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 Chapter 6

Kinetic Modeling of 1‐G Ethanol Fermentations

Samuel C. Oliveira, Dile P. Stremel,

Eduardo C. Dechechi and Félix M. Pereira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65460

Abstract
The most recent rise in demand for bioethanol, due mainly to economic and environ-
mental issues, has required highly productive and efficient processes. In this sense,
mathematical models play an important role in the design, optimization, and control of
bioreactors for ethanol production. Such bioreactors are generally modeled by a set of
first-order ordinary differential equations, which are derived from mass and energy
balances over bioreactors. Complementary equations have also been included to
describe fermentation kinetics, based on Monod equation with additional terms
accounting for inhibition effects linked to the substrate, products, and biomass. In this
chapter, a reasonable number of unstructured kinetic models of 1-G ethanol fermenta-
tions have been compiled and reviewed. Segregated models, as regards the physiologi-
cal state of the biomass (cell viability), have also been reviewed, and it was found that
some of the analyzed kinetic models are also applied to the modeling of second-gener-
ation ethanol production processes.

Keywords: ethanol fermentation, kinetic modeling, unstructured and unsegregated

models, inhibition phenomena, bioreactors

1. Introduction

The interest in producing industrial bioethanol essentially comes from economic and environ-
mental issues. Bioethanol can be produced from batch, fed-batch, and continuous processes, as
well as in some cases using flocculating yeasts [1–6].
The development of efficient control strategies for the main operating variables in ethanol
fermentations, such as pH, temperature, residual sugars concentration, agitation speed, foam
level, among others, requires accurate dynamic models. In addition, mathematical models are
important tools for the design, optimization, and control of bioreactors. Bioreactor models seek
to describe the overall performance of the bioreactor and consist of two submodels: a balance/

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
94 Fermentation Processes

transport submodel that describes mass and heat transfer within and between the various
phases of the bioreactor and a kinetic submodel that describes how the rates of the microor-
ganism's growth, substrate consumption, and product formation depend on the key local
environmental variables [7].
In ethanol fermentation, the main bioreactions can be summarized by the reductive pathway S
! X + P + CO2. According to this reaction, substrates S (glucose and fructose, which result
from hydrolysis of sucrose as the limiting substrate), in anaerobic conditions, are metabolized
to produce a yeast population X, ethanol P (mainly produced by yeast through the Embden-
Meyerhof-Parnas metabolic pathway), and carbon dioxide (CO2). The hydrolysis of sucrose
promoted by the invertase present in the yeast is not the limiting step of ethanol production in
industrial processes. The stoichiometry of ethanol-formation reaction from glucose is given by
the classical Gay-Lussac equation: C6H12O6 !2CH3CH2OH + 2CO2
According to Doran [8], both Saccharomyces cerevisiae yeast and Zymomonas mobilis bacteria
produce ethanol from glucose under anaerobic conditions without external electron acceptors.
The biomass yield from glucose is 0.11 g/g for yeast and 0.05 g/g for Z. mobilis. In both cases, the
nitrogen source is NH3, and the cell compositions are represented by the formula C1.8O0.5N0.2.
From these data, Doran [9] proposed the following stoichiometric equation for ethanol fermen-
tation (the values of stoichiometric coefficients a, b, c, d, e, and f are presented in Table 1):
aC6 H12 O6 þ bNH3 ! cCH1:8 O0:5 N0:2 þ dCO2 þ eH2 O þ f C2 H6 Oðmolar basisÞ

Kinetic modeling of growth, ethanol production, and substrate consumption by yeasts has been
traditionally conducted using an unsegregated and unstructured approach for the biomass. This
approach ignores the presence of individual cells and structural, functional, and compositional
aspects of the cell, describing the complex processes of growth, ethanol production, and sub-
strate consumption through simple kinetic equations [10–15]. Figure 1 shows a simplified
scheme of this approach for the ethanol fermentation process by yeasts and bacteria.

Microorganism Stoichiometric coefficients

a b c d e f

Yeast 1 0.16 0.81 1.75 0.35 1.72

Bacteria 1 0.074 0.37 1.89 0.17 1.87

Table 1. Stoichiometric coefficients for ethanol fermentation.

Figure 1. Kinetic modeling of ethanol fermentation based on an unsegregated and unstructured approach for cells (yeasts
or bacteria).
 Kinetic Modeling of 1‐G Ethanol Fermentations 95
http://dx.doi.org/10.5772/65460

2. Kinetics of cell growth and ethanol formation

In ethanol fermentation, the kinetics of growth and ethanol production are generally the
following:

μX ¼ f 1 ðSÞg1 ðPÞ (1)

μp ¼ f 2 ðSÞg2 ðPÞ (2)

where μX and μp, are, respectively, the specific rate of yeast growth and ethanol production,
whereas S and P represent the limiting substrate and ethanol concentrations.

2.1. Effect of substrate concentration

The functions f1(S) and f2(S) are generally of the Monod type [11], except when an inhibition
caused by high concentrations of substrate or diffusional limitations occurs due to high cell
concentrations.

μmax S
f ðSÞ ¼ ðMonod equationÞ (3)
KS þ S

The inhibition caused by the excess of substrate has generally been modeled by applying the
Andrews equation [16–19], though there are other types of equations that are less commonly
used [20].

μmax S
f ðSÞ ¼ ðAndrews equationÞ (4)
KS þ S þ S2 =KI

In the case of continuous processes operated near to the steady state, the inhibition concentra-
tions of the substrate are rarely identified. However, inhibitory concentrations can occur
during the start-up of these processes or in situations resulting from changes in the substrate
feed load.
Atala et al. [21], modeling the effect of temperature upon the kinetics of ethanol fermentation
with a high concentration of biomass in a continuous system with total cell retention, used an
inhibitory factor (IF) of the exponential type to describe the inhibitory effect of the substrate
upon the kinetics of cell growth, which was inserted in the expression of f(S), being f(S), in this
case, given by the Monod equation.

