A Critical Study of the Design and

Optimization of Ideal Chemostats in

Series

Group 6

Ahmed Osman 300073024

Azfar Azfar 8224174

Fatma Yilmaz 300021786

Shivam Parekh 300018212

William Fitzgerald 300082868

A report submitted to Jordan Nhan

and Dr. Jean-Philippe St-Pierre in partial fulfillment of the course

Biochemical Engineering

CHG 4381

Department of Chemical and Biological Engineering

Faculty of Engineering
University of Ottawa

Submitted on December 6th, 2021

Abstract
This study focuses on the design and optimization of two-stage Chemostat in series to synthesize

800,000 kg of proprietary product (P) annually. The growth of two novel strains (A and B) of

aerobic bacteria were modeled under standard conditions of 20℃ and a near neutral pH. Pilot

experimental data were leveraged to derive key kinetic parameters for both strains. The growth of

the bacteria was assumed to be limited by a single substrate and so Monod kinetics were deemed

to be valid. The two-stage Chemostat was designed by using functional governing mass balance

on Biomass (X), Substrate (S), Precursor (Sb) and Product (P). The selection of bacteria strain and

optimization was achieved by analyzing productivity (DX) against Dilution rate (D), profit against

the dilution rate and the optimal Dilution rate was used to assess a more industrially practical

working volume. It was determined that strain A was a more process and economically feasible

choice. The profit when using strain was $3.4 million per year. The total cost which primarily

consisted of substrate and precursor cost amassed to about $1.84 million per year. The revenue

which consisted of selling the product (P) and residual biomass (X) was $6.35 million per year.

Process considerations were also considered in determining that A was the better choice. It was

determined that the working volumes were 7866.58 and 18333.3 L for the first and second reactor

respectively. This was more inline with typical industrial values than the working volumes

determined for strain B which was approximately 53 L when operating near optimal biomass

productivity for the single stage ideal chemostat. Additionally, the dilution rates for strain B were

0.12712 and 0.06 h-1 for the first and second tank respectively. The dilution rate for the first tank

was selected just below the optimal value in the biomass productivity against dilution rate plot in

order to gain high productivity but also avoid stability issues arising from potential washout. The

dilution rate for the second tank was considerably lower than that of the first tank as lower dilution

rates are required to achieve product and higher profitability. Future considerations could include

i
using the structured model in deriving the kinetic and Monod parameters to better determine

conditions suited towards using either strain or mitigate any limits inherent in the unstructured

model.

ii
Table of Content

Abstract ............................................................................................................................................ i
Table of Content............................................................................................................................. iii
List of Figures ..................................................................................................................................v
List of Tables .................................................................................................................................. vi
Nomenclature ................................................................................................................................ vii
1. Introduction................................................................................................................................. 1
1.1 Fundamentals of Growth....................................................................................................... 1
1.2 The Practical Applications of the Chemostat........................................................................ 2
1.3 Project Objectives ................................................................................................................. 2
2.0 Data Analysis and Interpretation.............................................................................................. 3
2.1 Growth Kinetics Attained from Experimental Data ............................................................. 3
2.2 Rate of Product Formation .................................................................................................... 6
2.3 Determining Monod Kinetic Parameters ............................................................................ 10
2.3.1 Determining Endogenous Metabolism Constant and Biomass Yield .......................... 11
2.3.2 Determining the Maximum Specific Growth Rate and Saturation Constant ............... 13
3.0 Bioreactor Design ................................................................................................................... 15
3.1 Ideal Chemostat .................................................................................................................. 15
3.2 Chemostat in Series............................................................................................................. 17
3.3 Optimization of the Chemostats.......................................................................................... 21
3.3.1 Chemostat 1 Strain A Optimization ............................................................................. 22
3.3.2 Chemostat 1 Strain B Optimization ............................................................................. 23
3.3.3 Chemostat 2 Optimization ............................................................................................... 24
3.4 Final Design ........................................................................................................................ 30
3.4.1 Separation Strategy .......................................................................................................... 31
3.4.2 Sterilization Techniques................................................................................................... 31
4. Conclusion ................................................................................................................................ 32
5. References ................................................................................................................................. 33
6. Appendix ................................................................................................................................... 34
6.1 Derivation of Governing Equations .................................................................................... 34
6.2 Sample Calculations............................................................................................................ 37
6.2.1 Sample Calculation of Experimental Data Analysis .................................................... 37

iii
 6.2.2 Sample Calculations of Double Stage Chemostat ....................................................... 42
6.3 Task Allocation ................................................................................................................... 47
6.4 Ethics Form ......................................................................................................................... 48

iv
List of Figures
Figure 1: Plot of the reciprocal of the growth rate against the reciprocal of the substate
concentration, in the absence of the precursor. ............................................................................... 4
Figure 2: Plot of the reciprocal of the growth rate against the reciprocal of the substate
concentration, in the presence of the precursor. ............................................................................. 5
Figure 3: Rate of product formation against precursor concentration for both strains A and B ... 7
Figure 4: Plot of ln(qP/qPMax-qP) against the logarithmic precursor concentration. ....................... 9
Figure 5: Relationship of the Inverse Biomass Yield vs. the Inverse Dilution Rate for strain A
using an initial substrate concentration of 450 g/L ....................................................................... 12
Figure 6: Relationship of the Inverse Biomass Yield vs. the Inverse Dilution Rate for strain B
using an initial substrate concentration of 490 g/L ....................................................................... 12
Figure 7: Relationship of the Inverse Specific Growth Rate vs. the Inverse Effluent Substrate
Concentration for strain A using an initial substrate concentration of 450 g/L ........................... 13
Figure 8: Relationship of the Inverse Specific Growth Rate vs. the Inverse Effluent Substrate
Concentration for Strain B using an initial substrate concentration of 490 g/L ........................... 14
Figure 9: Schematic diagram for the ideal chemostat 1 ............................................................... 15
Figure 10: Schematic diagram of a two-stage chemostat in series 1 ............................................ 19
Figure 11: Biomass productivity against dilution rate in the first chemostat using strain A. ...... 23
Figure 12: Biomass productivity against dilution rate in the first chemostat using strain B ....... 24
Figure 13: Plot of annual profits against the dilution rate for strain A. ...................................... 26
Figure 14: Annual profits against precursor flowrates. ............................................................... 27
Figure 15: Plot of the dilution rate against product productivity. ................................................ 29
Figure 16: Schematic of the process flow diagram...................................................................... 30

v
List of Tables
Table 1: Growth kinetics in the absence of Precursor ................................................................... 3
Table 2: Growth kinetics in the presence of Precursor .................................................................. 5
Table 3: Monod parameters of both strain A and B in the presence vs absence of a precursor .... 6
Table 4:Data analysis for the specific rate of production for Strain A .......................................... 8
Table 5: Specific rate of production for Strain B ........................................................................... 9
Table 6: Rate parameters for Allosteric enzyme kinetics for Strains A and B ............................ 10
Table 7: Effect of the precursor flowrate variation on product formation rate, liquid volume, and
profits. ........................................................................................................................................... 28
Table 8: Effect of product productive dilution rates variation on working volume and profits .. 29
Table 9: Optimised design parameters of the double stage ideal chemostat. .............................. 30
Table 10: Constants and kinetic parameters pertaining to strain A. ............................................ 42

vi
Nomenclature
Variable Symbol Name of Variable Units

𝑫 Dilution Rate h-1

𝑫𝒎𝒂𝒙 Maximum Dilution Rate h-1

𝑫𝒐𝒑𝒕 Optimal Dilution Rate h-1
𝑭𝑶 Inlet Flowrate to First Reactor L/h
𝑭𝟏 Effluent Flowrate of First
Reactor L/h
𝑭𝒃𝒐 Influent Precursor Flowrate L/h
second reactor
𝑭𝟐 Effluent Flowrate of Second L/h
Reactor
𝝁𝒈 Gross Specific Growth Rate h-1
𝝁𝒎 Maximum Specific Growth Rate h-1
𝝁𝒏𝒆𝒕 Net Specific Growth Rate h-1
𝑽𝟏 Volume of First Reactor L
𝑽𝟐 Volume of Second Reactor L
𝑲𝒔 Saturation Constant gS/L
𝑲′′
𝒎 Allosteric Constant gS/L
𝒌𝒅 Specific death rate (endogenous h-1
metabolism constant)
𝒎𝒑 Mass Flowrate of Product Kg/h
n Number of Allosteric Sites -
P Effluent Flowrate of Product gP/L
from second reactor
𝒒𝟎𝟐 Specific rate of Oxygen gP/gX h
Consumption
OUR Oxygen Uptake Ratio Kcal*g/h*L
𝑸𝑮𝑹 Rate of Heat Generation Kcal/h
𝑿𝟎 Initial Biomass Concentration gX/L
𝑿𝟏 Steady State Biomass gX/L
Concentration in First Reactor
𝑿𝟐 Steady State Biomass gX/L
Concentration in Second Reactor
𝐒𝟎 Initial Substrate Concentrate fed gS/L
to First Reactor
𝐒𝟏 Steady State Substrate gS/L
Concentration in first Reactor
𝐒𝟐 Steady State Substrate gS/L
Concentration in second Reactor
𝐒𝒃𝒐 Initial Precursor Concentration g/L
fed to Second Reactor
𝐒𝒃𝟏 Steady State Precursor g/L
Concentration in Second Reactor
𝒀𝑷/𝑺 Product Yield on Substrate 𝒈𝑷/𝒈𝑺
𝒀𝑷/𝑿 Product Yield on Biomass 𝒈𝑷/𝒈𝑺

vii
 𝑨𝒑 Apparent biomass yield on 𝒈𝑿/𝒈𝑺
𝒀𝑿/𝑺
Substrate
𝒀𝑴
𝑿/𝑺 Maximum Biomass yield on 𝒈𝑿/𝒈𝑺
Substrate

viii
1. Introduction
The use of continuous culture systems for cell growth has been very effective in large industrial

processes as a means to synthesize products that may not be economically feasible or possible to

produce from conventional processes. Continuous culture systems present several key advantages

over conventional methods. The first can be seen in the higher efficiencies typically associated

with the strengths of primary products. Additionally continuous systems operate at constant

environmental conditions which allows for a considerable degree of uniformity in the resulting

products. It is important to state that continuous systems do have some drawbacks. There is a

susceptibility to contamination during long-term operations which can lead to shut down. There is

also strain instability in long-term operations. Additionally, continuous processes are not usually

fit for the production of secondary metabolites and display limited flexibility in producing variety

of products.

1.1 Fundamentals of Growth

Cell growth, product formation as well as the consumption of various reactants can be represented

as follow :

∑ 𝑆 + 𝑋 → ∑ 𝑃 + 𝑛𝑋 (1.1)

In the equation above, the substrate (S) is the reagent and is consumed to produce the Products

(P) and an increase in the number of cells (𝑛𝑋) as compared to the initial cell count (X). As is the

case for conventional reactors, the rate of cell growth and rate of product formation are dictated

by a plethora of kinetic models. Whether a particular model is well suited to represent a growth

rate depends on the system. For the scope of tsis report, these reactions are represented as

unstructured models which means the simplest model that adequately describes the system is

selected. Such systems include Monod , Tessier, Blackman, Contois and Moser kinetics.

