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Biochemical Engineering Journal 119 (2017) 4251

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

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 43

tions for cell maintenance [8]. Another common source of kinetic provided by the Faculty of Food Engineering/State University of
uctuation could be related to the variability of feedstock quality, Campinas and originally obtained from the Santa Adlia sugarcane
which is caused by the quality of molasses (related to the level of mill. The stock culture was maintained in YPD (10 g/L yeast extract,
exhaustion and sultation in the sugar production) and the quality 20 g/L peptone and 20 g/L dextrose) at 80 C with 30% (v/v) glyc-
of sugarcane juice, which may vary during the sugarcane season, erol.
depending on the variety, harvest period, climate conditions and
juice extraction procedure [9]. 2.2. Batch fermentations
Rossell et al. [10] recently developed a novel continuous VHG
ethanol fermentation by enhancing the engineering design and A stock culture was activated in liquid YPD medium at 33 C
operation process. Ethanol fermentation in multistage bioreactors under agitation (250 rpm) for 24 h on an Innova 44 orbital shaker
with a well-dened temperature prole was considered in this (New Brunswick, NJ, USA). Then, a sample was streaked onto
technology. Higher temperatures were considered in the initial YPD agar plates, incubated at 33 C for 48 h, and stored at 5 C.
stages to maximize conversion, and lower temperatures were con- The inoculum for all fermentations was prepared by transfer-
sidered in the later stages to minimize the inhibition and cellular ring three loops from agar plates to a new semi-synthetic liquid
damage from the high ethanol concentration. In addition, a dis- medium. The medium consisted of 2.30 g/L urea, 6.60 g/L K2 SO4 ,
tributed carbon source feeding was considered in the rst and 3.0 g/L KH2 PO4 , 0.50 g/L MgSO4 7H2 O, 1.0 g/L CaCl2 2H2 0, 5.0 g/L
second stages to avoid inhibition by high sugar concentrations. yeast extract, 25.44 mg/L trace elements (as detailed in Basso [12]),
The process also included intracellular detoxication through a sec- 3.0 ppm thiamine and 80.0 g/L TSAI. Then, the cells were incu-
ond centrifugation followed by a cell reactivation stage to promote bated at 33 C under agitation (250 rpm) for 12 h on the Innova 44
membrane recovery and enzymatic restoration. The primary goal of orbital shaker. Commercial crystal sugar was used as the source
this technology was to maintain active and viable cells for an entire of TSAI. After the inoculation period was complete, the cells were
harvest season, which is a critical condition to provide operating recovered using a Beckman Avanti J-26 XP centrifuge (JLA-16.250
stability and a high rate of sugar conversion to ethanol. rotor, 5509 g, 10 C, 15 min). The cells were diluted with sterilized
The primary challenge in the VHG fermentation process is to potable water, and this suspension was inoculated in a 7.5 L Bioo
determine the effect of temperature as well as high substrate and 115 bioreactor (New Brunswick, NJ, USA). The initial cell concen-
ethanol concentrations on cell growth kinetics, which clearly affect tration was approximately 1 g/L. The propagation was conducted
the performance of the fermentation process. Although different in fed-batch mode at 33 C and aerated with the carbon source
empirical studies were developed for improving VHG processes, limited to 18 g TSAI/L to minimize ethanol production through the
they lack a systematic model-based approach. A realistic kinetic Crabtree effect [13]. The agitation and airow were controlled by
model is required for the design, optimization and control of the dissolved oxygen (DO) control in cascade mode, with the dissolved
process. O2 concentration maintained above 25% air saturation. The propa-
Temperature is a crucial operating parameter due to its inu- gation medium was the same as that used for the inoculum, with
ence on the conversion of sugars to ethanol, which is an exothermic 180 g TSAI/L. After all sugars were consumed, to recover cells, the
reaction, i.e., heat is released. In the case of conventional fermen- fermented medium was centrifuged using a Beckman Avanti J-26
tation, a temperature control is required, often in the range of XP centrifuge (JLA-9.1000 rotor, 5509 g, 10 C, 15 min). The cells
3235 C [4]. For VHG fermentation, a quantitative understanding were re-suspended with sterilized potable water and refrigerated
of the effect of temperature on substrate consumption and ethanol at 5 C until inoculation for further fermentations. The fermentation
production rates must be investigated to dene the most suitable medium for the cell propagation was similar to the semi-synthetic
operating conditions using the denition of optimum temperature medium described above. The salt and nutrient requirements were
proles. Previous work [11] has addressed the effect of temperature varied according to the initial cell density. All batch fermentations
on ethanol production from sugarcane by S. cerevisiae, resulting were performed in a 3 L Bioo 115 bioreactor (New Brunswick, NJ,
in the formulation of a mechanistic model, which includes terms USA) under agitation at 200 rpm with a working volume of 2 L. The