IF ¼ ðe−KI S Þ (5)

Tsuji et al. [22] evaluating the performance of different ethanol fermentation systems (conven-
tional chemostat, multiple bioreactors, cell recycle bioreactor, extractive bioreactor, and
immobilized cell bioreactor) expressed the specific growth rate by an equation analogous to
Eq. (1):
96 Fermentation Processes

μX ¼ μ1 ðSÞμ2 ðPÞ (6)

One of the analyzed cases considered growth inhibition by substrate, represented by a hyper-
bolic equation:
 
μmax S Ki
μ1 ðSÞ ¼ (7)
KS þS Ki þS

Sousa and Teixeira [23] reported that one of the main disadvantages of the systems that use
flocculating cells (bacteria or yeast) is the reduced reaction rates caused by diffusional limita-
tions of the substrate within the flocs and that, in most cases, the diffusion rate is lower than
the reaction rate, which means that the process is controlled by diffusion. Sousa and Teixeira
[23] reported that it is generally accepted that yeast flocs are formed by a mediator cation
(usually Ca2+) from the interaction between protein and mannans on adjacent cell walls.
According to Sousa and Teixeira [23], one means through which to avoid diffusional limita-
tions within the flocs is by using polymeric additives, which act by widening the bridges
formed between adjacent cells.

Fontana et al. [24] reported that when the yeast flocs are suspended in a sucrose solution,
various phenomena occur simultaneously: the sugar penetrates by diffusion in the aggregates
and is hydrolyzed into glucose and fructose by an invertase that is primarily located on the
yeast's cell wall. These two sugars diffuse inside and outside of the particle and are fermented
in ethanol and CO2, which in turn diffuse back in the liquid medium.
Fontana et al. [24] assumed that the Fick's law was valid for the aggregate and that the
temporal variation of the concentration of each component involved in the transformation is
represented by the following equation:

∂ Ci ∂ 2 Ci
¼Def , i þ ∑ri (8)
∂t ∂ x2

where Ci is the concentration of the component i in the aggregate in time t and distance x as of
the floc surface; Def,i is the effective diffusion coefficient, while ∑ri is the sum of the consump-
tion or production rates of component i, which are given by Michaelis-Menten-type equations,
such as the following:
S
∑ri ¼ −rSmax ðSucroseÞ (9)
KS þ S
S G
∑ri ¼ þYG=S rSmax −rGmax ðGlucoseÞ (10)
KS þ S KG þ G

where S and G represent, respectively, the concentration of sucrose and glucose, while YG/S is
the conversion factor in glucose based on the hydrolyzed sucrose (YG/S = 0.505g-glucose/g-
sucrose). One relation identical to Eq. (10) can be obtained for fructose.
However, the theoretical descriptions of the diffusional resistances in systems that make use of
flocculating microorganisms are generally conducted by introducing a factor into the Monod
 Kinetic Modeling of 1‐G Ethanol Fermentations 97
http://dx.doi.org/10.5772/65460

equation that takes into account the reduction in growth rate due to mass transfer limitations.
One equation of this type is that proposed by Contois [25].

μmax S
f ðSÞ ¼ ðContois equationÞ (11)
KS X þ S

According to Menezes et al. [26], the Monod model is appropriate at low cell concentrations,
while the Contois model is more appropriate at high concentrations, given that the variable
saturation term, KSX, can describe the diffusional limitations present in high cell concentra-
tions. Oliveira et al. [27], modeling a continuous process of ethanol fermentation in a tower
bioreactor with recycling of flocculating yeasts, obtained a high value for KS, which was
attributed to the diffusional limitations caused by the high cell concentrations reached in the
bioreactor.

2.2. Effect of ethanol concentration

The dependence of μX and μP on the ethanol concentration is due to the fact that this product
has been reported in the literature to act as a noncompetitive inhibitor both for growth and its
own production [10, 28–32].
Noncompetitive inhibition is characterized by the fact that in the graph of 1/μX or 1/μp versus
1/S (Figure 2), for each ethanol concentration (P), straight lines with different slopes

Figure 2. Lineweaver-Burk graph for the specific rates of cell growth (μX) and ethanol production (μP) (adapted from
Aiba et al. [31]).
98 Fermentation Processes

   
K S, i 1
μmax, i gi ðPÞ , i ¼ 1; 2 and different intercepts μ ,i ¼ 1; 2 are obtained, but the same inter-
max, i gi ðPÞ

sections with the abscissa are maintained (−1/KS,i; i = 1, 2).

The molecular base of the mechanism through which the ethanol exerts an inhibitory effect
upon fermentation is complex so long as this component, which acts as a denaturing agent, not
only acts directly upon the proteins and causes an inactivation or inhibition of the enzymes
from the glycolytic pathway but can also act upon the integrity of the lipid membranes,
affecting the essential factors, including membrane components, such as transport proteins
and the enzymes linked to it [33].
Table 2 presents the main equations proposed for g1(P) and g2(P), which are first approxima-
tions of much more complicated effects [10, 28–32, 34–42].

 
Linear (L) gðPÞ ¼ 1− PPm (12)
n
Generalized nonlinear (GN)

gðPÞ ¼ 1− PPm (13)
 
Hyperbolic (H) gðPÞ ¼ KP
(14)
KP þP

0:5
Parabolic (P)

gðPÞ ¼ 1− PPm (15)

Exponential (E) gðPÞ ¼ ðe−KP P Þ (16)

Table 2. Types of commonly proposed equations to describe the inhibitory effect of ethanol upon μX and μP.