1
1.2 The Practical Applications of the Chemostat
The development of continuous cell growth systems has led to the advancement of novel processes

and products which are used in a wide breath of industries such as food, beverage, and

pharmaceuticals. Devices that are used for continuous cell growth can be categorized into three

major types: Chemostat, Turbidostat, and Plug Flow Reactor (PFR). Chemostats are used in

applications which necessitate constant chemical environments as well as constant feed and

effluent flowrates. Additionally, chemostats have only one limited nutrient substrate with all other

sources in excess. The presence of a sole limiting substrate allows for the models such as Monod

kinetics to be used. As such, the growth kinetics of cells are dictated by the limiting substrate. This

work will document the use of a two stage chemostat in series. Chemostats also serves as useful

research tools which can be used in designs of industrial processes when considering scaling up

factors. The is because the constant chemical conditions of the chemostat can allow steady state

date to be determined for a particular organism. This data can then be used to derive models which

relate to the organism’s metabolic and growth processes. Another application of chemostats can

be found in the enrichment of the mutant bacteria in cultures that show antibacterial resistance. An

example of industrial usage of multiple chemostats in series is in the synthetic production of

Ethanol. Due to the large-scale production of ethanol, the process has shown great economic

feasibility.

1.3 Project Objectives

This work concerns the design and optimization of two-stage chemostats. It relates to the

development of a recently developed of two proprietary strains of aerobic bacteria to produce a

sufficient number of products. The process occurs at a temperature of 20℃ and at a near neutral

pH since the bacteria is a Neutrophilic and Mesophilic strain. These bacteria depend on two sources

in the fermentation medium to facilitate cell growth and product formation. These include the

2
Substrate (S) which is used solely for cell growth and a precursor (Sb) which is internally

metabolized inside the bacteria to produce Product (P) via a series of metabolic reactions. The

Substrate and precursor are feedstock which are available at inlet concentrations of 500 g/L and

2000 g/L respectively and the required market share of the company in the production of product

is 800,000 kg/year. In order to develop the reactors, experimental data from pilot tests will be

gathered from an unknown reactor system in order to ascertain key kinetic parameters. Material

balances on cells (X), Substrate (S), Precursor (Sb) and Product will be derived to gather key kinetic

parameters to design the system. Optimization will be based on the strain and parameters which

yield the most favourable profit and viability for the application.

2.0 Data Analysis and Interpretation

2.1 Growth Kinetics Attained from Experimental Data

The total specific growth rates for both strain A and B were obtained over the course of multiple

experiments where the substrate concentration was varied. This was done both in the absence of a

precursor and in the presence of a precursor. As shown in Table 1, the concentration of the

substrate as well as the specific growth rate of both strain A and B are inverted for the case in

absence of the precursor.

Table 1: Growth kinetics in the absence of Precursor

Substrate Concentration Strain A ug Strain B ug 1/S 1/ugA 1/ugB

[g/L] [h-1] [h-1] [L/g] [h] [h]
0 0 0 - - -
0.956 0.3331 0.2551 1.046025 3.002101 3.920031
1.246 0.3965 0.3076 0.802568 2.522068 3.250975
1.688 0.4518 0.3597 0.592417 2.213369 2.780095
2.319 0.5127 0.4078 0.43122 1.950458 2.452182
4.942 0.6422 0.529 0.202347 1.557147 1.890359
9.781 0.7373 0.5951 0.102239 1.3563 1.68039
20.202 0.7829 0.639 0.0495 1.277302 1.564945

3
By plotting 1/μ g against 1/S for both strain A and B, the data can be linearized, which shows that

Monod kinetics apply for both strains. This can be observed in Figure 1.

4.5

3.5
y = 2.3301x + 1.4316
3 R² = 0.9985
1/ug (h)

2.5
Strain A
2
y = 1.7065x + 1.1961
1.5 Strain B
R² = 0.9987

1 Linear (Strain A)

0.5 Linear (Strain B)

0
0 0.2 0.4 0.6 0.8 1 1.2
1/S (L/g)

Figure 1: Plot of the reciprocal of the growth rate against the reciprocal of the substate concentration, in the
absence of the precursor.

Using the slope and y-intercept of the 1/μ g vs 1/S line, the μm and KS were found, as shown in

Equation (2.1) and (2.2).

1
μm = (2.1)
(𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)

𝐾𝑆 = 𝑢𝑚 ∗ (𝑆𝑙𝑜𝑝𝑒) (2.2)

These parameter values are necessary for Monod equation, which is a specific growth rate equation

that assumes substrate-limited growth. This is very important for future calculations and ultimately

the design of the chemostats.

The substrate concentration and specific growth rates for both strain A and B are also inverted for

the case in the presence of an inhibitor as seen in Table 2.

4
 Table 2: Growth kinetics in the presence of Precursor

Substrate Concentration Strain A ug Strain B ug 1/S 1/ugA 1/ugB

[g/L] [h-1] [h-1] [L/g] [h] [h]
0 0 0 - - -
0.956 0.011 0.0026 1.046025 90.90909 384.6154
1.246 0.0143 0.0031 0.802568 69.93007 322.5806
1.688 0.0192 0.0036 0.592417 52.08333 277.7778
2.319 0.0262 0.0041 0.43122 38.16794 243.9024
4.942 0.0539 0.0052 0.202347 18.55288 192.3077
9.781 0.1003 0.0059 0.102239 9.97009 169.4915
20.202 0.1838 0.0064 0.0495 5.440696 156.25

A plot of 1/μ g versus 1/S for both strain A and B is produce as seen in Figure 2. The linear nature

suggests than Monod kinetics still apply in the presence of the precursor.

450

400

350 y = 225.36x + 145.69

R² = 0.9993
300 Strain A
1/ug (h)

250 Strain B
200 Linear (Strain A)
150 Linear (Strain B)
100

50 y = 85.736x + 1.2063
R² = 1
0
0 0.2 0.4 0.6 0.8 1 1.2
1/S (L/g)

Figure 2: Plot of the reciprocal of the growth rate against the reciprocal of the substate concentration, in the
presence of the precursor.

This time in the presence of the precursor, the slope and y-intercept of the 1/μ g vs 1/S line are used

to solve μm and KS. These values are compared to the μm and KS values of both strain A and B in

the absence of a precursor as show in Table 3.

5
 Table 3: Monod parameters of both strain A and B in the presence vs absence of a precursor

Parameter Value in the Absence of Precursor Value in the Presence of Precursor

umA [h-1] 0.836050497 0.828981182
umB [h-1] 0.698519139 0.006863889
KSA [g S/L] 1.426720174 71.07353063
KSB [g S/L] 1.627619447 1.546846043

For strain A, the KS when the precursor is present is greater than when the precursor is absent and

the μm with the precursor present remains essentially the same as when the precursor is absent,

which indicates that the precursor is a competitive inhibitor. The KI can be found from the equation

below, which is the isolated KI value from the competitive inhibition equation for Ks,app. The solved

KI for strain A using competitive inhibition was 0.205 gS/L.

[𝐼]𝐾𝑠 (2.3)
𝐾𝐼 =
𝐾𝑠,𝑎𝑝𝑝 − 𝐾𝑠

For strain B, the KS remains essentially the constant when the precursor is present compared to

when it is absent and μm decreases significantly when the precursor is present, which indicates

non-competitive inhibition. The KI can be found from the equation below, which is the isolated KI

value from the non-competitive inhibition equation for μm,app. The solved KI for strain B using non-

competitive inhibition was 0.099 gS/L.

[𝐼]𝑢𝑚,𝑎𝑝𝑝 (2.3)
𝐾𝐼 =
𝑢𝑚 − 𝑢𝑚,𝑎𝑝𝑝

2.2 Rate of Product Formation

In this chemostat setup the rate of product formation was dependent on the concentration of

precursor. This precursor is utilized by both strains of the bacteria to metabolise product through

numerous intermediate steps, but the rate limiting step is based upon the precursor. Experiments

6
were performed using increasing precursor concentrations and initial product formation rates were

measured.

A plot of the Precursor concentration against the product formation of both strain A and B was

performed, and the following was observed in Figure 3 :

0.14

0.12
Product formation rate (gP/gX h)

0.1

0.08

0.06 Strain A

0.04 Strain B

0.02

0
0 5 10 15 20 25
Precursor Concentration Sb (g/L)
Figure 3: Rate of product formation against precursor concentration for both strains A and B

From the plot in Figure, it is observed that both strains have an S-shaped curve. For an S-shaped

curve at low precursor concentrations, the enzyme has an inactive conformation 2. When the

precursor concentration starts to increase, it binds to the enzyme and causes a conformational

change to the active site 2. After the binding of the first substrate the subsequent substrates can

more readily bind to the active site. This process is referred to as “cooperativity”, whereby the S-

shaped curve indicates co-operative binding 2. The conformational change of the active site leads

to an increased rate in substrate binding and reaction rates, but over a limited concentration of

substrate 2. When the active site is occupied, the curve starts to plateau as seen in Figure 3. The

7
product formation rate can be technically coined as a reaction rate; therefore, the product formation

rate of both strains can be followed through allosteric kinetics.

For both strains no substrate was present, allowing for growth rate to be negligible, in Table 4, are

the experimental data of strain A utilised in obtaining rate parameters.

Table 4:Data analysis for the specific rate of production for Strain A

Precursor
Concentration, Sb qp ln(qp/qpmax - qp) ln(Sb)
[g/L] [gP/gX·h]
0 0 - -
0.251 0.0003 -5.763099052 -1.38230234
0.512 0.0014 -4.211068837 -0.669430654
1.096 0.0063 -2.653688992 0.091667189
2.504 0.0259 -0.992822681 0.917889453
4.979 0.0579 0.423766272 1.605229068
10.121 0.0851 2.073583294 2.314612473
20.352 0.0958 - 3.013179187

Studies on the enzyme that metabolizes the precursor to the product, was found to structurally have

multiple active sites used for binding. Implying the enzyme is allosteric, this is observed if

precursor concentration is plotted against the initial product formation, the resulting plot gives an

allosteric pattern. Allosteric enzymes have a rate expression give by Equation (2.4) which can then

be linearized to form Equation (2.5).

𝑞𝑝𝑚𝑎𝑥 [𝑆]𝑛
𝑞𝑝 = " (2.4)
𝐾𝑚 + [𝑆]𝑛

𝑞𝑝 "
(2.5)
𝑙𝑛 ( ) = 𝑛𝑙𝑛(𝑆𝑏 ) + 𝑙𝑛(𝐾𝑚 )
𝑞𝑝𝑚𝑎𝑥 + 𝑞𝑝

By plotting ln(qp/qpmax - qp) against ln(Sb) allows for the determination of n and K”m. This plot

should yield a linear line for both strains as evidenced by Figure 4.