for the cell, substrate and ethanol inhibitions. The results reported experiments were performed using the initial cell and substrate
that the maximum specic growth rate increases as the temper- concentrations and at the temperatures shown in Table 1.
ature increases. After approximately 37 C, the maximum specic
growth rate begins to decrease. The maximum ethanol and cell con- 2.3. VHG fed-batch fermentation with cell recycling
centrations, i.e., the concentrations at which cell growth ceases, are
inversely related to the fermentation temperature. This model was The inoculum and cell propagation were conducted accord-
validated during batch fermentation under conventional fermen- ing to the aforementioned batch fermentation. The inoculum
tation conditions. medium contained 5.0 g/L urea, 1.1 g/L (NH4 )2 HPO4 (DAP), 1.0 g/ L
This study investigated the effect of temperature on ethanol MgSO4 7H2 O, 5.0 ppm ZnSO4 7H2 O, 3.0 ppm thiamine, 5.0 g/L yeast
production from sugarcane. For this purpose, a mechanistic extract and 80.0 g/L TSAI. The propagation medium contained
fermentation model was developed, with kinetic parameters deter- 5.0 g/L urea, 1.1 g/L DAP, 1.0 g/L MgSO4 7H2 O, 5.0 ppm ZnSO4 7H2 O
mined using a model-based optimization algorithm. The model was and 150.0 g/L TSAI. The TSAI source consisted of 79% (w/w) from
validated through experiments, including fed-batch fermentation sugarcane juice and 21% (w/w) from sugarcane molasses. The
with cell recycling, operating under VHG conditions. The proposed expected nal concentration was 400 g TSAI/L. Physicochemical
methodology drives the systematic development of an industrially treatment, referred to as clarication, was performed to reduce the
reliable mathematical model for VHG fermentation. 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
2. Material and methods 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
2.1. Microorganism [14]. Several minutes of rest were required for ake formation and
decantation. These conditions allowed the drag of soluble impuri-
This study used an unclassied Saccharomyces cerevisiae strain ties. Thus, a claried substrate with high quality, minimal turbidity
cultivated in the Bioprocess Development Laboratory at CTBE, 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- was performed on the cells by adding sulfuric acid (2 M) through
strate required for each fermentation were sterilized at 121 C for a silicon septum until a pH of 2.5 was reached. The acid treat-
30 min. The VHG ethanol fermentation was based on the patent ment conditions were 30 C, 0.2 LPM of air (positive pressure to
WO2014078924A1 of CNPEM/CTBE [10]. A schematic representa- avoid contamination) and 600 rpm for 30 min. This cell suspension
tion of the process is shown in Fig. 1. Fed-batch fermentations was centrifuged as previously described. The cells were then re-
were performed sequentially with cell recycling under six differ- suspended with cooled reactivation medium that contained carbon,
ent conditions. Under each condition, ve fermentation cycles were nitrogen and phosphate sources. This cell suspension was trans-
performed. All stages shown in Fig. 1 are performed in each cycle. ferred back to the bioreactor vessel for the cellular reactivation
Under the rst condition, referred to as the reference condition, stage. After this stage was complete, a new fed-batch fermentation
the fermentations were performed in the 3 L Bioo 115 bioreac- was performed. In the reactivation medium, sugarcane substrate
tor (New Brunswick, NJ, USA), with a working volume of 2 L at and DAP were used to supply carbon, phosphorus and nitrogen
200 rpm and temperature controlled over the range of 2834 C. for membrane restoration and cellular activity recovery. The cell
The oxidation-reduction potential (ORP), pH, capacitance and DO concentration ranged from 80 to 100 g/L (dry basis) in a medium
were monitored. A capacitance system (Aber Instruments, UK) was with 100 g/L TSAI and 3.1 g/L DAP. The operating parameters of the
correlated with viable cell concentrations. The fed-batch started bioreactor with a working volume of 0.9 L were 600 rpm, 33 C, and
with an initial cell suspension volume of 0.9 L at a concentration 2 L/min of air, which produced an environment with an ORP over
of approximately 80 g/L (dry basis), followed by substrate feeding the range of +50100 mV. The residence time was 60 min.
for 5 h. The temperature was set according to the ethanol con- To determine the robustness, stability and reproducibility of
centration. The temperature started at 34 C and ended at 28 C the VHG ethanol fermentation described above, changes to cer-
until all sugar was consumed, producing an ethanol concentra- tain key parameters, such as the DAP concentration, temperature
tion of approximately 120 g/L. The fermentation time for each cycle during fermentation, micro-aeration rate and acid treatment, were
was 11.5 h. A micro-aeration rate of 0.2 vvm was required when investigated. Table 2 summarizes the conditions studied.
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 2.4. Analytical methods
were re-suspended with 0.9 L of sterilized potable water, and then,
they were transferred back to the bioreactor vessel. Acid treatment 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 45

