FACULTY OF ENGINEERING AND BUILT ENVIRONMENT

3rd YEAR INTEGRATED PROJECT

(CHEMICAL ENGINEERING
PROGRAMME) SEMESTER II 2020/2021

TITLE: PRODUCTION OF DAPTOMYCIN FROM STREPTOMYCES

ROSEOSPORUS

COURSE CODES: KKKR3583, KKKR3733, KKKR3723, KKKR3713

LECTURERS: KKKR3583: PROF. MADYA TS. EN. NOORHISHAM TAN

KOFLI
TS. DR. AHMAD RAZI OTHMAN
KKKR3733: PROF. MADYA IR. DR. HASSIMI ABU HASAN
PROF. MADYA TS. EN. NOORHISHAM TAN
KOFLI
DR. DARMAN NORDIN
DR. MUHAMMAD ZULHAZIMAN MAT SALLEH
KKKR3723: PROF. MADYA IR. DR. SHUHAIDA HARUN
IR. DR. TEOW YEIT HAAN
KKKR3713: PROF. MADYA DR. NORLIZA ABD. RAHMAN
DR. JARINAH MOHD ALI

GROUP: KK3

SUBMISSION 27TH MAY 2021

DATE:
MEMBERS: ATHIRAH HASNI BINTI ANUAR (A168096)
NURSYAHIRAH BINTI ZUBIR (A167179)
HARIZ HAZMI BIN SAIFUL BASAR (A167599)
NOR ZULAIKHA AFIQAH ABD RASID (A177266)
SALSABILA THANESYA PUTRI (A184717)
 CHAPTER IV

MASS AND ENERGY BALANCE

4.1 MASS BALANCE

A mass balance is used to compare the inputs and outputs of the process. The mass that
enters a system must equal to either the mass that is flow out the system or accumulate
within the system. The feed materials in this process are dextrin, glucose, ammonium
nitrate and oxygen gas to produce daptomycin as the product and carbon dioxide and
water as waste. Six major unit operations are involved in this process including seed
fermenter, fed batch bio-fermenter, multichamber bowl centrifuges, HPLC and lastly
freeze-dryer. Error! Reference source not found. shows the molecular weight of the
substances.

Table 4.1 The molecular weight of the substances

MW
Substance
(kg/kmole)
Dextrin, X C18H32O16 504.40
Glucose, G C6H12O6 180
Ammonium Nitrate, A NH4NO3 80
Oxygen, O O2 16.0
Carbon dioxide, C CO2 44.01
Daptomycin, D C72H101N17O26 1620.7
Water, W H2O 18.01

(Source: PubChem)
STOICHIOMETRY EQUATION

The chemical equation of the production of daptomycin is as shown below:

C6H12O6 + α1O2 + α2C18H32O16 + α3NH4NO3 → α4C72H101N17O26 + α5CO2 + α6H2O

In production of daptomycin, there are 7 species and 4 elements.

Degree of freedom = 7 - 4 = 3

Balance of elements:
C: -18 α2 + 72 α4 + α5 = 6 ……… (1)
N: -α3 + 17 α4 = 0 ……… (2)
H: -32 α2 - 4 α3 + 101 α4 + 2 α6 = 12 ……… (3)
O: -2 α1 - 16 α2 - 3 α3 + 26 α4 + 2 α5 + α6 = 6 ……… (4)

Yield of yeast on glucose, YY/G = 0.5065 (I. Son Ng et. al 2013)

The stoichiometry was determined by using MATLAB :
α1 = 17.5165
α2 = 1.7898
α3 = 8.6105
α4 = 0.5065
α5 = 28.0263
α6 = 26.2788

Hence, given the chemical equation of:

C6H12O6 + 17.5165O2 + 1.7898C18H32O16 + 8.6105NH4NO3 → 0.5065C72H101N17O26

+ 28.0263CO2 + 26.2788H2O
4.1.1 Mass Balance on Fed Batch Bio-fermenter (F-102)

Fed-batch fermentation was performed at 30°C with tank pressure of 0.05 MPa.
Dissolved oxygen was maintained above 30% of the saturated dissolved oxygen
concentration by automatically adjusting the agitation speed above 450 rpm. The
bacteria were fermented in the medium in seed fermenter (F-101) prior reacted in the
fed batch bio-fermenter (F-102). All the assumptions, rate of reaction and other
calculations are provided below.