8
 3

2 y = 2.094x - 2.8582
R² = 0.9995
1

0
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
-1
ln(qP/qPMax-qP

y = 2.0459x - 2.8941
-2 R² = 0.9992
Strain A
-3
Strain B
-4

-5

-6

-7 ln(Sb)
Figure 4: Plot of ln(qP/qPMax-qP) against the logarithmic precursor concentration.

Utilizing the slope and y-intercept values which is shown in Equation (2.5) and (2.6).

𝑛 = 𝑆𝑙𝑜𝑝𝑒 (2.5)

"
𝐾𝑚 = 𝑒 −𝑦−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 (2.6)

Plotting and calculating rate parameters were down for the obtained experimental data of Strain B,

shown in Table 5. Same restrictions were utilised in obtaining the data as was in Strain A

experiments.

Table 5: Specific rate of production for Strain B

Precursor
Concentration, Sb qp ln(qp/qp_max - qp) ln(Sb)
[g/L] [gP/gX·h]
0 0 - -
0.251 0.0004 -5.653365989 -1.38230234
0.512 0.0016 -4.256498842 -0.669430654
1.096 0.0068 -2.762426972 0.091667189
2.504 0.0288 -1.090477438 0.917889453
4.979 0.0664 0.322414879 1.605229068
10.121 0.1004 1.96298741 2.314612473

9
 20.352 0.1145 - 3.013179187

Rate parameters obtained for both parameters were compared and used for the determination of

which strain to optimize within chemostat setup. Parameters are shown in Table 6.

Table 6: Rate parameters for Allosteric enzyme kinetics for Strains A and B

Parameter Strain A Strain B

n 2.093995077 2.045883449
K”m (g/L) 17.42996972 18.06763873

It was seen that the strain A had a higher cooperativity coefficient (n) then strain B, but they were
relatively similar. Which is expected since the enzyme should have same number of binding sites
in both experiments. K”m was determined to be higher in strain B, which indicates that the strain
A has a larger binding affinity.

2.3 Determining Monod Kinetic Parameters

The kinetics represented by the Monod model have subtracted limited growth and depend on the

substrate concentration (S), saturation constant (𝐾𝑠 ) and the maximum specific rate of growth (𝜇𝑔 ).

The maximum specific growth rate and saturation constant are determined from the experimental

data summarized in Table 2 above with an initial substrate concentration (S0) of 450 g/L and 490

g/L for strain A and B respectively. In using the Monod model there are several inherent

assumptions which are made. The most pertinent assumption was that the systems abide by

substrate-limited growth whereby there is only a single limiting substrate. Thus, all other nutrient

sources present in the chemostat are assumed to have negligible effect on the cell growth. As there

is substrate limited growth it can be surmised that there is a slower growth and that there is a

smaller cell population density. This is to ensure that any toxic compounds present in the reactor

will have limited hinderance on cell growth. Additionally, the Monod model can offer an analogy

to the Michaelis-Menten relations for single enzymes processes. This can be seen in Equation (2.7)

10
below where 𝜇𝑔 is the specific growth rate, 𝜇𝑚 is the maximum specific growth rate, 𝐾𝑠 is the

saturation constant and S is the substrate concentration.

𝜇𝑚 𝑆 (2.7)
𝜇𝑔 =
𝐾𝑠 + 𝑆

2.3.1 Determining Endogenous Metabolism Constant and Biomass Yield

The endogenous metabolism constant as well as the maximum biomass yield can be found by

creating a plotting the inverse of the apparent biomass yield (1/Yapp) vs. the hydraulic residence

time (1/D). The equation of the plot can be expressed as

1 𝑘𝑑 1 1 (2.8)
𝐴𝑝𝑝 = 𝑀 (𝐷 ) + 𝑀
𝑌𝑋/𝑆 𝑌𝑋/𝑆 𝑌𝑋/𝑆

𝐴𝑝𝑝
In order to calculate the apparent biomass yield (𝑌𝑋/𝑆 ) the raw values or the cell and substrate

concentrations as well as the initial substrate concentrations are taken. So, the apparent yield can

be expressed as:

𝐴𝑝𝑝 ∆𝑋 𝑋 (2.9)
𝑌𝑋/𝑆 = =
∆𝑆 𝑆0 − 𝑆

It is important to note that in the numerator (∆𝑋) is stated as 𝑋, due to a sterile medium assumption
1 1
(𝑋0=0). The relationship between 𝐴𝑝𝑝 against for strain A and B are seen below.
𝑌𝑋/𝑆 𝐷

11
 7

4 y = 0.1211x + 2.3628
1/Yx/s

R² = 1
3

0
0 5 10 15 20 25 30 35
1/D (h)

Figure 5: Relationship of the Inverse Biomass Yield vs. the Inverse Dilution Rate for strain A using an initial
substrate concentration of 450 g/L

40

35

30

25
1/Yx/s

20 y = 0.1008x + 3.2258
R² = 1
15

10

0
0 50 100 150 200 250 300 350
1/D (h)

Figure 6: Relationship of the Inverse Biomass Yield vs. the Inverse Dilution Rate for strain B using an initial
substrate concentration of 490 g/L

The y -intercept of the plots seen in Figure 5 and 6 above represent the inverse of the maximum

biomass yield. It was found by using linear regression that the y-intercepts for strain A and B were

2.3626 and 3.2258 respectively. As such, the maximum biomass yield was found to be 0.4233 for

strain A and 0.31 for strain B. Next the slope of the plots represents the maintenance coefficient.

For strain A it was found that the maintenance coefficient was 0.12211 gS/gX-h and for strain B

12
the maintenance coefficient was 0.1008 gS/gX-h. Finally, the product of the maintenance

coefficient and the maximum biomass yield is the endogenous metabolism constant (Kd). It was

calculated that the endogenous metabolism constant was 0.0517 h-1 and 0.03125 h-1.

2.3.2 Determining the Maximum Specific Growth Rate and Saturation Constant
The maximum biomass yield and the endogenous metabolism constant can be used to linearize the

Monod equation to ascertain the maximum specific growth rate and saturation constant. The model

can be extended from Equation 2.10 to account for non-negligible cell death during intracellular

substrate consumption and non-endogenous metabolism. This results in Equation (2.10)

𝜇𝑚 𝑆 (2.10)
𝜇𝑔 = 𝐷 + 𝑘𝑑
𝐾𝑠 + 𝑆

Equation (X) can then be rearranged to be expressed as:

1 1 𝑘𝑠 1 1 (2.11)
= =( ) +
𝜇𝑔 𝐷 + 𝑘𝑑 𝜇𝑚 𝑆 𝜇𝑚
The graphical representation of Equation (2.11) above can be seen for both strains below in Figure

7 and 8.

14
y = 25.522x + 5.5782
12 R² = 0.9754

10
1/(D+kd) (h)

0
0 0.05 0.1 0.15 0.2 0.25 0.3
1/S (L/g)

Figure 7: Relationship of the Inverse Specific Growth Rate vs. the Inverse Effluent Substrate Concentration for
strain A using an initial substrate concentration of 450 g/L

13
 35

30

25
1/(D+kd) (h)

20

y = 18.549x + 20.458
15
R² = 0.9999

10

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
1/S (L/g)

Figure 8: Relationship of the Inverse Specific Growth Rate vs. the Inverse Effluent Substrate Concentration for
Strain B using an initial substrate concentration of 490 g/L

The y-intercept of the line of best fit in the above figures represents the inverse of the maximum

specific growth rate. From Figure 7 it can be seen that the y-intercept is 5.5782 h and so the

maximum specific growth rate for strain A is 0.17927 h-1. Similarly, from Figure 8 the y-intercept

is 20.458 h and since strain B maximum apparent specific growth rate for strain B is 0.17927 h-1

due to non-competitive inhibition. The actual value for strain B’s maximum specific growth is

calculated by using the initial precursor concentration of 40 g/L and is found to be 19.751 h-1. The

product of the slope and the previously calculated maximum specific growth rate represents the

saturation constant (Ks). For strain A, this value is apparent due to competitive inhibition. The

apparent saturation constant for strain was found to be 4.575 g/L and by using the initial precursor

concentration of 200 g/L the true saturation constant is found to be 0.00468 g/L. Due to non-

competitive inhibition, the product of the slope and the maximum apparent specific growth rate

yields the true saturation constant. For strain B it was found to be 0.907 g S/L.

14
3.0 Bioreactor Design
3.1 Ideal Chemostat
For a continuous culture, an ideal chemostat is used, where there is a constant feed and effluent

flowrate. The chemostat used for cell growth and is limited by the substrate, S, introduced at an

initial concertation S0. The operation in the chemostat is assumed to have a sterile feed media and

to operate under steady state conditions. Steady state conditions can be assumed because cell

growth rate which depends on S will increase with substrate availability and as S is consumed,

growth rate decreases accordingly. The first reactor is used exclusively for cell growth therefore

having negligible product formation.

Figure 9: Schematic diagram for the ideal chemostat 1

A material balance around the chemostat shown in Figure 9 can be written in the general mass

balance form, “in - out + generation - consumption = accumulation”. The application of the

equation is presented in Equation (3.1), where F is the flowrate, X0 is initial biomass concertation

and VR is the liquid volume in the tank.

𝑑𝑋1
𝐹0 𝑋0 − 𝐹1 𝑋1 + 𝑉𝑅1 𝜇𝑔1 𝑋1 − 𝑉𝑅1 𝑘𝑑 𝑋1 = 𝑉𝑅1 (3.1)
𝑑𝑡

15
As a result of the assumption made previously, the feed being sterile results in negligible the initial

biomass concertation. Furthermore, the bioreactor is expected to operate predominantly under

steady state conditions, reducing the derivative term to zero, giving Equation (3.2).

−𝐹1 𝑋1 + 𝑉𝑅1 𝜇𝑔1 𝑋1 − 𝑉𝑅1 𝑘𝑑 𝑋1 = 0 (3.2)

The dilution rate, D, is defined as the inverse of the residence time in the bioreactor which is

defines as D=F/VR. Dividing all terms in Equation (3.2) by the culture volume and using the

dilution rate relationship, Equation (3.2) is rearranged, obtaining Equation (3.3) below, which then

can be further simplified to Equation (3.4).

𝑋1 (−𝐷1 + 𝜇𝑔1 − 𝑘𝑑 ) = 0 (3.3)

𝜇𝑔1 = 𝐷1 + 𝑘𝑑 (3.4)

The bioreactor also consists of substrate used for cells growth. A mass balance on substate

concentration presented in Equation (3.5) where cell death and growth are expressed in terms of

the maximum yield coefficient. The mass balance is this presented below, where S is the substate

concentration, YX/SM is the yield of biomass in terms of substrate, qp and YP/S are the rate of product

formation and yield coefficient of product, respectively.

