C H A P T E R

15
Fermentation and biochemical
engineering: principles and applications
Leone Mazzeo, Vincenzo Piemonte*
University Campus Biomedico of Rome, Faculty of Engineering, Rome, Italy
* Corresponding author. e-mail address: v.piemonte@unicampus.it

1. Introduction Enzyme theory is mainly based on different

physical reaction models that follow quite well
Industrial fermentation is a chemical engi- all the experimental data: examples of the
neering term used to describe the processes
that utilize a chemical change induced by a
living organism or enzyme, in particular bacte-
ria, yeasts, molds, or fungi, that produces a spe-
cific product [1]. Although in the biochemical
context the word “fermentation” describes the
anaerobic metabolic process of partial oxidation
of organic compounds, in the industrial field it
refers to either aerobic or anaerobic processes.
The fermentation technique was used long
before humankind discovered, or understood,
microbes, and it was adopted mainly for the pro-
duction of drinks containing the same active
compound: ethanol. Nowadays fermentation is
a developed technology used in many sectors,
such as food, pharmaceutical, biofuel, and
biopolymer production (Fig. 15.1).
There are two main characters in industrial
fermentation: the enzymes and the cell cultures.
Both of them will be treated in this chapter with a FIGURE 15.1 A picture of industrial scale bioreactor
particular focus on their kinetics. fermenters.

Catalysis, Green Chemistry and Sustainable Energy

https://doi.org/10.1016/B978-0-444-64337-7.00015-X 261 Copyright © 2020 Elsevier B.V. All rights reserved.
262 15. Fermentation and biochemical engineering: principles and applications
estimation of the parameters of such models will
be given. Cell theory, different from enzyme the-
ory, is mainly based on experimental tests
followed by the ad hoc formulation of models.
Engineers always work to make their life easier,
and the most-used expression adopted to
describe cell population growth is suspiciously
similar to the one used for enzymes.
Finally, a general description of the most-
used reactors will be given and typical
examples of the cell-populated bioreactors will
be analyzed. The purpose of this chapter is to
transmit a basic knowledge of bioreactor
modeling and prepare the reader to face future
bioreactor design work.
2. Enzymes
Enzymes are globular proteins that act as bio-
logical catalysts: they are able to increase the rate
of a chemical reaction without being consumed
and without affecting the thermodynamic
equilibrium of the reaction itself (respecting the
definition of catalysts) (Fig. 15.2).
Before getting into all the mechanisms and
related models used to describe the kinetics of
a catalyzed enzymatic reaction, it is important
to remind the reader of the terminology used
for this specific topic:
FIGURE 15.2 The action of a catalyst (left) lowers the activation energy of the chemical reaction and (right) makes it
possible for molecules with low internal energy to react (right).
IV. Selected examples and case history
• Substrate: This is a term used to indicate a
reactant involved in an enzymatic reaction.
• Active site: This is a specific area of the
enzyme on which the substrate is able to
connect, giving a substrateeenzyme complex.
• Cofactor: This is a nonprotein compound that
connects with an inactive protein (the
apoenzyme) to form a catalytically active
complex (the holoenzyme), commonly called
enzyme. Cofactors can be made of metal ions
or complex organic macromolecules.
• Turnover number: This is a typical index of
the enzyme’s catalytic activity. It is defined as
the maximal number of molecules of
substrate converted to product per active site
per unit time when the enzyme is saturated
with substrate [2].
• Units: These are also called units of activity
and are commonly used to express the
concentration of enzymes in a solution. A unit
is defined as the amount of catalytic activity
per amount of enzyme. The necessity of
defining such an unusual unit measure arises
from the impossibility of completely isolating
enzymes from a protein mixture.
The number of enzymes that is possible to find
in nature is incredibly high and every single one
has a unique and complex chemical structure.
Because of this it has been preferred to classify en-
zymes by the particular class of reaction in which
 2. Enzymes 263
TABLE 15.1 International classification of enzymes [3].

Class Class name Type of reaction catalyzed

1 Oxidoreductases Transfer of electrons (hydride ions or H atoms)

2 Transferases Group transfer reactions
3 Hydrolases Hydrolysis reactions (transfer of functional groups to water)

4 Lyases Cleavage of CeC, CeO, CeN, or other bonds by elimination, leaving double bonds or rings,
or addition of groups of double bonds
5 Isomerases Transfer of groups within molecules to yield isomeric forms

6 Ligases Formation of CeC, CeS, CeO, and CeN bonds by condensation reactions coupled to
cleavage of ATP or similar cofactor

they are involved. Six main classes of reactions
and six types of enzymes have been identified
and are reported in Table 15.1.
2.1 Enzyme kinetics
Several investigations into the mechanisms
that enzymes adopt to catalyze biological reac-
tions have been done. All the results of such
studies agree on the existence of a substratee
enzyme complex as the first step of the enzymatic
reaction: the substrate links with the active site of
the enzyme. As is easy to see in Fig. 15.3, the
three-dimensional configuration of the enzyme
plays a critical role in the enzymatic action, devel-
oping its selectivity for a specific substrate.
How enzymes increase the rate of a biochem-
ical reaction is still a research topic, but two pos-
itive effects have been highlighted:
1. Proximity effect: This is the ability of the
enzyme to “collect” the substrates in its active
site.
2. Orientation effect: The substrates in the
active site are oriented in the proper way to
interact with each other.
The design of any type of reactor requires
knowledge of the kinetics expression that

FIGURE 15.3 Focus on the substrateeenzyme complex. In this picture, the enzyme hexokinase, which is responsible for
transforming glucose and ATP into glucose 6-phosphate and ADP, is represented.

IV. Selected examples and case history

264 15. Fermentation and biochemical engineering: principles and applications

describes the rate of the reaction. With the 2. The substrateeenzyme complex releases the
growing utilization of enzymes in the medical, product. This step is assumed to be
pharmaceutical, and industrial fields, the neces- irreversible:
sity of finding a simple way, in line with all the
experimental data, to reproduce biological reac- k2
ES ! E þ P: (15.2)
tions catalyzed by enzymes is becoming more
and more important. As usual, many assump- Since every step of the mechanism is consid-
tions and exceptions hide behind every formula; ered an elementary reaction:
for this reason, in the following paragraphs a dp ds
schematic guide is given, to prevent confusion v¼ ¼  ¼ k2 ðesÞ; (15.3)
dt dt
between all the different cases and their range
of application. se k1
¼ ¼ Km ; (15.4)
ðesÞ k1
2.1.1 MichaeliseMenten kinetics
e þ ðesÞ ¼ e0 : (15.5)
From the pieces of information known about
enzymes at the time, Michaelis, Menten, and Here, s, e, and (es) indicate the concentrations of the
Henri in 1902 proposed the following mecha- compounds S,E, and ES, respectively, while the
nism of reaction and its related model, from parameter Km is the dissociation constant and e0
which they derived (Fig. 15.4) the famous is the initial concentration of the enzyme. Rear-
MichaeliseMenten kinetics expression: ranging all the equations it is easy to obtain the
MichaeliseMenten rate equation plotted in Fig. 15.5:
1. The substrate links with the enzyme, forming
the substrateeenzyme complex. This step is
s
supposed to be in equilibrium: v ¼ vmax ; vmax ¼ k2 e0 : (15.6)
Km þ s
k1
S þ E # ES: (15.1) Another way to describe the same situation
k1 was developed by Briggs and Haldane. They

FIGURE 15.4 A schematic of the hydrolysis of sucrose as an example of a MichaeliseMenten reaction mechanism.

