ve-step proce-
dure is proposed to estimate the kinetic parameters for the current
model.
In Step 1, an identiability analysis of the kinetic model was per-
formed, which was achieved by the calculation of a meta-model
based on Plackett-Burman (PB) design [22], capable of providing
all the required sensitivity information. For this analysis, the inu-
ence of kinetic parameters (max , Xmax , Pmax , Yx , Yp/x , Ks , Ki , ms ,
m, n, mp ) on responses X, S and P (data at a single timepoint) was
evaluated using a PB design for eleven factors. In the PB design,
the simulations involve systematically varying all kinetic parame-
ters within 10% of nominal values reported elsewhere [23]. The
simulation aims to determine the subset of model parameters with
the largest effect on the model prediction, which are modeled as
temperature-dependent.
The simulation results were evaluated using the Pareto chart,
which indicates each of the estimated effects of the parameters on
X, S and P in decreasing order of magnitude. The size of each bar
is relative to the standardized effect, calculated as the estimated
effect divided by its standard error, which is equivalent to estimate
a t-statistic for each effect. In addition, a horizontal line (critical t-
value) represents the effects limit of signicance. Bars extending
beyond that line relate to effects that are statistically signicant at
the 95% signicance level.
The parameter estimation largely depends on the initial guesses
of the parameter values due to the nonlinear nature of the kinetic
model, which contains multiple parameters. Thus, in Step 2 the
initial guesses of the temperature-dependent parameters to be
estimated are obtained from a previous study [9,11,20,23,24]. The
remaining parameters were xed at values given by Atala et al. [24].
This information is incorporated into the model along with the ini-
tial conditions for X, S and P. Hence, in Step 3, the kinetic model
written in FORTRAN is integrated using the routine LSODE [25] to
obtain the time-course concentrations of X, S and P. Next, in Step 4,
an objective function J() is dened as the least square error given
by Eq. (11):

3 
Np
(yexp (t j ) ysim (t j ))2
J() = i i
(11)
2
yexpi max
i=1 j=1
where  is a vector containing all the temperature-dependent
parameters, yexpi (t j ) is the ith measured concentration, i.e., the mea-
sured concentrations of the cell, substrate and ethanol at sampling
time j, ysimi (t j ) and yexpi max are the concentrations computed by
the kinetic model and maximum measured concentration, respec-
tively, and Np is the number of sampling points.
In Step 5, a nonlinear optimization problem (NP) is formulated,
as shown in Eq. (12), which minimizes the objective function J():
Min J()
Subject to :
NP : (12)
Eqs. (3-8)
lb  ub
where  lb and  ub are the lower- and upper-bound vectors for the
temperature-dependent parameter vector , respectively.
In this study, a hybrid optimization algorithm based on the
genetic algorithm (GA) and the Quasi-Newton method (QN) [11] is
adopted to solve Eq. (12). The hybrid algorithm comprises a thor-
ough exploration of the search space ( lb   ub ) and nding the
neighborhood of the global optimum through the GA. Then, the
hybrid algorithm, starting from the best estimate of the GA ( best ),
uses gradient information from the QN method to accelerate the
convergence toward the global optimum, which involves determin-
ing the optimum values for the parameters ( optimum ) that produce
the best t between the measured concentrations of X, S and P
and their corresponding computed concentrations from the kinetic
model while minimizing J().
The execution of Steps 15 generated a set of optimal values for
the temperature-dependent parameters for each temperature con-
sidered (24, 27, 30, 33 and 36 C). Then, the set of optimal values for
each parameter was adjusted to Eqs. (9) or (10). This adjustment
yielded the best constants A, B, C and D for each temperature-
dependent parameter. Once the kinetic model was established, the
constants were ne-tuned using the GA such that J was minimized.
The nonlinear optimization problem (Eq. (12)) was imple-
mented in a FORTRAN routine on a 3.6 GHz Intel(R) Core(TM)
i7-4790 CPU.
3.4. Mass balance equations for predicting VHG fed-batch
fermentation
After the kinetic parameters were estimated, the kinetic rates rx ,
rs and rp that incorporate these parameters were used in a mecha-
nistic fed-batch model with the mass balance equations described
in Eqs. (13)(15). Thus, the kinetic study provides a basis to allow for
the dynamic simulation of the VHG ethanol fermentation process
through a model that is sensitive to inhibition factors and changes
in temperature:
dX F
= rx X (13)
dt V
dS F
= (S f S) rs (14)
dt V
dP F
= rp P (15)
dt V
where F denotes the feed ow rate (L/h), V denotes the working
volume (L) of the bioreactor, and Sf denotes the total sugar concen-
tration (g/L) in the feed. The reaction rates rx , rs and rp are the same
as those obtained from the previous batch experiments.
During model simulation, it was assumed that the sugars are fed
at a xed F, and the measured V is an input data at each integration
time during the simulation.
4. Results and discussion
4.1. Identiability analysis using the PB design
The parameter estimation procedure considered iterative steps
with an evaluation of the model reliability at several points. These
evaluations included the identiability analysis of the kinetic model
using the calculation of a meta-model based on the Plackett-
Burman (PB) statistical design. Fig. 2AC shows the Pareto charts
obtained from PB design for responses X, S and P, respectively.
According to the estimated effects, Pmax , Yx and Yp/x were consid-
ered statistically signicant for all responses. In addition, max was
considered statistically signicant for S, and its absolute standard
effect on X is close to the cut-off line for signicant effects. The
parameters mp and ms appeared relevant only for P with very small
effects (nine times smaller) compared to those for Yx and Yp/x .
In this study, the statistically signicant parameters max , Pmax ,
Yp/x and Yx were modeled as functions of the fermentation tem-
perature. In addition, the parameters Xmax and ki were modeled
as a function of temperature based on previous studies [11,20,24].
The remaining parameters were xed as follows [24]: Ks = 4.1 g/L,
mp = 0.1 g/(g h), ms = 0.2 g/(g h), m = 1.0 and n = 1.5.
 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251 49

