See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/339617023

Chymosin

Research Proposal · March 2020

CITATIONS READS

0 2,663

1 author:

Amanina Rozi
Universiti Kebangsaan Malaysia
2 PUBLICATIONS   0 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

PRODUCTION OF CHYMOSIN View project

All content following this page was uploaded by Amanina Rozi on 02 March 2020.

The user has requested enhancement of the downloaded file.

 1

CHAPTER I

INTRODUCTION

1.1 INTRODUCTION OF CHYMOSIN

Rennet is a natural complex of enzymes which responsible for curdling milk and
separate the curds and whey, allowing it to be retained for a longer period in stomach.
According to the McGraw-Hill Encyclopedia of Science and Technology, Rennet is
extracted from the inner mucosa of the fourth stomach chamber of young calves. To
obtain rennet, stomach of calve was then dried and cleaned, cut into small pieces and
put into an extraction solution, which will be filtered after several days. Industrially,
rennet is used in making cheese, yogurts, junket, a soft, pudding-like dessert.

The major component in rennet is chymosin, also known as rennin. Rennet may
contain 50%–95% chymosin depending on the age of the animal (Addis et al., 2008).
Chymosin is a complex compound, a protein formed of a single chain of 323 amino
acids with intramolecular disulfide linkages. There are chymosins A and B which only
differ by one amino acid in the polypeptide chain, where at position 286, chymosin A
has an aspartic acid whereas chymosin B has a glycine (TD Luerce, 2014). Chymosin
is secreted as an inactive proenzyme called prochymosin and it is being most active in
acidic environment. It catalyses the breakdown of proteins to form smaller molecules.

Chymosin is replaced by pepsin as the calf ages, however, pepsin has the
tendency to result in higher fat losses because the curd formed has a more open, looser
structure compare with that formed with chymosin. There is more or less consensus of
 2

opinion in favour of chymosin as the enzyme for cheese making. Given that chymosin
production from calf rumen is limited, alternative methods are needed to produce
sufficient bovine chymosin to meet the increasing global demand, that led to the
production of chymosin through recombinant DNA technology. The cloned chymosin
can be produced by different microorganisms and there is no major distinction could be
detected among cheeses that made with natural enzyme and cloned chymosin.

1.2 PHYSICAL AND CHEMICAL PROPERTIES OF CHYMOSIN

Chymosin has molecular weight of 35.6 kDa when it is brought to pH 4.2 result in an
active form of chymosin. The maximum storage stability of chymosin is at 4°C between
pH 3.0 and 6.2. Meanwhile, chymosin exhibits optimum stability at pH between 5.3 and
6.3, the enzyme is moderately stable at pH 2.0, but the enzyme is unstable at pH around
3.5 and above 6.5, clotting activity is not observed at pH 7.0. Besides, the optimum
temperature for chymosin is between 44-55°C but it is stable up to 50°C and a relative
milk-clotting activity of 50% is recorded when the temperature is raised to 60°C.
(Biotechnol. 30, 243-258 2010)

Table 1.1 Characteristic and Properties of Chymosin

Charateristic Properties
Molecular Weight(kDa) 35.6
Optimum pH 5.3-6.3
Optimum Temperature(°C) 44-55°C

1.3 USAGE OF CHYMOSIN

Chymosin also known as rennin is a proteolytic enzyme that is related to pepsin

synthesized by chief cells in the stomach of young mammals. It is secreted as an inactive
proenzyme called prochymosin that is activated on exposure to acid. Chymosin role in
digestion is to curdle or coagulate milk in the stomach which is a process that
 3

considerably prominence in the very young animals’ stomachs (Bowen). It also

functions as a peptidase that catalyzes the breakdown of proteins to form smaller
molecules (Clegg, 2017). Chymosin precisely hydrolyze the Phe105-Met106 bond of
micelle-stabilizing protein, K-casein, leading to coagulation in the presence of Ca2+ at
Temperature > 20°C (2003). If milk were not coagulated, it would rapidly flow through
the stomach and miss the opportunity for initial digestion of its proteins.

Chymosin is a vital industrial enzyme as it is broadly used in cheesemaking.