𝑉𝑅1 (𝜇𝑔1 − 𝑘𝑑 )𝑋1 𝑉𝑅1 𝑞𝑃 𝑋1 𝑑𝑆1

𝐹𝑆0 − 𝐹1 𝑆1 − − = 𝑉𝑅1
𝑌𝑋 𝑀 𝑌𝑃 𝑑𝑡 (3.5)
𝑆 𝑆

The first reactor has negligible product formation since product is to be formed in the second

reactor. The purpose of the bioreactor system is to produce a desired quantity of a secondary

metabolite product, P 1. For production to occur, enough biomass must be transferred to the second

reactor. Growth and product formation may require different bioreactor conditions (pH,
16
temperature, etc.), therefore using separate reactors for cell growth and product formation will

allow for easier control of optimal conditions 1. Once again, the reactor is assumed to operate under

steady state, leading to no accumulation in the tank, yielding the following equation.

𝑉𝑅1 (𝜇𝑔1 − 𝑘𝑑 )𝑋1

𝐹𝑆0 − 𝐹1 𝑆1 − =0
𝑌𝑋 𝑀 (3.6)
𝑆

Dividing by the volume of the first reactor and using the dilution relation gives the following:
(𝜇𝑔1 − 𝑘𝑑 )𝑋1
𝐷1 (𝑆0 − 𝑆1 ) − =0
𝑌𝑋 𝑀 (3.7)
𝑆

Isolating for biomass concentration in the first tank yields the following equation, which will be

used in the design of the first bioreactor to determine cell concertation as a function of varying

dilution rate.

𝑌𝑋 𝑀 𝐷1
𝑋1 = × (𝑆0 − 𝑆1 ) × ( ) (3.8)
𝑆 𝐷1 + 𝑘𝑑

As growth is limited by a single substrate, the Monod equation for growth rate can be applied to

chemostat 1. Using the relationship given in Equation (3.4), the Monod equation, isolated for

substrate concentration is presented in Equation (3.9).

𝐾𝑠 (𝐷 + 𝑘𝑑 )
𝑆= (3.9)
𝜇𝑚 − 𝑘𝑑 − 𝐷

3.2 Chemostat in Series

In fermentation processes associated with the production of secondary metabolites (e.g.,

antibiotics), the growth stage and the product formation stage should be separated, since each stage

optimally operates at different conditions and environments, in that they operate at different

17
pressures, temperatures, dissolved oxygen levels, and they require varying nutrients and medium

compositions 1. Secondary products are most produced through growing cells, because often

product formation is repressed by growth 1. Moreover, the precursor and inducers might act as

inhibitors for cell growth rates, hence, it is recommended to operate a multistage system when

different optimal conditions are required for growth and product formation. Therefore, multistage

chemostat systems often result in varying products and cell physiology 1.

In a double stage chemostat an inlet to the first reactor is fed to the system and the effluent stream

of the first reactor is the inlet to the subsequent reactor. The second reactor in this case also has an

additional inlet stream, namely the precursor stream. The precursor is a starting molecule that will

be modified by cells through a series of chemical reactions with the aid of enzymes to assist in

producing a desired product. The Precursor serves as the starting point for the transformation of

biomass into products. Furthermore, the outlet of the second reactor is the final effluent stream.

The two control volumes of the system considered are the first tank which was primarily concerned

with growth formation, and the second tank which was concerned with product formation through

the precursor, these control volumes are highlighted in Figure 10. Similarly, to the previous

section’s material balance on the single stage ideal chemostat, mass balances were performed on

a double stage chemostat in series for the substrate, biomass, precursor, and product

concentrations. The material balances on the first tank are derived from the previous section, but

there is no product formation since the precursor is only fed to the second reactor.

18
 Figure 10: Schematic diagram of a two-stage chemostat in series 1

Similarly, to the first reactor, accumulation in the reactor was assumed to be negligible, in that the

reactor operates at steady state conditions (i.e., negligible derivative term in all mass balances).

With this assumption in mind, the biomass material balance can be shown as follows:

𝑑𝑋2 (3.10)
𝐹𝑋1 − (𝐹𝑏0 + 𝐹)𝑋2 + 𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 = 𝑉𝑅2 =0
𝑑𝑡

Where 𝑋1 is the effluent biomass concentration of the first tank, 𝑋2 is the effluent biomass

concentration of the second tank, 𝜇𝑔2 is the growth specific growth rate in the second tank which

depends on the substrate and precursor concentrations in the second tank. Furthermore, 𝑘𝑑 is a

constant term which accounts for death rate and endogenous metabolism, and its value depends on

the utilized strain the reactor. Moreover, Equation (3.10) can be simplified further by dividing the

equation with the working volume of the second reactor and the effluent biomass concentration,

𝑋2. The Equation (3.11), simplifies to the following:

𝐹 ∗ 𝑋1 (3.11)
𝐷2 − − 𝜇2 = 0
𝑉𝑅2 ∗ 𝑋2

19
The substrate concentration material balance is performed using the previous assumption of

steady-state and knowing that the substrate is only concerned with growth formation (i.e.,

negligible product formation), the substate material balance is given by the following:

𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝑑𝑆2 (3.12)

𝐹𝑆1 − (𝐹 + 𝐹𝑏0 ) ∗ 𝑆2 − = 𝑉𝑅2 =0
𝑌𝑋 𝑀 𝑑𝑡
𝑆

Similarly, the previous Equation (3.12) can be simplified further by dividing the equation with the

working volume of the second reactor and the dilution rate.

(𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝐹𝑆1 (3.13)

𝑆2 + − =0
𝑌𝑀 𝐷2 𝑉𝑅2
𝐷2 𝑆𝑋

The precursor material balance is done, analogously to the substrate material balance, however, it

is performed separately since the precursor is a different species than the substrate. Moreover, the

precursor is only concerned with product formation (i.e., negligible product rate), the precursor

material balance is given by the following:

𝑉𝑅2 𝑞𝑃 𝑋2 𝑑𝑆𝑏1 (3.14)

𝐹𝑏0 𝑆𝑏0 − (𝐹 + 𝐹𝑏0 ) ∗ 𝑆𝑏1 − = 𝑉𝑅2 =0
𝑌𝑃 𝑑𝑡
𝑆

The previous Equation (3.14) can be simplified by dividing by the working volume and the

dilution rate of the second reactor, as seen below:

𝑞𝑃 𝑋2 𝐹𝑏0 𝑆𝑏0 (3.15)

𝑆𝑏1 + − =0
𝑌𝑃 𝐷2 𝑉𝑅2
𝐷2 𝑆

The last mass balance pertains to the product concentration material balance, which is outlined

below:

𝑑𝑋2 (3.16)
𝐹𝑃0 − (𝐹𝑏0 + 𝐹)𝑃 + 𝑞𝑝 ∗ 𝑋2 ∗ 𝑉𝑅2 = 𝑉𝑅2 =0
𝑑𝑡

20
Dividing the equation by the working volume of the second reactor and isolating for the product

concentration, simplifies the Equation (3.16) to the following:

𝑞𝑝 𝑋2 (3.17)
𝑃=
𝐷2

3.3 Optimization of the Chemostats

Having ascertained the key kinetic growth parameters from various provided experimental data

pertaining to both cellular strains, the ideal chemostat in series was designed through the aid of the

governing mass balance equations of the system in combination with the obtained parameters. The

first chemostat reactor will mimic the ideal single-stage chemostat, however with no product

formation in the first chemostat since the precursor stream was added to the second stage, to

separate product formation stage. Hence, the modeling and optimization of the first chemostat is

relatively simple since there is no inhibition or product formation and the optimization will be

performed based on the biomass productivity which yields the maximum growth rate. However,

the second reactor requires more computation due to the presence of inhibition, interdependent

parameters, and more governing equations, which will be solved for and optimized through various

variations sets seen in Section 3.3.2.

In chemostat 1, substrate concentration was kept at 500 g/L in the solution feed into and the

flowrate was kept constant a 1000 L/h, throughout the optimization. Chemostat 1 is used to

increase biomass, for cells to produce product in chemostat 2 and be sold upon exiting in the

effluent of chemostat 2. Since no product is formed in chemostat 1, the cells are the only

constituent that could theoretically be sold in attempt to make a profit, however they are desired

to be used in chemostat 2 for product formation with the rest being sold as by-products. Biomass

production is the primary purpose of chemostat 1, thus by optimizing for biomass productivity, the

overall profit will eventually be greater. The cost of cooling and using electricity is negligible

21
compared to the cost of substrate inputted and no product is formed, which means that the net

profit of chemostat 1 is essentially the cost of the substate. However, since biomass is produced,

it is still possible for the overall system of chemostats to have a net positive profit.

It is desired to optimize the biomass productivity (DX) in chemostat 1. Since productivity is the

multiplication of the dilution rate and biomass, the corresponding biomass must be calculated for

every dilution rate. The biomass concentration is dependent on substrate concentration due to the

mass balance derivation, which meant that substrate concentration needed to also be calculated at

every dilatation rate. The specific growth rate for each dilution rate is calculated as well, as it is

necessary for the calculation of the oxygen uptake rate (OUR). The equation that is used to solve

biomass concentration can be seen below. This equation is attained from a mass balance on

substrate, after a mass balance on biomass was performed prior.

𝑌𝑋 𝑀 𝐷1
𝑋1 = × (𝑆0 − 𝑆1 ) × ( ) (3.18)
𝑆 𝐷1 + 𝑘𝑑

3.3.1 Chemostat 1 Strain A Optimization

For the optimization of chemostat 1 , the dilution rate was initially selected as a value close to 0

and was increased in equal increments. A maximum biomass productivity close to 19.05 g·L-1·h-1

, at a dilution rate of 0.127 h-1 was observed. Using this value as an initial estimate, the maximum

biomass productivity was solved while ensuring that substrate and cell concentration remained

within a constraint above a value of 0. It can be seen through Figure 11, the optimal biomass

productivity is observed at a dilution rate of 0.12718 h-1, yielding a biomass productivity of 19.06

g·L-1·h-1, with a culture volume of 7862.2 L. The residence time for the given model is close to 8

hours.

22
 19.059

Biomass Productivity DX (g/Lh)

19.058

19.057

19.056

19.055

19.054

19.053
0.1271 0.12712 0.12714 0.12716 0.12718 0.1272 0.12722 0.12724 0.12726 0.12728
Dilution Rate D (h-1)

Figure 11: Biomass productivity against dilution rate in the first chemostat using strain A.

3.3.2 Chemostat 1 Strain B Optimization

Biomass productivity is optimized by varying the dilution rate (D) using solver. Initially dilution

rates where manually put into the simulator to observe the effect on the biomass productivity and

it was found that there was a peak in DX when the D value was between 18 and 19 h-1. Solver was

then used to find that the optimum dilution rate was approximately 18.88 h-1. This was slightly

before the washout point, which occurred at a dilution rate of approximately 19.72 h-1.