4.2. Batch fermentation and kinetic study results

A series of batch fermentation experiments was performed

using temperatures of 24, 27, 30, 33 and 36 C and different initial
concentrations of X and S, as shown in Table 1. The ethanol produc-
tivity for all fermentations typically resulted in high values due to
the rapid conversion of sugars into ethanol and other co-products.
The fermentation conditions at 24 C with a low substrate concen-
tration were unfavorable for conversion to ethanol; this resulted in
a lower productivity and yield. In contrast, a temperature of 33 C
led to a higher productivity and yield, as expected [26].
The initial conditions and temperatures of the batch experi-
ments were used to perform the previously described parameter
estimation procedure. A set of optimal values of the temperature-
dependent parameters for each temperature considered was
generated. Fig. 3 shows the behavior of the optimal values of the
kinetic parameters at 24, 27, 30, 33 and 36 C (solid symbols). Then,
the constants A and B in the Arrhenius-type Eq. (9) were adjusted for
the temperature-dependent kinetic parameters Xmax , Pmax , Yp/x and
Yx and the constants A, B, C and D in Eq. (10) for the parameters max
and ki . As in previous studies [9,11,20], Eq. (10) was used instead of
the Arrhenius-type equation to accurately describe the nonlinear
inuence of temperature on some kinetic parameters. The opti-
mal values of the constants and the correlation coefcient (R2 ) for
each adjustment are shown in Table 4. Fig. 3 shows the t using
Eqs. (9) and (10) (solid curves). These data clearly demonstrate Fig. 5. RSD (%) to evaluate the goodness-of-t of the (A) kinetic model and (B) fed-
the inuence of temperature on the kinetic parameters. As max batch model against VHG fermentations with cell recycling.

increases from 24 to 33 C, multiplication and metabolism begin

to be inhibited at higher temperatures. Xmax and Pmax decreased by
mean values of approximately 1.2 and 1.4 g/L, respectively, for each
3 C decrease in temperature, suggesting that tolerance to ethanol
and cell concentrations increases at low temperatures. In addition,
Yp/x and Ki exhibited lower values at higher temperatures, whereas
the cell yield Yx decreases at lower temperatures, which could lead
to signicant changes in the substrate consumption rate. Previ-
ous studies of ethanol fermentation from sugarcane juice, molasses
[9,27] and bagasse hydrolysates [20] as substrates have shown that
the yields are temperature dependent; temperatures above 34 C
begin to favor glycerol formation. Ethanol plants have higher losses
at higher temperatures due to the ethanol dragged with CO2 .
The computed proles (from the model described by Eqs. Fig. 6. Performance parameters of fermentation.