F1 = 0.045 kg/h F6 = 0.292 kg/h

F1G = 0.045 kg/h F6O = 0.016 kg/h
F6C = 0.276 kg/h
F2 = 0.227 kg/h
F2X = 0.227 kg/h

F3 = 0.173 kg/h
F3A = 0.173 kg/h

F4 = 0.045 kg/h F7 = 0.339 kg/h

F4SR = 0.045 kg/h F7G = 0.00498 kg/h
F7X = 0.025 kg/h
F7A = 0.019 kg/h
F7D = 0.184 kg/h
F5 = 0.141 kg/h F7W = 0.106 kg/h
F5O = 0.141 kg/h

Figure 4.1 Fed Batch Bio-fermenter (F-102)

Assumptions: 1. The system is a steady-state process.

2. All feed streams and draw streams are pumped into and out of the
system continuously.
Material Balance of Dextrin

N2X = α2N1G
N2X = (1.7898) (2.514×10-4) = 4.500 ×10-4 kmole/h
F2X = 4.500 ×10-4 × 504.40 = 0.227 kg/h
N2X = N7X – α2rV
4.500 ×10-4 = N7X – (-1.7898) (2.238 × 10−9) (105)
N7X = 4.950 ×10-5 kmole/h
F7X = (4.950 ×10-5) (504.40) = 0.025 kg/h

Material Balance of Ammonium Nitrate

N3A = α3N1G
N3A = (8.6105) (2.514×10-4) = 2.165×10-3 kmole/h
F3A = 2.165×10-3 × 80 = 0.173 kg/h
N3A = N7A – α3rV
2.165×10-3 = N7A – (-8.6105) (2.238 × 10−9) (105)
N7A = 2.381 × 10−4 kmole/h
F7A = (2.381 × 10−4) (80) = 0.019 kg/h

Material Balance of Daptomycin

0 = N7D – α4rV
0 = N7D – (0.5065) (2.238 × 10−9) (105)
N7D = 1.133 × 10−4 kmole/h
F7D = (1.133 × 10−4) (1620.7) = 0.184 kg/h

Material Balance of Carbon Dioxide

0 = N6C – α5rV
0 = N6C – (28.0263) (2.238 × 10−9) (105)
N6C = 6.271×10−3 kmole/h
F6C = (6.271×10−3 ) (44.01) = 0.276 kg/h
Material Balance of Water

0 = N7W – α6rV
0 = N7W – (26.2788) (2.238 × 10−9) (105)
N7W = 5.880 × 10−3 kmole/h
F7W = (5.880 × 10−3) (18.01) = 0.106 kg/h

Table 4.2 Inlet stream table of fed-batch bio-fermenter (F-102)

Mass flow rate of Inlet Inlet Inlet Inlet Inlet
components (kg/h) stream 1 stream 2 stream 3 stream 4 stream 5
Glucose, G 0.045 0 0 0 0
Dextrin, X 0 0.227 0 0 0
Ammonium Nitrate, A 0 0 0.173 0 0
S. Roseosporus 0 0 0 0.045 0
Daptomcin, D 0 0 0 0 0
Oxygen, O 0 0 0 0 0.141
Carbon Dioxide, C 0 0 0 0 0
Water, W 0 0 0 0 0
Total 0.045 0.227 0.173 0.045 0.141

Table 4.3 Outlet stream table of fed-batch bio-fermenter (F-102)

Mass flow rate of components
Outlet stream 6 Outlet stream 7
(kg/h)
Glucose, G 0 0.00498
Dextrin, X 0 0.025
Ammonium Nitrate, A 0 0.019
Daptomcin, D 0 0.184
Oxygen, O 0.016 0
Carbon Dioxide, C 0.276 0
Water, W 0 0.106
Total 0.292 0.339

F1 + F2 + F3 + F4 + F5 = F6 + F7
0.045 + 0.227 + 0.173 + 0.045 + 0.141 = 0.292 + 0.339
0.631 = 0.631
(Balanced)
4.1.2 Mass Balance on Multichamber Bowl Centrifuge 1 (S-101)

Supernatant liquor is obtained after the fermentation broth is centrifuged by using the
multi chamber bowl centrifuge. The daptomycin enters an organic phase after the
supernatant liquor is extracted by an organic solvent, sodium acetate-acetic acid buffer
solution is used to perform re-extraction to reach a water phase to obtain strip-extraction
liquid.
F9 = 0.192 kg/h
F9G = 0.0002 kg/h
F9X = 0.0023 kg/h
F8 = 0.339 kg/h
F9A = 0.00027 kg/h
F8G = 0.0048 kg/h
F9D = 0.184 kg/h
F8X = 0.0025 kg/h
F9W = 0.0053 kg/h
F8A = 0.019 kg/h
F8D = 0.184 kg/h
F8W = 0.106 kg/h F10 = 0.147 kg/h
F10G = 0.00478 kg/h
F10X = 0.023 kg/h
F10A = 0.019 kg/h
F10D = 0.00015 kg/h
F10W = 0.101 kg/h