Furthermore, operating slightly below the optimum dilution rate at 18.83 h-1 yielded a biomass

concentration of 163.129 g/L. A plot of DX versus D was produced using dilution rates that varied

from 18.8 to 18.95 h-1 to highlight the parabolic curve and determine a value for the dilution rate,

which is slightly below the optimum dilution rate, this can be seen in Figure 12.

23
 3072.4
3072.2

Biomass Productivity (g/Lh)

3072
3071.8
3071.6
3071.4
3071.2
3071
3070.8
18.78 18.8 18.82 18.84 18.86 18.88 18.9 18.92 18.94 18.96
Dilution Rate D (h-1)

Figure 12: Biomass productivity against dilution rate in the first chemostat using strain B

The operating dilution rate should be selected to be slightly below the optimal dilution rate to

maintain system stability. However, the volume of the tank when operating slightly below Dopt is

approximately 53 L, which is impractical and unfeasible for reactor of this scale because at the

given flowrate the residence time would be less than 3.2 minutes. This would not allow sufficient

time for the cells to multiply significantly meaning that there would be no point of using strain B

for chemostat 1. For this reason, strain B was not selected to be used in the chemostats in series.

3.3.3 Chemostat 2 Optimization

The second set of optimizations pertain to the second stage chemostat, which requires rigorously

more complex equations due to the presence of a precursor stream which serves to aid in product

formation and acts as an inhibitor on both strains. The optimizations for the second tank will be

based and centered primarily on profits, while also accounting for optimum product productivity

and ensuring the required production of the product is met or slightly exceeded.

In the second tank the effluent substrate, biomass, and precursor concentrations are unknown, and

they are indirectly interdependent on one another, however, using the previously derived mass

balances for the corresponding concentrations, initial guesses can be applied to the three

24
concentrations. Excel’s solver can be utilized to set the objective which is the summation of the

three equations to 0, by changing the three concentration variables, subject to the constraint that

each separate equation is also 0. The three equations are highlighted below:

𝐹 ∗ 𝑋1 (3.19)
𝐷2 − − 𝜇2 = 0
𝑉𝑅2 ∗ 𝑋2

(𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝐹𝑆1 (3.20)

𝑆2 + 𝑀 − =0
𝑌 𝐷2 𝑉𝑅2
𝐷2 𝑆𝑋

𝑞𝑃 𝑋2 𝐹𝑏0 𝑆𝑏0 (3.21)

𝑆𝑏1 + − =0
𝑌𝑃 𝐷2 𝑉𝑅2
𝐷2 𝑆

Since the second reactor is concerned with product formation, it is desired to operate at low dilution

rates, much below the optimum dilution rates required for the formation biomass 1. Therefore, the

dilution rate was varied at low values, with an initial guess of 100 L/h for the precursor flowrate.

To determine the optimum dilution rate based on profitability, a plot of the dilution rate against

the corresponding annual profits were conducted as seen in Figure 13. The dilution rate was varied

through trial and error and narrowed down until the annual profitability and product productivity

was seen to decrease and display a parabolic maximum curve. The determined range for the

dilution rate was varied from 0.02 to 0.37 h-1.

25
 3.45
3.4
3.35
Profit (106 $/Year)

3.3
3.25
3.2
3.15
3.1
3.05
3
0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.12
Dilution Rate (h-1)

Figure 13: Plot of annual profits against the dilution rate for strain A.

As seen from Figure 13, the dilution rate which yields maximum annual profitability of

approximately $ 3.4 million CAD, corresponds to the dilution rate of 0.06 h-1. Therefore, the

dilution rate of 0.06 h-1 was determined to be the most profitable dilution rate under which

subsequent optimizations for the precursor flowrate and product productivity would be performed.

However, the optimizations should be performed in conjunction with meeting the obligated

product production of 800,000 kg/year. The typical stream factor (i.e., the fraction which the plant

operates in a given year) of a plant is around 0.95, to account for scheduled maintenance,

downtime, and cleaning equipment 3. Therefore, the required product production accounting for a

stream factor of 0.95 is 842,105 kg/year, or analogously 96,130 g/hour of product production rate.

To assess an optimal precursor flowrate, since initially it was a guess, the dilution rate was kept at

the previously determined 0.06 h-1, which yields maximum profitability. The flowrate was varied

from 100 to 3600 L/h. Furthermore, the optimal precursor flowrate depends on the profitability it

yields and if it meets the required production or slightly exceeds it. It was determined that using a

26
precursor flow rate of 600 L/h and upwards leads to profit deficits. A plot of the annual profit

against the precursor flowrate is showcased in the following Figure 14.

7.5

6.5
Profit (106 $/year)

5.5

4.5

3.5

3
100 150 200 250 300 350 400
Precursor Flowrate (L/h)

Figure 14: Annual profits against precursor flowrates.

Varying the flowrate from 100 to 400 L/h, it can be seen from Figure 14 that at approximately

210 L/h yields the maximum annual profit of $ 6.9 million dollars. However, since the dilution

rate was kept constant throughout the precursor flowrate optimization set, an increased flowrate

leads to a larger reactor volume due to the proportionality between the two variables. This in turn

leads to a higher capital cost when the precursor flowrate is increased. Furthermore, increasing the

flowrate of the precursor more than required can lead to a surplus amount of annual product that

exceeds the company’s market share for the sale of the product, which means that the company

will have to hold more inventory to sell the excess product at a later fiscal stage. Therefore, the

company will have to compromise on operating with the profitable flowrate, to not drastically

exceed the required market share and to lower the capital costs associated with the reactor. The

following trend is highlighted in Table 7.

27
 Table 7: Effect of the precursor flowrate variation on product formation rate, liquid volume, and profits.

Working
volume of
Product formation second reactor
Precursor flowrate Fb0 (L/h) rate mp (kg/year) V2 (L) Profit (106 $/year)
100 1129638.56 18333.33 3.40
200 2081728.55 20000 6.88
300 2093812.61 21666.66667 5.21
400 2083429.69 23333.33333 3.41
500 2072878.10 25000 1.60

As seen in Table 7, as the precursor flowrate increases, the product formation rate, and working

volume increase. The profitability decreases after a flowrate of 200 L/h; therefore, it is desired to

operate slightly below the most profitable flowrate (i.e., 200 L/h) to reduce capital costs associated

with increasing reactor volumes and to reduce excess product formation. Hence, the precursor

flowrate will be operated at 100 L/h, however, this results in an excess 287,533.29 kg of product

annually since the required product formation, accounting for stream factor, is 842,105.26 kg of

product annually.

The final set of Optimization performed pertains to product productivity, whereby the dilution rate

was varied until a maximum productivity was reached, this can be observed in Figure 15, where

the dilution rate was varied from 0.24 to 0.37 h-1. It is observed that the optimal dilution rate for

productivity is 0.28 h-1 which yields a productivity of 13.07 (gP*L-1*h-1).

28
 13.0705

13.07
Product Productivity DP (gP/L*h)

13.0695

13.069

13.0685

13.068

13.0675

13.067

13.0665

13.066

13.0655
0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.36 0.38
Dilution Rate D (h-1)

Figure 15: Plot of the dilution rate against product productivity.

However, it was observed from the profitability analysis that operating at or near the maximum

product productivity leads to an unprofitable system with a loss deficit, due to the decreased

product concentrations at high dilution rates. The following trend is highlighted in Table 8

Table 8: Effect of product productive dilution rates variation on working volume and profits

Working volume of Product productivity

Dilution rate D2 (h ) -1
second reactor V2 (L) 5
Profit (10 $/year) (g P/L h)
0.24 4583.333 1.69 13.066
0.28 3928.571 -2.40 13.070
0.32 3437.5 -5.46 13.069
0.36 3055.556 -7.84 13.068
0.37 2972.973 -8.35 13.067

From Table 8, it is observed that operating near the maximum product productivity is not profitable

for most cases, due to the lower product concentration at these dilution rates, which means lower

sale of product and ultimately an unprofitable system. Even though the reactor volume is

significantly smaller compared to volumes corresponding to lower dilution rates, this will

ultimately not counteract the significant losses from unprofitability in the long run. Hence, it was

29
determined that optimizing the system based on product productivity, although it would lower

capital costs, it is unfeasible due to two factors: unprofitability and the system not reaching the

required market share product specifications outlined by the company.

3.4 Final Design

Figure 16: Schematic of the process flow diagram

The optimised design parameters of the system are displayed below in Table 9.

Table 9: Optimised design parameters of the double stage ideal chemostat.

Parameter Value
Strain A
D1 0.12712 (h-1)
F0/F1 1000 (L/h)
VR1 7866.56 (L)
X1 149.92 (g/L)
S1 1.80 (g/L)
D2 0.06 (h-1)
VR2 18,333.33 (L)
Fb0 100 (L/h)
S2 0.041 (g/L)
Sb1 4.20 (g/L)
X2 136.96 (g/L)
Profit 3.26*106 $/year

30
3.4.1 Separation Strategy
After the production of the desired product from the second stage chemostat, a separation process

must be implemented downstream to allow for further purification and recovery of the desired

product, this is essential since the following process is a commercial one 1. The product is assumed

to be an intracellular one. Hence, the cells are required to be disrupted to release the intracellular

product 1. Therefore, the main processing steps for separating intracellular products are cell

separation, which is performed through centrifugation in the cyclone, cell disruption through

agitation with an abrasive compound (i.e., Al2O3). Further product processing includes primary

isolation with precipitation and purification through a series of chromatography towers.

3.4.2 Sterilization Techniques

When considering the design of a chemostat, it is essential to account for possible contamination.

There are significant economic consequences of contamination in bioreactors. Contamination may

result in a loss of productivity, wash out, and considerably expensive methods for product

purification. To avoid such consequences, sterilization techniques may be used to ensure minimal

contamination. The sterilization techniques used must consider the operation requirements of the

chemostat system since a strain of aerobic bacteria is being used at pH near 7 and a constant room

temperature of 20°C. Since large volumes of air are being supplied for the growth of the aerobic

bacteria, a method of gas sterilization must be used. Sterilization of gasses can be achieved using

surface filters, which essentially remove particles larger than the membrane pore size through a

sieve effect. The filter must also be steam sterilized. For sterilization of process liquids, filter

sterilization, where microporous filters are used and may be preferred if the strain of bacteria is

heat sensitive. The filter itself must also be sterilized to prevent further contamination. In the case

where the bacterial strain may survive at high temperatures, continuous heat sterilization may be

31
used by steam injection or indirect heat exchangers. All equipment must also be sterilized. Process

vessel and equipment may be heat sterilized using close to high-pressure steam 4.