(3)(10)) of the cell, substrate and ethanol concentrations at 27,

30 and 33 C and with different initial concentrations are shown
in Fig. 4AC, respectively. The measured concentrations used for
the parameter estimation are also shown in these gures for com-
parison. The results illustrate that the model effectively tracks the
desired trajectory of the measured data.
A more rigorous assessment of the goodness-of-t between the
measured and computed data can be conducted using residual stan-
dard deviation (RSD), as suggested by Cleran et al. [28]. Recent
studies [29,30] have adopted RSD as a measure of the prediction
accuracy of proposed models. RSD is written as a percentage of the
average of the measured data, yexp , dened by Eq. (16):
0.5

Np
Np
1
(yexp (t j ) ysim (t j ))2
j=1
RSD(%) = 100
yexp
where yexpi (t j ) represents the ith measured concentration, i.e., the
measured concentrations of the cell, substrate and ethanol at sam-
pling time j, ysimi (t j ) are the concentrations computed by the model
and Np is the number of sampling points.
Fig. 5A illustrates that the model predictions were particularly
accurate, as determined by the RSD (%). These results demonstrate
that the model can be used to predict the dynamic behavior under
the current batch conditions. Therefore, the kinetic model that
incorporates inhibition terms and temperature-dependence func-
tions can be used to predict the concentrations of X, S and P at
changing reaction temperatures, as well as with different initial
concentrations of X and S. This model can handle temperature uc-
tuations between 24 and 36 C.
These kinetic results were further used to predict the dynamic
behavior of X, S and P in VHG ethanol fermentation.
4.3. Prediction of VHG fed-batch experiments with cell recycling
VHG fed-batch fermentations with cell recycling were per-
formed sequentially under six different conditions, as described in
Table 2. Five fermentation cycles were performed under each con-
dition. All stages described in Fig. 1 are conducted for each cycle.
(16) Fig. 6 shows the average fermentation performance for each condi-
tion in terms of yield, productivity and nal ethanol concentration.
Condition 1 (the reference condition, described in detail in Sec-
tion 2.3) obtained a yield of 84%, a productivity of 10.2 g/L h and
an ethanol concentration of 120 g/L (average values for ve fed-
batch fermentations). After condition 1 was tested, a second set of
ve fed-batch fermentations were conducted (condition 2) with the
aim of improving the fermentation performance by varying the DAP
concentration in the reactivation stage to a value above the refer-
50 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251

Table 4
Optimal values of the constants in Eqs. (9) and (10) for temperature-dependent kinetic parameters.

Parameter Equation R2 A B C D

max (9) 0.98 0.92273 45.20986 1.26669 38.29429

Xmax (10) 0.99 57.89562 4.89193
Pmax (10) 0.98 102.12366 3.58256
Yp/x (10) 0.99 18.27807 30.6689
Yx (10) 0.96 0.01965 36.15008
ki (9) 0.99 1.29176 15.68355 1.28591 15.76083
Concentraons of X, S and P

(A) (B)

Concentraons of X, S and P
210 210
184 184
158 158
131 131
(g/L)

(g/L)
105 105
79 79
53 53
26 26
0 0
0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35340 45
Cycle
50 455 60
Cycle
65 570 0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35340 45
Cycle
50 455 60
Cycle
65 570
(C)
Concentraons of X, S and P

(D)

Concentraons of X, S and P
210 210
184 184
158 158
131 131
(g/L)

(g/L)
105 105
79 79
53 53
26 26
0 0
0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35340 45
Cycle
50 455 60
Cycle
65 570 0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35 340 45
Cycle
50 455 60
Cycle
65 570
(E) (F)
Concentraons of X, S and P

Concentraons of X, S and P

210 210
184 184
158 158
131 131
(g/L)

(g/L)