This is because it retained milk for longer periods as it converts liquid milk to a
semisolid like cottage cheese. Traditionally, chymosin is obtained from the stomach
lining of unwean mammals which in turn requires slicing open the baby mammal itself.
This means it will kill the infants. However, in the 1970s, anticipating a crisis of supply
and demand, researchers turned to a then-unprecedented technology in food science:
genetically modified organisms. The result was a genetically altered bacterium that
produced cheesemaking enzymes in a beaker, instead of an animal's stomach known as
the recombinant chymosin. This revolutionize the cheese industry. Nowadays, majority
of cheese is made with enzymes produced not by baby animals, but genetically modified
microbes. Some of the types of cheese manufactured including Cheddar, Edam, Tilsit,
Italian, Manchego, Colby and Gouda. (Garg & Johri, 1995)

This recombinant chymosin which replaced the traditionally produced chymosin

from the baby mammals’ abomasum is identical to the young animals’ chymosin in
properties and applicability. Shifting the source of chymosin was not only to avoid the
slaughtering of young animals but to rather having a more consistent outcome in
fulfilling the high demands of cheese around the globe.
 4

CHAPTER II

ECONOMIC ISSUE ON PRODUCTION OF CHYMOSIN

2.1 SUPPLY AND DEMAND OF CHYMOSIN

Chymosin is generally is used in dairy industry as a milk-clothing agent for the

manufacture of quality cheeses with good texture and great taste. High demand of
cheese production worldwide leads to the increasing of chymosin demand.
Geographically, the global largest market share in terms of revenue is North America
over the forecast period 2016-2013. Meanwhile, Asia Pacific, especially China is
expected to be the fastest growing region for chymosin market due to growing dairy
and food and beverage industries. (ResearchAndMarkets 2018) Chymosin market also
grows in Europe, Germany, Russia and rest d Middle East and Africa.

The global market of Chymosin volume estimated at 135.33 kiloton in 1999 and
it have registering CAGR of 4% between 1999 to 2018 (Neelakantan et al., 1999) and
expected to grow at CAGR of 4.3% from 2019 to 2025. (Chymosin Market Report
2019). The chart below shows the supply and demand of chymosin from 2000 to 2025.
 5

Figure 2.1 Graph of Supply and Demand of Chymosin

2.2 PRICE OF CHYMOSIN

Since North America is the dominant region in producing rennet, the price will be set
in USD for convenience. The graph illustrates average monthly price for rennet in
United States throughout the year of 2018. Rennet price in 2018 starts lower compared
to the end of the year 2017 but increased considerably up until April. The price then
dropped from May until July before increasing steadily until the end of the year (Rennet
and Acid Casein 2019).

7
6
Price(USD/ton)

5
4
3
2
1
0

Month

Figure 2.2 Graph of Price of Rennet Throughout the Year 2018

 6

2.3 COMPANIES PRODUCING CHYMOSIN

The chymosin segment is estimated to account for the largest share in 2017. Though its
major application is in cheese production, chymosin is also used in halal, organic, and
GMO-free products. As the consumption of cheese is increasing, the companies
manufacturing chymosin is growing substantially, especially in developing
countries(Dairy Enzymes Market, 2018).

Table 2.1 List of Companies Producing Chymosin

Company Info
Mayasan AS Mayasan AS’s fermented rennet is pure chymosin enzyme named
Renmax® produced from special strains through controlled
fermentation. Renmax® is available in several activities and in liquid and
powder forms. Other than fermented rennet, Mayasan AS also manufactures
Renna® which is their DNA recombinant chymosin produced through
continuous extraction and membrane column filtration process from bobby
calf stomachs. Renna® is available in liquid and powder forms with different
activities.
Maxiren Maxiren® XDS is Maxiren latest innovation in cheese coagulants. It is a
bovine chymosin offering extended textural shelf life in cheese while
maintaining lower proteolysis. It has the highest specificity on the market for
improved texture and sliceability, to improve plant efficiency and to reduce
carbon footprint.
Danisco Dupont Dupont speciality is making the traditional cheese appeal by customizing
chymosin formulation which is their Carlina™ Animal Rennet range that is
available in various package sizes.
Chr. Hansen Holding Chr. Hansen’s range of pure fermentation-produced chymosin (FPC) are
ideal milk-clotting enzymes delivering superior properties for cheese and
whey such as balanced flavour and texture. Chr. Hansen claims that their
product increased cheese yield and have a better process control.
Renco New Zealand RENCO Natural Calf Rennet is purified by an ion exchange process,
RENCO Natural Calf Rennet is naturally high in chymosin (>93%),
providing a superior yield with flavour and texture benefits. RENCO Natural
Calf Rennet can be supplied in both liquid and powder forms of varying
strengths and pack sizes.

