A New Approach to Model the Influence of Stirring Intensity

on Ethanol Production by a Flocculant Yeast Grown on

Cashew Apple Juice

Andréa da Silva Pereira,1 Alvaro Daniel Teles Pinheiro,2 Maria Valderez Ponte Rocha,1
Luciana Rocha B. Gonçalves * and Samuel Jorge Marques Cartaxo1*
1

s-Graduaça
1. Programa de Po ~o em Engenharia Quımica, Universidade Federal do Ceara , Fortaleza, CE, Brazil
^
2. Departamento de Agrotecnologia e Ciencias Sociais, Universidade Federal Rural do Semiarido, Mossoro, RN, Brazil

In this work, a rigorous mathematical model was developed, aiming to address a major flaw inherent in most fermentation models found in the
literature, which is the inability to accurately account for mass transfer effects. This model was based on the hypothesis of the existence of a stagnant
film involving the cell where the mass transfer rate of the substrate flowing from the medium to the cell surface is equal to the rate of substrate
consumption by the cells. The model was used to explore the influence of stirring speed, substrate, and initial cell concentrations and temperature
on ethanol production by the flocculant yeast Saccharomyces cerevisiae CCA008, grown on cashew apple juice. Model parameters were estimated
and validated against experimental data. The experimental data was divided into two sets: one for parameter optimization using non-linear
Marquardt least-squares method; and the other to validate the final form of the model equations. Results have shown that the model herein
proposed was capable of accurately describing the production of ethanol by S. cerevisiae flocculant yeast considering the influence of operational
conditions, especially the effect of the stirring speed on the fermentation rate.

Keywords: cashew apple juice, initial cell concentration, stirring speed, mathematical model

INTRODUCTION speed is essential to avoid cells assuming a flocculated state during

fermentation.[9]

A
lcoholic fermentation allows for the production of ethanol,
which is currently a major substitute for gasoline.[1–5] The
global production of biofuels has grown constantly during
the last decade from 16 billion L in 2000 to around 110 billion L in
2013.[3] Although a less optimistic outlook is expected, biofuels
production is expected to grow 16 % from 2016–2022, mainly
due to supportive policy in Brazil and the U.S.A. as well as the
rising demand for fuel in Asia.[4]
In this context, cashew apple juice appears to be a promising
alternative raw material for the production of ethanol by
fermentation.[5] Thus, since the potential of the fruit is not being
fully explored, the use of this coproduct for the production of
ethanol will not only bring economic benefits to the process but
also solve its disposal issues, adding value to the production chain
of cashews (nut and apple).[6]
In these processes, yeasts are mostly used as microorganisms
and Saccharomyces is one of the most important for the industry in
addition to being one of the most studied groups by the scientific
community.[7] They are facultative microorganisms, i.e., when in
aerobiosis they transform part of the sugar in biomass, CO2, and
water, and when in anaerobiosis, they convert sugar into ethanol
and CO2. Thus, O2 transfer to the medium is undesirable for
producing ethanol.
The flocculant yeast Saccharomyces cerevisiae notoriously forms
little floccules that are deposited on the bottom of the fermenter at
the end of the reaction. A beneficial effect of this is that the
microorganism can be easily separated from the fermented
medium, leading to a reduction in process costs, as medium
centrifugation becomes unnecessary.[8] A drawback of floccula-
tion is that it may cause loss of productivity in the fermentation
process due to the barrier imposed by the transport of reactants
and metabolite products across the flocs. Therefore, the stirring
S. cerevisiae CCA008 must be non-flocculent during ethanol
fermentation, which makes the use of mechanical agitation
mandatory in order to avoid lowering the yield of the conversion of
sugars into ethanol. Stirring speed must be at a sufficient level to
homogenize the medium, without promoting oxygen transfer to
the reaction medium. Therefore, the influence of stirring speed on
the reaction medium containing the flocculant S. cerevisiae is
necessary to evaluate its influence on the fermentation parame-
ters. It is important to point out that flocculant yeasts present low
levels of cells growth, which may be associated with the mass
transfer limitations of glucose in the flocs.[10] Another fact that
should be taken into account, to ease the production of ethanol, is
the concentration of yeasts in the bioreactor because it is
experimentally observed that the higher the cell concentration,
the faster the fermentation, with higher productivity and better
control against contaminants.[11] On the other hand, high cell
densities may favour flocculation requiring a full understanding of
the phenomena.[12]
Although, there is a significant amount of fermentative models
that are available, a prevalent deficiency in them is the inability to
account for the stirring speed influence, which indicates a major
flaw in representing the physics of mass transfer processes that are
* Author to whom correspondence may be addressed.
E-mail address: lrg@ufc.br (L. R. B. Gonçalves);
samuel@ufc.br (S. J. M. Cartaxo)
97:1253–1262,
Can. J. Chem. Eng. 9999:1–10, 2019
2018
© 2018 Canadian Society for Chemical Engineering
DOI 10.1002/cjce.23419
Published online in Wiley Online Library
(wileyonlinelibrary.com).