105 105
79 79
53 53
26 26
0 0
0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35340 45
Cycle
50 455 60
Cycle
65 570 0 Cycle
5 101 15
Cycle
20 225 30
Cycle
35340 45
Cycle
50 455 60
Cycle
65 570
Fig. 7. Experimental data from the VHG fed-batch fermentations with cell recycling (Cells (); Substrate () and Ethanol ()) compared with simulated concentration time
curves () under Condition A) 1, B) 2, C) 3, D) 4, E) 5 and F) 6 in Table 2. The fermentation time for each cycle was 11.5 h.

ence condition (the other operating conditions remained the same
as the reference conditions). A slightly better fermentation perfor-
mance than the reference condition was obtained, with a yield of
87%, a productivity of 10.3 g/L h and an ethanol concentration of
122 g/L. For condition 3, the temperature at the nal stage during
the exhaustion of sugar conversion was slightly above the nal ref-
erence temperature. As shown in Fig. 6, this condition was the most
efcient process in terms of fermentation yield, with a value of 90%.
However, the productivity of 10.2 g/L h and ethanol concentration
of 120 g/L were lower than those of condition 2. In condition 4, in
which the micro-aeration rate was reduced while maintaining the
same temperature prole of condition 2, the fermentation perfor-
mance was the poorest of the examined conditions, with a yield
of 84%, a productivity of 9.8 g/L h and an ethanol concentration
of 115 g/L. An attempt was made to remedy this poor fermenta-
tion performance situation in condition 5, which used the same
operating parameters as those in condition 3. However, despite a
slight improvement (a yield of 84%, a productivity of 9.9 g/L h and an
ethanol concentration of 117 g/L), the fermentation performance of
condition 3 could not be reproduced. Thus, a micro-aeration rate
of 0.2 vvm at the end of the fermentation should be maintained to
avoid a decrease in the fermentation performance. Finally, in con-
dition 6, nitric acid was used instead of sulfuric acid. This condition
provided a yield of 88%, a productivity of 10.4 g/L h and an ethanol
concentration of 124 g/L. Hence, this condition was the most favor-
able in terms of productivity and ethanol concentration.
These results demonstrate that all average productivities were
approximately 10 g/L h while maintaining yields between 84% and
90%. These productivity values are high compared to the typical
industrial value of 5.5 g/L h in which the yield is 89% working in
continuous mode. The unexpected low value of yield is related to
the residual sugar at the end of fermentation, indicating that more
time is required for its consumption.
The fed-batch model can describe the dynamics of cell growth,
substrate consumption and ethanol production during VHG fer-
mentation under varying conditions (Table 2). These results are
shown in Fig. 7. The corresponding assessment using RSD (%) is
shown in Fig. 5B. This proposed model could provide a basis for the
design and operation of VHG ethanol fermentation at an industrial
scale.
The model assumed that the ethanol production rate, rp , is
consistent with the growth and non-growth associated prod-
uct, as described by Eq. (7). However, above the Pmax value in
VHG fermentation, rp is consistent only with the non-growth
associated product. Pmax is a temperature-dependent parameter;
therefore, it has different values for different temperatures, as
 E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251
shown in Fig. 3. Alfenore et al. [31] also observed these two phases
(growth and non-growth associated ethanol production) during
VHG ethanol fermentation. The phase in which ethanol produc-
tion was disconnected from cell growth was observed when the
ethanol concentration in the medium reached values of approxi-
mately 100 g/L. A nutrient feeding and aeration strategy was used
for higher ethanol concentrations to avoid loss of cell membrane
integrity because ethanol acts as a chaotropic solute in the aqueous