Figure 4.2 Multichamber Bowl Centrifuge 1 (S-101)

Table 4.4 Stream table of multichamber bowl centrifuge 1 (S-101)

Mass flow rate of components
Inlet stream 8 Outlet stream 9 Outlet stream 10
(kg/h)
Glucose, G 0.00498 0.00020 0.00478
Dextrin, X 0.025 0.0023 0.023
Ammonium Nitrate, A 0.019 0.00027 0.019
Daptomcin, D 0.184 0.184 0.00015
Oxygen, O 0 0 0
Carbon Dioxide, C 0 0 0
Water, W 0.106 0.0053 0.101
Total 0.339 0.192 0.147

F8 = F9 + F10
0.339 = 0.192 + 0.147
0.339 = 0.339
(Balanced)
4.1.3 Mass Balance on Multichamber Bowl Centrifuge 2 (S-102)

Supernatant liquor is obtained after the fermentation broth is centrifuged by using the
multi chamber bowl centrifuge. The daptomycin enters an organic phase after the
supernatant liquor is extracted by an organic solvent, sodium acetate-acetic acid buffer
solution is used to perform re-extraction to reach a water phase to obtain strip-extraction
liquid.

F9 = 0.192 kg/h
F9G = 0.0002 kg/h
F11 = 0.00767 kg/h
F9X = 0.0023 kg/h
F11G = 0.0002 kg/h
F9A = 0.00027 kg/h
F11X = 0.0023 kg/h
F9D = 0.184 kg/h
F11A = 0.000078 kg/h
F9W = 0.0053 kg/h
F11W = 0.0051 kg/h

F11 = 0.184 kg/h

F11A = 0.00019 kg/h
F11D = 0.18353 kg/h
F11W = 0.00025 kg/h

Figure 4.3 Multichamber Bowl Centrifuge 2 (S-102)

Table 4.6 Stream table of multichamber bowl centrifuge 2 (S-102)

Mass flow rate of components
Inlet stream 9 Outlet stream 11 Outlet stream 12
(kg/h)
Glucose, G 0.00020 0.00020 0
Dextrin, X 0.0023 0.0023 0
Ammonium Nitrate, A 0.00027 0.000078 0.00019
Daptomcin, D 0.184 0 0.18353
Oxygen, O 0 0 0
Carbon Dioxide, C 0 0 0
Water, W 0.0053 0.0051 0.00025
Total 0.192 0.00767 0.184

F9 = F11 + F12
0.192 = 0.00767 + 0.184
0.192 = 0.192
(Balanced)
4.1.4 Mass Balance on HPLC (C-101)

The strip-extraction liquid is purified through hydrophobic interaction chromatography

to extract daptomycin from the fermentation broth. Organic solvent extraction and resin
chromatography technology is applied comprehensively, and the daptomycin with
purity more than 85% can be separated and purified from the fermentation broth by
using the method (Luo Bo Te et.al 2013).

F12 = 0.184 kg/h F15 = 0.028 kg/h

F12A = 0.00019 kg/h F15A = 0.000054 kg/h
F12D = 0.18353 kg/h F15D = 0.028 kg/h
F12W = 0.00025 kg/h F15W = 0.000123 kg/h

F15 = 0.1563 kg/h

F14A = 0.000137 kg/h
F14D = 0.156 kg/h
F14W = 0.000132 kg/h

Figure 4.4 HPLC (C-101)

Table 4.6 Stream table of HPLC (C-101)

Mass flow rate of components
Inlet stream 12 Outlet stream 14 Outlet stream 15
(kg/h)
Glucose, G 0 0 0
Dextrin, X 0 0 0
Ammonium Nitrate, A 0.00019 0.000054 0.000137
Daptomcin, D 0.18353 0.028 0.156
Oxygen, O 0 0 0
Carbon Dioxide, C 0 0 0
Water, W 0.00025 0.000123 0.000132
Total 0.184 0.028 0.1563

F12 = F14 + F15

0.184 = 0.028 + 0.1563
0.184 = 0.184
(Balanced)
4.1.5 Mass Balance on Freeze-dryer (D-101)

The liquid daptomycin can be converted to the solid pharmaceutical composition

through lyophilization process by using freeze-dryer. Lyophilization works by
freezing the material, then reducing the pressure and adding heat at 40°C to allow the
frozen water in the material to sublimate (Tepzz & Connor 2018).