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involved in the fermentation reaction. Research reported in
literature relates mostly to statistical models, hybrid models,
and control adaptive algorithms based on experimental data.
Saraceno et al.[13] brings a hybrid neural approach to model the
batch fermentation of ricotta cheese whey to ethanol, which
represents the influence of temperature, pH, initial substrate
concentration, and stirring speed with an average percentage error
lower than 10 %. Pinheiro[14] presents a statistical approach as a
reliable alternative to model, optimize, and study the interactive
effect of the operational conditions that were evaluated for the
alcoholic fermentation process of cashew apple juice. Yao et al.[15]
proposes a new relationship that represent the L-lysine formation
rate and a mathematical description that explicitly relates the
process state variables to the agitation speed using combined
kinetic study and material balances.
To tackle the mass transfer problem, in this work the
mechanistic modelling approach is used, which favours the
understanding of the particularities of the process and its kinetic
parameters, giving to them physical meaning and grounded
rationalization. Furthermore, this makes it possible to adapt the
model according to the system under investigation by calibrating
the relevant parameters, which increases its range of applicability
remarkably.
A comprehensive model should cover the various operational
variables that affect the performance of the fermentative process,
such as initial substrate concentration (sugar), temperature, initial
cell concentration, and stirring speed.[16] In this context, this work
seeks to propose a new mechanistic model that includes all of the
operational conditions cited above. The capability to represent
mass transfer-related phenomena with reasonable accuracy is
critical for scale-up applications, since the mixing characteristics
inside the reactor are strongly dependent on its size in the case of
flocculant yeasts.
It should be emphasized that the modelling approach presented
herein can be used to embed mass transfer effects in possibly any
strictly kinetic fermentation model, provided that the necessary
hypotheses remain sustained. In this particular case, the
fermentation model presented in this paper was built upon a
purely kinetic model from the same source of the experimental
data used for validation.[17]
EXPERIMENTAL
Experimental Data
Fermentation was conducted in a 1 L bench-scale bioreactor
(Tec-Bio, Model 1.5, Tecnal, SP, Brazil), at stirring speed of
150 rpm without aeration, and with a working volume of 750 mL.
The bioreactor was inoculated with Saccharomyces cerevisiae
CCA008 for ethanol production. The operation conditions of
the fermentative essays are shown in Table 1, where initial
cell concentration varied from 4–10 g/L and stirring speed
from 80–800 rpm during a period of 10 h. Samples were
taken at pre-defined intervals and analyzed for cell growth,
pH, substrate concentration, and ethanol concentration. The
detailed procedure can be found in Pinheiro et al.[17]
MATHEMATICAL MODELLING
The Mass Transfer Problem
A strictly mechanistic model for describing fermentation and
considering the kinetics and mass transfer phenomena may be
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presently out of reach, in the sense that every single model
parameter is deduced through solid analytical argumentation.
However, the inclusion of more physics in the mathematical
model both reduces the number of parameters and provides
justification for their existence, since these parameters arise
naturally from conservation principles or other physical laws.
In the field of fermentation, prevalent models are derived from
the principle of mass conservation associated with chemical
kinetic rules adapted to the realm of biochemistry or cell biology.
Such a view reduces the complexity of the fermentation process to
a chain of reaction steps, in which the product of each step is
immediately available to be the reactant of the next step without
delay. While this approximation is quite close to reality for
homogeneous chemical systems, it may seriously fail in heteroge-
neous biochemical processes due to the existence of significant
mass transport barriers between each reaction step.
A more complete description needs to deal with both kinetics
and mass transport resistances, and, thus, our approach consists of
incorporating the mass transfer effects between the reaction steps
that are classically represented in the currently available
fermentation models.
The mass transfer process may affect the fermentation metabo-
lism in two ways: by diminishing the entrance of substrate into the
cell; or by retarding the removal of metabolite products from the
cell. This model treats only the first situation. This is a reasonable
assumption when the substrate entrance controls the whole
