Laboratory 1: Batch Kinetic Fermentation

Introduction

Figure 1.1: Phases of a typical growth curve of E.coli in a batch culture

In a batch culture, there is neither input fed nor output produced during the fermentation
process. Initially, the medium culture is inoculated with the microorganism. The growth
constantly rises until at certain level, where it is inhibited because of the dropping substrate
concentration and the presence of toxic metabolites. During the growth of the cells in a batch
fermentation, they follow a growth curve analogous to the one shown in Figure 1. The growth
curve contains four distinct regions known as phases. They are known as Lag Phase, Log
Phase, Stationary Phase, and Death Phase.

Lag phase occurs between inoculation and the maximum growth rate. It is the first major
phase of microbial growth in a batch fermentation process. It is also a phase of adaptation of
the cells to their new environment. During this phase, there is only a minimal increase in cell
density. In certain types of fermentation, this phase may not even happened at all. [1]
There
are two sub phases in the lag phase. In the first phase, there is no growth acknowledged while
in the second sub phase, also known as acceleration phase, a constant growth begins.
The second phase is the exponential phase. The cells begin to multiply with their maximum
growth rate. This phase is crucial to determine the maximum growth rate, μ max and doubling
time, d since the growth at this time is the most constant and ideal. This is the second major
phase in a batch fermentation process. It is also known as the logarithmic growth phase.
During this phase, cells have adjusted to their new environment. They are dividing at a
constant rate resulting in an exponential increase in the number of cells present. This is
known as the specific growth rate and can be shown mathematically by the first order kinetics
as the following:

dX
=( µ−k d ) X
dt

Where: X is the cell concentration

t is time
µ is the cell growth rate
k d is the cell death rate

For (µ−k d ), it can be substitute as µnet . The cell death rate may be negligible only if it is
significantly smaller than the cell growth rate. Next, the growth curve during this phase can
be projected using the Monod batch kinetics:

µmax S
µ=
Ks+S

Where: µ is the specific cell growth rate (hr-1)

µmax is the maximum specific cell growth rate (hr-1)
S is the substrate concentration (g/L)
1
K s is the saturation constant (g/L) where it is equal to S when µ is equal to µ
2 max
Before stationary phase, there is a phase called retardation phase. This is the period between
the exponential and stationary phase. It is also can be called as the phase prior to the growth
becomes stationary. Several factors that hinder the growth are substrate concentration,
reduced dissolved oxygen tension (DOT), pH changes and presence of inhibiting metabolites.

After retardation phase, the growth phase arrives at the stationary phase which is the third
Figure 2.2: A typical Monod batch kinetics graph
major phase in a batch fermentation process. During this phase, the growth becomes constant
for a period of time before it decreases. This is due to the number of cells that are multiplying
and dying at the same rate. The cells’ death may be due to several factors such as the lack of
growth nutrients, excess of toxic substances, and stress due to recombinant gene. Primary
metabolite or growth associated production cease to exist. While secondary metabolite or
non-growth associated production may continue to run.

Finally, the fourth major phase is the death phase or decline phase. The growth declines from
its stationary phase due to the cells lysis. This is shown by the decrease amount of the viable
cell. In this phase, the cells’ death rate is greater than the cells’ multiplication rate. It can be
shown using the first order kinetics, similar to the exponential phase:

dX
=−k d X
dt

Procedure

1. The unit procedure chosen is a batch vessel procedure in a fermenter.

2. In the task tab, batch is chosen as the mode of operation.
3. Again in the task tab, the pure component was registered. They were biomass, carbon
dioxide, impurity, MAB, media, nitrogen, oxygen, and water.
4. Next, connect mode was chosen and three streams were added at the input, while a single
stream was added at the main output. Another output stream was added at the top output
as the vent.
5. The input streams were labelled ‘media & biomass’, ‘air’, and ‘water’. Meanwhile the
main output was labelled ‘MAB’, and the vent was labelled ‘carbon dioxide’.
6. Each input stream was double-clicked and their respective mass composition was
inserted.
7. At the fermenter, the operation data was added as follows; CHARGE-1, CHARGE-2,
HEAT-1, FERMENT-1, COOL-1, AND TRANSFER-OUT-1.
8. At the CHARGE-1 data, 10,000 kg/batch of water was charged using water to the
fermenter. The setup duration required in this charge operation was 5 minutes.
Meanwhile, the processing time was calculated to be 100 L/minute.
9. Next, at the CHARGE-2 data, 500 kg/batch of a feed stream consisting of 50 kg of
Biomass and 450 kg of Media was charged. This step required 20 minutes of setup
duration and the processing time was calculated to be 10 kg/minute.
10. At the HEAT-1 data, the fermenter was heated to 37°C using steam at a heating rate of
0.5°C/minute. This step required 5 minutes of setup time. Also, at the vent/emission tab,
carbon dioxide stream was automatically selected.
11. Next, for the FERMENT-1 data, the vessel went through the fermentation for 24 hours.
Chilled water was used as the heat transfer agent to ensure the fermenter was at a final
temperature of 37°C. CO2 produced in the fermenter was emitted through the vent. Also,
air is selected as the broth aeration.
12. ‘R’ symbol was clicked to insert the Monod’s kinetic information using the given
stoichiometric data in the reaction tab. Other information were also added such as S1-
Term was selected, media as the substrate and rate reference component, Biomass as the
B-term, and 0.2 h-1 as the µmax value.
13. At the COOL-1 data, the fermenter was cooled to a final temperature of 5°C using Glycol
at a cooling rate of 0.5°C/minute. It required a setup time of 5 minutes.
14. Finally, at the TRANSFER-OUT-1 data, the fermented broth was transferred out from the
fermenter for further product separations for 5 hours of process time.
Process Flow (P&ID)