Table 2
Process conditions for VHG fed-batch ethanol fermentation.

Condition Parameter assessed Description of conditions

1 Reference 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

2 DAP DAP concentration is increased to 5.6 g/L


3 Fermentation temperature Beginning at 34 C and ending at 30 C. This last temperature was set
when the ethanol concentration reached 108 g/L
4 Fermentation temperature and Beginning at 34 C and ending at 30 C. The micro-aeration rate is
micro-aeration reduced by half when ethanol concentration reached 108 g/L
5 Fermentation temperature and Returned to the same condition as that used in 3
micro-aeration
6 Acid treatment 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
Fig. 2. Pareto chart of the effects for the concentration of: A) cell, B) substrate and
C) ethanol (at a 95% signicance level). 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
uid chromatography (HPLC). Details are provided elsewhere [15].
validation (RMSECV). The outliers were removed based on F-values
Measurements of the same components in the VHG fed-batch fer-
obtained from the OPUS software calculator. Statistics calculated
mentations were also taken via HPLC. However, HPLC was applied
for the calibrations included the coefcient of determination in
only for the rst and last samples during fermentation. Mea-
cross validation (R2 ), the RMSECV, the bias (systematic averaged
surements of the remaining samples, the feeding substrate and
deviation between the true and predicted values), the number of
the fermented medium concentration were conducted using mid-
PLS factors (rank), and the residual prediction deviation (RPD). Glu-
infrared (MIR) spectroscopy. The technique aimed to use a rapid and
cose and fructose were analyzed using only one calibration curve
accurate method for analyzing VHG ethanol fermentation samples
by reducing sugars (glucose plus fructose). The statistical results for
instead of using HPLC. The cellular viability and budding were ana-
the PLS calibrations developed using the VHG fermentation sam-
lyzed by staining the cells with methylene blue [16] and counted
ples are shown in Table 3. The obtained calibration curves can be
using a Neubauer chamber in an optical microscope (Eclipse CI-S;
used to quantitatively determine these fermentation compounds
Nikon, Japan) for samples at the end of the fermentations. The dry
in VHG fermentation samples with high accuracy (R2 > 99, RPD > 5,
cell concentration was determined gravimetrically after centrifug-
RMSECV (%) < 5). RPD values greater than 5 are considered appro-
ing, washing two times with Milli-Q water and drying at 80 C until
priate for quality control and routine analysis [17].
a constant weight was obtained in an analytical balance.
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 where rx , rs and rp represent the cell growth, substrate consumption
and ethanol production reaction rates, respectively (g/(L h)).
The fermentative yield was calculated using Eq. (1), which is During VHG ethanol fermentation, the cell is exposed to stress
based on the by-products method initially proposed by Finguerut barriers, including osmotic stress due to high substrate concen-
[18]: tration, ethanol stress at the end of fermentation and high cell
densities inside the bioreactor. To simulate these phenomena in
Yield =
100 terms of the physiological state of the cell, substrate saturation
Aacetic Alactic
1.0032 + 0.54
P
(X + G + Sresidual ) + 0.504 P
+ 0.525 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
where P is the ethanol produced, X is the cell produced, G
Eq. (6), respectively).
is the glycerol produced, Sresidual is the TSAI residual, Aacetic is the
acetic acid produced and Alactic is the lactic acid produced.
S P
 n  X
m
The denition of yield based on the by-products method was rx = max exp(Ki S) 1 1 X (6)
Ks + S Pmax Xmax
chosen because it is less sensitive to variations in the specic
weight of the fermented medium (which requires an accurate mea- In the above equation, max denotes the maximum specic growth
surement) compared with the conventional method. This method, rate (h1 ), Ks denotes the substrate saturation constant (g/L), Ki
currently used in ethanol plants in Brazil, consists of measuring the denotes the substrate inhibition parameter (g/L), Xmax denotes the
formation of the main by-products (cells, acetic acid, lactic acid and cell concentration when cell growth ceases (g/L), Pmax denotes the
CO2 ) as well as unconverted sugars related to ethanol formation. ethanol concentration when cell growth ceases (g/L) and m and
The stoichiometric relationship among the acids and CO2 was based n denote parameters related to cellular and product inhibitions,
on empirical data from the ethanol plants. respectively.
The volumetric productivity was calculated according to Eq. (2): Cells meet their energy requirements for growth and mainte-
P nance by producing ethanol. Maintenance is the energy required
Productivity = (2) to support cell function, other than that required for cell synthe-
V.t
sis. For instance, repairing active cell material involves a protein
where P represents the ethanol produced in g, V is the nal volume
synthesis system, among others. The ethanol production rate, rp ,
of the fermented medium in L and t is the total fermentation time
is consistent with the growth and non-growth associated product
in h.
model proposed by Luedeking and Piret [19] and is represented in
Eq. (7):