The type of inhibition that affects cell growth is not mandatorily the same as that which affects
ethanol production, as it is necessary to determine separately each effect, as proposed by
Oliveira et al. [27]. According to Bonomi et al. [17], one of the major difficulties during the
development of a mathematical model that fits experimental data of ethanol fermentation is
the definition of the type of product inhibition exhibited by the yeast's metabolism. Bonomi
et al. [17] reported that this inhibition is characterized by the behavior of the specific growth
and production rates with an increase in ethanol concentration, while holding constant the
substrate concentration. When developing a mathematical model for a batch system of ethanol
production, Bonomi et al. [17] set three values for substrate concentration and determined the
corresponding pairs of values (μX, P) and (μP, P) in each fermentation test. These points—(μX,
P) and (μP, P)—were then plotted to define the types of existing relations between the specific
rates and ethanol concentration which, in this case, were both exponential. The values of the
specific growth and ethanol production rates were calculated based on experimental data,
using the geometric approach proposed by Le Duy and Zajic [43].
The conceptual limitation of the hyperbolic and exponential inhibition is that they predict cell
growth and production for all of the ethanol concentrations, even though many experimental
tests have shown that cell growth and production cease upon reaching a given high concen-
tration of ethanol [44]. The models of linear, generalized nonlinear, and parabolic inhibition
consider that there is a determined concentration of ethanol above which growth and produc-
tion do not occur. In these models, the Pm parameters represent the ethanol concentrations for
which the growth and production processes are completely interrupted.
 Kinetic Modeling of 1‐G Ethanol Fermentations 99
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In the linear, generalized nonlinear, and parabolic models, the exponents of the term (1−P/Pm)
are called by Levenspiel [40] as “toxic powers.” The values of toxic powers are indicative of
how the term of inhibition (1−P/Pm) strongly affects the specific growth and ethanol production
rates. With the rise in toxic power, the intensity of inhibition increases for a determined ethanol
concentration.
In the hyperbolic and exponential inhibition models, the KP parameters do not admit a phys-
ical meaning and can be considered simple empirical constants that apparently depend on the
cultivation mode: batch or continuous [31, 32, 37]. Aiba and Shoda [32] argued that the fact
that the hyperbolic inhibition constant of the specific growth rate (KP) has been lower in a
batch culture (KP = 16.0 g/L) than in a continuous culture (KP =5 5.0 g/L) suggests the possibility
that a chemical affinity of ethanol for a key participating enzyme in cell growth appeared in
batch experiments. By contrast, the fact that the hyperbolic inhibition constant of the specific
ethanol production rate (K′P) has been lower in continuous cultures (K′P = 12.5 g/L) than in
batch cultures (K′P = 71.5 g/L) suggests that the ethanol inhibition upon another key enzyme
responsible for the fermentation activity was more expressive in continuous experiments.
Another inhibition model commonly used in the literature is that proposed by Luong [37]:

 β
P
gðPÞ ¼ 1− (17)
Pm

where Pm continues to be the ethanol concentration above which no growth or production can
occur, and β is an empirical constant.
One different proposal to describe the inhibitory effects of ethanol upon μX and μP was
presented by Wang and Sheu [45] when they applied multiobjective optimization methods to
estimate the parameters of kinetic models of batch and fed-batch processes for ethanol pro-
duction, using one yeast that is highly tolerant to ethanol (Saccharomyces diastaticus). In their
study, the kinetic models for the specific rate of cell growth and product formation were
represented as follows:

   
μm S KP
μX ¼ (18)
KS þ S þ S2 =KIS KP þ P þ P2 =KIP
! 0
!
νm S KP
μP ¼ 0 0 0 0 (19)
KS þ S þ S2 =KIS KP þ P þ P2 =KIP

Olaoye and Kolawole [46], modeling the kinetics of ethanol fermentation in batch culture of
Kluyveromyces marxianus, used a semiempirical approach to describe the fermentation process.
To model the temporal profile of the biomass concentration, the authors inserted Eq. (20) in the
cell mass balance (dX/dt = μXX) and analytically integrated the resulting equation to obtain the
so-called logistic growth curve (Eq. 21). The ethanol concentration was described, directly
applying the modified Gompertz equation (Eq. 22), which represented the empirical part of
the proposed mathematical model. The authors did not report the values of the model's
parameters.
100 Fermentation Processes

 
X
μX ¼ μm 1− (20)
Xm

Xm
X¼   ; X0 ¼ Xð0Þ (21)
Xm −X0
1þ X0 e−μm t
  
Prm exp ð1Þ
P ¼ Pm exp − exp ðλ−tÞ þ 1 (22)
Pm

where Pm and Prm are, respectively, the maximum concentration and maximum productivity
of ethanol; λ is the time of duration of the lag phase, anterior to the exponential phase of
ethanol production.
The logistic equation has been used to model fermentation kinetics due to its mathematical
simplicity. According to Mitchell [7], the logistic equation can, many times in a single equation,
offer an adequate approximation of the entire growth curve, including the lag phase and the
cessation of growth in the latter stages of fermentation.

2.3. Effect of cell concentration

The inhibition models presented thus far have been sufficient to satisfactorily describe a large
number of fermentations. However, in continuous processes with cell recycling, high cell
densities are obtained in the fermenter, and the consideration of other factors, such as the
inhibition caused by the excess of biomass, may well be necessary for a better description of
the bioprocess kinetic behavior.
The inhibition of cell growth by cell concentrations has been modeled using the following
generalized equation [44]:
X δ
 
hðXÞ ¼ 1− (23)
Xmax

where Xmax is the maximum cell concentration that would be reached if ideal conditions for
growth were observed, that is, an adequate supply of nutrients and the absence of inhibitory
effects [44]. Analogous to the term of inhibition caused by the product, δ indicates the intensity
of the inhibition due to the high cell concentrations.