4. Conclusion
A successful design is depicted of a two-stage chemostat setup that can synthesize the desired
800,000 kg annually of product P. The design utilised two continuous flowing stirred tanks, with
the precursor Sb being added to the secondary chemostat and additional centrifuge and mixing tank
to aid in processing the product separation. With the availability of two novel strains of an aerobic
bacteria used as the biocatalyst, the depicted design utilised the optimal strain that was able to
maximise profit. By graphically analysing raw experimental data, the kinetic parameters of both
available strains were determined. Which were then used into determination of the optimal design
parameters of the two-stage chemostat.

Through economic analysis of determining if the overall setup was making high revenue while
minimising cost associated with the system. Varying optimization studies were done for various
parameters to maximize the overall profit of the system, allowing for the selection of the best value
associated with each parameter. The first chemostat of the two-stage system required for the
optimization of the initial dilution rate to obtain the best biomass productivity of the system, with
other parameters being a dependent on these values. The result determined based on the values
that made the most realistic sense were that of strain A, with a dilution rate of 0.12712 h-1, a culture
volume of 7866.56 L, a biomass concentration of 149.92 g/L, substrate concentration of 1.80 g/L.
Values of substrate concentration and flow rate were kept constant at 500 g/L and 1000 L/h
respectively. Strain B optimized results had a working volume of 53 L, which resulted in a 3.2
minutes of residence time and was determined to be unrealistic. The second chemostat had three
independent variables to be optimized, effluent substrate, biomass, and precursor concentrations.
Optimization through maximizing profits and required amount of product the resulting parameters
achieved were a dilution rate of 0.06 h-1, a culture volume of 18,333.33 L, a precursor flow rate of
100 L/h, an effluent substrate concentration of 0.041 g/L, a precursor concentration of 4.2 g/L and
a biomass concentration of 136.96 g/L. The combination of both optimizations resulted in the
system having a theoretical profit of $3,264,416 annually.

32
5. References

[1] Shuler, M. L. (2018). Bioprocess Engineering: Basic concepts (Second). Pearson.

[2] The Biology Project Department of Biochemistry and Molecular Biophysics University of
Arizona. (2004, October). Energy, enzymes, and catalysis problem set. Retrieved
December 6, 2021, from
http://www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/14t.
html.

[3] Turton, R., Bailie, R. C., & Whiting, W. B. (2015). Analysis, synthesis, and design of
Chemical Processes. Pearson Education International.

[4] Kuila, Arindam, and Vinay Sharma. “Sterilization Techniques Used in Fermentation
Processes.” Principles and Applications of Fermentation Technology, Scrivener
Publishing, Beverly, 2018.

33
6. Appendix
6.1 Derivation of Governing Equations
First Tank
a. Material Balance on Cell Concentration (X)
𝑑𝑋1 (6.1)
𝐹0 𝑋0 − 𝐹1 𝑋1 + 𝑉𝑅1 𝜇𝑔1 𝑋1 − 𝑉𝑅1 𝑘𝑑 𝑋1 = 𝑉𝑅1
𝑑𝑡

Assuming steady state conditions and sterile feed, Equation (1) reduces to the following:
−𝐹1 𝑋1 + 𝑉𝑅1 𝜇𝑔1 𝑋1 − 𝑉𝑅1 𝑘𝑑 𝑋1 = 0 (6.2)

Dividing by the volume of the first reactor, gives the following:

𝑋1 (−𝐷1 + 𝜇𝑔1 − 𝑘𝑑 ) = 0 (6.3)

Where the following equation can be simplified into:

𝜇𝑔1 = 𝐷1 + 𝑘𝑑 (6.4)

The gross specific growth rate follows Monod kinetics with no inhibition, since there is no
precursor stream in the first tank, applying the Monod equation leads to the following:
𝜇𝑀 𝑆1 (6.5)
= 𝐷1 + 𝑘𝑑
𝐾𝑆 + 𝑆1

Isolating for the effluent substrate concentration, yields the following equation:

𝐾𝑠 ∗ (𝐷1 + 𝑘𝑑 ) (6.6)
𝑆1 =
(𝑢𝑚 − 𝑘𝑑 − 𝐷1 )

b. Material Balance on Substrate Concentration (S)

𝑉𝑅1 (𝜇𝑔1 − 𝑘𝑑 )𝑋1 𝑉𝑅1 𝑞𝑃 𝑋1 𝑑𝑆1 (6.7)
𝐹𝑆0 − 𝐹1 𝑆1 − − = 𝑉𝑅1
𝑌𝑋 𝑀 𝑌𝑃 𝑑𝑡
𝑆 𝑆

Assuming steady state conditions and negligible product formation in the first tank since the
substrate S is only used for growth, the equation is reduced to the following:
𝑉𝑅1 (𝜇𝑔1 − 𝑘𝑑 )𝑋1 (6.8)
𝐹𝑆0 − 𝐹1 𝑆1 − 𝑀 =0
𝑌𝑋
𝑆

Dividing by the volume of the first reactor, gives the following:

(𝜇𝑔1 − 𝑘𝑑 )𝑋1 (6.9)
𝐷1 (𝑆0 − 𝑆1 ) − =0
𝑌𝑋 𝑀
𝑆

The biomass concentration term, X1 can be isolated as follows:

34
 𝑌𝑋 𝑀 𝐷1 (6.10)
𝑋1 = ∗ (𝑆0 − 𝑆1 ) ∗ ( )
𝑆 𝐷1 + 𝑘𝑑

Second Tank
a. Material Balance on Cell Concentration (X)
𝑑𝑋2 (6.11)
𝐹1 𝑋1 − (𝐹𝑏0 + 𝐹1 )𝑋2 + 𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 = 𝑉𝑅2
𝑑𝑡

Assuming steady state conditions, Equation (8) reduces to the following:

𝐹1 𝑋1 − (𝐹𝑏0 + 𝐹1 )𝑋2 + 𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 = 0 (6.12)

Dividing by the volume of the second reactor, gives the following:

𝐹1 (6.13)
𝑋 − 𝐷2 𝑋2 + (𝜇𝑔2 − 𝑘𝑑 )𝑋2 = 0
𝑉𝑅2 1

Dividing by the cell concentration X2, reduces the previous equation into:
𝐹1 ∗ 𝑋1 (6.14)
− 𝐷2 + (𝜇𝑔2 − 𝑘𝑑 ) = 0
𝑉𝑅2 ∗ 𝑋2

The previous equation can be written in the form, (first equation used in Solver):
𝐹1 ∗ 𝑋1 (6.15)
𝐷2 − − 𝜇2 = 0
𝑉𝑅2 ∗ 𝑋2

b. Material Balance on Precursor Concentration (Sb)

𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝑉𝑅2 𝑞𝑃 𝑋2 𝑑𝑆𝑏1 (6.16)
𝐹𝑏0 𝑆𝑏0 − (𝐹 + 𝐹𝑏0 ) ∗ 𝑆𝑏1 − − = 𝑉𝑅2
𝑌𝑋 𝑀 𝑌𝑃 𝑑𝑡
𝑆 𝑆

Assuming steady state conditions and negligible growth since the precursor is only used for
product formation, the equation is reduced to the following:
𝑉𝑅2 𝑞𝑃 𝑋2 (6.17)
𝐹𝑏0 𝑆𝑏0 − (𝐹1 + 𝐹𝑏0 ) ∗ 𝑆𝑏1 − =0
𝑌𝑃
𝑆

Dividing by the volume of the second reactor, gives the following:

𝐹𝑏0 𝑆𝑏0 𝑞𝑃 𝑋2 (6.18)
− 𝐷2 ∗ 𝑆𝑏1 − =0
𝑉𝑅2 𝑌𝑃
𝑆

Dividing by the dilution rate, D2, gives the following, (second equation used in Solver):
𝑞𝑃 𝑋2 𝐹𝑏0 𝑆𝑏0 (6.19)
𝑆𝑏1 + − =0
𝑌𝑃 𝐷2 𝑉𝑅2
𝐷2 𝑆

c. Material Balance on Substrate Concentration (S)

35
 𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝑉𝑅2 𝑞𝑃 𝑋2 𝑑𝑆2 (6.20)
𝐹1 𝑆1 − (𝐹1 + 𝐹𝑏0 ) ∗ 𝑆2 − − = 𝑉𝑅2
𝑌𝑋 𝑀 𝑌𝑃 𝑑𝑡
𝑆 𝑆

Assuming steady state conditions and negligible product formation since the substrate is only
used for growth, the equation is reduced to the following:
𝑉𝑅2 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 (6.21)
𝐹1 𝑆1 − (𝐹1 + 𝐹𝑏0 ) ∗ 𝑆2 − 𝑀 =0
𝑌𝑋
𝑆

Dividing by the volume of the second reactor, gives the following:

𝐹1 𝑆1 (𝜇𝑔2 − 𝑘𝑑 )𝑋2 (6.22)
− 𝐷2 𝑆2 − =0
𝑉𝑅2 𝑌𝑋 𝑀
𝑆

The previous equation can be simplified to the following, (third equation used in Solver):
(𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝐹1 𝑆1 (6.23)
𝑆2 + − = 0
𝑌𝑀 𝐷2 𝑉𝑅2
𝐷2 𝑆𝑋

d. Material Balance on Product Concentration (P)

𝑑𝑋2 (6.24)
𝐹1 𝑃0 − (𝐹𝑏0 + 𝐹1 )𝑃 + 𝑞𝑝 ∗ 𝑋2 ∗ 𝑉𝑅2 = 𝑉𝑅2
𝑑𝑡

Assuming steady state conditions and no initial product, the equation is reduced to the following:
(𝐹𝑏0 + 𝐹1 )𝑃 + 𝑞𝑝 ∗ 𝑋2 ∗ 𝑉𝑅2 = 0 (6.25)

Dividing by the volume of the second reactor, gives the following:

𝐷2 𝑃 + 𝑞𝑝 𝑋2 = 0 (6.26)

The previous equation can be simplified to the following:

𝑞𝑝 𝑋2 (6.27)
𝑃=
𝐷2

36
6.2 Sample Calculations
6.2.1 Sample Calculation of Experimental Data Analysis
Determining Growth Kinetic Parameters

As outlined in Section 2.1 of the report, the growth kinetic parameters were plotting the inverse

of the gross specific growth (𝜇𝑔 ) against the inverse substrate concentration in absence and

presence of the inhibitor.