process, i.e., the rate of metabolites’ removal is much higher than
the availability of substrate in the cell cytoplasm.
The obvious entry point for the inclusion of the substrate mass
transfer resistance is the mass conservation equation for the
substrate, described in detail in the next section. This feature is
remarkable because in order to adapt a specific kinetic model to a
given yeast or another microorganism, one will just need to
change the differential equation for the substrate.
Reactor Mass Balances
Cell concentration and stirring intensity are both relevant
variables for the fermentation system studied here since the
microorganism used in the validation experimental data is a
flocculent yeast (S. cerevisiae), which tends to form floccules that
can deposit at the bottom of the fermenter.[17] This creates zones
with higher and lower cell concentrations, interfering in the mass
transfer. The intensity of the stirring speed applied to the reaction
and the initial cell concentration are essential to maintain the
floccules in suspension during the fermentation in a homogeneous
medium.[12]
Another fact that should be highlighted is that the metabolic
behaviour of biomass for the production of ethanol may
change according to the yeast concentration in the bioreactor
Table 1. Batch fermentation operating conditions
Entry S0 (g/L) T (8C) X0 (g/L) Agitation (rpm)
1 100 34 4 150
2 100 34 5 150
3 100 34 8 150
4 100 34 10 150
5 110 34 7 80
6 110 34 4 300
7 110 34 5 490
8 110 34 5 650
9 110 34 5 800
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(experimentally observed). Although the former model did not 1
ds ¼ kmix  ð4Þ
take into account the cellular metabolism, a parameter to absorb Imix n
shifts promoted by metabolism based on the substrate will be
incorporated. ks
JS ¼   ðS  S Þ  Imix n ð5Þ
The model considers that there is a stagnant film around the cell, kmix
leading to a lower concentration of substrate; in other words, the
available substrate (CS ) for the reaction is lower than the bulk The mass transfer rate of substrate is obtained by multiplying its
substrate concentration (CS), as shown in Figure 1. The flux by the total surface of cells per solution volume. The
simplifying hypotheses used in the development of this model superficial area of cells is proportional to its concentration,
are shown as follows: according to Equation (7). By combining Equations (5), (6), and
 unstructured non-segregated kinetic model, in which the (7), it is possible to express the substrate transfer rates
microorganism is taken as a simple reactant; (Equation (8)). The parameters can be grouped in a single bmix
 the reaction kinetic is a function of substrate and product parameter, so Equation (9) proposes the substrate transfer rate in
concentrations: Ghose and Tyagi’s model;[17] the stagnated film:
 temperature dependence is represented by the modified
Arrhenius equation;[17] jS ¼ J S  A x ð6Þ
 existence of a stagnated film (mass boundary layer) involving
the cell; Ax / X ! Ax ¼ ax  X ð7Þ
 mass transfer rate of substrate flowing from medium to the cell
surface is equal to the rate of substrate consumption by cells; dS ks : ax
¼  ðS  S Þ  Imix n  X ð8Þ
 substrate concentration on the cell surface is an exponential dt kmix
decay function of cell concentration because there is a
dependence between concentration of cell and resistance to dS
¼ bmix  ðS  S Þ  Imix n  X ð9Þ
the mass transfer; and dt
 it is assumed that the thickness of the boundary layer (ds) is a
function of the stirring speed following a power law. As mentioned in the simplifying hypotheses, the substrate that
The substrate flux through the boundary layer is defined reaches the surface of the cells is consumed to produce ethanol.
analogically by Fick’s law (Equation (1)), where the substrate flux Therefore, mass transfer and reaction are consecutive steps of the
is proportional to the mean approximate gradient: process; in other words, they occur in series (see Figure 1). Making
an analogy between the cause and effect model controlled by an
~ ~ a
J A ¼ Da  rC ð1Þ appropriate resistance (Equation (10)), we obtain the resistance to
the mass transfer (Equation (13)) and the resistance to the reaction
ðS  S Þ (Equation (16)):
JS ¼ ks  ð2Þ
ds cause
effect ¼ ¼ conductance  cause ð10Þ
resistance
The stirring speed lowers the thickness of the boundary layer
(ds ) in such way that we can assume an inverse proportionality
relation. It is assumed that ds may vary with the stirring speed
following a power law, as shown in Equation (4). Substituting
Equation (4) in Equation (2), the substrate flux through the
stagnated film as function of the stirring speed is obtained
(Equation (5)):
Table 2. Mathematical model proposed
ds / ðstirring speedÞ1 ! ds / ðImix Þ1 ð3Þ
Mathematical Model
dXv
dt ¼ m  X v  k d  Xv
dXd
¼ k d  Xv
dt  
mix :mS
dS
dt ¼  mmmix þmS  Xv
dP
dt ¼ Y P=X  m  Xv
X T ¼ Xv þ X d
mmix ¼ bmix  ðS  S Þ  Imix n
 
