ERT 416/3
CHAPTER 7: UPSTREAM PROCESSING
IN BIOPROCESS PLANT
MISS. RAHIMAH BINTI OTHMAN
(Email: rahimah@unimap.edu.my)
COURSE OUTCOME 1 CO1)
CHAPTER 7 : Upstream Processing In Bioprocess Plant.
CLASSIFY the elements of bioprocesses; LIST typical unit operations in bioprocess
like sterilization, fermentation, enzymatic reactions, extraction and filtration or
crystallization and DEFINE the unit procedure involved for the process design.
UNDERSTAND and LIST typical upstream steps like preparation of medium, the
sterilization of the raw materials and the inoculums preparation. DECIDE and
DESIGN suitable upstream processing units for the design process based on
stoichiometry, thermodynamics, separation and reaction engineering principles.
CHAPTER 8 : Bioreaction Design In Bioprocess Plant.
CHAPTER 9 : Downstream Processing In Bioprocess Plant.
CHAPTER 10: Integrated Bioseparation Scheme for Product
Isolation, Purification and Formulation Units
For Bioprocess.
OUTLINES
1. LIST of typical unit operations in bioprocess plant
system.
2. DEFINITION of the unit procedure involved for the
process design.
3. UNDERSTANDING the typical upstream steps like
preparation of medium, the sterilization of the raw
materials and the inoculums preparation.
4. SELECTION and DESIGN PROCEDURE on the
suitable upstream processing units for the design
process based on stoichiometry, thermodynamics,
separation and reaction engineering principles.
GENERALIZED VIEW OF
BIOPROCESS
RAW MATERIALS
UPSTREAM PROCESSES
Inoculum
Preparation
Equipment
Sterilization
Media Formulation
and
Sterilization
BIOREACTOR - FERMENTER
Reaction Kinetics
and Bioactivity
Transport Phenomena
and Fluid Properties
Instrumentation
and Control
DOWNSTREAM PROCESSES
Separation
Recovery and
Purification
Waste Recovery,
Reuse and Treatment
THE BOTTOM LINE
REGULATION
ECONOMICS
HEALTH AND SAFETY
LIST OF TYPICAL UNIT OPERATIONS IN
BIOPROCESS PLANT SYSTEM
1. Sterilization
2. Fermentation
3. Enzymatic
reactions
4. Extraction and
filtration
5. Crystallization
DEFINITION OF THE UNIT PROCEDURES
FOR THE PROCESS DESIGN
1. Sterilization
Definition: Sterilization (or sterilisation) is a term referring
to any process that eliminates (removes) or kills all forms of
life, including transmissible agents (such as fungi, bacteria,
viruses, spore forms, etc.) present on a surface, contained in a
fluid, in medication, or in a compound such as biological
culture media.
Sterilization can be achieved by applying the proper
combinations of heat, chemicals, irradiation, high pressure, and
filtration.
AUTOCLAVE
DEFINITION OF THE UNIT PROCEDURES
FOR THE PROCESS DESIGN
1. Sterilization
(i) Sterilization of Liquid Media
a) Batch Sterilization of Liquid Media
b) Continuous Sterilization of Liquid Media
c) Kinetics of Thermal Death of Microorganisms
d) Examples of Design for Continuous Liquid
Medium Sterilization in a Tubular Sterilizer
(ii) Air Sterilization by Fibrous Bed Filters
 Mechanisms of Air Filtration
 Design of Fibrous Packed Beds
 Example of Design of Fibrous Packed Bed for
Air Sterilization
Schematic Diagram
of Continous
Sterilizer System.
Overview of
Sterilizer Phases
Fibrous Packed Bed for Air Sterilization
1. STERILIZATION OF
LIQUID MEDIA
 The liquid media which contains all
essential nutrients for cell growth is:
 First heat sterilized with steam,
then;
 Cooled down before introduction
into the bioreactor vessel
 Two types of sterilization:
 Batch sterilization
(see Fig. 5.1, and Table 5.1 for
corresponding temperature profile)
 Continuous sterilization
(see Fig. 5.2a, 5.2b)
(i-a) Batch Sterilization of Liquid Media
FIG. 5.1 Types of equipment for batch sterilization of media. [Adopted from S.
Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical
Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 254].
TABLE 5.1. Temperature-Time Profile in Batch Sterilization. [Adopted from S. Aiba,
A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering,
2nd Ed., Academic Press, Inc., New York (1973) 254].
(i-b) Continuous Sterilization of Liquid Media
 Two types of continuous sterilization:
 Direct steam injection sterilizer
(see Fig. 5.2a)
 Plate heat exchanger sterilizer
(see Fig. 5.2b)
FIG. 5.2a Direct steam injection type of continuous sterilization of liquid media.
[Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In
Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].
FIG. 5.2b Plate heat exchanger type of continuous sterilization of liquid media.
[Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In
Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].
Fig. 5.3a and 5.3b show the temperature-time
profiles for the two types of continuous
sterilization.
FIG. 5.3a Sterilization temperature vs. time profile for direct steam injection
continuous sterilizer. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis.
“Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc.,
New York (1973) 258].
FIG. 5.3b Sterilization temperature vs. time profile for plate heat exchanger
sterilizer. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media
Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New
York (1973) 257].
(i-c) Kinetics of Thermal Death of Microorganisms
Heat is used to kill:


Contaminant microorganisms
Spores
- present in a liquid nutrient medium.
The destruction of microorganisms by heat means:

Loss of Viability of these microorganisms and
spores.
 The thermal death of microorganisms follow
first order kinetics given by Eq. 5.1.
dN/dt = -kN……………………...(5.1)
Where:
N = Number of viable microorganisms
t = Sterilization time, min
k = Thermal death rate constant, min-1
If at time t0 = 0, N = N0, then integration of
Eq.5.1 results in Eq. 5.2.
N = N0 e-kt ………………………(5.2)
Also:
ln(N/N0) = -kt ………………….(5.3)
 The term decimal reduction time, D, is used to
characterize the death rate constant.
 D is defined as the sterilization time required to
reduce the original number of viable cells
by
one tenth.
N/N0 = 1/10 = e-kD
ln(0.10) = -Kd
D = 2.303/k……………………………(5.4)
 Fig. 4.4 and 4.5 shows typical data of N/N0
vs. sterilization time for spores of Bacillus
stearothermophillus, one of the hardest
spores to kill, and vegetative cells of E. coli
FIG. 4.4 Typical thermal death rate data for spores of Bacillus stearothermophilus Fs 7954 in
distilled water where N = number of viable spores at any time, N0 = original number of viable
spores. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In
Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 241].
FIG. 4.5 Typical death rate data for E. coli in buffer, where N = number of viable
spores at any time, N0 = original number of viable spores. [Adopted from S. Aiba,
A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering,
2nd Ed., Academic Press, Inc., New York (1973) 241].
 The thermal death rate constant k is given
by Eq. 5.5 and follows the typical Arrhenius
equation.
K = A e-E/RT………………………(5.5)
Where:
A = empirical constant
E = Activation energy for thermal death
of microorganism
T = Absolute temperature, oK
R = Gas constant = 1.98 cal/g mole oK
 Fig. 4.6 and 4.7 shows the Arrhenius plots
of k for spores of B. stearothermophilus,
and vegetative cells of E. coli, respectively.
FIG. 4.6 Correlation of isothermal death rate data for spores of Bacillus stearothermophilus
Fs 7954, where k = reaction rate constant and T = absolute temperature. Value of E
(activation energy) = 68.7 kcal/ g mole. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis.
“Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York
(1973) 242].
FIG. 4.7 Correlation of isothermal death rate data with temperature for E. coli,
where k = reaction rate constant and T = absolute temperature. Value of E
(activation energy) = 127 kcal/g mole. [Adopted from S. Aiba, A.E. Humphrey and
N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic
Press, Inc., New York (1973) 243].
For spores of B. stearothermophilus, the
following kinetic parameters apply:

A = 7.94 x 1038 min-1

E = 68.7 x 103 cal/g mole
The higher the value of E, the more
difficult it is to kill by thermal denaturation a
microorganism or spore.
The value of activation energy, E, due to
thermal denaturation (death) for vegetative
microbial cells and spores is in the range
of E = 50 to 100 kcal/g mole.
For the thermal denaturation of enzymes,
vitamins, and other fragile nutrients, the
activation energy, E, is in the range of
E = 2 to 20 kcal/ g mole.
For a given liquid medium containing
both, it is easier (faster) to denature
thermally, enzymes and vitamins and other
nutrients, and more difficult (slower) to
denature (kill) vegetative cells.
In order to find the value of k for any system
(spores and vegetative cells, nutrients) it is
important to know both A
and E in the Arrhenius Eq. 5.5.
 Sterilization at relatively high temperatures
with short sterilization times is highly
desirable because it favours the fast killing
of vegetative cells and spores with minimal
denaturation of nutrients present in the
liquid medium.
BATCH STERILIZATION OF LIQUID MEDIA
During batch sterilization:


Both temperature and time change
Also k changes with time, since k = f (T)
Table 4.1 shows the sterilization temperature
as a;
function of time for batch sterilization
using different types of heat transfer and
cooling.
dN/dt = -kN = -Ae-E/RT N…(5.6)
Integrating Eq. 5.6 from t0 = 0, N = N0 to any
time t = t and N = N, we get Eq. (5.7).
ln(N0/N) = 0t kdt = A 0t e-E/RTdt ….....(5.7)
We define:
= ln (N0/N)………………………….(5.8)
 In sterilization design:


Is used as a criterion of design.
Specifies the level of sterilization
required for a liquid nutrient
medium.
During batch sterilization, there are three
periods of sterilization:

Heating of the liquid medium period

Holding at constant temperature period

Cooling period
During each period, a separate value of
Total
= ln(N0/N) =
heating
+
holding
+

heating
= ln(N0/N1) = 0t1 kdt

holding
= ln(N1/N2) = t1t2 kdt

cooling
is calculated:
cooling………(5.9)
= ln(N2/N) = t2t3 kdt
Where:
N = No. of contaminants after sterilization
N0 = No. of contaminants before sterilization
N1 = No. of contaminants after heating period t1
N2 = No. of contaminants after holding period t2
t1, t2, t3 = Sterilization times during, heating, holding and cooling.
 Total batch sterilization time, t, is given
by Eq. 5.10.
t = t1 + t2 + t3 .………………..(5.10)
EXAMPLE OF BATCH STERILIZATION
Calculate the total degree of batch
sterilization, total, for a liquid medium inside
a bioreactor vessel, which reaches
maximum temperature 120 oC, and then
cooled off. Assume that the liquid medium
contains spores of B. stearothermophilus,
and the initial total number is N0 = 6 x 1012
spores. The temperature vs. time profile
during batch sterilization is given below.
t (min) T1 (oC)
0
30
10
50
30
90
36
100
43
110
50
120
55
120
58
110
63
100
70
90
102
60
120
44
140
30
For spores of B. stearothermophilus:
k = 7.94 x 1038 exp[(-68.8 x 103)/RT] min-1
R = 1.98 cal/g mole oK
FIG. 4.8 Batch sterilization: k and T vs. t ; example calculation. Area under the
curve k vs. t is total degree of sterilization, total. [Adopted from S. Aiba, A.E.
Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd
Ed., Academic Press, Inc., New York (1973) 256].
Fig. 4.8 shows the temperature-time profile
and the value of k as a function of
T [i.e. k = f (T)] as given in the previous
slide.
 Examining Fig. 4.8, it is also evident that
the values of k are a function of t [i.e. k = f
(t)], ranging between 0 to 34 min, and
between 64 to 140 min. Therefore, the area
under the curve k (min-1) vs. t (min) is the
graphical integration, which gives:


140 kdt = 33.8
=
ln(N
/N)
=

total
0
0
N = N0/exp(33.8) = 6x1012/4.77698x104
= 1.256x10-2
CONTINUOUS STERILIZATION OF
LIQUID MEDIA
 Fig. 4.2a and 4.2b show the two most
common types of continuous sterilizers
used with steam to carry out the sterilization
of liquid fermentation media.
 In both systems, the liquid medium is
heated rapidly the desired high temperature
either by direct steam injection or by plate
heat exchangers and then it goes through a
holding section, which is a tube of given
diameter and length to give the desired
residence (holding) sterilization time:

The holding tubular section is well
insulated and it is held at the same
sterilization temperature along its length.