3. Modeling and parameter estimation
rp = Yp/x rx + mp X (7)
First, the temperature-dependent kinetic model for ethanol
fermentation was developed with experimental data from batch where Yp/x denotes the LuedekingPiret growth-associated
experiments. The model was expanded to simulate fed-batch constant (g/g) and mp denotes the LuedekingPiret non-growth-
ethanol fermentation operated under VHG conditions and vali- associated constant (g/(g h)).
dated against experimental observations. Similar to the ethanol production rate, the LuedekingPiret
equation was proposed for the substrate consumption rate, rs , as
3.1. Kinetic and theoretical aspects of batch fermentation shown in Eq. (8), by considering that the substrate consumed is
not only related to cell growth but is also used for other processes
A mechanistic model that describes the kinetics of the concen- requiring energy:
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), rs = rx /Yx + ms X (8)
was applied to the ethanol fermentation process [11]:
dX where Yx and ms are the cell yield (g/g) and maintenance parameter
= rx (3) (g/(g h)), respectively.
dt
The kinetic model is formed using Eqs. (3)(8), which contain
dS eleven kinetic parameters. The model should include or be able to
= rs (4)
dt estimate these parameters for estimating rx , rs and rp .
dP The reaction rates of the cell are affected by different factors,
= rp (5)
dt 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 convergence toward the global optimum, which involves determin-
ing the optimum values for the parameters ( optimum ) that produce
An accurate estimation of model parameters is a major issue in the best t between the measured concentrations of X, S and P
the development of mechanistic models. Here, a ve-step proce- and their corresponding computed concentrations from the kinetic
dure is proposed to estimate the kinetic parameters for the current model while minimizing J().
model. The execution of Steps 15 generated a set of optimal values for
In Step 1, an identiability analysis of the kinetic model was per- the temperature-dependent parameters for each temperature con-
formed, which was achieved by the calculation of a meta-model sidered (24, 27, 30, 33 and 36 C). Then, the set of optimal values for
based on Plackett-Burman (PB) design [22], capable of providing each parameter was adjusted to Eqs. (9) or (10). This adjustment
all the required sensitivity information. For this analysis, the inu- yielded the best constants A, B, C and D for each temperature-
ence of kinetic parameters (max , Xmax , Pmax , Yx , Yp/x , Ks , Ki , ms , dependent parameter. Once the kinetic model was established, the
m, n, mp ) on responses X, S and P (data at a single timepoint) was constants were ne-tuned using the GA such that J was minimized.
evaluated using a PB design for eleven factors. In the PB design, The nonlinear optimization problem (Eq. (12)) was imple-
the simulations involve systematically varying all kinetic parame- mented in a FORTRAN routine on a 3.6 GHz Intel(R) Core(TM)
ters within 10% of nominal values reported elsewhere [23]. The i7-4790 CPU.
simulation aims to determine the subset of model parameters with
the largest effect on the model prediction, which are modeled as
3.4. Mass balance equations for predicting VHG fed-batch
temperature-dependent.
fermentation
The simulation results were evaluated using the Pareto chart,
which indicates each of the estimated effects of the parameters on
After the kinetic parameters were estimated, the kinetic rates rx ,
X, S and P in decreasing order of magnitude. The size of each bar
rs and rp that incorporate these parameters were used in a mecha-
is relative to the standardized effect, calculated as the estimated
nistic fed-batch model with the mass balance equations described
effect divided by its standard error, which is equivalent to estimate
in Eqs. (13)(15). Thus, the kinetic study provides a basis to allow for
a t-statistic for each effect. In addition, a horizontal line (critical t-
the dynamic simulation of the VHG ethanol fermentation process
value) represents the effects limit of signicance. Bars extending
through a model that is sensitive to inhibition factors and changes
beyond that line relate to effects that are statistically signicant at
in temperature:
the 95% signicance level.
The parameter estimation largely depends on the initial guesses dX F
of the parameter values due to the nonlinear nature of the kinetic = rx X (13)
dt V
model, which contains multiple parameters. Thus, in Step 2 the
initial guesses of the temperature-dependent parameters to be dS F
= (S f S) rs (14)
estimated are obtained from a previous study [9,11,20,23,24]. The dt V
remaining parameters were xed at values given by Atala et al. [24].
dP F
This information is incorporated into the model along with the ini- = rp P (15)
tial conditions for X, S and P. Hence, in Step 3, the kinetic model dt V
written in FORTRAN is integrated using the routine LSODE [25] to where F denotes the feed ow rate (L/h), V denotes the working
obtain the time-course concentrations of X, S and P. Next, in Step 4, volume (L) of the bioreactor, and Sf denotes the total sugar concen-
an objective function J() is dened as the least square error given tration (g/L) in the feed. The reaction rates rx , rs and rp are the same
by Eq. (11): as those obtained from the previous batch experiments.
During model simulation, it was assumed that the sugars are fed