mS ¼ Y mX=S þ ms
S ¼ h  S
h ¼ a  eðbXv Þ
   
mmax S
m ¼ f ðS; P; T Þ ¼ 2  1  Pmax
P
Ks þSþSK
i

mmax ¼ f ðT Þ ¼ A1  eð =T Þ þ C 1  eð =T Þ
B1 D1

¼ f ðT Þ ¼ A  eð =T Þ þ C  eð =T Þ
B2 D2
YX=S 2 2
Figure 1. Substrate concentration around the cell and resistances for mass
Y P=X ¼ f ðT Þ ¼ A3  eð =T Þ þ C 3  eð =T Þ
B3 D3

transfer.

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Figure 2. Schematic diagram of the reactors: (a) 1 L reactor; and (b) 14 L reactor.

 
Mass transport: dS  dS 
¼ ð18Þ
dt mass dt reaction
effect conductance cause  
z}|{ dS Xv mmix  mS
dS ¼ zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl}|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{ z}|{ ð11Þ ¼ ¼  Xv ð19Þ
bmix  ðS  S Þ  Imix n  Xv dt Req mmix þ mS
dt

mmix ¼ bmix  ðS  S Þ  Imix n ð12Þ From the general equation for substrate consumption, consid-
ering influence of mass transfer and the kinetic of the reaction, it
1 was time to estimate substrate concentration in the surface of the
Rmix ¼ ð13Þ
mmix cell (S ). The easiest way to relate S is by using Equation (20),
where 0 < h < 1, once the surface concentration is a function of
Reaction kinetic: the instant substrate concentration in the mean:

conductance cause S ¼ h :S ð20Þ

effect
z}|{ zfflfflfflfflfflfflfflfflfflfflfflfflffl}|fflfflfflfflfflfflfflfflfflfflfflfflffl{
 
dS ¼ m z}|{ ð14Þ
It was observed from experimental data that h varies with cell
 þ ms  Xv
dt YX=S concentration, which is justified by the fact that the higher the cell
concentration then the higher the mass transfer resistance. It is
 
m also a way to consider the metabolism of cells, which in some
mS ¼  þ ms ð15Þ concentrations may require more or less substrate.
YX=S
Linear, exponential, and power laws were tested to estimate the
1 parameter h with dependence of cell concentration; however, the
Rc ¼ ð16Þ exponential model allowed the best fit, see Equation (21):
mS

h ¼ a:eðb:Xv Þ ð21Þ
By combining the resistances in series, the equivalent resistance
is obtained, shown in Equation (17). As the mass rate of substrate
in the stagnated film is equal to the substrate consumption rate, Table 2 presents the equations for the model herein proposed,
Equation (18) can be applied, resulting in Equation (19): using the model presented by Pinheiro et al.[17] improved with the
influence of initial substrate and cell concentrations, temperature,
1 1 m þ mS and stirring speed.
Req ¼ Rmix þ Rc ¼ þ ¼ mix ð17Þ
mmix mS mmix  mS