Jarzebski et al. [47], modeling a continuous ethanol fermentation process with high yeast
concentrations in a membrane filtration module system, used the following expressions for
μX and μP, in which other formats can be observed for the terms that describe the inhibitory
effects of the biomass itself:

  "  A1 # "  A2 #

μ0 S P X
μX ¼ 1− 1− (24)
KS þ S Pm Xm

μP ¼ a exp ð−bXÞ (25)

 Kinetic Modeling of 1‐G Ethanol Fermentations 101
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In cases occurring inhibition of cell growth and product formation by the biomass itself, the
expressions of the specific growth and ethanol production rates must be augmented to incor-
porate these inhibitory effects, that is,

μX ¼ f 1 ðSÞg1 ðPÞh1 ðXÞ (26)

μP ¼ f 2 ðSÞg2 ðPÞh2 ðXÞ (27)

As regards the procedures of many authors using a kinetic expression for μP detached from μX,
Bu'Lock et al. [10] reported that this does not mean that there is no association between
these rates, so long as the ethanol production has been commonly reported in the literature as
a process associated with growth. Bu'Lock et al. [10] justified the adoption of such a procedure
due to the simplicity and to the better adaptation of such equations to experimental data.
In relating the kinetics of ethanol production to the kinetics of cell growth, the procedure has
been to apply the Luedeking-Piret model:

μP ¼ αμX þ β (28)

The Luedeking-Piret model is based on the classification of products of the fermentation

process as associated (α > 0, β = 0), nonassociated (α = 0, β > 0), and partially associated with
cell growth (α > 0, β > 0) [12]. Since ethanol is a product of the primary metabolism of the
yeasts, the majority of described cases assumes α > 0 and β = 0, as reported by Oliveira et al.
[19] when they modeled the batch ethanol production, using the expression μP = αμX, with α =
4.17 g/g. However, it is possible to find descriptions in the literature of ethanol production,
parts associated and not associated with cell growth, that is, α > 0 and β > 0, as is the case with
that reported by Guidini et al. [18] that used the following equation to describe μP in a fed-
batch ethanol fermentation process with flocculating yeasts (S. cerevisiae):


Y P=S
μP ¼ μX þ b (29)
YX=S
|fflfflfflffl{zfflfflfflffl}
α

Rivera et al. [48], modeling a fed-batch ethanol fermentation process with a strain of industrial
yeast (S. cerevisiae), used a modified version of the Luedeking and Piret model in which the β
coefficient is given as a function of the substrate concentration (S):


βm S
μP ¼ YP=X μX þ (30)
KβS þ S
|fflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflffl}
β

Ghosh and Ramachandran [49], analyzing the effect of in situ product removal on the stability
and performance of a continuous bioreactor with cell separator for ethanol production, empha-
sized the use of the Luedeking-Piret model to represent the kinetics of product formation.
102 Fermentation Processes

In Table 3, typical kinetic parameter values for ethanol fermentation are presented [10, 19, 28–
32, 34–41].
Table 3 shows large variations in the values of kinetic parameters, demonstrating that these
parameters are strongly dependent upon the operational conditions for which they were
adjusted, from the culture medium and from the microorganisms used in the fermentation.

Oliveira et al. [50] analyzed the scale-up effects on kinetic parameters and predictions of a
mathematical model developed for a continuous process, in small scale, of ethanol fermenta-
tion in a tower bioreactor with flocculating yeast recycling, and concluded that the scale-up
did not affect the parameter values and that the model continued to be valid to describe the
process in the newly investigated scale.
Although the great majority of mathematical models reviewed thus far have been developed
for free cell systems, these are equally valid for naturally or artificially immobilized cell
systems. However, physiological changes in microbial cells caused by immobilization can
significantly affect the values of the kinetic parameters of such models. Moreover, internal
and external diffusion effects in microbial particles and flocs can affect the fermentation
kinetics. Admassu et al. [38], modeling the hydrodynamics and the profile of product concen-
tration in a tower fermenter for the continuous production of ethanol with flocculating yeasts,
reported that the growth and reaction rates for these flocculating microorganisms are fre-
quently limited by mass transfer.

Vicente et al. [51], developing a new technique to measure kinetics and mass transfer param-
eters in flocs of S. cerevisiae, modeled the kinetics of oxygen consumption using the following
equation:

Parameters of f1(S), f2(S) h(X) Parameters of g1(P) g2(P)

L GN P H E

μmax,1 (/h) 0.11–0.56

μmax,2 (g/g/h) 0.21–1.90
KS,1 (g/L) 0.07–0.57
KS,2 (g/L) 0.33–60.0

Pm,1 (g/L) 87.0–95.0 73.0–87.5 93.6

Pm,2 (g/L) 114.0–135.0 87.5 99.0
n1 (–) 0.41–4.0
n2 (–) 0.41
Kp,1 (L/g or g/L) 16.0–105.2 0.016–0.029
Kp,2 (L/g or g/L) 12.5–71.5 0.015–0.094
Xmax (g/L) 100.0–330.0

Table 3. Typical values of kinetics parameters in ethanol fermentation.