IV. Selected examples and case history

 2. Enzymes 265

FIGURE 15.5 (A) Plot of the MichaliseMenten equation. (B) MichaeliseMenten kinetics of the chimeric enzyme CelA--
SSO1949-CelA at pH 3 and 80 C, in which the velocity has been divided by the constant enzymatic concentration [4].

solved numerically the set of equations relative of them are mainly based upon a linear transfor-
to the reaction scheme given earlier (Eqs. 15.1 mation of Eq. (15.4) (Table 15.2). The most
and 15.2): important linearization methods are the ones
8 indroduced by Langmuir and Lineweaver-Burk.
>
> ds
>
> v ¼  ¼ k1 se  k1 ðesÞ An example of the estimation of Michaelise
>
> dt
>
> Menten parameters is given: sago starch hydro-
< dðesÞ lysis using amyloglucosidase derived from
¼ k1 se  ðk1 þ k2 ÞðesÞ : (15.7)
> dt
> Aspergillus niger [6,7].
>
>
>
> e þ es ¼ e0 In general, the Langmuir plot appears to be
>
>
: more accurate than LineweavereBurk. This is
I:C: sð0Þ ¼ s0 ðesÞð0Þ ¼ 0 because the maximum experimental error
From the results shown in Fig. 15.6 it is notice-
able that if the ratio e0/s0 is low enough, an
approximation of quasiesteady state can be
done, setting:
dðesÞ
¼ 0: (15.8)
dt
After a few calculations it is easy to find that
even when imposing the quasi-steady-state con-
dition the expression of MichaeliseMenten ki-
netics it is obtained again. This time Km ¼
k1 þk2
k1
from the model, but nothing changes
from the experimental point of view: the same
equation can be used to fit experimental data
and describe the substrate consumption rate.
Several strategies have been developed to FIGURE 15.6 Result plot of the set of Eq. (15.5). In this
evaluate MichaeliseMenten parameters, and all case the ratio e0/s0 is low enough that after a short time the
quasi-steady-state condition, Eq. (15.6), is established.

IV. Selected examples and case history

266 15. Fermentation and biochemical engineering: principles and applications

TABLE 15.2 Common linear transformation which deactivate cholinesterases (enzymes

methods to evaluate enzyme kinetics that are an integral part of nerve
parameters [5,6]. transmission).
Model Equation Slope and intercept • Reversible inhibitors: This type of inhibition
is often employed by cells as a control
LineweavereBurk 1
¼ Km 1
þ vmax
1
Slope vKmax
m 1
, intercept vmax
v vmax s mechanism to achieve an efficient use of
Langmuir s
v
¼ 1
vmax
s þ vKmax
m
Slope 1
vmax
, intercept vKmax
m
nutrients. A common example is so-called
feedback inhibition (Fig. 15.7), and in this case it
is the product that plays the role of inhibitor.
happens to be at a low concentration of sub-
strate, having a significant impact on the
2.1.2.1 Reversible inhibition modeling
LineweavereBurk plot slope. Therefore, even
though the regression coefficient of The model used to face the presence of an in-
LineweavereBurk (e.i. R2 ¼ 0.9687) is higher hibitor is again represented by the Michaelise
than that of Langmuir (e.i. R2 ¼ 0.926), the sec- Menten equation (Eq. 15.4):
ond method should be preferred to the first [8]. app s
v ¼ vmax app : (15.9)
Km þs
2.1.2 Enzymatic activity inhibition
In this case, the maximum reaction velocity
As for all catalysts, the activity of enzymes is a ðvapp
max Þ and the MichaeliseMenten constant
crucial aspect that must be taken into account in app
ðKm Þ depend also on the concentration of the in-
every enzymatic reactor design. There are some hibitor (i) and the dissociation constants of the
chemical species called inhibitors, able to connect reactions in which the inhibitor is involved (Ki).
to the enzymes and modify their activity. Some- The most common types of inhibition action
times such activity alteration is permanent, are [8]:
sometime it is just temporary, and, depending
on this aspect, it is possible to divide the inhibi- • Competitive: The inhibitor adsorbs at the
tors into two families: substrate binding site. In this case, two types
of complexes are formed: enzymeeinhibitor
• Irreversible inhibitors: These are poisons and (EI) and enzymeesubstrate (ES); complex EI
a group of compounds called nerve gases, has no enzyme activity.

FIGURE 15.7 Typical feedback inhibition scheme. The product of every reaction constitutes a (intermediate) substrate for
the following one involving another enzyme. The end product inhibits the activity of the first enzyme involved in the reaction
chain.

IV. Selected examples and case history

 2. Enzymes
• Uncompetitive: The inhibitor binds only to
the ES complex; it does not interfere with the
binding of substrate to the active site but
prevents the dissociation of the ES complex: it
results in the dependence of the inhibition on
only the inhibitor concentration and its Ki
value.
• Noncompetitive: The
enzymeeinhibitoresubstrate (EIS) complex is
unable to dissociate to give a product of
reaction. In this case, inhibitor binds to E or to
the ES complex. The binding of the inhibitor
to the enzyme reduces its activity but does not
affect the binding of substrate. As a result, the
extent of the inhibition depends on only the
concentration of the inhibitor (i).
• Mixed: This action is generated by the
combination of different types of inhibition.
Table 15.3 summarizes the cases described
above and correlates, for each case, the respec-
app
tive expression of vapp
max and Km .
Usually, to recognize the presence of an inhib-
itor, a comparison of the LineweavereBurk plots
267
with and without the inhibitor species is done
(Figs. 15.8 and 15.9).
2.1.2.2 Other factors influencing enzyme activity
The enzyme’s structural or chemical state can
be affected by many other factors having conse-
quences on their catalytic activity [11]:
• pH
• temperature
• fluid forces (hydrodynamic forces,
hydrostatic pressure, and interfacial tension)
• chemical agents (such as alcohol, urea, and
hydrogen peroxide)
• irradiation (light, sound, ionizing radiation)
2.1.2.3 Influence of pH
The active sites of enzymes are often consti-
tuted by ionizable groups (basic, neutral, and
acidic groups) that can be positively or nega-
tively charged depending on the pH. According
to this, it has been noticed experimentally that
there exists a certain pH at which the activity
of an enzyme is maximum (Fig. 15.10). Several

TABLE 15.3 Typology of inhibition attack and related expression of the MichaeliseMenten constants.