Table 4. Statistical average analysis for the model that considers the
Table 3. Estimated parameters for the model that considers the effect effect of stirring
of stirring
  Cell Substrate Product Fermentation
bmix L  g1 h1 rpmn n h (%)
eexp ð%Þ > 8.86 14.05 13.00 13.40
2.201 0.365 87.2 RSD 0.72 9.00 4.72

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 Table 5. Estimated parameters for the model that considers the effect Table 6. Statistical average analysis for the model that considers the
of initial concentration of cells and stirring effect of initial concentration of cells and stirring
  Cell Substrate Product Fermentation
bmix L  g1 h1 rpmn n a b
eexp ð%Þ > 9.57 10.16 9.77 9.77
2.201 0.365 2.600 0.282
RSD 0.78 6.49 3.57

Parameter Estimation vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

u
u Sm 2  ðn  pÞ
e >t Pn=3 h exp
2 exp
2 exp
2 ð25Þ
exp
The Marquardt algorithm was used to estimate the kinetic
parameters by nonlinear least squares fitting.[18] The results Ftab  j Xj þ Sj þ Pj 
obtained from the model integration for the cell and substrate and
product concentrations were compared with the experimental
data, using an optimization routine until a minimal error of 103,
between the experimental and simulated values, was achieved.
The mathematical model was implemented in Python/IPython
Notebook 2.7.8, associated with some of its scientific modules.

Accuracy Assessment
The prediction quality of the kinetic model was evaluated through
graphical analysis, consistency of physical parameters, and by the
calculation of the deviation residual standard deviation (RSD
(%)). Equation (22) shows the RSD suggested by Cleran et al.:[19]
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Xn 2
i¼1 i
ðy exp  yi cal Þ
RSD ¼ ð22Þ
n

The calculation of the variance error between the experimental

data and theoretical data is one of the ways to discriminate the
models (modified F-test).[20] Using Equation (25), it is possible to
estimate the experimental error:

Pn=3 h
2
2
2 i
j Xj exp  Xj cal þ Sj exp  Sj cal þ Pj exp  Pj cal
Sm 2 ¼
np
ð23Þ

Pn=3 h
2
2
2 i
e2 j Xj exp þ Sj exp þ Pj exp
Se 2 ¼ ð24Þ
n  nv

Figure 4. Experimental and simulated data for test 1: (closed circles) cell
concentration (g  L1); (closed square) substrate concentration (g  L1);
Figure 3. Substrate transport efficiency by stagnated film as a function of (closed triangles) ethanol concentration (g  L1); (—) model; and (---)
cell concentration. confidence interval with 90 % significance level for the experimental data.