Fig. 4.3a and 4.3b give approximate
temperature-time profiles for the steam
injection and plate heat exchanger types
respectively.
NOTE: The direct steam injection gives
much faster rise in temperature but, the
original liquid medium is being diluted by
the amount of the steam condensate
during the injection of the steam.
Therefore, an enthalpy and mass balance is
required at the steam injection nozzle.
 The problem: design and size both the
diameter and length of the tubular holding
section which is held at a given temperature
assuming a desired degree of sterilization,
using the thermal rate constant and its
Arrhenius relationship for spores of B.
stearothermophilus, which is one of the
hardest spores to kill by steam sterilization.

NOTE: In both the injection type and
plate exchanger type of continuous
sterilizers, it is required to design
(size-up) the length and
diameter of
the
tubular holding section.
3. Enzymatic Reactions
3. Enzymatic Reactions
1. Enzymes are proteins that catalyze (i.e., increase
the rates of) chemical reactions.
2. In enzymatic reactions, the molecules at the
beginning of the process are called substrates, and
the enzyme converts them into different molecules,
called the products.
3. Almost all processes in a biological cell need
enzymes to occur at significant rates.
4. Since enzymes are selective for their substrates and
speed up only a few reactions from among many
possibilities, the set of enzymes made in a cell
determines which metabolic pathways occur in that
cell.
The Enzymatic Reactions Flow Diagram
The energies of the stages of a chemical reaction. Substrates need a
lot of energy to reach a transition state, which then decays into
products. The enzyme stabilizes the transition state, reducing the
energy needed to form products.
Industrial Application
Application
Food processing
Amylases catalyze the release
of simple sugars from starch
Enzymes used
Amylases from fungi and
plants.
Proteases
Enzymes from barley are
released during the mashing
stage of beer production.
Industrially produced barley
enzymes
Brewing industry
Germinating barley used for
malt.
Amylase, glucanases,
proteases
Betaglucanases and
arabinoxylanases
Amyloglucosidase and
pullulanases
Proteases
Uses
Production of sugars from starch, such
as in making high-fructose corn syrup.In
baking, catalyze breakdown of starch in
the flour to sugar. Yeast fermentation of
sugar produces the carbon dioxide that
raises the dough.
Biscuit manufacturers use them to lower
the protein level of flour.
They degrade starch and proteins to
produce simple sugar, amino acids and
peptides that are used by yeast for
fermentation.
Widely used in the brewing process to
substitute for the natural enzymes found
in barley.
Split polysaccharides and proteins in the
malt.
Improve the wort and beer filtration
characteristics.
Low-calorie beer and adjustment of
fermentability.
Remove cloudiness produced during
storage of beers.
Industrial Application
Application
Dairy industry
Roquefort cheese
Enzymes used
Uses
Rennin, derived from the
stomachs of young ruminant
animals (like calves and lambs).
Manufacture of cheese, used to
hydrolyze protein.
Microbially produced enzyme
Now finding increasing use in
the dairy industry.
Lipases
Is implemented during the
production of Roquefort cheese
to enhance the ripening of the
blue-mould cheese.
Lactases
Break down lactose to glucose
and galactose.
Amylases, amyloglucosideases
and glucoamylases
Glucose isomerase
Glucose
Starch industry
Fructose
Converts glucose into fructose in
production of high fructose
syrups from starchy materials.
These syrups have enhanced
sweetening properties and lower
calorific values than sucrose for
the same level of sweetness.
Industrial Application
Application
Enzymes used
Amylases, Xylanases,
Cellulases and ligninases
Degrade starch to lower viscosity,
aiding sizing and coating paper.
Xylanases reduce bleach required
for decolorising; cellulases smooth
fibers, enhance water drainage, and
promote ink removal; lipases reduce
pitch and lignin-degrading enzymes
remove lignin to soften paper.
Cellulases
Used to break down cellulose into
sugars that can be fermented (see
cellulosic ethanol).
Ligninases
Use of lignin waste
Restriction enzymes, DNA
ligase and polymerases
Used to manipulate DNA in genetic
engineering, important in
pharmacology, agriculture and
medicine. Essential for restriction
digestion and the polymerase chain
reaction. Molecular biology is also
important in forensic science.
Paper industry
A paper mill in South Carolina.
Bio fuel industry
Cellulose in 3D
Molecular biology
Part of the DNA double helix.
Uses
Thank you
Prepared by,
MISS RAHIMAH OTHMAN