Model

Type Equations Dissociation constantsa Constant expressions

Competitive E þ S4ES Ks max ¼ vmax ¼ k2 e0

vapp
E þ I4EI Ki  
k2 i
ES ! E þ P Kmapp ¼ Ks 1 þ
Ki
 
Uncompetitive E þ S4ES Ks i
ES þ I4EIS max ¼ k2 e0 = 1 þ
vapp
Ki Ki
k2  
ES ! E þ P
i
Kmapp ¼ Ks = 1 þ
Ki
 
Noncompetitive E þ S4ES Ks i
vapp ¼ k e = 1 þ
EI þ S4EIS Ks
max 2 0
Ki
E þ I4EI
Ki Kmapp ¼ Ks
ES þ I4EIS
k2 Ki
ES ! E þ P

a
Remember that for the noncompetitive type, the inhibitor and the substrate do not influence each other as to affinity for the complex with the enzyme. For
this reason the dissociation constants are just Ks and Ki.

IV. Selected examples and case history

268 15. Fermentation and biochemical engineering: principles and applications

FIGURE 15.8 Qualitative overview by means of LineweavereBurk plot of different cases of inhibition [9]. (A) Competitive
inhibitor, (B) noncompetitive inhibitor, (C) uncompetitive inhibitor, (D) mixed inhibitor.

models have been proposed to study this phe-

nomenon; a simple one is described as follows:
H þ H þ
E #þ E #þ E2 : (15.10)
þH þH
K1 K2

Here, K1 and K2 are the equilibrium constant of

the reactions. Starting from Eq. (15.8), it is easy
to calculate the fraction of active enzyme and
its maximum (pKi is defined as log(Ki)):
1
y ¼ ; (15.11)
1 þ hþ =K1 þ K2 =hþ
1
ðpHÞoptimum ¼ ðpK1 þ pK2 Þ: (15.12)
2

2.1.2.4 Influence of temperature

FIGURE 15.9 LineweavereBurk plot of pancreatic lipase The temperature has a very big impact on the
in the absence and presence of an ethanolic extract of Oncoba protein structure of enzymes: raising the temper-
spinosa. It shows a mixed inhibition behavior [10]. ature gives energy to the atoms. Eventually, they

IV. Selected examples and case history

 2. Enzymes 269

FIGURE 15.10 Activity detection of two commercial b-galactosidases (Lactozym and Maxilact) depending on pH. Two
types of models have been used to fit the data (M1 and M2). Further information in [12].

acquire enough energy to overcome the weak in-

teractions holding the global protein structure
together, and enzyme deactivation follows. The
deactivation may be of two types:
• Reversible: Ea # Ei, where the equilibrium
constant of the reaction is eeai ¼ Kd ðTÞ ¼
 
exp DG
RT
d
. Adopting again the mechanism
described by Eqs. (15.1) and (15.2), this time
the quantity of active enzyme is no longer e0,
but it is:
FIGURE 15.11 Effect of temperature on the activity of
ea ¼ e0  ei ¼ e0  Kd ea Bentong ginger protease [13,14].
e0 (15.13)
ea ¼ :
1 þ Kd
maximum velocity changes with respect to tem-
The maximum rate of reaction can be perature (Fig. 15.11).
described now as: k
• Irreversible: Ea /Ed ; here multiple choices
e0
vmax ¼ k2 ðTÞ : (15.14) can be made to describe the kinetics of this
1 þ Kd ðTÞ reaction, depending on the selected order of
The different contributions, depending on reaction itself. One strategy is to assume
temperature, of k2 and Kd establish how the different orders of reaction and evaluate the

IV. Selected examples and case history

270 15. Fermentation and biochemical engineering: principles and applications
best one by means of an experimental data
fitting. What is certain is that the activity of
the enzyme will decrease with increase in the
temperature.
3. Cellular kinetics
Life requires adequate environmental condi-
tions: in a cell culture the properties of the liquid
medium may be responsible of the life behavior
of the cells themselves. To completely describe
the cellular kinetics is fundamental to recog-
nizing that two interacting systems are involved:
• the biological phase consisting of a cell
population
• the environmental phase or growth medium
3.1 System description
Once it is understood that the system is
composed of a biological part and an environ-
mental one, the focus should be moved to how
the two sides interact with each other. To
grow, reproduce themselves, and survive, the
cells require the consumption of nutrients (sub-
strate) taken from the environment. Usually
such action is followed by the release of an end
product of the cell’s metabolic process.
Increasing their biomass, cells change the me-
dium properties:
• Temperature: Cells usually release heat,
raising the environmental temperature.
• Composition: This changes as a consequence
of the product ejected from the cells into the
environment. In this case such products may
alter the pH of the medium or even
participate in chemical reactions.
• Rheological properties (viscosity, etc.).
Environmental modifications also have a big
impact on cellular activity, closing the interac-
tion cycle. From a physical state point of view,
the cellular environment is often a multiphase
IV. Selected examples and case history
system consisting of a liquid with dispersed
gas bubbles, a liquideliquid system in which
immiscible fluids are present, or a three-phase
system (liquideliquidegas).
3.2 Growth medium model
simplifications
Cellular growth is followed by many physical
and chemical phenomena, the description of
which is complicated due to the enormous quan-
tity of variables involved. In common engineer-
ing usage, to study the complexity of the
system, several parameters are imposed as
“fixed” to detect one by one the effects of
different agents. In this case, the growth is
formulated so that all components but one are
present at sufficiently high concentrations that
could be considered not to change in quantity.
Hence, a single component becomes the rate-
limiting nutrient and with respect to just this
compound we consider the effects of medium
concentration on cell growth kinetics.
3.3 Cell model simplifications
The cell population is a heterogeneous collec-
tion of single cells: every cell is different from the
others depending on its actual internal composi-
tion and with respect to the particular life func-
tion that it is developing (i.e., growth,
reproduction) or its age. It is clear that a rigorous
description of such situation is complex but
sometimes useful. Here are listed the usual sim-
plifications adopted to model the system (see
also Table 15.4):
• Structured segregated model: This is the case
in which the system is considered as it is, with
a multicomponent heterogeneous description
of cells. Thus there are no simplifications.
• Structured unsegregated model: In this case
the multicomponent nature of the cells is still
considered, but all cells have the same
 3. Cellular kinetics 271
TABLE 15.4 Schematic classification of cellular sys- composition, which is the average of the
tem simplification. population.
Unstructured Structured
• Unstructured segregated model: Here, in the
balance equations, is considered just the
Unsegregated quantity of biomass without taking into
account the presence of every single
component in the cells, even if they still
compose a heterogeneous system. This
simplification is the result of the balanced
growth hypothesis, which assumes that the
average cellular composition is not affected
Most idealized case by the proliferation of the cell population.
Segregated • Unstructured unsegregated model: This is
the most idealized model, in which there are
both the average and the balanced growth
hypotheses.