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SCALE-UP OF THE FERMENTER
A usual criteria for scaling-up an anaerobic bioprocess is the
constancy of power in the unaerated system per medium volume
ratio (P/V).[21] Thus, P/V criteria was used and applied when the
estimation of mass transfer rates was necessary. Afterwards, the
proposed model was used to foresee the experimental data
obtained in a 14 L reactor after the scale-up, maintaining the
same initial substrate concentration, temperature, and initial cell
concentration conditions. Therefore, the objective was to ensure
that the mathematical approach is capable to predict the
fermentation behaviour in many production levels and estimate
the stirring speed required for given process conditions.
Figure 5. Experimental and simulated data for test 5: (closed circles) cell
concentration (g  L1); (closed square) substrate concentration (g  L1);
(closed triangles) ethanol concentration (g  L1); (—) model; and (---)
confidence interval with 90 % significance level for the experimental data.
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In order to make the scale-up from a 1 L to a 14 L usable volume
fermenter, the first step was to guarantee the geometric similarity
between systems (see Figure 2). After that, the scale-up relation
that was chosen (P/V) was developed. With the new stirring
conditions in hand, experimental runs were performed
with the purpose of validating the mathematical model for the
scaling-up.
The criteria used to scale-up was the constancy of power in the
unaerated system per medium volume ratio (P/V), once it had
been indicated that it was necessary to keep the mass transfer ratio
constant. Therefore, power per volume in the 1 L reactor (reactor
1) should be equal to the power per volume of the 14 L reactor
(reactor 2):
Figure 6. Validation model using an initial cell concentration of 5 g/L and
stirring speed of 650 rpm: (closed circles) cell concentration (g  L1);
(closed square) substrate concentration (g  L1); (closed triangles) ethanol
concentration (g  L1); and (—) model.
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  23
Di1
Table 7. Operating conditions for reactors 1 and 2 N2 ¼ N1 ð34Þ
Di2
Volume S0 Temperature X0 Agitation
Reactor (L) (g/L) (8C) (g/L) (rpm)
For Reynolds numbers of 104 and above, the mixing-time factor
1 1 110 34 6 150 N.tT is constant, and since time tT is assumed constant, speed N
2 14 97 will be the same in both vessels. By substituting Equation (35) into
Equation (34), Equation (36) is obtained, which predicts the
    mixing-time for the new system:
P P
¼ ð26Þ  
V 1 V 2 tT1
N2 ¼ N1 ð35Þ
tT2
The power number (Np) for the unaerated systems is shown in
Equation (27), and the dimensionless number relates the required  23
Di2
power to homogenize the medium (P), the stirring speed (N), the tT2 ¼ tT1 ð36Þ
Di1
impeller diameter (Di), and the fluid density (r). Np keeps
approximately constant in different scales when the same impeller
is used, considering turbulent regime. Therefore, Equation (28)
expresses the required power for a certain type of impeller. Doran[21] RESULTS AND DISCUSSION
presents Equations (29) and (30) to model bioreactors with Rushton
turbine impellers, as employed in the fermenters herein studied: Initial Cell Concentration and Stirring Speed
P Table 3 presents the parameters of the specific mass transfer rate
Np ¼ ð27Þ estimated for assays 5, 6, 7, and 9, keeping the free substrate portion
rN Di 5
3
as constant as the cell. It can be observed that 87.2 % of the
substrate present in the fermentative medium reaches the surface
P ¼ rN3 Di 5 Np ð28Þ of the cell, since part of it transformed into ethanol by the reaction.
Table 4 shows the statistical analysis of the simulations
DT comprising all assays for the model with influence of stirring
¼ 3 ! DT ¼ 3Di ð29Þ
Di speed and for parameter h constant (substrate transfer efficiency
through the boundary layer). One can observe that RSDs values
HL and apparent experimental errors are high, which indicates that
¼ 3 ! HL ¼ 3Di ð30Þ
Di

The occupied volume of the cylindrical fermenter (V) is

obtained by Equation (31) as a function of the tank diameters
(DT) and the liquid height inside it (HL). By replacing
Equations (29) and (30) in Equation (31), the volume as a
function of the impeller diameter is obtained (Equation (32)):

DT 2
V¼p HL ð31Þ
4

27 Di 3
V¼p ð32Þ
4

Finally, replacing Equations (28) and (32) in Equation (26)

results in Equation (34), i.e., the prediction of stirring speed of the
scaled-up fermenter (reactor 2). Therefore, the consumption of
power per volume is maintained constant for both reactors:
! !
rN3 Di 5 Np rN3 Di 5 Np
27 Di 3
¼ 27 Di 3
ð33Þ
p 4 1
p 4 2

Table 8. Estimates of specific mass transfer velocity parameters for

reactors 1 and 2
 
Reactor bmix L  g1 h1 rpmn (%)

1 2.201 55.0 Figure 7. Profile of specifi

Profile specificc mass
mass transfer
transfervelocity
velocityfor:
for (a)
(a) reactor
reactor 1;
1 and (b)
2 0.055 13.7 reactor 2.