 Kinetic Modeling of 1‐G Ethanol Fermentations 103
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p1 C
rC ¼ − X (31)
p2 þ C

where rC is the oxygen consumption rate (mg O2/(L h)) from which the specific rate of
respiration qO can be calculated, C is the dissolved oxygen concentration (mg O2/L), X is the
active biomass concentration (g/L), p1 corresponds to qO,m (mg O2/(g h)), qO,m and p2 corre-
sponds to Km (mg O2/L).
According to Vicente et al. [51], although Eq. (31) represents a Monod-type kinetic model, the
calculated values of p1 and p2 are only apparent and have no direct relationship with the usual
kinetic parameters. Vicente et al. [51] argue that the designations qO,m and Km were not used
because they are generally applied to suspended free cell cultures and that, in this case, cell
aggregates were studied, which significantly change the overall behavior of the system and,
therefore, the meaning of such parameters.

3. Kinetics of substrate consumption

The kinetics of substrate consumption can generally be described by the Herbert-Pirt model,
according to which the substrate is consumed for cell growth and maintenance and the
production of a specific product [26]:
μ μ
μS ¼ X þ P þ m (32)
Y X=S Y P=S

where Y*X/S and Y*P/S are, respectively, the stoichiometric coefficients of substrate conversion
in cells and product based on the substrate consumed exclusively for each process.
Substrate consumption for cell maintenance refers to the substrate used in the generation of
energy for distinct growth functions, such as the maintenance of the concentration gradients
between the interior and exterior environment of the cell (osmotic work), synthesis of the cell
components that are being continuously degraded, among others [12].
Equation (32) considers that the specific rate of cell maintenance m is a constant, hypothesis,
which Ramkrishna et al. [52] do not adopt. According to these authors, the cells suffer a
process of degradation in stages in which, at the first stage, the cells would lose their cell
viability and, at a second stage, they would die if their maintenance requirements were not
attended. To recover the viability, the nonviable cells would need a substrate that would be the
same used for growth (exogenous substrate) or an internally stored substrate (endogenous
substrate). From these considerations, the following modification in the mathematical repre-
sentation of the metabolism of maintenance can be introduced, in turn substituting the con-
stant term m in Eq. (32) by a Monod-type expression [26, 53]:

ζmax S
ζ¼ (33)
KS, m þ S
104 Fermentation Processes

Equation (33) shows that, at high concentrations of substrate, there is a predominance of

exogenous metabolism with ζmax!m when S>>KS,m, whereas at low concentrations, the
endogenous metabolism predominates with ζ!0 when S!0.
Generally, the kinetics of substrate consumption is not described using the Herbert-Pirt model
to its full extent. The more common approach is the use of apparent coefficients of substrate
conversion in cells (YX/S) and ethanol (YP/S), relating μS to μX or to μp by means of these
coefficients (Eqs. (34) and (35)). Another approach to describe μS is that represented by
Eq. (36).
μX
μS ¼ (34)
YX=S
μP
μS ¼ (35)
Y P=S
μX
μS ¼ þm (36)
YX=S

Applications of these approaches can be found in the studies listed in Table 4.

Sinclair and Kristiansen [12] emphasize the importance of not confusing the stoichiometric
coefficients with apparent coefficients as is normally reported in the literature. The stoichio-
metric coefficient is a constant that depends on the chemical equation, relating the substrates
and the products (Y*P/S = 0.511g-ethanol/g-glucose in the fermentation of glucose to ethanol).
The apparent coefficient is the ratio of the mass of a product formed by the total mass of a
consumed substrate, which could be participating in multiple reactions, forming a variety of
products, including new cells. In this sense, the following definitions for the stoichiometric and
apparent coefficients are convenient:

Study μS Reference

Optimization of an industrial bioprocess of ethanol fermentation with multiple stages and cell Eq. (34) [54]
recycle, using techniques of factorial design and response surface analysis in combination with
phenomenological modeling and simulation
Ethanol fermentation modeling in a tower bioreactor with flocculating yeasts Eq. (35) [38]
Analysis of the steady-state stability and modeling of the dynamic behavior of a continuous ethanol Eq. (35) [36]
fermentation process in a gas-lift tower bioreactor with high cell densities
Bifurcation analysis of two continuous membrane fermentor configurations for ethanol production Eq. (36) [55]
Modeling, simulation, and analysis of an ethanol fermentation process with control structure in Eq. (36) [56]
industrial scale
Modeling of a fed-batch ethanol fermentation process with a strain of industrial yeast Eq. (36) [48]
(Saccharomyces cerevisiae)
Modeling of a fed-batch ethanol fermentation process with flocculating yeasts (S. cerevisiae) Eq. (36) [18]

Table 4. Mathematical models used for modeling of substrate-consumption kinetics in 1-G ethanol fermentation
processes.
 Kinetic Modeling of 1‐G Ethanol Fermentations 105
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Mass of new cells formed

•YX=S ¼ (37)
Substrate mass consumed only for the formation of new cells

Mass of new cells formed

•YX=S ¼ (38)
Total mass of substrate consumed
Mass of product formed
•YP=S ¼ (39)
Substrate mass consumed only for the formation of product

Mass of product formed

•YP=S ¼ (40)
Total mass of substrate consumed

Oliveira et al. [57], modeling a continuous ethanol fermentation process in a two-stage tower
bioreactor cascade with flocculating yeast recycle, used simplified (μS = μP/YP/S) and general-
ized (μS ¼ μX =YX=S þ μP =YP=S þ m) kinetic expressions to describe μS and obtained similar
predictions of the state variables by both employed approaches.
Bonomi et al. [17], modeling the ethanol production using cassava hydrolyzate in a batch
bioreactor, defined the following equation for the mass balance of substrate:
 