F17 = 0.003 kg/h

F17A = 0.00027 kg/h
F17W = 0.000132 kg/h

F15 = 0.1563 kg/h

F15A = 0.000137 kg/h
F18 = 0.156 kg/h
F15D = 0.156 kg/h
F18D = 0.156 kg/h
F15W = 0.000132 kg/h

Figure 4.5 Freeze-dryer (D-101)

Table 4.7 Stream table of freeze-dryer (D-101)

Mass flow rate of components
Inlet stream 15 Outlet stream 17 Outlet stream 18
(kg/h)
Glucose, G 0 0 0
Dextrin, X 0 0 0
Ammonium Nitrate, A 0.000137 0.00027 0
Daptomcin, D 0.156 0 0.156
Oxygen, O 0 0 0
Carbon Dioxide, C 0 0 0
Water, W 0.000132 0.000132 0
Total 0.1563 0.0003 0.156

F15 = F17 + F18

0.1563 = 0.0003 + 0.156
0.1563 = 0.1563
(Balanced)
4.1.6 Comparison of material balance of fermenter between manual calculation
and using Superpro

Table 4.8 Comparison between manual and Superpro calculation of material balance
Manual Superpro
Percentage
Substance Calculation Calculation
Error
(g/h) (g/h)
Glucose 4.98 44.6936 88%
Dextrin 25.00 226.2627 88%
Ammonium Nitrate 19.00 172.4371 88%
Daptomycin 184.00 0.6704 89%
Oxygen 16.00 0.0513 89%
Carbon Dioxide 276.00 0.9129 88%
Water 106 0.3866 89%
Total 631 445.4173 42%

Figure 4.6 Calculation of mass balance using superpro

Percentage Error

!"## %&'()"*+ '% #,-+)-)'.!"## %&'()"*+ '% /"0,"& 1"&1,&"*2'0

=! !× 100%
!"## %&'()"*+ '% #,-+)-)'

From the above comparison, the error obtained was quite higher since there is no
Streptomyces roseosporus in inlet stream at Superpro while it was included in the
manual calculation. It can be concluded that the error occurred due to lack of
information in Superpro.
4.2 ENERGY BALANCE

The concept of energy conservation as expressed by an energy balance equation is

central to chemical engineering calculations. Similar to mass balance studied
previously, a balance on energy is crucial to solving many problems (Levicky, 2018).
The First Law of Thermodynamics is a statement of energy conservation. Energy cannot
be created nor destroyed, but it can be converted from one state to another.
The calculation of energy balance is to determine the heat change of the unit system.
Bio-fermenter and centrifuge are the unit operation selected and the energy balance for
those units are calculated for this project.

Table 4.9 Heat of formation of the substances

ΔHRx
Substance
(kJ/kmole)
Dextrin, X C18H32O16 -2 256 350

Glucose, G C6H12O6 -1 260 000

Ammonium Nitrate, A NH4NO3 -350 260

Oxygen, O O2 0

Carbon dioxide, C CO2 -393 500

Daptomycin, D C72H101N17O26 -1 865 610

Water, W H2O -285 800

(Source: STLCC, ACTG, Chemeo)

Table 4.10 Specific heat of capacity of the substances

CP
Substance
(kJ/mole.K)
Dextrin, X C18H32O16 1481.53

Glucose, G C6H12O6 115.00

Ammonium Nitrate, A NH4NO3 139.27

Oxygen, O O2 29.38

Carbon dioxide, C CO2 36.94

Daptomycin, D C72H101N17O26 1688.16

Water, W H2O 75.38

(Source: GSU, Chemeo)

4.2.1 Energy Balance on Fed Batch Bio-fermenter (F-102)

Figure below shows the fed batch bio-fermenter. The input components enter the bio-
fermenter and undergo fed batch fermentation at 30ºC and tank pressure of 0.05 MPa.
The system is assumed to be steady state, where all the feed and draw streams are
pumped into and out of the system continuously.