3.4 Cell life cycle

Actual case When an inoculum of living cells is seeded into
• indicates a cellular component. a liquid medium and nothing is removed or
added to the culture (batch cultivation), the
quantity of biomass changes typically as is re-
ported by the plot in Fig. 15.12.

FIGURE 15.12 Typical growth curve for batch cell cultivation.

IV. Selected examples and case history

272 15. Fermentation and biochemical engineering: principles and applications
As represented in Fig. 15.12 four phases are
typically present in cell growth:
• Lag phase: The cells need an amount of time to
synthesize the cofactors (coenzymes, ions) and
enzymes able to metabolize the nutrient, so
when a new medium is applied on the culture
or when the cells are transferred to a new
environment the lag phase occurs. Special care
must be taken when a cell population is
transferred into a larger volume of medium
since the outward diffusion of cofactors into
the new medium may cause a delay in cellular
growth. Sometimes it is possible to observe
multiple lag phases due to the exhaustion of
one nutrient and the reorganization of the cell
to absorb its carbon supply from another
source. This phenomenon is called diauxic
growth (Fig. 15.13).
• Logarithmic phase: Also called exponential
growth, because it has an exponential trend,
this is the phase in which the cell biomass
simply grows. The description of this
phenomenon is often accomplished by means
of an unstructured and unsegregated model
in which the cell mass generation rate is:
ðrx Þ ¼ mobs x; (15.15)
FIGURE 15.13 Typical cellular diauxic growth plot.
IV. Selected examples and case history
where x is the cell mass per unit culture volume,
s is the concentration of the limiting nutrient, and
mobs is the observed specific growth rate of the cells,
which is a constant since all substrates necessary
for growth are present in excess. When there is a
limiting nutrient present, mobs ¼ f(s), where s is
the concentration of the limiting nutrient. All
the phenomena that may influence the cell
mass growth are included in mobs(s). In this chap-
ter only unstructured and unsegregated models
will be used.
• Stationary phase: In this phase the rate of
production of newborn cells is equal to the rate
of death and the net quantity of cells does not
change. It happens that the nutrients are
almost finished and the living population uses
the components of dead cells to sustain itself.
• Death phase: This is usually an exponential
decay in the number of living cells due to the
exhaustion of the nutrient resources.
3.5 Monod growth kinetics
It has already been mentioned that the rate of
biomass generation for an unstructured and un-
segregated model is usually written as Eq.
(15.15). The parameter mobs is usually expressed
as a combination of parameters that may influ-
ence the generation of biomass and, in the pres-
ence of a limiting nutrient, it may be written as:
mobs ðsÞ ¼ mðsÞ þ k: (15.16)
Here k represents a generic kinetic constant of
other possible mechanisms having an impact
on biomass growth. For example, in the case of
cellular death by mechanical agitation occurring
in the reactor, k < 0 (called the death kinetic con-
stant) is introduced to consider this phenome-
non. Several ways are used to express the
parameter m(s), the specific growth rate of cells.
The most common one is the Monod equation:
mmax s
mðsÞ ¼ : (15.17)
Ks þ s
 3. Cellular kinetics 273
TABLE 15.5 A list of the variations of Monod’s is not related to any physical model. As for
equation to model other possible enzyme activity, the cellular growth ability
dependence of cell growth on substrate, may change according to different inhibition
product, and more than one limiting
nutrient. types or because of the presence of more than
one limiting nutrient (Table 15.5 and Fig. 15.14):
Type Equation

Substrate inhibition mðsÞ ¼ mmax Ki þsþss 2 =Kp

Product inhibition mðsÞ ¼ mmax Kisþs
Kp 3.6 Substrate consumption
Kp þp

More than one limiting nutrient mðsÞ ¼ mmax K1sþs

1 s2
Y The substrate consumption rate is always
1 K2 þs2
Here, s is the concentration of the limiting
nutrient, Ks represents the substrate concentra-
tion value at which the specific growth rate is
half its maximum, and mmax is the maximum spe-
cific growth rate achievable when s [ Ks. This
condition is almost always satisfied, since the
value of Ks is often rather small. This explains
also why in the plot in Fig. 15.12 the growth is
simply exponential. Although Eq. (15.16) is
very similar to that of Michaelis and Menten
(Eq. 15.17), it is just an experimental law and it
related to the amount of biomass; in fact,
commonly it is:
ð  rs Þ ¼ qðsÞx; (15.18)
where q(s) is the observed specific consumption rate
of substrate. Before giving some typical formulas
of q(s) we must define the observed growth yield of
the organism Yx/s, which is the ratio of the
biomass produced with respect to the substrate
consumed. The consumption of substrate (Ds)
is partially dedicated to the production of new
cell material (Ds)G and partially to the mainte-
nance of the cells (Ds)M [16].

FIGURE 15.14 An example of using the Monod expression to model the behavior of the cyanobacterium Synechocystis sp.
PCC6803 having as nitrogen as the limiting nutrient. Here for msyn,LI is identified the maximum specific rate modified by the
effect of LI (light irradiation) [15].

IV. Selected examples and case history

274 15. Fermentation and biochemical engineering: principles and applications

Calling Dx the amount of biomass produced: role in growth, development, or

Dx Dx
Yx=s ¼ ¼ ;
Ds ðDsÞG þ ðDsÞM
Dx
YG ¼ : (15.20)
ðDsÞG
YG is termed the “true growth yield” because
it is considered to be the “true” stoichiometric
factor of the reaction that produces biomass
from the substrate, since it takes into account
only the amount of substrate used by the cells
to grow. Because YG is usually a constant [10],
it is often used instead of Yx/s, while the terms
related to maintenance may be included in the
expression of q(s) or mobs(s) as is reported in
Table 15.6.
A combined model of the two listed in
Table 15.6 can be found in Ref. [17].
3.7 Product formation
reproduction. However, they play a role in
(15.19) ecological functions like defense mechanisms,
serve as antibiotics, and produce pigments
[18]. An additional quantity of substrate
(energy) is needed to produce them.
As for substrate consumption, and also for
product formation, it is always possible to define
an observed formation yield of product Yp/s related
to the mass of product formed with respect to
the total amount of substrate consumed. Only
in the case in which the product is a secondary
metabolite is it possible to define a “true” product
yield YP, which takes into account the additional
quantity of substrate used to form the product
(Dssm).
Dp Dp
Yp=s ¼ ¼ ; (15.21)
Ds ðDsÞG þ ðDsÞM þ ðDsÞsm
Dp
YP ¼ : (15.22)
ðDsÞsm

Products may belong to two different classes It is common to relate the production forma-
of metabolites: tion rate and the amount of biomass as follows:

• Primary metabolites are directly involved in ðrp Þ ¼ qp x (15.23)

the growth, development, and reproduction
of living organisms. No additional quantity of
substrate (energy) is needed to produce them. 3.8 Example of observed and “true” yield
• Secondary metabolites are produced through mass coefficients of organism growth/
the modification of primary metabolites. They product formation and related models
are formed near the stationary phase of
See: Table 15.7.
growth. Secondary metabolites do not play a

TABLE 15.6 Different cell growth-substrate 4. Ideal bioreactors

consumption-specific rate models.