dS 1 1 1
¼− μ Xþ μ X (41)
dt 2 YX=S X Y P=S P

According to these authors, the definition of the apparent coefficients YX/S and YP/S guarantee
that the terms μX X=Y X=S and μP X=YP=S are equal; this equality was also reported by Aiba et al.
[31] and Ghose and Tyagi [28]. Bonomi et al. [17] argue that the two terms are not exactly equal
due to the fact that the calculated values of YX/S and YP/S are affected by different experimental
errors and the values of μX and μP are calculated using estimates of other parameters of the
model. These authors justify the introduction of the average among the aforementioned terms
in Eq. (41), as a means through which to minimize the propagation of errors discussed above.
By contrast, Jin et al. [42], modeling the kinetics of batch fermentation for ethanol production
with S. cerevisiae immobilized in calcium alginate gel, presented the following mass balance
equation for the substrate, without the introduction of the 1/2 factor in the equation:
 
dS 1 1
¼− μ Xþ μ X (42)
dt YX=S X YP=S P

One equation like μS ¼ μX =YX=S þ μP =YP=S was also employed by Marginean et al. [58] to model,
simulate, and develop proportional integral derivative (PID) control strategies for temperature
and the pH of an ethanol production process in a continuous stirred tank reactor (CSTR).
A different proposal was presented by Limtong et al. [35] to model a continuous process of
ethanol fermentation in a tower bioreactor with recycle of flocculating yeasts. The authors
determined linear relations between the product concentration (P (g/L)) and the specific rates
of glucose consumption (μS) and ethanol production (μP). The ratios between the
corresponding angular and linear coefficients of the straight lines (1.63/3.74 and 0.020/0.046)
106 Fermentation Processes

provide a reasonable estimate of the value of YP/S (=0.43 g/g), which demonstrates the consis-
tency of such relations (Eqs. (43) and (44)).

μS ¼ −0:046P þ 3:74ðg=g=hÞ (43)

μP ¼ −0:020P þ 1:63ðg=g=hÞ (44)

Another situation to be analyzed is when there is more than one fermentable sugar present in the
medium, as is the case in the production of beer. Ramirez [59], modeling the dynamic of batch beer
fermentation, considered the glucose (G), maltose (M), and maltotriose (N) to be the three majority
sugars contained in the fermentative medium. The specific consumption rates of these sugars were
described by equations that exhibit a kinetic pattern of preferential use of these substrates, that is,
the preferred sugar (G) is first used until its complete exhaustion; next, the second sugar (M), of
intermediate preference, is consumed; and lastly, the third sugar (N), the least preferred, is con-
sumed. According to Ramirez [59], this pattern of sequential use is modeled by inserting terms of
inhibition of the consumption of a less preferential sugar by one or more preferential sugars in
such a way that the specific consumption rates of these sugars μi are given by

VGG
μG ¼ (45)
KG þ G
0
!
VMM KG
μM ¼ 0 (46)
K M þ M KG þ G

0
! 0
!
VNN KG KM
μN ¼ 0 0 (47)
KN þ N K G þ G KM þ M

where Vi is the maximum specific consumption rate of the sugar i (g/g/h), Ki is the saturation
constant for the sugar i (g/L), and K'i is the constant of inhibition caused by the sugar i (g/L).
Additionally, the specific rates of cell growth (μX) and ethanol production (μE) were given by
the following equations [59]:

μX ¼ RXG μG þ RXM μM þ RXN μN (48)

μE ¼ REG μG þ REM μM þ REN μN (49)

where RXi and REi are, respectively, the stoichiometric yield of the biomass and ethanol per
gram of sugar i consumed (g/g).
A similar approach was employed by Lee et al. [60] when they modeled the batch ethanol
production by S. cerevisiae from a mixture of glucose and maltose. One term ξ was included in
the equation of μM to represent the glucose repression effect upon the maltose consumption.
The final set of the mathematical model equations is presented as follows, highlighting the
prediction of diauxic growth in the expression of μX and the production of ethanol from the
two sugars in the expression of μE.
 Kinetic Modeling of 1‐G Ethanol Fermentations 107
http://dx.doi.org/10.5772/65460

μG, max G μM, max Mξ

 
dX
¼ μX X ¼ þ ηX (50)
dt KG þ G KM þ M

μG, max G
 
dG
¼ −μG X ¼ − η X (51)
dt YX=G ðKG þ GÞ

μM, max Mξ
 
dM
¼ −μM ¼ − η X (52)
dt YX=M ðKM þ MÞ

YE=G μG, max G YE=M μM, max Mξ

 
dE
¼ μE X ¼ þ ηX (53)
dt YX=G ðKG þ GÞ YX=M ðKM þ MÞ
   
X E
η ¼ 1− 1− (54)
Xmax Emax

1
ξ¼ (55)
1 þ G=ki

4. Loss of cell viability

The loss of cell viability during continuous ethanol fermentation processes with high cell
density has been observed by many authors; however, few studies consider this phenomenon
in the kinetic modeling of the process.
Jarzebski et al. [47] studied a continuous system of ethanol fermentation consisting of a perfect
mixture reactor and a filter with a membrane for separation and posterior recycling of the cells
for the fermenter. These authors compared the predictions of an intrinsic model, in which the
loss of cell viability was considered, with the predictions of a modified nonintrinsic model
where this phenomenon was not considered. The authors concluded that the predictions
provided by the two models were similar, and for proposals of simulation and additional
analyses of the process, both models could be used. The intrinsic model was thus called
because the substrate and ethanol concentrations in this model are defined as regards a
corrected volume that neglects the volume occupied by the cells in systems with high cell
densities. Monbouquette [61] presented a detailed mathematical development for the formula-
tion of mass balance equations in terms of these intrinsic concentrations.
Lafforgue-Delorme et al. [62] studying a system similar to that of Jarzebski et al. [47] pointed
out the need to consider other factors other than the dilution rate and concentration of yeasts
that would be important for the modeling of processes with high cell densities, as is the case of
continuous ethanol fermentations with cell recycling. These authors developed a model con-
sidering the following aspects: dilution and yeast purge, broth viscosity, filter plugging, limi-
tation by substrate, physiological state of the yeasts (cell viability), and inhibition phenomena
linked both to ethanol and biomass. They also introduced the concept of steric “stress,”
according to which, at high cell densities, there would be a reduction in the specific growth
rate due to the lack of space for cell division. The effects of inhibition, owing to high cell
108 Fermentation Processes

concentrations and steric stress, were described, respectively, by the terms KX/(KX + XV) and
(1−X/Xm), where KX is an empirical constant, Xv is the viable cell concentration, and X is the
total concentration of cells (viable + nonviable). The final expressions of μX and μP in the
proposed model are given by Eqs. (56) and (57). The predictions of the model agreed satisfac-
torily with the experimental data both for the operation of the bioreactor with total recycle as
well as for the operation with partial recycle.
     