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the model does not obey adequate limits based on 10 %
experimental mean errors for fermentative processes.[22]
Therefore, the influence of initial cell concentration on the
estimated parameters was investigated, and one can conclude that
X0 directly affects the concentration of substrate available on the
surface to react. This fact may be explained by the higher
resistance to the mass transfer when the cell concentration is
higher, making the medium more susceptible to the formation of
zones with a higher concentration of the yeast S. cerevisiae due to
its flocculation in the bottom of the fermenter.
From essays 1, 2, 3, and 4, the parameters of exponential decay
of h were estimated with instant cell concentration, shown in
Table 5. Figure 3 illustrates the behaviour of h for the range from
3–10 g/L where it can be observed that at low concentrations of
cell (X < 4 g/L) the substrate concentration on the surface is
superior to 80 % of the free substrate in the medium; however, at
high concentrations (X > 7 g/L) the efficiency of the mass transfer
of the substrate that reaches the cells surface is inferior to 40 %,
significantly affecting the substrate concentration profiles and,
consequently, the product concentration profiles.
It is important to emphasize that the graph decreases with an
increasing cell concentration, however, the reliability of the
parameter is guaranteed only from the range of the experiments
(4–10 g/L).
The assays were simulated for new parameters, and the
statistical analyses are shown in Table 6. One can verify that
the values of RSDs and apparent errors of tests were lowered
Figure 8. Simulation of the mechanistic model for scaling with: (a) mmix estimated for reactor 1; and (b) mmix estimated for reactor 2: (closed circles) cell
concentration (g  L1); (closed square) substrate concentration (g  L1); (closed triangles) ethanol concentration (g  L1); (—) model; and (---)
confidence interval with 90 % significance level for the experimental data.
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and are comprised by the adequate limits from the modified
F Test. Therefore, the range of utilization of Model 4 will be from
80–800 rpm stirring speed and reduced to an initial cell
concentration from 4–10 g/L.
Figures 4 and 5, respectively, show the experimental simulated
profiles for assays 1 and 5. It is observed that the model
satisfactorily describes the behaviour of the assays, as was already
proven in the statistical analysis (see Table 6).
Model Validation
The operational conditions of test 8 (X0 ¼ 5 g/L and stirring
speed ¼ 650 rpm) was simulated and compared to the experi-
mental data obtained in the same conditions, as a means of
validating the proposed model. It is important to highlight that this
experimental assay was not used to estimate the model
parameters; however, it is comprised in the ranges of initial cell
concentration (4–10 g/L) and stirring speed (80–800 rpm) that
were investigated.
The model validation was performed by an inspection of the
behaviour of the concentration profiles and statistical analysis for
assay 8 (not used in the model parameters estimation), shown in
Figure 6.
It is known that typical experimental errors, for measured
variables in fermentative processes, is approximately 10 %, due to
the lack of reproducibility and natural fluctuations of the
process.[23] Therefore, the model can be considered statistically
adequate since eapparent < 10 %.
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 RSDs and apparent experimental errors (eapparent ¼ 9:77 %) The criteria of constant power per unit volume (P/V) proved to
are low and are comprised by adequate limits of the model for all be an adequate strategy for scaling-up. However, it was necessary
concentration profiles. Thus, a satisfactory mechanistical model to estimate mass transfer specific rates for the 14 L reactor once it
is proposed to represent the influence of the initial concentration presented homogenization problems caused by the deposition of
of the substrate and cells, as well as temperature and speed of yeast floccules in the bottom of the fermenter at the beginning of
agitation. the reaction.
The statistical analysis, model validation, and system scale-up
Scale-Up steps showed that the model is quantitatively adequate and
After choosing the operational conditions within the validity of the flexible to describe the alcoholic fermentation of cashew apple
proposed models for the 1 L fermenter (Di1 ¼ 9:5 cm), the stirring juice by S. cerevisiae flocculant, however, the mass transfer