phase of the membrane system and induces changes in its structure
and uidization of lipid bilayers [5].
In this work, the application of micro-aeration when the
ethanol concentration was approximately equal to Pmax , as well as
with appropriate temperature and nutrient supplementation, was
essential to overcome the inhibitory and detrimental effect of high
ethanol concentrations. These operating conditions, along with the
process conguration shown in Fig. 1 (where a second centrifuga-
tion and a cell treatment step were incorporated, in contrast to the
conventional process), render ethanol fermentation feasible under
VHG conditions.
5. Conclusions
This study demonstrated how a systematic model-based
approach can be applied to address kinetic model development.
The resulting model can predict cell growth and ethanol produc-
tion from sugarcane under different VHG conditions. An advanced
temperature-dependent parameter estimation procedure was used
to guarantee the accuracy of the proposed model. The inuence of
temperature on fermentation kinetic behavior was also explicitly
demonstrated. The VHG fermentation results demonstrated that an
appropriate temperature prole, micro-aeration around the Pmax
value and nutrient supplementation during cell treatment produce
yields of up to 90%, with an ethanol productivity of 10.2 g/L h.
Acknowledgements
The authors acknowledge British Petroleum Biofuels (BP), the
Brazilian Bioethanol Science and Technology Laboratory/Brazilian
Center of Research in Energy and Materials (CTBE/CNPEM) and
FAPESP (process number 2016/01785-0) for their nancial support,
assistance regarding the use of facilities, sharing expertise and the
opportunity to develop this work.
References
[1] EPE, Brazilian Energy Balance Year 2014, Empresa de Pesquisa Energtica
(EPE), Rio de Janeiro, 2015, pp. 292 (in Portuguese).
[2] R. Zeidan, C. Boechat, A. Fleury, Developing a sustainability credit score
system, J. Bus. Ethics 127 (2015) 283296.
[3] S. Brethauer, C.E. Wyman, Review: continuous hydrolysis and fermentation
for cellulosic ethanol production, Bioresour. Technol. 101 (2010) 48624874.
[4] L.C. Basso, H.V. de Amorim, A.J. de Oliveira, M.L. Lopes, Yeast selection for fuel
ethanol production in Brazil, FEMS Yeast Res. 8 (2008) 11551163.
[5] J.A. Cray, A. Stevenson, P. Ball, S.B. Bankar, E.C. Eleutherio, T.C. Ezeji, R.S.
Singhal, J.M. Thevelein, D.J. Timson, J.E. Hallsworth, Chaotropicity: a key factor
in product tolerance of biofuel-producing microorganisms, Curr. Opin.
Biotechnol. 33 (2015) 228259.
[6] C.J. Cassan, J. Riess, F. Jolibert, P. Taillandier, Optimization of very high gravity
fermentation process for ethanol production from industrial sugar beet syrup,
Biomass Bioenergy 70 (2014) 165173.
[7] P. Puligundla, D. Smogrovicova, V.S. Obulam, S. Ko, Very high gravity (VHG)
ethanolic brewing and fermentation: a research update, J. Ind. Microbiol.
Biotechnol. 38 (2011) 11331144.
51
[8] L. Benbadis, M. Cot, M. Rigoulet, J. Francois, Isolation of two cell populations
from yeast during high-level alcoholic fermentation that resemble quiescent
and nonquiescent cells from the stationary phase on glucose, FEMS Yeast Res.
9 (2009) 11721186.
[9] E.C. Rivera, A.C. Costa, R.R. Andrade, D.I.P. Atala, F. Maugeri Filho, R. Maciel
Filho, Development of adaptive modeling techniques to describe the
temperature-dependent kinetics of biotechnological processes, Biochem. Eng.
J. 36 (2007) 157166.
[10] C.E.V. Rossell, J. Nolasco Junior, C.K. Yamakawa, Processo e equipamento para
fermentaco continua multiestgio com recuperaco, reativaco, e reciclo de
fermento para obtenco de vinhos com alto teor alcolico. Patent
WO2014078924A1, 2012.
[11] E.C. Rivera, A.C. Costa, D.I.P. Atala, F. Maugeri, M.R.W. Maciel, R. Maciel Filho,
Evaluation of optimization techniques for parameter estimation: application
to ethanol fermentation considering the effect of temperature, Proc. Biochem.
41 (2006) 16821687.