Absence of Inhibitor

The maximum specific growth rate can be related to the double reciprocal plot (1/ug vs 1/S) as:

1
𝜇𝑚𝐴 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

The y-intercept of the line of the best for strain A was found to be 1.1961 h and so:

1
𝜇𝑚𝐴 =
1.1961

𝝁𝒎𝑨 = 𝟎. 𝟖𝟑𝟔 𝐡−𝟏

The saturation constant can be related to the double reciprocal plot as:

𝐾𝑠𝐴 = 𝜇𝑚𝐴 × 𝑠𝑙𝑜𝑝𝑒

The slope for the line of the best for strain A was found to be 1.7065 and so:

𝐾𝑠𝐴 = 0.8361 × 1.705

𝒈𝑺
𝑲𝒔𝑨 = 𝟏. 𝟒𝟐𝟕
𝑳

Prescence of Inhibitor

The maximum specific growth rate can be related to the double reciprocal plot (1/ug vs 1/S) in the

presence of inhibitor as:

1
𝜇𝑚𝐴 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

The y-intercept of the line of the best for strain A was found to be 1.2063 h and so:

37
 1
𝜇𝑚𝐴 =
1.2063

𝝁𝒎𝑨 = 𝟎. 𝟖𝟐𝟗𝐡−𝟏

The saturation constant can be related to the double reciprocal plot as:

𝐾𝑠𝐴 = 𝜇𝑚𝐴 × 𝑠𝑙𝑜𝑝𝑒

The slope for the line of the best for strain A in the presence of precursor inhibitor was found to

be 85.736 and so:

𝐾𝑠𝐴 = 0.829 × 85.736

𝒈𝑺
𝑲𝒔𝑨 = 𝟕𝟏. 𝟎𝟕𝟒
𝑳

Determining Inhibitor Saturation Constant

It was determined that strain A undergoes competitive inhibition as the Um is virtually the same

and KsA is increase when precursor inhibitor is added. As such the KI for strain A can be expressed

as:

𝐾𝑠𝐴 (𝑤𝑖𝑡ℎ 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛)

𝐾𝐼𝐴 = 𝑆𝑏 ( − 1)−1
𝐾𝑠𝐴 (𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛)

71.074
𝐾𝐼𝐴 = 10( − 1)−1
1.427
𝒈𝑺
𝑲𝑰𝑨 = 𝟎. 𝟐𝟎𝟒
𝑳

Determining the Allosteric Kinetic Parameters

As outlined in Section 2.2 of the report, the allosteric kinetic parameters were found by plotting

ln(qp/qpmax - qp) against ln(Sb). Please refer to report Table 4 for the calculated values. The slope

of this plot (Figure 4) represents the number of allosteric sites or the degree of allostery (n). The

slope for the strain as was found as:

𝐧 = 𝐬𝐥𝐨𝐩𝐞 = 𝟐. 𝟎𝟗𝟒

38
Additionally, the allosteric saturation constant (𝑘′′𝑚 ) can be related to the plot as:

𝑘′′𝑚 = 𝑒−𝑦−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 = 𝑒−𝑛

The y-intercept of the line of best fit for strain A was found to be -2.8582. Thus, for strain A the

allosteric saturation constant (𝑘′′𝑚 ) was calculated as:

𝑘′′𝑚 = 𝑒−(−2.8582)

𝒌′′𝒎 = 𝟏𝟕. 𝟒𝟑

Determining the Monod Kinetic Parameters

As outlined in Section 2.3 of the report the kinetic parameters for the Monod model were found by

plotting a number of relationships related to the experimental data found in Table 5 and 6.

Maintenance Coefficient

By generating the reciprocal plot of 1/𝑌𝑋/𝑆 against 1/D the maintenance coefficient (mA) and

endogenous metabolism constant can be found.

The slope of the double reciprocal plot represents the maintenance coefficient such that:

𝑚𝑠𝐴 = 𝑠𝑙𝑜𝑝𝑒

The slope of the line of best fit was found to be 0.12211 as so:

𝒈𝒔 ∙ 𝒉−𝟏
𝒎𝒔𝑨 = 𝟎. 𝟏𝟐𝟐𝟏𝟏
(𝒈𝑿)

Maximum Biomass Yield

The y-intercept of the double reciprocal plot represents the inverse of the maximum yield of

biomass on substrate (𝑌𝑚

𝑥/𝑠 ) such that:

1
𝑌𝑚
𝑥/𝑠 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

It was found from the line of best fit for strain A that the y-intercept was 2.3626 such that:

39
 1
𝑌𝑚
𝑥/𝑠 =
2.3623
𝒈𝑿
𝒀𝒎
𝒙/𝒔 = 𝟎. 𝟒𝟐𝟑
𝒈𝑺

Endogenous Metabolism Constant

The product of the maintenance coefficient and the maximum biomass yield represents the

endogenous metabolism constant (𝑘𝑑𝐴 ):

𝑘𝑑𝐴 = 𝑌𝑚
𝑥/𝑠 × 𝑚𝑠𝐴

𝑘𝑑𝐴 = 0.423 × 0.12211

𝒌𝒅𝑨 = 𝟎. 𝟎𝟓𝟏𝟕 𝒉−𝟏

1 1
By plotting the double reciprocal plot of 𝐷+𝑘 vs 𝑆 (Figure 7) several important parameters can
𝑑

be found:

Maximum Specific Growth Rate

The maximum specific growth rate (𝜇𝑚𝐴 ) can be related to the double reciprocal plot as the inverse

of the y-intercept such that:

1
𝜇𝑚𝐴 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

The y-intercept of the line of best fit for strain A was found to be 5.5782 so that:

1
𝜇𝑚𝐴 =
5.5782

𝝁𝒎𝑨 = 𝟎. 𝟏𝟕𝟗 𝐡−𝟏

Apparent Saturation constant

𝑎𝑝𝑝
The Apparent Saturation constant (𝐾𝑆𝐴 ) can be related to the maximum specific growth rate (𝜇𝑚𝐴 )

and slope of that plot such that:

𝑎𝑝𝑝
𝐾𝑆𝐴 = 𝑆𝑙𝑜𝑝𝑒 × 𝜇𝑚𝐴

40
The slope of the line of best fit for strain A was found to be 25.522 such that:
𝑎𝑝𝑝
𝐾𝑆𝐴 = 25.522 × 0.17927

𝒂𝒑𝒑 𝒈𝑺
𝑲𝑺𝑨 = 𝟒. 𝟓𝟕𝟓
𝑳
Actual Saturation constant

Due to Strain A being subjected to Competitive inhibition by precursor (Sb), the saturation

constant found in the previous step merely represents the apparent value. Thus, the true saturation
𝑎𝑝𝑝
constant (𝐾𝑆𝐴 ) must relate 𝐾𝑆𝐴 the initial precursor concentration (Sbo of 200 g/L).
𝑎𝑝𝑝
𝐾𝑆𝐴
𝐾𝑆𝐴 =
1 + 𝑆𝑏0 (𝐾𝐼𝐴 )

4.575
𝐾𝑆𝐴 =
1 + 100(0.2049)

𝒈𝑺
𝑲𝑺𝑨 = 𝟎. 𝟎𝟎𝟒𝟔𝟖
𝑳

41
6.2.2 Sample Calculations of Double Stage Chemostat
Sample calculations for the two-stage ideal chemostat were performed using strain A as the

biocatalyst bacteria, with an initial substrate concentration of 500 g/L, a feed inlet of 1000 L/h,

and a dilution rate of 0.12712 h-1 for the first reactor. Furthermore, a precursor concentration of

2000 g/L, precursor flowrate of 100 L/h, and a dilution rate of 0.06 h-1 were used for the second

reactor. The kinetic parameters for strain A are showcased in Table 10.

Table 10: Constants and kinetic parameters pertaining to strain A.

Strain Strain A
YMx/s 0.423263
kd (h-1) 0.051685
Ks (g S/L) 0.004681
KI (h-1) 0.204851
µm (h-1) 0.179269
n (allosteric sites) 2.093995
k''m (g/L) 17.42997
YX/O2 1.1
YP/Sb 0.66
qPMax (g P/ g X* h) 0.0958

Chemostat 1
Effluent substrate concentration
𝐾𝑠𝐴 ∗ (𝐷1 + 𝑘𝑑𝐴 )
𝑆1 =
(𝑢𝑚𝐴 − 𝑘𝑑𝐴 − 𝐷1 )

𝑔
0.00468 ( 𝐿 ) ∗ (0.12712 (ℎ−1 ) + 0.05168(ℎ−1 )) 𝑔
𝑆1 = = 1.801 ( )
(0.17927(ℎ−1 ) − 0.05168(ℎ−1 ) − 0.12712 (ℎ−1 )) 𝐿
Specific growth rate
𝑢𝑚𝐴 ∗ 𝑆1
𝑢𝑔1 =
(𝐾𝑠𝐴 + 𝑆1 )

𝑔
0.17927(ℎ−1 ) ∗ 1.801 ( 𝐿 )
−1
𝑢𝑔1 = 𝑔 𝑔 = 0.1788 (ℎ )
(0.00468 ( 𝐿 ) + 1.801 ( 𝐿 ))

Net Specific growth rate

𝑢1 = 𝑢𝑔1 − 𝑘𝑑𝐴

𝑢1 = 0.1788 (ℎ−1 ) − 0.05168 (ℎ−1 ) = 0.12712 (ℎ−1 )

42
Effluent biomass concentration
𝑌𝑋 𝑀 𝐷1
𝑋1 = ∗ (𝑆0 − 𝑆1 ) ∗ ( )
𝑆 𝐷1 + 𝑘𝑑𝐴

𝑔 𝑔 0.12712 (ℎ−1 ) 𝑔
𝑋1 = 0.423 ∗ (500 ( ) − 1.801 ( )) ∗ ( ) = 149.92 ( )
𝐿 𝐿 0.12712 (ℎ−1 ) + 0.05168(ℎ−1 ) 𝐿
Biomass Productivity
𝐷𝑋 = 𝐷1 ∗ 𝑋1

𝑔 𝑔
𝐷𝑋 = 0.12712 (ℎ−1 ) ∗ 149.92 ( ) = 19.06 ( )
𝐿 𝐿∗ℎ
Volume of Reactor
𝐹0
𝑉𝑅1 =
𝐷1

1000 (𝐿/ℎ)
𝑉𝑅1 = = 7861.64 (𝐿)
0.12712 (ℎ−1 )
Cost of Substrate
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 𝐹0 ∗ 𝑆0 ∗ 𝐶𝑜𝑠𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑝𝑒𝑟 𝑘𝑔

𝐿 𝑔 $ 1 𝑘𝑔 $
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 1000 ( ) ∗ 500 ( ) ∗ 0.25 ( ) ∗ ( ) = 125 ( )
ℎ 𝐿 𝑘𝑔 1000 𝑔 ℎ
Power Required
𝑃𝑜𝑤𝑒𝑟 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 = 𝑉𝑅1 ∗ 𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑓𝑜𝑟 𝑎𝑔𝑖𝑡𝑎𝑡𝑖𝑜𝑛

𝐾𝑊 1 𝑚3
𝑃𝑜𝑤𝑒𝑟 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 = 7861.64 (𝐿) ∗ 5 ( ) ∗ ( ) = 39.33 (𝐾𝑊)
𝑚3 1000 𝐿
Cost of Electricity
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 ∗ 𝐸𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑐𝑜𝑠𝑡 𝑝𝑒𝑟 𝐾𝑊ℎ

$ $
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 39.33 (𝐾𝑊) ∗ 0.14 ( ) = 5.50 ( )
𝐾𝑊ℎ ℎ𝑟
Total Costs
𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑠𝑡𝑠 = 𝐶𝑜𝑠𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 + 𝐶𝑜𝑠𝑡 𝑜𝑓 𝐸𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦

$ $ 𝑑𝑎𝑦𝑠 ℎ $
𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑠𝑡𝑠 = (125 ( ) + 5.50 ( )) ∗ 365 ( ) ∗ 24 ( ) = 1.095 ∗ 106 ( )
ℎ ℎ 𝑦𝑒𝑎𝑟 𝑑𝑎𝑦 𝑦𝑒𝑎𝑟
Profit
𝑃𝑟𝑜𝑓𝑖𝑡𝑠 = 𝑅𝑒𝑣𝑒𝑛𝑢𝑒𝑠 − 𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑠𝑡𝑠

$
𝑃𝑟𝑜𝑓𝑖𝑡𝑠 = 𝑅𝑒𝑣𝑒𝑛𝑢𝑒𝑠 − 𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑠𝑡𝑠 = −1.095 ∗ 106 ( )
𝑦𝑒𝑎𝑟

43
Chemostat 2

In the second stage chemostat, there are three unknowns, namely S2, Sb1, X2, and there are three

equations pertaining to the mass balance of the precursor, substrate, and biomass material balances

derived in the governing equations. Hence, initial guesses can be taken and applied to S2 and X2

from S1 and X1. For Sb1 an arbitrary guess of Sb0 can be applied. Utilizing Excel’s Solver feature

the three previously derived equations can be summed and set to 0 on the objective tab by changing

S2, Sb1, X2 subject to the constraint that each individual equation is equal to 0.