Figure 3: The Piping & Instrumentation Design (P&ID) of the batch vessel fermenter

1. The unit procedure chosen is a batch vessel in a fermenter labelled P-1/FR-101.

2. At the task tab, the pure component was registered. They were biomass, carbon dioxide,
impurity, MAB, media, nitrogen, oxygen, and water.
3. There are three streams at the input, while there is only a single stream at the main output.
Another output stream was added at the top output as the vent.
4. Each input streams were labelled ‘media & biomass’, ‘air’, and ‘water’. Meanwhile at the
output stream, the main output was labelled ‘MAB’, and the vent was labelled ‘carbon
dioxide’.
5. At the fermenter, the operation data are as follows; CHARGE-1, CHARGE-2, HEAT-1,
FERMENT-1, COOL-1, AND TRANSFER-OUT-1.
6. At the CHARGE-1 data, 10,000 kg/batch of water is charged using water to the
fermenter. The setup duration in this charge operation is 5 minutes. Meanwhile, the
processing time calculated is 100 L/minute.
7. Next, at the CHARGE-2 data, 500 kg/batch of the feed stream consisting of 50 kg of
Biomass and 450 kg of Media was charged. This step required 20 minutes of setup
duration and the processing time calculated is 10 kg/minute.
8. At the HEAT-1 data, the fermenter was heated to 37°C using steam at a heating rate of
0.5°C/minute. This step required 5 minutes of setup time. Also, at the vent/emission tab,
carbon dioxide stream was automatically selected.
9. Next, for the FERMENT-1 data, the vessel went through the fermentation for 24 hours.
Chilled water was used as the heat transfer agent to ensure the fermenter was at a final
temperature of 37°C. CO2 produced in the fermenter was emitted through the vent. Also,
air is selected as the broth aeration.
10. ‘R’ symbol was clicked to insert the Monod’s kinetic information using the given
stoichiometric data in the reaction tab. Other information were also added such as S1-
Term was selected, media as the substrate and rate reference component, Biomass as the
B-term, and 0.2 h-1 as the µmax value.
11. At the COOL-1 data, the fermenter was cooled to a final temperature of 5°C using Glycol
at a cooling rate of 0.5°C/minute. It required a setup time of 5 minutes.
12. Finally, at the TRANSFER-OUT-1 data, the fermented broth was transferred out from the
fermenter for further product separations for 5 hours of process time.

Results and Discussion

The unit procedure chosen is a batch vessel procedure in a fermenter. The fermenter itself is
labelled as P-1/FR-101 with a vessel volume of 13249.57L. In this simulation laboratory,
only one fermenter was used. The unit cost is $965,000. The major equipment specification
& FOB cost which includes both the fermenter and unlisted equipment is $1,206,000. The
Total Plant Direct Cost (TPDC) is $3,884,000 and the Total Plant Indirect Cost (TPIC) is
$2,330,000. The Total Plant Cost (TPC) which is the sum of TPDC & TPIC is $6,215,000.
The Contractor’s Fee & Contingency (CFC) is 932,000. The Direct Fixed Capital Cost (DFC)
which is the sum of TPC & CFC is $ 7,147,000. The annual labour cost for an operator is
$1,129,801 with an annual amount of 16,374 hours. The annual utilities cost is $37,561
which includes Standard Power, Steam and Glycol. The annual operating cost which includes
labour-dependent, facility-dependent, laboratory, and utilities is $2,684,000.

In this simulation laboratory, the annual operating time is calculated as 7,911.28 hours with a
recipe batch/cycle time of 33.81 hours. The number of batches per year is 234 batches. The
total mass of materials which includes air, biomass, media, and water is 20,194.75 kg/batch
or 4,725,571 kg/year. For the input, media & biomass is 500 kg/batch, air is 9694.75
kg/batch, water is 10,000 kg/batch. The total input is 20,194.75 kg/batch. For the output, the
carbon dioxide is 9,707.34 kg/batch, biomass is 176 kg/batch, impurity is 45 kg/batch, water
is 10,270 kg/batch, and the main bio-product monoclonal antibody (MAB) is 9 kg/batch. The
total output is 20,207.34 kg/batch. Inside the fermenter FR-101, the operation starts with the
operation CHARGE-1 at 0.00 hour and ends at 1.76 hour. Next, the operation CHARGE-2
starts at 1.76 hour and ends at 2.93 hour. Right after that, operation HEAT-1 starts at 2.93
hour and ends at 3.41 hour. Next, the actual fermentation process, operation FERMENT-1
starts at 3.41 hour and ends at 27.41 hour. After that, the operation COOL-1 starts at 27.41
hour and ends at 28.56 hour. Finally, the operation TRANSFER-OUT-1 occurs which starts
at 28.56 hour and ends at 33.81 hour. It also marks the end time for the fermenter.