[12] T.O. Basso, Improvement of Alcoholic Fermentation in Saccharomyces
cerevisiae by Evolutionary Engineering. Ph.D. Thesis, University of So Paulo,
Brazil, 2011 (in Portuguese).
[13] J.P. Barford, R.J. Hall, An examination of the crabtree effect in Saccharomyces
cerevisiae: the role of respiratory adaptation, J. Gen. Microbiol. 114 (1979)
267275.
[14] COPERSUCAR (Cooperative of Sugarcane, Sugar and Ethanol Producers of the
State of Sao Paulo), Fermentation, Technical Report (in Portuguese), So
Paulo, Brazil (1987).
[15] E.C. Rivera, C.K. Yamakawa, M.H. Garcia, V.C. Geraldo, C.E.V. Rossell, R. Maciel
Filho, A. Bonomi, A procedure for estimation of fermentation kinetic
parameters in fed-batch bioethanol production process with cell recycle,
Chem. Eng. Trans. 32 (2013) 13691374.
[16] S.S. Lee, F.M. Robinson, H.Y. Wong, Rapid determination of yeast viability,
Biotechnol. Bioeng. Symp. 11 (1981) 641649.
[17] Y. Huang, J. Carragher, D. Cozzolino, Measurement of fructose glucose,
maltose and sucrose in barley malt using attenuated total reectance
mid-infrared spectroscopy, Food Anal. Methods 9 (2015) 10791085.
[18] J.I. Finguerut, Research Project in Public Policy (PPPP) Ethanol, I Technological
Workshop on Obtaining Ethanol, University of So Paulo, Lorena, SP, 2006 (in
Portuguese).
[19] R. Luedeking, E.L. Piret, A kinetic study of the lactic acid fermentation. Batch
process at controlled pH, Biotechnol. Bioeng. 1 (1959) 393412.
[20] R.R. Andrade, F. Maugeri Filho, R. Maciel Filho, A.C. Costa, Kinetics of ethanol
production from sugarcane bagasse enzymatic hydrolysate concentrated with
molasses under cell recycle, Bioresour. Technol. 130 (2013) 351359.
[21] M.J. Torija, N. Rozs, M. Poblet, J.M. Guillamn, A. Mas, Effects of fermentation
temperature on the strain population of Saccharomyces cerevisiae, Int. J. Food
Microbiol. 80 (2003) 4753.
[22] R.R. Plackett, J.P. Burman, The design of optimum multifactorial experiments,
Biometrika 33 (1946) 305325.
[23] R.R. Andrade, E.C. Rivera, A.C. Costa, D.I.P. Atala, F. Maugeri Filho, R. Maciel
Filho, Estimation of temperature dependent parameters of a batch alcoholic
fermentation process, Appl. Biochem. Biotechnol. 136140 (2007) 753763.
[24] D.I.P. Atala, A.C. Costa, R. Maciel Filho, F. Maugeri Filho, Kinetics of ethanol
fermentation with high biomass concentration considering the effect of
temperature, Appl. Biochem. Biotechnol. 9193 (2001) 353365.
[25] K. Radhakrishnan, A. Hindmarsh, Description and Use of LSODE, the Livermore
Solver for Differential Equations, NASA Reference Publication, 1993, pp. 132.
[26] S.R. Andrietta, Modeling, Simulation and Control of Industrial Scale Processes
for Continuous Alcoholic Fermentation. Ph.D. Thesis, State University of
Campinas, Brazil, 1994 (in Portuguese).
[27] M. Phisalaphong, N. Srirattana, W. Tanthapanichakoon, Mathematical model
to investigate temperature effect of kinetic parameters of ethanol
fermentation, Biochem. Eng. J. 28 (2006) 3643.
[28] Y. Cleran, J. Thibault, A. Cheruy, G. Corrieu, Comparison of prediction
performances between models obtained by the group method of data
handling and neural networks for the alcoholic fermentation rate in enology,
J. Ferment. Bioeng. 71 (1991) 356362.
[29] D. Farias, R.R. Andrade, F. Maugeri Filho, Kinetic modeling of ethanol
production by Scheffersomyces stipitis from Xylose, Appl. Biochem. Biotechnol.
172 (2014) 361379.
[30] G.H.S.F. Ponce, J. Moreira Neto, S.S. De Jesus, J.C.C. Miranda, R. Maciel Filho,
R.R. Andrade, M.R. Wolf Maciel, Sugarcane molasses fermentation with in situ
gas stripping using low and moderate sugar concentrations for ethanol
production: experimental data and modeling, Biochem. Eng. J. 110 (2016)
152161.
[31] S. Alfenore, X. Cameleyre, L. Benbadis, C. Bideaux, J.L. Uribelarrea, G. Goma, C.
Molina-Jouve, S.E. Guillouet, Aeration strategy: a need for very high ethanol
performance in Saccharomyces cerevisiae fed-batch process, Appl. Microbiol.
Biotechnol. 63 (2004) 537542.