3 
Np
(yexp (t j ) ysim (t j ))2
J() = i i
(11) at a xed F, and the measured V is an input data at each integration
2
yexpi max
time during the simulation.
i=1 j=1

where  is a vector containing all the temperature-dependent


4. Results and discussion
parameters, yexpi (t j ) is the ith measured concentration, i.e., the mea-
sured concentrations of the cell, substrate and ethanol at sampling
4.1. Identiability analysis using the PB design
time j, ysimi (t j ) and yexpi max are the concentrations computed by
the kinetic model and maximum measured concentration, respec-
The parameter estimation procedure considered iterative steps
tively, and Np is the number of sampling points.
with an evaluation of the model reliability at several points. These
In Step 5, a nonlinear optimization problem (NP) is formulated,
evaluations included the identiability analysis of the kinetic model
as shown in Eq. (12), which minimizes the objective function J():
using the calculation of a meta-model based on the Plackett-

Min J() Burman (PB) statistical design. Fig. 2AC shows the Pareto charts
Subject to : obtained from PB design for responses X, S and P, respectively.
NP : (12) According to the estimated effects, Pmax , Yx and Yp/x were consid-

Eqs. (3-8)
ered statistically signicant for all responses. In addition, max was
lb  ub considered statistically signicant for S, and its absolute standard
where  lb and  ub are the lower- and upper-bound vectors for the effect on X is close to the cut-off line for signicant effects. The
temperature-dependent parameter vector , respectively. parameters mp and ms appeared relevant only for P with very small
In this study, a hybrid optimization algorithm based on the effects (nine times smaller) compared to those for Yx and Yp/x .
genetic algorithm (GA) and the Quasi-Newton method (QN) [11] is In this study, the statistically signicant parameters max , Pmax ,
adopted to solve Eq. (12). The hybrid algorithm comprises a thor- Yp/x and Yx were modeled as functions of the fermentation tem-
ough exploration of the search space ( lb   ub ) and nding the perature. In addition, the parameters Xmax and ki were modeled
neighborhood of the global optimum through the GA. Then, the as a function of temperature based on previous studies [11,20,24].
hybrid algorithm, starting from the best estimate of the GA ( best ), The remaining parameters were xed as follows [24]: Ks = 4.1 g/L,
uses gradient information from the QN method to accelerate the 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 the current batch conditions. Therefore, the kinetic model that
in Fig. 4AC, respectively. The measured concentrations used for incorporates inhibition terms and temperature-dependence func-
the parameter estimation are also shown in these gures for com- tions can be used to predict the concentrations of X, S and P at
parison. The results illustrate that the model effectively tracks the changing reaction temperatures, as well as with different initial
desired trajectory of the measured data. concentrations of X and S. This model can handle temperature uc-
A more rigorous assessment of the goodness-of-t between the tuations between 24 and 36 C.
measured and computed data can be conducted using residual stan- These kinetic results were further used to predict the dynamic
dard deviation (RSD), as suggested by Cleran et al. [28]. Recent behavior of X, S and P in VHG ethanol fermentation.
studies [29,30] have adopted RSD as a measure of the prediction
accuracy of proposed models. RSD is written as a percentage of the 4.3. Prediction of VHG fed-batch experiments with cell recycling
average of the measured data, yexp , dened by Eq. (16):
0.5 VHG fed-batch fermentations with cell recycling were per-