𝐹 ∗ 𝑋1
𝐷2 − − 𝜇2 = 0
𝑉𝑅2 ∗ 𝑋2

(𝜇𝑔2 − 𝑘𝑑 )𝑋2 𝐹𝑆1

𝑆2 + 𝑀 − =0
𝑌 𝐷2 𝑉𝑅2
𝐷2 𝑆𝑋

𝑞𝑃 𝑋2 𝐹𝑏0 𝑆𝑏0
𝑆𝑏1 + − =0
𝑌
𝐷2 𝑆𝑃 𝐷2 𝑉𝑅2

The obtained values from solver for S2, Sb1, X2 are 0.041 g/L, 4.20 g/L, and 135.96 respectively.

Specific growth rate

Since the precursor, is present in the second tank, the specific growth rate follows competitive

inhibition kinetics.

𝑢𝑚𝐴 ∗ 𝑆2
𝑢𝑔2 =
𝑆
(𝐾𝑠𝐴 (1 + 𝐾𝑏1 ) + 𝑆2 ))
𝐼𝐴

𝑔
0.17927 (ℎ−1 ) ∗ 0.041 ( )
𝑢𝑔2 = 𝐿 = 0.052 (ℎ−1 )
𝑔
4.20 ( )
𝑔
(0.00468 ( ) ∗ (1 + 𝐿 ) + 0.041 (𝑔)))
𝐿 𝑔 𝐿
0.205 ( )
𝐿
Net specific growth rate
𝑢1 = 𝑢𝑔2 − 𝑘𝑑𝐴

𝑢1 = 0.052 (ℎ−1 ) − 0.05168 (ℎ−1 ) = 2.96 ∗ 10−4 (ℎ−1 )

Product formation rate
44
Product formation rate follows Allosteric kinetics.
𝑞𝑝_𝑚𝑎𝑥 [𝑆𝑏1 ]𝑛
𝑞𝑝 = "
𝐾𝑚 + [𝑆𝑏1 ]𝑛

𝑔 2.094
0.0958 (ℎ−1 ) ∗ (4.20 ( 𝐿 )) 𝑔𝑃
𝑞𝑝 = = 0.0514 ( )
g 𝑔 2.094 𝑔𝑋ℎ
17.43 (L) + (4.20 ( 𝐿 ))
Product concentration
𝑞𝑃 𝑋2
𝑃=
𝐷2

𝑔𝑃 𝑔
0.0514 ( ) ∗ 135.96 𝑔
𝑔𝑋ℎ 𝐿
𝑃= −1
= 117.231 ( )
0.06 (h ) 𝐿

Volume of reactor
𝐹0 + 𝐹𝑏0
𝑉𝑅2 =
𝐷2

𝐿 𝐿
1000 ( ) + 100 ( )
𝑉𝑅2 = ℎ ℎ = 18333.3 (𝐿)
0.06 (h−1 )

Biomass productivity
𝐷𝑋 = 𝐷2 ∗ 𝑋2

𝑔 𝑔
𝐷𝑋 = 0.06 (ℎ−1 ) ∗ 135.96 ( ) = 8.22 ( )
𝐿 𝐿∗ℎ
Product productivity
𝐷𝑃 = 𝐷2 ∗ 𝑃

𝑔 𝑔
𝐷𝑋 = 0.06 (ℎ−1 ) ∗ 117.231 ( ) = 7.034 ( )
𝐿 𝐿∗ℎ
Cost of precursor
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑝𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 𝐹𝑏0 ∗ 𝑆𝑏0 ∗ 𝐶𝑜𝑠𝑡 𝑜𝑓 𝑝𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟 𝑝𝑒𝑟 𝑘𝑔

𝐿 𝑔 $ 1 𝑘𝑔 $
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑝𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 100 ( ) ∗ 2000 ( ) ∗ 0.99 ( ) ∗ ( ) = 198 ( )
ℎ 𝐿 𝑘𝑔 1000 𝑔 ℎ
Power required
𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 = 𝑉𝑅2 ∗ 𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑓𝑜𝑟 𝑎𝑔𝑖𝑡𝑎𝑡𝑖𝑜𝑛

𝐾𝑊 1 𝑚3
𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 = 18333.3 (𝐿) ∗ 5 ( ) ∗ ( ) = 91.67 (𝐾𝑊)
𝑚3 1000 𝐿
Cost of electricity

45
 𝐶𝑜𝑠𝑡 𝑜𝑓 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 𝑃𝑜𝑤𝑒𝑟 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 ∗ 𝐸𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑐𝑜𝑠𝑡 𝑝𝑒𝑟 𝐾𝑊ℎ

$ $
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 91.67 (𝐾𝑊) ∗ 0.14 ( ) = 12.83 ( )
𝐾𝑊ℎ ℎ𝑟

Total costs
𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑠𝑡𝑠 = 𝐶𝑜𝑠𝑡𝑠 𝑜𝑓 𝑅𝑒𝑎𝑐𝑡𝑜𝑟 1 + 𝐶𝑜𝑠𝑡 𝑜𝑓 𝐸𝑙𝑒𝑐𝑡𝑟𝑖𝑐𝑖𝑡𝑦 + 𝐶𝑜𝑠𝑡 𝑜𝑓 𝑃𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟

$ $ $ 𝑑𝑎𝑦𝑠 ℎ
𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑠𝑡𝑠 = 1.095 ∗ 106 ( ) + (12.83 ( ) + 198 ( )) ∗ 365 ( ) ∗ 24 ( )
𝑦𝑒𝑎𝑟 ℎ𝑟 ℎ 𝑦𝑒𝑎𝑟 𝑑𝑎𝑦
$
= 2.942 ∗ 106 ( )
𝑦𝑒𝑎𝑟

Sale of biomass
𝑆𝑎𝑙𝑒 𝑜𝑓 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 𝐹2 ∗ 𝑋2 ∗ 𝑆𝑎𝑙𝑒 𝑜𝑓 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑝𝑒𝑟 𝑘𝑔

𝐿 𝑔 $ 1 𝑘𝑔
𝐶𝑜𝑠𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑝𝑒𝑟 ℎ𝑜𝑢𝑟 = 1100 ( ) ∗ 135.96 ( ) ∗ 0.11 ( ) ∗ ( )
ℎ 𝐿 𝑘𝑔 1000 𝑔
$
= 16.45 ( )
ℎ
Sale of product
𝑆𝑎𝑙𝑒 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 = 𝐹2 ∗ 𝑃 ∗ 𝑆𝑎𝑙𝑒 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑝𝑒𝑟 𝑘𝑔

𝐿 𝑔 $ 1 𝑘𝑔 $
𝑆𝑎𝑙𝑒 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 = 1100 ( ) ∗ 117.231 ( ) ∗ 5.49 ( ) ∗ ( ) = 707.96 ( )
ℎ 𝐿 𝑘𝑔 1000 𝑔 ℎ

Total revenues
𝑇𝑜𝑡𝑎𝑙 𝑟𝑒𝑣𝑒𝑛𝑢𝑒𝑠 = 𝑆𝑎𝑙𝑒 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 + 𝑆𝑎𝑙𝑒 𝑜𝑓 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

$ $ 𝑑𝑎𝑦𝑠 ℎ $
𝑇𝑜𝑡𝑎𝑙 𝑟𝑒𝑣𝑒𝑛𝑢𝑒𝑠 = (707.96 ( ) + 16.45 ( )) ∗ 365 ( ) ∗ 24 ( ) = 6.35 ∗ 106 ( )
ℎ ℎ 𝑦𝑒𝑎𝑟 𝑑𝑎𝑦 𝑦𝑒𝑎𝑟

Profits
𝑃𝑟𝑜𝑓𝑖𝑡𝑠 = 𝑇𝑜𝑡𝑎𝑙 𝑟𝑒𝑣𝑒𝑛𝑢𝑒𝑠 − 𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑠𝑡𝑠

$ $ $
𝑃𝑟𝑜𝑓𝑖𝑡𝑠 = 6.35 ∗ 106 ( ) − 2.942 ∗ 106 ( ) = 3.408 ∗ 106 ( )
𝑦𝑒𝑎𝑟 𝑦𝑒𝑎𝑟 𝑦𝑒𝑎𝑟

46
6.3 Task Allocation

Report Lead Co- Editor(s)

Author(s) Author(s)
1. Abstract SP
2. Nomenclature SP
3. Introduction SP
4. Data Analysis and Interpretation SP (25%),
FY (25%),
WF
(25%),
AA (25%)
5. Bioreactor Design, Optimization and AO
Discussion (33.3%),
FY
(33.3%),
WF
(33.3%)
6. Conclusions AA
7. Derivation of Governing Equations AO
8. Sample Calculations AO(50%),
SP (50%)

Simulator Lead Contributor(s) Co-

Contributor(s)
1. Data Analysis – Table 1 & 2 AO (50%), FY (50%)
2. Data Analysis – Table 3 AO
3. Data Analysis – Table 4 & 5 AO (50%), SP (50%)
4. Economic Analysis – Table 7 & 8 AO
5. Simulator – Base Case SP (20%), FY
(20%), WF (20%),
AA (20%), AO (20%)
6. Simulator – Optimization AO, FY

Legend:
• AO = Ahmed Osman
• AA = Azfar Azfar
• FY = Fatma Yilmaz
• SP = Shivam Parekh
• WF = William Fitzgerald

47
6.4 Ethics Form

48