μmax S P KX X
μX ¼ 1− 1− (56)
KS þ S Pm KX þ XV Xm
 
K P XV
μP ¼ μP m exp − (57)
D

Augusto [63], investigating the influence of the specific rate of oxygen consumption in a continu-
ous ethanol fermentation with high cell density, established the range of 0.1–0.8 mmol O2/(g-cell h)
as being that which the oxygen participates in the metabolism as a micronutrient that is
essential to the synthesis of the cell membrane compounds, which would in turn increase the
tolerance of the membrane to ethanol and to other inhibitors produced in the fermentation.
The greatest tolerance resulted in a lower specific rate of cell death and in a greater efficiency of
substrate conversion in ethanol due to the reduction in the value of the maintenance coefficient
by the activation of the oxidative catabolic pathway. According to this author, this range of
oxygen consumption for which the positive effects of this nutrient are observed in the biocon-
version of the substrate would be dependent on the microorganism used, on the fermentation
medium, and on the mode in which the process is conducted (batch, fed-batch, and continu-
ous). To calculate the many parameters of fermentation, Augusto [63] segregated the microbial
population into viable and nonviable cells, this procedure being possible due to the availability
of experimental measures of the concentration of each type of cell separately.
Hojo et al. [41], studying the ethanol production with a strain of flocculating yeast in CSTR
with and without cell recycle, concluded that the cell viability was of utmost importance in
developing the mathematical model of the process with cell recycle and that cell death is a
phenomenon that should be considered in the kinetic modeling of prolonged continuous
fermentations in cases in which the hydraulic residence time is high. The kinetic expressions
for the specific rates of cell growth (μX), substrate consumption (μS), ethanol production (μP),
and cell death (μd) were represented by
P n
   
μmax S
μX ¼ 1−  ; μmax ¼ 0:6=h−1 , KS ¼ 0:57 g=L , P ¼ 80 g=L, n ¼ 1:8 (58)
KS þ S P
μX
μS ¼ ; Yg ¼ 0:014 g=g (59)
Yg

μP ¼ A þ BμS ; A ¼ 0:065 g=g=h; B ¼ 2:24 g=g (60)

μd ¼ kd ; kd ¼ 0:0054 h−1 (61)

In the aforementioned works, it was considered that the microbial population consisted solely
of viable and nonviable cells, with the latter being incapable of growing and producing the
 Kinetic Modeling of 1‐G Ethanol Fermentations 109
http://dx.doi.org/10.5772/65460

desired product. Although inactive in both processes, it was assumed that the nonviable cells
remained intact, which means that cell lysis phenomenon was not considered.
For Borzani [64], when intending to apply such an approach, the segregation of the microbial
population must be performed considering the active and inactive cells in the growth process,
as well as the active and inactive cells in the production process. According to Borzani [64], this
differentiation is justified by the fact that a cell that is considered to be active in a given process
may not be active in another, or vice-versa. Though quite realistic, this approach is rarely
applied, given the enormous experimental difficulty to quantify the concentration of each
group of cells separately.
Using an approach that is quite similar to that suggested by Borzani [64], Ghommidh et al.
[65], modeling the oscillatory behavior of Z. mobilis in continuous cultures for ethanol produc-
tion, segregated the microbial population in three distinct groups: viable cells that grow and
produce ethanol (Xv), nonviable cells that do not grow but produce ethanol (Xnv), and dead
cells (Xd). The processes of ethanol production, cell growth, loss of viability, and cell death
were represented according to the scheme shown in Figure 3.

Starting from the scheme proposed by Ghommidh et al. [65], Jarzebski [66] modeled the
oscillatory behavior of the state variables X, S, and P in a continuous ethanol fermentation
process with S. cerevisiae, introducing the concept of combined effect of inhibition by substrate
and ethanol simultaneously, since, according to that author, the inhibition by substrate would
depend on the ethanol concentration and vice-versa. Taking into account this combined effect
of inhibition, Jarzebski [66] proposed the following equations to describe the specific rates of
viable cell growth (μv), conversion of viable cells into nonviable cells (μnv), and cell death (μd):
   
μmax S P S
μv ¼ 1− for P < Pc ðK2 þ SÞ=S (62)
K1 þ S Pc K2 þ S
  !
μmax S P S 0
μnv ¼ 1− 0 −μv for P < Pc ðK2 þ SÞ=S (63)
K1 þ S Pc K2 þ S

Figure 3. Schematic representation of the cell processes involved in ethanol production by Zymomonas mobilis in contin-
uous cultures, according to the model proposed by Ghommidh et al. [65].
110 Fermentation Processes

0
μd ¼ −μv for P < Pc ðK2 þ SÞ=S (64)

Watt et al. [67], using the mathematical model proposed by Jarzebski [66], simulated the
continuous ethanol fermentation process for different feed volumetric flow rates and substrate
concentrations in the feed stream.