(Source: Mordor Intelligence, 2019)

 7

2.4 PLANT CAPACITY

Based on the global demand and supply, by the year 2025, the production rate of plant
is estimated as below:

Demand = 40 321 000 ton/year

Supply = 37 741 000 ton/year

Demand-Supply = 40 321 000 ton/year – 37 741 000 ton/year

= 2 580 000 ton/year

= 2 580 000 000 kg/year

This shows that Chymosin demand will be 2580000 tonnes more than the
supply. The production of Chymosin is lower than global demand. Since our plant is
planning to cater 0.0025% of the world’s demand of Chymosin, the calculation for our
plant capacity is as follows:

Production time a day = 24 hours

Maintenance = 4 days/month

= 1152 hours/year

Production time a year = (365 days x 24 hours) - 1152 hours

= 7608 hours

(0.0025)(2580000000kg)
Plant capacity =
7608h

= 850 kg/h

= 7446 ton/year
 8

CHAPTER III

PROCESS DESCRIPTION OF CHYMOSIN

3.1 PREPARATION OF RECOMBINANT E. COLI

In the process of the production of chymosin, a piece of tissue is extracted from the calf
fourth stomach and the DNA which is Cym gene, is isolated from the cells. It is
important to use DNeasy blood and tissue kit which provides purification and isolation
of total DNA from animal blood and tissue from cells, bacteria, or other
microorganisms. After isolated process, it is essential to amplify the Cym gene using
PCR that performed using a degenerate sense primer primer 5’-TGG CAA AAT GTA
CCC ACT GA-3’ and an anti-sense primer 5’-ATG TGT GCA TGT GTG TGT GC-
3(Emtage et al., 1983) Amplifications need to have 850 bp (base pairs) of the chymosin
gene.

After that, the gene sequences were introduced into plasmids and transferred it
into bacterial host cell E. coli K-12 strain HB101.The bacteria were treated with
penicillin to select the antibiotic resistance bacteria. (Valeria et al, 2019).

3.2 CULTIVATION OF RECOMBINANT E. COLI

First, E. coli K-12 strain HB101 growth in seed fermentor to multiply at 37℃ in M9
medium which supplemented with glucose and ammonium nitrate until fully consumed
by the bacteria (Emtage et al, 1983). In the seed fermentor oxygen will be supplied
because E. coli is facultative anaerobic which means it will growth at optimum with
 9

existence of oxygen. The bacteria can grow at pH 6-11 and the optimum pH at 8.5
(Rathgeber et al, 2005). Then after this cultivation, E. coli will transfer to main
fermentor to start produced chymosin as final product.

3.3 DOWN STREAM PROCESS

First, E. coli was transfer to main fermentor to further growth at 37℃ in medium
supplemented with glucose, ammonium nitrate to maintaining and development of the
recombinantion DNA of E. coli.

C6H12O6 + 0.398NH4NO3 + 1.898O2 → 3.186CH1.94O0.52N0.25 + 2.814CO2 +3.706H2O

The next step is transferred it into centrifuge that function to disrupt the cell and
produce lysis at 600-1000 bar of pressure. After that, put lysis in a rotary filter to
separate the lysis with different phases which contained biomass and prochymosin. The
biomass produced is being discharged and the prochymosin were being transfer to a
urea tank. The prochymosin were washed with 9M urea in urea tank and was purified
by elution with a buffer containing 0.5 M sodium chloride in chromatography. In
chromatograph, filtration occur to separate chymosin and other waste material which
are waste water, ammonium nitrate and glucose. Refer to Process Flow Diagram (PFD)
that show the flow of the process.
10
 11

CHAPTER IV

MASS BALANCE AND ENERGY BALANCE

4.1 STOICHIOMETRY EQUATION

The chemical equation is shown as below:

C6H12O6 + α1NH4NO3 + α2O2 → α3CH1.94O0.52N0.25 + α4CO2 +α5H2O (4.1)

The unknown coefficient of substance is denoted as α.