For one batch cycle, the mass of bio-product monoclonal antibody (MAB) produced is 9.00
kg and the Carbon Dioxide vented is 314.90 kg. The media was converted 100% without
leaving any amount at the output stream. Meanwhile for biomass, it increases to a final mass
of 176.00 kg/batch at the output.

Below is the Materials & Streams Report:

1. OVERALL PROCESS DATA

Annual Operating Time :7,911.28h

Recipe Batch Time :33.81h
Recipe Cycle Time :33.81h
Number of Batches per Year :234.00
MP = Undefined

2.1 STARTING MATERIAL REQUIREMENTS (per Section)

Section Starting Active Amount Molar Mass Yield Gross Mass Yield
Material Product Needed (kg Yield (%) (%)
Sin/kg MP) (%)
Main (none) (none) Unknown Unknown Unknown Unknown
section
Sin = Section Starting Material, Aout = Section Active Product

2.2 BULK MATERIALS (Entire Process)

Material Mass per year (kg/year) Mass per batch (kg/batch)

Air 2,268,571.00 9,694.75
Biomass 11,700.00 50.00
Media 105,300.00 450.00
Water 2,340,000 10,000.00
TOTAL 4,725,571 20,194.75
2.3 BULK MATERIALS (per Material)

Procedure % Total Mass per year Mass per batch

(kg/year) (kg/batch)
Main Section (Main Branch)
P-1 100.00 2,268,571.00 9,694.75
TOTAL 100.00 2,268,571.00 9,694.75
Air

Procedure % Total Mass per year Mass per batch

(kg/year) (kg/batch)
Main Section (Main Branch)
P-1 100.00 11,700.00 50.00
TOTAL 100.00 11,700.00 50.00
Biomass

Media

Procedure % Total Mass per year Mass per batch

(kg/year) (kg/batch)
Main Section (Main Branch)
P-1 100.00 105,300.00 450.00
TOTAL 100.00

Procedure % Total Mass per year Mass per batch

(kg/year) (kg/batch)
Main Section (Main Branch)
P-1 100.00 2,340,000 10,000.00
TOTAL 100.00 2,340,000 10,000.00
Water

3. STREAM DETAILS

4. OVERALL COMPONENT BALANCE (kg/batch)

5. EQUIPMENT CONTENTS

Below is the Economic Evaluation Report:

Questions:

1. Calculate the amount of air to be supplied for the aerobic fermentation process.
For one batch process, the amount of air supplied for the aerobic fermentation process is
9694.74974 kg.
2. Determine the size of the fermenter necessary for this batch process.
The fermenter size must be at least 13249.57 L of volume.
3. What is the composition of vent & how much CO2 is produced in the batch process?
The composition of vent is as below:

Component Mass Composition (%)

Carbon dioxide 3.2440
Nitrogen 76.7118
 Oxygen 20.0443

For one batch process, the CO2 produced is 314.90150 kg.

4. What is the composition of the broth and how much MAB is produced per batch?
The composition of broth is as below:

Component Mass Composition (%)

Biomass 1.6762
Impurity 0.4286
MAB 0.0857
Water 97.8095

For one batch process, the MAB produced is 9 kg.

5. How long is the heating and cooling operation required?
Heating operation = 3.41 h – 2.93 h = 0.48 h
Cooling operation = 28.56 h – 27.41 h = 1.15 h
Heating operation required 0.48 h or 28.8 minutes, while cooling operation required 1.15
h or 65.4 minutes.
6. What is the total time required to complete one batch? If the total time per batch requires
36 hours, what is the maximum duration that the transfer out operation can prolong?
The total time required to complete one batch is 33.81 hours. If the total time per batch
requires 36 hours, the maximum duration that the transfer out operation can prolong is 36
h – 33.81 h = 2.19 hours.
7. Below is the Graph of Biomass Concentration (g/L) vs Time (h):
 Graph of Biomass Concentration (g/L) vs Time (h)
18

16

14
Biomass Concentration (g/L)

12

10

0
0 5 10 15 20 25

Time (h)

Concentration Biomass (g/L)

Graph of MAB Concentration (g/L) vs Time (h)

0.9

0.8

0.7
MAB Concentration (g/L)

0.6

0.5

0.4

0.3

0.2

0.1

0
0 5 10 15 20 25

Time (h)

Concentration MAB (g/L)

 Graph of Media Concentration (g/L) vs Time (h)
45

40

35
Media Concentration (g/L)

30

25

20

15

10

0
0 5 10 15 20 25

Time (h)

Concentration Media (g/L)