Model Equation A bioreactor is a process unit in which

biochemical reactions occur. Commonly bioreac-
Herbert model mobs ðsÞ ¼ mðsÞ  c
(endogenous metabolism)
tors are isothermal. The meaning of the adjective
1 ideal may be different depending on the reactor
qðsÞ ¼ mðsÞ
YG
typology. The most important reactors are the
Pirt model mobs ðsÞ ¼ mðsÞ following (see also Table 15.8):
1
qðsÞ ¼
YG
mðsÞ þ m • The batch stirred tank reactor (BSTR) is a
closed system; there are no mass flows

IV. Selected examples and case history

 5. Typical industrial fermentation setup 275
TABLE 15.7 A useful example for understanding the difference between the observed yield and the “true” yield for
product formation and biomass growth.

Assumed stoichiometric equations Modela

Observed Overall equation for growth and production dx

dt
¼ mobs ðsÞx
mass yield sS þ nN þ oO2 / X þ pP þ wH2O þ eCO2 dp
¼
Yp=s dx
pM dt Yx=s dt
Yx=s ¼ sM
Mx
; Yp=s ¼ sMsp 1 dp
s
 ds
dt
¼ 1 dx
Yx=s dt
¼ Yp=s dt

“True” mass (1) Growth equation (From Pirt’s maintenance model)

yield dx
¼ mðsÞx
s1S þ n1N þ o1O2 / X þ w1H2O þ e1CO2 dt
dp
¼ qp x
dt  
(2) Product formation equation  ds ¼ 1
mðsÞ þ m þ
qp
x
dt YG YP
s2S þ n2N þ o2O2 / P þ w2H2O þ e2CO2

(3) Maintenance equation

s3S þ n3N þ o3O2 / maintenance þ w3H2O þ e3CO2
M
YG ¼ sM x
1 Ms
; YP ¼ s2 Mp s

Here S is the carbon source, N the nitrogen source, X the cell mass, and P the product; s, n, o, w, and e are the stoichiometric coefficients of the
reactions; Mx, Mp, and Ms are the molecular weights of the cells, products, and substrates, respectively. It is important to remember that the
“true” yield model is related only to describe the production of a secondary metabolite. For further details see Ref. [19].
a
Material balances are assumed to be for a constant volume batch fermentor. For the equations in the model obtained from the estimation of the “true” yield
coefficient, there is assumed to be no cell death related to the mechanical agitation in the reactor. See Chapter 4 for details.

entering of coming out from it, but it can
eventually exchange heat with the
environment. An ideal BSTR has the property
of perfect fluid mixing. It represents the
typical case of a cell culture.
• The fed-BSTR is a semi-open system; in fact,
mass can enter but not exit the reactor. It is an
enhancement of the closed batch process in
which the substrate is added in increments as
the fermentation progresses. An ideal fed-
BSTR has the property of perfect fluid
mixing.
• The continuous stirred tank reactor (CSTR)
is an open system. An ideal CSTR has the
property of perfect fluid mixing. A classical
CSTR bioreactor is individualized in the
chemostat configuration, in which a sterile
nutrient solution is added to the bioreactor
continuously and an equivalent amount of
converted nutrient solution with
microorganisms is simultaneously taken out
of the system [20].
• The plug flow reactor (PFR) is an open
system. The fluid passing through a PFR has a
flat velocity profile; in this sense a PFR is an
ideal reactor. This condition happens
typically when a fluid moves through a pipe
with a sufficiently large Reynolds number
(Re > 2100).
5. Typical industrial fermentation setup
Two recurrent industrial biological reactor
configurations will be analyzed in this section:
the chemostat and the aerobic reactors. In the
first typology, the mathematical dynamic model
used to describe it helps in understanding the
possibility of the occurrence of unstable working
sates of the reactor; in the second typology, oxy-
gen must be supplied to the cell population:
particular attention is given to the transfer rate
expression of oxygen from the gaseous phase
to the liquid phase.

IV. Selected examples and case history

276 15. Fermentation and biochemical engineering: principles and applications

TABLE 15.8 Equations used for a general description of the principal ideal
reactors.

Equations System

VR ¼ constant
dx
dt
¼ ðrx Þ
ds
dt
¼  ðrs Þ
dp
dt
¼ ðrp Þ

Batch stirred tank reactor

a
Total mass balance
dðVR Þ
dt
¼ Q
Components mass balance

dðVR xÞ
dt
¼ ðrx ÞVR þ QðtÞxf
dðVR sÞ
dt
¼  ðrs ÞVR þ QðtÞsf
dðVR pÞ
dt
¼ ðrp ÞVR þ QðtÞpf

Fed-batch stirred tank reactor

Steady state

VR ¼ constant
Q(x  xf) ¼ (rx)VR
Q(s  sf) ¼ (rs)VR
Q(p  pf) ¼ (rp)VR

Continuous stirred tank reactor

Steady state
u dx
dz
¼ ðrx Þ
ds
u dz ¼  ðrs Þ
u dp
dz
¼ ðrp Þ

Plug flow reactor

a
The density of the inlet fluid is assumed to be the same as that of the fluid inside the reactor.