Figure 4.7 Fed Batch Bio-fermenter (F-102)

Glucose conversion: 89%

Table 4.11 The N, ΔHRx and Cp values for components in Stream 1

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Glucose 8.100 -1 260 000 115.00
Total 8.100 -1 260 000 115.00

Table 4.12 The N, ΔHRx and Cp values for components in Stream 2

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Dextrin 114.50 -2 256 350 1481.53
Total 114.50 -2 256 350 1481.53
 Table 4.13 The N, ΔHRx and Cp values for components in Stream 3
Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Ammonium nitrate 13.84 -350 260 139.27
Total 13.84 -350 260 139.27

Table 4.14 The N, ΔHRx and Cp values for components in Stream 4

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
S. Roseosporus 188.8 N/A N/A
Total 188.8 N/A N/A

Table 4.15 The N, ΔHRx and Cp values for components in Stream 5

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Oxygen 2.256 0 29.38
Total 2.256 0 29.38

Table 4.16 The N, ΔHRx and Cp values for components in Stream 6

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Oxygen 0.256 0 29.38
Carbon Dioxide 12.15 -393 500 36.94
Total 12.41 -393 500 66.32

Table 4.17 The N, ΔHRx and Cp values for components in Stream 7

Component N (kmol/h) ΔHRx (kJ/kmol) Cp (kJ/kmol.K)
Glucose 0.8964 -1 260 000 115.00
Dextrin 12.61 -2 256 350 1481.53
Ammonium nitrate 1.520 -350 260 139.27
Daptomycin 298.2 -1 865 610 1688.16
Water 1.909 -285 800 75.38
Total 315.1 -6 018 020 3499.3

Calculation to find Q:

ΔHºRx = - 6 018 020 - (- 1 260 000 + (-2 256 350) + (-350260))

= -2 151 410 kJ/kmol
Δ Cp = 3499.3 - (115.0 + 1481.53 + 139.27 + 29.38)
Σ ϕiCpi(T-Ti) = 1734.12 kJ/kmol.K
ΔHRx = -2 151 410 kJ/kmol + 1734.12 kJ/kmol.K (373 – 298) K
= -2 021 351 kJ/kmol
= [1(115.0) + 1(1481.53) + 1(139.27) + 1(1688.53) + 1(75.38)] *
(373- 353) K
= 69 986 kJ/kmol
Q = (8.10 kmol/h)*(69 986 kJ/kmol) + (-2 021 351 kJ/kmol)*
(8.10 kmol/h)*(0.90)
= -1.42 x 107 kJ/h

Q is a negative value. Thus, the reaction is an exothermic process. Hence, heat is

released from the system to the surroundings.

4.1.2 Mass Balance on Multichamber Bowl Centrifuge 1 (S-101)

Fermentation broth is centrifuged in this process and supernatant liquid. Daptomycin

enters an organic phase after the supernatant liquor is extracted by an organic solvent
at 30ºC.

Figure 4.8 Multichamber Bowl Centrifuge 1 (S-101)

 Table 4.18 The N, ΔHRx and Cp values for components in Stream 8
Component N (kmol/h) ΔHRx (kJ/kmol) ΔH (kJ/h)
Glucose 0.8964 -1 260 000 -1 129 464
Dextrin 12.61 -2 256 350 -28 452 574
Ammonium nitrate 1.520 -350 260 -532 395
Daptomycin 298.2 -1 865 610 -556 324 902
Water 1.909 -285 800 -545 592
Total 315.1 -6 018 020 -586 984 927

Table 4.19 The N, ΔHRx and Cp values for components in Stream 9

Component N (kmol/h) ΔHRx (kJ/kmol) ΔH (kJ/h)
Glucose 0.0360 -1 260 000 -45360
Dextrin 1.160 -2 256 350 -2 617 366
Ammonium nitrate 0.0216 -350 260 -7565.62
Daptomycin 298.2 -1 865 610 -556 324 902
Water 0.0955 -285 800 -27293.9
Total 299.5 -6 018 020 -559 022 488

Table 4.20 The N, ΔHRx and Cp values for components in Stream 10

Component N (kmol/h) ΔHRx (kJ/kmol) ΔH (kJ/h)
Glucose 0.8640 -1 260 000 -1 084 104
Dextrin 11.60 -2 256 350 -26 173 660
Ammonium nitrate 1.520 -350 260 -532 395
Daptomycin 0.2431 -1 865 610 -453 530
Water 1.819 -285 800 -519 870
Total 16.04 -6 018 020 -28 763 559

Calculation for Q:

Since the centrifuge does not involve any chemical reactions, the energy balance is
calculated as:
Q = Noutlet ΔHºRx,outlet - Ninlet ΔHºRx,inlet
Q = ΔHºRx,outlet - ΔHºRx,inlet
Q = [(-559 022 488) + (-28 763 559)] - (-586 984 927)
Q = -8.01 x 105 kJ/h
Q is a negative value. Thus, the reaction is an exothermic process. Hence, heat is
released from the system to the surroundings.