Np formed sequentially under six different conditions, as described in
Np
1
(yexp (t j ) ysim (t j ))2 Table 2. Five fermentation cycles were performed under each con-
j=1 dition. All stages described in Fig. 1 are conducted for each cycle.
RSD(%) = 100 (16) Fig. 6 shows the average fermentation performance for each condi-
yexp
tion in terms of yield, productivity and nal ethanol concentration.
where yexpi (t j ) represents the ith measured concentration, i.e., the Condition 1 (the reference condition, described in detail in Sec-
measured concentrations of the cell, substrate and ethanol at sam- tion 2.3) obtained a yield of 84%, a productivity of 10.2 g/L h and
pling time j, ysimi (t j ) are the concentrations computed by the model an ethanol concentration of 120 g/L (average values for ve fed-
and Np is the number of sampling points. batch fermentations). After condition 1 was tested, a second set of
Fig. 5A illustrates that the model predictions were particularly ve fed-batch fermentations were conducted (condition 2) with the
accurate, as determined by the RSD (%). These results demonstrate aim of improving the fermentation performance by varying the DAP
that the model can be used to predict the dynamic behavior under 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 provided a yield of 88%, a productivity of 10.4 g/L h and an ethanol
as the reference conditions). A slightly better fermentation perfor- concentration of 124 g/L. Hence, this condition was the most favor-
mance than the reference condition was obtained, with a yield of able in terms of productivity and ethanol concentration.
87%, a productivity of 10.3 g/L h and an ethanol concentration of These results demonstrate that all average productivities were
122 g/L. For condition 3, the temperature at the nal stage during approximately 10 g/L h while maintaining yields between 84% and
the exhaustion of sugar conversion was slightly above the nal ref- 90%. These productivity values are high compared to the typical
erence temperature. As shown in Fig. 6, this condition was the most industrial value of 5.5 g/L h in which the yield is 89% working in
efcient process in terms of fermentation yield, with a value of 90%. continuous mode. The unexpected low value of yield is related to
However, the productivity of 10.2 g/L h and ethanol concentration the residual sugar at the end of fermentation, indicating that more
of 120 g/L were lower than those of condition 2. In condition 4, in time is required for its consumption.
which the micro-aeration rate was reduced while maintaining the The fed-batch model can describe the dynamics of cell growth,
same temperature prole of condition 2, the fermentation perfor- substrate consumption and ethanol production during VHG fer-
mance was the poorest of the examined conditions, with a yield mentation under varying conditions (Table 2). These results are
of 84%, a productivity of 9.8 g/L h and an ethanol concentration shown in Fig. 7. The corresponding assessment using RSD (%) is
of 115 g/L. An attempt was made to remedy this poor fermenta- shown in Fig. 5B. This proposed model could provide a basis for the
tion performance situation in condition 5, which used the same design and operation of VHG ethanol fermentation at an industrial
operating parameters as those in condition 3. However, despite a scale.
slight improvement (a yield of 84%, a productivity of 9.9 g/L h and an The model assumed that the ethanol production rate, rp , is
ethanol concentration of 117 g/L), the fermentation performance of consistent with the growth and non-growth associated prod-
condition 3 could not be reproduced. Thus, a micro-aeration rate uct, as described by Eq. (7). However, above the Pmax value in
of 0.2 vvm at the end of the fermentation should be maintained to VHG fermentation, rp is consistent only with the non-growth
avoid a decrease in the fermentation performance. Finally, in con- associated product. Pmax is a temperature-dependent parameter;
dition 6, nitric acid was used instead of sulfuric acid. This condition therefore, it has different values for different temperatures, as
E. Ccopa Rivera et al. / Biochemical Engineering Journal 119 (2017) 4251 51

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