IV. Selected examples and case history

 5. Typical industrial fermentation setup 277

5.1 Chemostat: focus on the washout

condition and productivity
The chemostat was originally introduced in the
1950s as a method to culture a bacterial population
at a reduced growth rate for an indefinite period
and is the most widely used approach to establish
steady-state culture for various applications [21].
The chemostat configuration may be simply rep-
resented as a conventional CSTR configuration.
To describe the system with the correct model
the following assumptions are made:
• There is perfect mixing in the reactor: the ideal
CSTR situation.
• Only one nutrient constitutes the limiting
substrate: m(s) can be expressed by the FIGURE 15.15 Space phase to visualize the system dy-
standard Monod equation, Eq. (15.17). namics and the washout conditions.
• The “true” yield mass stoichiometric
coefficients and Pirt’s model are used: YG, YP.
not desired, and the stationary state should be
• The death of cells caused by the mechanical
reached by ensuring the second one. A clear
agitation of the impeller is neglected:
vision of the situation may be illustrated with a
mobs(s) ¼ m(s).
space state graph (Fig. 15.15), which takes into
• The feed stream is sterile: xf ¼ 0.
consideration the dynamics of the system
• The reactor is isotherm.
following the nonstationary equation:
Using the set of equations for an ideal CSTR in  
s
steady state, already expressed in Table 15.8, and x_ ¼ mmax  D x: (15.27)
rearranging them with all the assumptions Ks þ s
described above it is easy to obtain: There is a situation in which it is always
s m(s) < D and the washout is the only stationary
Qx ¼ mmax xVR ; (15.24) state possible:
Ks þ s
  D > mðsf Þ: (15.28)
1 s
Qðs  sf Þ ¼  m þ m xVR : In fact m(sf) is the maximum rate possible ac-
YG max Ks þ s
cording to the Monod equation. Bearing this dis-
(15.25) cussion in mind, the maximum value that D may
Calling D ¼ VQR the dilution rate, it is possible have to not induce the system to wash out is:
to rewrite Eq. (15.24) as: mmax sf
  Dmax ¼ mðsf Þ ¼ : (15.29)
s Ks þ sf
0 ¼ mmax  D x: (15.26)
Ks þ s In continuous cultivation the objective is the
It is noticeable that Eq. (15.26) has not got a production of cells, and for this reason it is
unique stationary point; in fact, the equation is important to focus on the rate of cell production
satisfied for x ¼ 0 and m(s) ¼ D. Obviously the per unit reaction volume: the productivity,
first condition, called the washout condition, is expressed by Dx.

IV. Selected examples and case history

278 15. Fermentation and biochemical engineering: principles and applications

The design of an aerobic reactor is focused on

the determination of the quantity of oxygen to
supply. Let us introduce again the equations
for an ideal steady-state CSTR reactor
(Table 15.8):
Cell balance:
Qðx  xf Þ ¼ ðrx ÞVR ; (15.30)
Oxygen balance in the liquid phase:
 
Q Cin
O2  C O2
þ qtO2 VR ¼ ðrO2 ÞVR ; (15.31)
FIGURE 15.16 Steady states in continuous culture:
mmax ¼ 2.38 L/h; sf ¼ 1100 mg/L; YG ¼ 0.45; Ks ¼ 35 mg/L;
Carbon source substrate balance:
m ¼ 0.05 [22].
Qðsf  sÞ ¼ ðrs ÞVR ; (15.32)

The results of the system of Eqs. (15.24) where the addition flux term qtO2
expresses the
e(15.25) and the productivity are plotted in the amount of oxygen transferred from the gas
graph shown in Fig. 15.16. phase, given by breathed air, to the liquid phase.
The evaluation of qtO2 is given according to the
mass transfer film theory (Fig. 15.17), which
gives:
5.2 Aerobic reactors: cellular respiration 
Organisms may be divided into three main qtO2 ¼ KOL aðCO2  CO2 (15.33)
classes depending on their ability to live with  2
or without oxygen consumption [23]: L
Here a is the gas bubble surface to reactor
L3
• Obligate anaerobes: These are prokaryotic liquid volume ratio, KOL is the overall liquidegas
microorganisms (Clostridium sp., the soil mass transfer coefficient, and CO2 is the oxygen
bacteria Actinomyces, and the methanogenic concentration in the liquid phase in equilibrium
bacteria) for which molecular oxygen is a
toxic substance.
• Obligate aerobes: These are organisms that
require molecular oxygen as the terminal
oxidizing agent to fulfill their energetic needs,
such as humankind.
• Facultative anaerobes: These are organisms
that are able to grow either in the presence or
in the absence of oxygen (e.g., Saccharomyces
cerevisiae, the “brewer’s yeast”).
It is obvious that an amount of oxygen must
be delivered to the reactor if it is necessary for
the survival of the cell population. In such case,
oxygen consumption is typically coupled FIGURE 15.17 Film theory sketch: visualization of the
directly to the amount of carbon source substrate gaseliquid interphase and the local mass transfer coefficients
consumed (commonly glucose). kG, for the gaseous film, and kL for the liquid one.

IV. Selected examples and case history

 5. Typical industrial fermentation setup
with the bulk of the gas phase. Remember that
the gaseliquid equilibrium equation is given
by Henry’s law:
yO 2 P
CO2 ¼ ; (15.34)
HO2 ;mix
where yO2 P is the oxygen partial pressure and
HO2 ;mix is the Henry constant of oxygen in the
liquid mixture of the reactor (which in biological
reactors, in many cases, is just considered the
Henry constant of oxygen in water for
simplicity).
The relation between the global mass transfer
and the local ones is simply:
1 1 1 CO2  3KO2 :
¼ þ :
KOL kG HO2 ;mix kL
Several models are proposed to express the
specific growth rate for cells m(s) when the oxy-
gen constitutes a limiting nutrient. Remembering
also that the consumption of oxygen is related to
the assimilation of a carbon source, the most
used expression of m(s) is again the Monod equa-
tion for multiple substrates (Table 15.2). It hap-
pens often with oxygen that the cellular specific
279
growth rate is almost zero when the oxygen con-
centration stands below a threshold value. In
these cases, the Monod formula is not completely
correct and other models may be used, such as
the modified MonodeGompertz model [24]:
  
s KO2  CO2
mðs; CO2 Þ ¼ mmax exp  exp :
Ks þ s KO2 =2
(15.36)
To avoid easy oscillations in the cell popula-
tion quantity in the reactor, it is useful to work
under conditions in which the oxygen is no
longer a limiting nutrient. This situation happens
when:
(15.37)
(15.35)
All the situations explained above are sum-
marized in Fig. 15.18, a study of desulfurization
by means of the sulfur-oxidizing bacteria Acidi-
thiobacillus thiooxidans [24], in which:
- an oxygen threshold value was identified,
calculated to be 0.4 mg/L;
- the data of the specific growth rate depending
on the other substrate (for the specific case of
H2S) were obtained under the conditions of

FIGURE 15.18 Experimental data of the cellular specific growth rate for the sulfur oxidizing bacteria Acidithiobacillus thi-
ooxidans. (A) The concentration of oxygen in the liquid is kept constant at a value at which it is no longer considered a limiting
nutrient and the dependence of the specific grow rate upon the concentration of sulfide in the liquid has been detected. (B) With
a constant concentration of sulfide in the liquid, the dependence of the specific growth rate upon the concentration of oxygen in
the liquid has been detected [24].