speed estimation was performed for the 14 L reactor (Di2 ¼ 21 cm) parameters are dependent on the volume of the fermenter.
using Equation (34). With the operational conditions in hand for
both cases (Table 7), the model can be simulated and validated
NOMENCLATURE
with the new experimental data.
It was also verified that the mixing time (Equation (35)) for Da diffusivity (m2  s1)
reactor 2 is 70 % higher than for reactor 1. Therefore, reactor 1 Ftab tabled value for the Fisher Test F
presents a better homogenization system and, consequently, a Imix stirring speed (rpm)
lower resistance to the mass transfer, indicating a more efficient Ja mass flux of the reagent to (g  m2  s1)
substrate consumption. JS substrate flux (g  m2  s1)
Therefore, a new estimation for the mass transfer rate for the jS substrate rate (g  s1)
mechanistic model is necessary, once the reaction kinetics are not ks mass transfer coefficient (m2  s1)
modified by the system geometry. kd specific rate cell death (h1)
Table 8 compares the estimated parameters for reactors 1 and 2, kmix proportionality constant for stirring
maintaining the same power law for the influence of agitation. A Ks substrate saturation constant (g  L1)
significant decrease in bmix for reactor 2 can be observed, i.e., an Ki substrate inhibition constant (g  L1)
average value of 13.7 % of available substrate arriving on the mS specific rate cell maintenance (h1)
cell’s surface to be converted into ethanol,. n number of experimental points
The behaviour of the mass transfer specific rate (mmix) during nv number of variables estimated
fermentation is illustrated in Figure 7. One can note that the sugar p number of model parameters
transfer resistance to the cells is significantly larger in the 14 L P product concentration (g  L1)
reactor, evidencing that there is a concentrated zone formed by the Pmax product concentration when cell growth ceases (g  L1)
deposition of S. cerevisiae flocculant in the bottom of the Rmix resistance to the mass transfer (h)
fermenter. Rc resistance to the reaction (h)
Figure 8 compares the simulated concentration profiles for the S free substrate concentration (g  L1)
model with experimental data obtained in the 14 L reactor. The S available substrate concentration for reaction (g  L1)
mechanistic model uses mmix estimated from the data of reactors 1 S0 initial substrate concentration (g  L1)
and 2. It is noted that due to the longer mixing time in the 14 L t time (h)
reactor, the substrate consumption and, consequently, the ethanol Xv cell alive concentration (g  L1)
production are delayed, as shown in Figure 8a. Xd cell dead concentration (g  L1)
It can be observed in Figure 8b that changing the value of the XT cell total concentration (g  L1)
mass transfer specific rate allowed the model to describe the yi experimental values
experimental data after the scale-up, confirming that the model ypi calculated values
sufficiently absorbed the physical changes of the process due to the YP/X yield of product based on cell growth (g  g1)
increase in the volume of the fermenter. YX/S yield of cell growth based on substrate consumption
(g  g1)
YP/S yield of product based on substrate consumption
(g  g1)
CONCLUSION
bmix coefficient
of mass transfer

of substrate in the stagnated
Some hypotheses were fundamental to the development of a film L  g1 h1 rpmn
satisfactory mechanistic model for the production of ethanol with eexp apparent experimental error
the influence of initial cell concentration and stirring speed, which h efficiency of substrate concentration at the boundary
were as follows: i) the existence of a stagnated fluid at the surface layer
of cells, triggering mass transfer resistance; and ii) substrate mS specific rate of reaction (h1)
transfer rates reaching the cell surface were equal to the rates of mmax maximum specific growth rate of cells (h1)
substrate consumption by cells. mmix specific rate of the mass transfer (h1)
The mathematical model herein proposed presented an m specific growth rate of cells (g  L1  h1)
innovation to describe the influence of the operation conditions ds thickness of the boundary layer (m)
that were derived from physical knowledge of the fermentative
process. It was verified that the model is adequate for ranges
of substrate concentration from 70–170 g/L, temperature from
26–42 8C, cell concentration from 4–10 g/L, and stirring ACKNOWLEDGEMENTS
speed from 80–800 rpm with an apparent mean error for the This work was supported by Fundaç~ao Cearense de Apoio ao
fermentation of eexp > 9:77 %. Desenvolvimento Cientıfico e Tecnolo
gico, Conselho Nacional

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