The mathematical modeling of ethanol fermentation processes in which the loss of cell
viability occurs is generally conducted by dividing the cell population into two distinct
groups: viable cells (Xv) which would be growing and producing ethanol and nonviable or
dead cells (Xd), which would be inactive in both processes [68]. The conversion rate from the
viable to the nonviable cells is considered to be the first order regarding the concentration of
viable cells [12]. The specific rates of cell growth, ethanol production, substrate consump-
tion, and loss of cell viability are defined as regards the viable cell concentration, which refer
to the effectively active cells in all of these processes. Mass balance equations for viable and
nonviable cells are developed separately. The mass balance equations for ethanol and sub-
strate are similar to those of the conventional model (model that does not incorporate the
loss of cell viability) with the previously discussed modifications in the terms involving the
specific rates.

Based on these premises, Oliveira et al. [69] developed a mathematical model for a continu-
ous ethanol fermentation process in a tower bioreactor with recycle of flocculating yeasts, in
which the loss of cell viability was considered and the predictions of this model were
compared with those of the conventional model. Both models provide similar predictions
and were equally appropriate for the fermentation process modeling. Later, in another
publication, the authors analyzed the scale-up effects on the kinetic parameters and on the
predictions of the modified model, and found changes in the values of some of the parame-
ters [70]. In addition, the predictions of the modified model agreed better with the experi-
mental data than did those of the conventional model, especially for the cell concentration
variable.

A better description of the fermentation process by the modified model is always the
desired result, primarily in those cases in which the levels of cell viability are significantly
different than 100%. The cell viability level in ethanol fermentations with high yeast densi-
ties has been reported as being strongly dependent on the rate of aeration imposed upon the
system [34], varying from 40% to 90% [10, 34, 38, 71]. Under anaerobic conditions, unsatu-
rated fatty acids are not synthesized and the yeasts become more sensitive to ethanol [72].
However, the high levels of cell viability in aerated systems are achieved at the expense of
the reduction in ethanol yields [70]. Thus, the rate of aeration is an important variable to be
optimized in these systems, seeking to provide an adequate level of oxygen dissolved in the
medium [70].
Other aforementioned works in which the segregated approach, regarding cell viability, was
applied to describe the microbial population are as follows: Kalil et al. [54], Atala et al. [21],
Costa Filho et al. [56], Nelson and Hamzah [53], and Watt et al. [67].
 Kinetic Modeling of 1‐G Ethanol Fermentations 111
http://dx.doi.org/10.5772/65460

5. Conclusions

The facility to model the kinetics of ethanol fermentation processes is due to the fact that the
governing factors of these processes (limitation by substrate, inhibition, loss of cell viability,
death, among others) are well known and that they have a large quantity of mathematical
models that have already been developed and made available within the literature.
The present work compiles, in a single publication, a reasonable quantity of kinetic models
that are potentially applicable to the adjustment of experimental data of ethanol fermentation
processes obtained under the broadest and most varied operating conditions. The models can
also be applied to the production processes of another generation, such as is the case of
obtaining ethanol from lignocellulosic feedstocks (second-generation bioethanol) for which
the literature presents the use of such models as being confirmed by the following recent
publications:

• Scott et al. [73]: Attainable region analysis for continuous production of second-generation
bioethanol.
• Vásquez et al. [74]: Modeling of a simultaneous saccharification and fermentation process
for ethanol production from lignocellulosic wastes by K. marxianus.

• Liu et al. [75]: Fermentation Process Modeling with Levenberg-Marquardt Algorithm and
Runge-Kutta Method on Ethanol Production by S. cerevisiae.

In general, many fermentation studies have confirmed that the unstructured models poorly
describe dynamic experiments in which composition and biomass activity change [13, 15]. By
contrast, the use of a more detailed approach of cell metabolism, aimed at better describing the
dynamic behavior of the process, can lead to the development of structured models containing
a large number of variables and parameters. In these cases, the parameter estimation can
become a difficult task due to the large experimental effort required and to the need to apply
complex numerical methods, which can lead to obtaining parameter values without physical
meaning. To illustrate such a scenario, Rivera et al. [76] used a structured model to interpret
experimental data of a tower bioreactor for ethanol production by immobilized S. cerevisiae.
The model contains 34 kinetic parameters and 9 parameters related to the glycolytic and
respiratory (tricarboxylic acid [TCA]) pathways. Thus, greater experimental and computa-
tional efforts would be required to estimate the parameters associated with this mathematical
model.

The class of structured models that are potentially useful is formed by simply applying the
structured formulation, through which the description of the quantity and of the biomass
properties is performed by using two or three variables, resulting in the so-called two- or
three-compartment models. These models combine a better description of the system's
behavior with a reasonable mathematical complexity and a smaller number of parameters
[77].
112 Fermentation Processes

Therefore, it is important to balance the complexity of the model with its identification and to
seek expressions that are as simple as possible and that are capable of accurately describing the
process in both dynamic and steady states [69].

Author details

Samuel C. Oliveira1*, Dile P. Stremel2, Eduardo C. Dechechi3 and Félix M. Pereira4

*Address all correspondence to: [email protected]

1 Department of Bioprocesses and Biotechnology, School of Pharmaceutical Sciences (FCF),

São Paulo State University (UNESP), Araraquara-SP, Brazil

2 Department of Engineering and Forestry Technology, Federal University of Paraná (UFPR),

Curitiba-PR, Brazil

3 CECE/PTI-Department of Engineering and Sciences at Itaipu Research Park (PTI), Western

Paraná State University (UNIOESTE), Foz do Iguaçu-PR, Brazil

4 Department of Chemical Engineering, Engineering School of Lorena (EEL), University of

São Paulo (USP), Lorena-SP, Brazil

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