IV. Selected examples and case history

280 15. Fermentation and biochemical engineering: principles and applications
Eq. (15.37) and, in fact, from the study KO2 ¼
1:10 mg=L and graph (A) of Fig. 15.18 was
made with CO2 y6 mg=L.
6. Bioethanol production
A typical example of employing both enzy-
matic and cellular technologies is the production
of bioethanol. Fossil fuel depletion has become a
great concern as the world population is
increasing at an alarming rate. Current concerns
such as global warming, depletion of fossil fuels,
and increasing price of petroleum-based fuels
have forced the search for alternative and cost-
effective energy sources with fewer greenhouse
gas emissions. Research into the development
of renewable and sustainable fuels has recog-
nized bioethanol as a viable alternative to fossil
fuels, owing to its low toxicity, biodegradability,
and ability to effectively blend with gasoline
without any engine modifications [25].
Depending on the raw materials used for the
bioethanol production, two main classes of
such product may be identified:
• First-generation bioethanol is made via a
traditional production approach using raw
FIGURE 15.19 Classification of cell pretreatment methods [26]. EFAC, Electroflotation by alternating current.
IV. Selected examples and case history
materials belonging to the food industry
(such as sugar cane, sorghum, and corn).
• Second-generation bioethanol was developed
to substitute food-based feedstock with raw
materials that are almost all waste products.
Five classes of such raw materials may be
defined: (1) wood residues, (2) municipal solid
waste, (3) agriculture residues, (4) dedicated
energy crops, and (5) microalgal biomass.
The bioethanol production process is
composed of three macrounits common to all
the types of raw materials:
1. Pretreatment: It is necessary to break the cell
wall membrane of the biomass and permit the
release of all the cell contents (carbohydrates
for bioethanol production, lipids for biodiesel
production). It is extremely important, since it
affects the efficiency and consequently the
cost of the whole process. Many different
pretreatment techniques have been
developed (Fig. 15.19) and the most
appropriate one to adopt depends on the
particular biomass selected and the
configuration of the plant.
2. Hydrolysis: Starch, lignocellulose, or, better,
polymeric carbohydrates and polyphenolic
lignin are not directly fermentable by most
 6. Bioethanol production
yeasts to produce bioethanol. Accordingly,
the hydrolysis of such compounds must be
carried out to obtain fermentable monomeric
sugars. Although acid hydrolysis constitutes
a valid strategy, hydrolysis run by means of
enzymes is known to be more cost effective
due to the mild operative temperature and
pH conditions. While cellulase, the term used
to describe the active material that
depolymerizes cellulose, is a complex mixture
of many different enzymes that are identified
by their generating organism (e.g.,
Trichoderma reesei cellulases), a more precise
classification is available for starch-degrading
enzymes (Fig. 15.20). Further information
may be found in Refs. [26,27].
3. Fermentation: This is the step in which a cell
culture, having monomeric sugars as a
nutrient source, generates ethanol as a
primary metabolite product. Historically, the
most commonly used microbe has been yeast,
and among the yeasts, S. cerevisiae, which can
produce ethanol to give concentrations as
high as 18% of the fermentation broth, is the
preferred one for most ethanol fermentation
[27].
The steps of hydrolysis and fermentation are
both conducted in bioreactors and are disposed
in sequence. Bearing this in mind, there are
several considerations to make about the
different process design choices that can be
done according to the specific requirements of
the plant.
FIGURE 15.20 Classification of starch-degrading enzymes [27].
IV. Selected examples and case history
281
6.1 Separate hydrolysis and fermentation
This situation presents enzymatic hydrolysis
and fermentation conducted in separate reactors.
In such configurations each step can be per-
formed at optimum conditions. In particular,
the enzymes are able to produce more substrate
for the yeast fermentation. On the other hand,
hydrolysis products inhibit the activities of the
cellulases so the rate of hydrolysis is progres-
sively reduced.
6.2 Simultaneous saccharification and
fermentation
The simultaneous saccharification and
fermentation (SSF) configuration consists of a
unique reactor in which both hydrolysis and
fermentation occur. Adopting this type of solu-
tion, the inhibition problem found in separate
hydrolysis and fermentation (SHF) disappears,
since glucose and cellobiose are progressively
consumed during their production. As a draw-
back, since the temperature of maximum enzy-
matic activity differs from the optimal one of
yeast growth, one of the two functions (or both
of them) will not operate under its optimum con-
ditions. As an example, T. reesei cellulase has an
optimum activity at 55 C, while S. cerevisiae ac-
tivity is optimum at 37 C.
The two configurations explained above may
be improved by the introduction of microbes
(genetically modified) able to ferment simulta-
neously pentose and hexose sugars, increasing
282 15. Fermentation and biochemical engineering: principles and applications
the ethanol production yield. In such a way the
issue involving S. cerevisiae, commonly unable
to ferment pentose sugars like xylose and arabi-
nose, was overcome [28]. Since in such case a
contemporaneous fermentation of different
types of monomeric sugars takes place, the pro-
cesses involving this technique are called simul-
taneous saccharification and cofermentation or
separate hydrolysis and cofermentation,
depending on whether the SSF or SHF configura-
tion is chosen, respectively.
6.3 Example: general second-generation
ethanol production case
A schematic representation of a possible pro-
cess flow diagram for second-generation bio-
ethanol production is illustrated in Fig. 15.21.
First of all, it is an SHF configuration with a
hydrolysis batch reactor and a fermentation fed-
batch reactor. Both reactors are drawn with a
pump around the loop with cooling water heat
exchange, which is a technique used to control
the temperature of the reactors. After the
hydrolysis reactor, a filter is inserted to effectuate
a solideliquid separation to obtain a lignin-rich
solid fraction, destined for a combustor, and a
FIGURE 15.21 Example of a process flow diagram for the production of bioethanol.
IV. Selected examples and case history
liquid sugar-rich phase, which sugar concentra-
tion is monitored by the evaporator, directed to
the fermentor. The fermentation bioreactor is
inoculated from an inoculum seed train. This is
required to avoid the shock of inserting a cell cul-
ture into a new or bigger volume environment,
which may cause undesired behaviors of the cells.
Finally, the content of the fermentation reactor is
collected into a storage tank, which permits all the
following units to work continuously.
7. Conclusion and future trends
In this chapter, an overview of the theoretical
key aspects of the design of both enzymatic and
cellular reactors was given. Each section is fol-
lowed by an example that applies the concepts
explained.
The development of biobased reactors has a
very old origin, and, over the centuries, the inter-
est in this fascinating kind of technology
continued to grow (Table 15.9).
Nowadays, the development of industrial
technologies is mainly focused on moving to-
ward the “green chemistry” concept. Green
chemistry requires the reduction, or elimination,
of the use (or generation) of hazardous
 List of abbreviations and acronyms 283
TABLE 15.9 Main events involving bioreactors in history [29].

Year Event

4000e3000 BC Baking, brewing

2000 BC Ethanol production and distillation (China)
1923 Commercial production of citric acid (Pfizer, USA)

1940s Production of penicillin by fermentation (USA)

1950s Design and scale-up of large aerated fermentors
1970s More than 100 new drugs and vaccines produced by bioprocesses
1980s Control of fed-batch bioreactors

substances in the design, manufacture, and
application of chemical products. It is noticeable
how the utilization of bioreactors fits perfectly
into such definition: in fact, such units operate
at low temperature and pressure (typically
T ¼ 37 C and P ¼ 1 atm), reducing the consump-
tion of energy in the plants, avoiding the waste
of chemicals, and guaranteeing safer working
conditions (Fig. 15.22).
According to some data on the year 2013, the
number of companies involved in the utilization
of bio-based reactors was already more than 200
[23]. This highlights how the potentialities of
such technology are well known and how, for
the near future, its rapid development is ex-
pected, in line with the ideal of green chemistry.
List of abbreviations and acronyms
SHF separate hydrolysis and fermentation
SSF simultaneous saccharification and fermentation

FIGURE 15.22 An example of the substitution of a conventional scrubber unit for the removal of H2S from a biogas stream
with a biological reactor. The bacteria Acidithiobacillus thiooxidans are responsible of the oxidation of H2S to obtain living energy.

IV. Selected examples and case history

284 15. Fermentation and biochemical engineering: principles and applications

List of symbols qtO2 flux of oxygen, from the gaseous phase to the liquid
phase, per unit volume of liquid in the reactor
R gas constant
a gas bubble surface to reactor liquid volume ratio
(rp) rate of product formation per unit volume of liquid
CO2 concentration of oxygen in the liquid
in in the reactor
CO concentration of oxygen entering a CSTR reactor

2
(Lrs) rate of substrate consumption per unit volume of
CO concentration of oxygen in the liquid phase in
2
liquid in the reactor
equilibrium with the bulk of the gas phase
(rx) rate of biomass formation per unit volume of liquid
c maintenance term in Herbert model
in the reactor
D dilution rate
ðLrO2 Þ rate of oxygen consumption per unit volume of
Dmax maximum dilution rate
liquid in the reactor
E enzyme element in the reaction formula
S substrate in the reaction formula
ES substrateeenzyme complex in the reaction formula
s substrate concentration
EI inhibitoreenzyme complex in the reaction formula
sf feed substrate concentration
EIS inhibitoresubstrateeenzyme complex in the reac-
T absolute temperature
tion formula
u mean velocity in the PFR reactor
Ea active enzyme element in the reaction formula
VR volume of liquid in the reactor
Ed denatured enzyme element in the reaction formula
X biomass in the reaction formula
Ei inactive enzyme element in the reaction formula
x biomass concentration
e enzyme concentration
xf feed biomass concentration
e0 initial enzyme concentration
YG “true” growth yield: stoichiometric factor of the
(es) substrateeenzyme complex concentration
reaction that produces biomass from substrate
DGd denaturation Gibbs free energy variation
YP “true” product yield: stoichiometric factor of the
HO2 ;mix Henry constant of oxygen in a mixed liquid solution
reaction that generates product from substrate for
I inhibitor in the reaction formula
secondary metabolites
i inhibitor concentration
Yp/s observed product yield formation
K1 enzyme first reduction equilibrium constant
Yx/s observed biomass yield formation
K2 enzyme second reduction equilibrium constant
yL fraction of active enzymes
Kd equilibrium constant of enzyme inactivation with
yO2 P oxygen partial pressure
temperature
Ki gas inhibitoreenzyme dissociation constant
Km MichaeliseMenten dissociation constant
app
Km apparent Michaelis-Menten dissociation constant
KOL overall liquidegas mass transfer coefficient Greek symbols
KP product concentration that reduces to one-half the
cellular specific growth rate m(s) biomass specific growth rate
KS substrate concentration at which the cellular specific mmax maximal biomass specific growth rate
growth rate is half its maximum mobs observed biomass specific growth rate
KO2 oxygen Monod constant n reaction rate
k1 kinetic constant for the production of the nmax maximum reaction velocity
substrateeenzyme complex napp
max maximum apparent reaction velocity
kL1 kinetic constant for the dissociation of the
substrateeenzyme complex
k2 kinetic constant for the formation of the product
kG gas mass transfer coefficient
References
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IV. Selected examples and case history

 Further reading
[5] M. Emmanuel, Papamichael, G.T. Leonidas, Enzyme
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Enzyme technology, Springer, India, 2006.
[6] P.M. Doran, Bioprocess Engineering Principles, 7, Aca-
demic Press Ltd., London, 1995.
[7] L.W. Lai, C.L. Teo, S. Wahidin, M. Suffian, M. Annuar,
Determination of enzyme kinetic parameters on sago
starch hydrolysis by linearized graphical methhods,
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(2014) 527e533.
[8] R. Eisenthal, A. Cornish-Bowden, The direct linear: a
new graphical procedure for estimating enzyme kinetic
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721e730.
[9] O.D. Lopina, Enzyme inhibitors and activators.
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[10] P. Kuma, R. Reddy, S. Babu, Inhibitory effects of
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litus (a-Amylase and a-Glucosidase) and obesity
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2155-6156.1000781.
[11] J.E. Bailey, D.F. Ollis, Biochemical engineering funda-
mentals, Mc Graw Hill.
[12] E. Jurado, F. Camacho, G. Luz on, J.M. Vicaria, Kinetic
models of activity for b-galactosidases: influence of
pH, ionic concentration and temperature, Enzyme
and Microbial Technology 34 (2004) 33e40.
[13] A. Nafi, F.H. Ling, J. Bakar 1, H.M. Ghazali, Partial
characterization of an enzymatic extract from Bentong
Ginger (Zingiber officinale var. Bentong), Molecules 19
(2014) 12336e12348, https://doi.org/10.3390/
molecules190812336.
[14] P. Held, K. Raymond, Determination of Algal Cell
Lipids Using Nile Red Using Microplates to Monitor
Neutral Lipids in Chlorella Vulgaris, Applications
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[15] H. Kim, S. Park, B.E. Rittmann, Multi-component ki-
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[17] G. Wang, W.M. Post, A theoretical reassessment of mi-
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IV. Selected examples and case history
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[18] Lakna, "Difference Between Primary and Secondary
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[19] J. Hong, Yield coefficients for cell mass and product
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Further reading
[1] W.W. Cleland, The kinetics of enzyme-catalyzed reac-
tions with two or more substrates or products. II. Inhibi-
tion: nomenclature and theory, Biochimica et Biophysica
Acta 67 (1963) 173e187, https://doi.org/10.1016/0926-
6569(63)90226-8.