Element balance:

C: 6 = α3 + α4 ----------- (1)

H: 12 = -4α1 + 1.94α3 +2α5-----------(2)

O: 6=-3α1 - 2α2 + 0.52α3 + 2α4 + α5--------------(3)

N: 2α1 = 0.25α3-------------(4)
 12

Degree of Freedom= No of unknown – No. of element

=5–4

=1

There are 5 unknowns need to be determined which are α1, α2, α3, α4 and α5,
since the equation only have 4 elements C, H, O and N thus 4 independent elemental
equations can be formed. Another one equation can be obtained by using the yield of
product with respect to substrate.

Yield of glucose = 0.456 (Journal of Bacteriology, 2017)

𝛼3 M𝑟 biomass
= 0.456
Mr glucose

𝛼3 (25.76)
= 0.456
180

𝛼3 = 3.186

From (4),

-2α1 = 0.25α3

2α1 = 0.25(3.186)

α1 = 0.398

From (1),

6 = α3 + α4

6=3.186 + α4
 13

α4 = 2.814

From (2),

12 = -4α1 + 1.94α3 +2α5

12=-4(0.398) + 1.94 (3.186) + 2α5

α5 = 3.706

From (3),

6=-3α1 - 2α2 + 0.52α3 + 2α4 + α5

6=-3(0.398) - 2 α2 + 0.52 (3.186) + 2(2.814) +3.706

α2 = 1.898

Thus, the balance stoichiometry equation obtained is:

C6H12O6 + 0.398NH4NO3 + 1.898O2 → 3.186CH1.94O0.52N0.25 + 2.814CO2 +3.706H2O (4.2)

 14

4.2 MASS BALANCE

From the calculation in Chapter II, our plant capacity production per year is 850 kg/h.

In order to calculate the material balance, we need to know the molecular weight
(MW) of each chemical species (PubChem, 2019) that participate in this reaction.

MWG, (C6H12O6) = 180.0 kg/kmole

MWAN, (NH4NO3) = 80.0 kg/kmole

MWO2 ,(O2) = 32.0 kg/kmole

MWB, (CH1.94O0.52N0.25) = 25.76 kg/kmole

MWW, (H2O) = 18.0 kg/kmole

MWN2 , (N2 ) = 28.0 kg/kmole

MWCO2 , (CO2 ) = 44.0 kg/kmole

4.2.1 Main Fermentor (F-101)

The bioreactor is an enclosed volume in which fermentation reaction take place and it
is designed to ensure that the reaction proceeds with highest efficiency towards desired
output products, producing the highest yield of product. The reaction take place in
which the E. coli undergoes aerobic respiration to produce biomass that contains
prochymosin. The side products of this reaction carbon dioxide and water. The
conversion of glucose in the main fermentor is 1.0. The rate of the fermentation reaction
is 34.52.
 15

T1= 298.15 K
F1O2 = 2096.8 kg/h T4 =310.15 K
F1N2 = 7887.9 kg/h F4CO2 = 4274.5 kg/h

T2 = 298.15 K F4N2 = 7887.9 kg/h

F2AN = 1099.2 kg/h

T3 = 298.15 K T5 =310.15 K
F3G = 6214.1 kg/h F5B = 2833.3 kg/h
F5W = 2303.0 kg/h

Figure 4.1 Main Fermentor (F-102)

C6H12O6 + 0.398NH4NO3 + 1.898O2 → 3.186CH1.94O0.52N0.25 + 2.814CO2 +3.706H2O (4.2)

Assume molar flowrate inlet for glucose, NinG = 34.52 kmol/h, as a basis

Glucose conversion, x = 1.0

NinG 𝑥𝐺
𝑟=
−𝛼𝐺

34.52(1.0)
=
−(−1)

= 34.52

Glucose balance:

N3G = N5G - αGr

= 0 – (-0.398) (34.52)
 16

= 34.52 kmol/h

F3G = 6214.1 kg/h

Ammonium Nitrate balance:

N2AN = N5AN - αANr

= 0 - (-0.398) (34.52)

= 13.74 kmol/h

F2AN = 1099.2 kg/h

Oxygen balance:

N1O2 = N4O2 - O2 r

= 0 - (-1.898) (34.52)

= 65.52 kmol/h

F1O2 = 2096.8 kg/h

Nitrogen gas balance:

F1N2 = F4N2 =7887.9 kg/h

Biomass balance:

NinB = N5B - αBr

0 = N5B - (3.186) (34.52)

 17

N5B = 109.99 kmol/h

F5B = 2833.3 kg/h

Carbon Dioxide balance:

NinCO2 = N4CO2 - CO2 r

0 = N4CO2- (2.814) (34.52)

N4CO2= 97.15 kmol/h

F4CO2 = 4274.79 kg/h

Water balance:

NinW = N5W - αWr

NinW = N5W - (3.706) (34.52)

N5W = 127.94 kmol/h

F5W = 2303.0 kg/h

 18

Table 4.1 The Mass and Molar Flowrate of the Component in Main Fermentor

COMPONENT INLET STREAM 1 INLET STREAM 2 INLET STREAM 3

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 6214.1 1
Ammonium Nitrate 0 0 1099.2 1 0 0
Oxygen 2096.8 0.21 0 0 0 0
Nitrogen 7887.9 0.79 0 0 0 0
Biomass 0 0 0 0 0 0
Carbon Dioxide 0 0 0 0 0 0
Water 0 0 0 0 0 0
Total 9984.7 1 1099.2 1 6214.1 1

Table 4.2 The Mass and Molar Flowrate of the Component in Main Fermentor

COMPONENT OUTLET STREAM 4 OUTLET STREAM 5

Mass Flowrate Mass Fraction Mass Flowrate Mass Fraction
(kg/h) (kg/h)
Glucose 0 0 0 0
Ammonium Nitrate 0 0 0 0
Oxygen 0 0 0 0
Nitrogen 7887.9 0.3515 0 0
Biomass 0 0 2833.3 0.5516
Carbon Dioxide 4274.5 0.6485 0 0
Water 0 0 2303.0 0.4484
Total 12162.4 1 5136.3 1

Material balance:

F1O2 + F1N2 + F2AN+ F3G = F4CO2 + F4N2 + F5W+ F5B

2096.8 + 7887.9 + 1099.2 + 6214.1 = 4274.5 + 7887.9 + 2303.0 + 2833.3

17298.0 kg/h = 17298.7 kg/h (balanced!)

 19

4.2.2 Rotary Filter 1 (R-101)

Rotary filter 1 is chosen to be used as phase separator as the product from main
fermentor outlet stream contains solid and liquid. Rotary filter I is used to separate the
biomass from the biomass. Stream 8 is the top product of rotary filter that consists of
impure prochymosin. Stream 7 is the bottom product of rotary filter which is the
wsatewater, the side product from this process.

T6 = 310.15 K
T8 = 310.15 K
F6B = 2833.3 kg/h
F8B = 2833.3 kg/h
F6W = 2303.0 kg/h

T7 = 310.15 K
F7W = 2303.0 kg/h

Figure 4.2 Rotary Filter 1 (R-101)

Table 4.3 Mass flowrate of the Component in Rotary Filter 1

COMPONENT INLET STREAM 6 INLET STREAM 7 INLET STREAM 3

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 0 0
Ammonium Nitrate 0 0 0 0 0 0
Biomass 2833.3 0.5516 0 0 2833.3 1
Carbon Dioxide 0 0 0 0 0 0
Water 2303.0 0.4484 2303.0 1 0 0
Total 5136.3 1 2303.0 1 2833.3 1

F6W = F7W = 2303.0 kg/h

F6B = F8B = 2833.3 kg/h

 20

4.2.3 Centrifuge (C-201)

Centrifuge is used to produce lysis which contains biomass and prochymosin. Stream 8
consist of biomass enters the centrifuge, the cell will disrupt by pressure supplied and
produce the lysis. Stream 9 is the outlet of centrifuge which contains 30% of biomass
and 70% of prochymosin.

T9 = 310.15 K
T8 = 310.15 K F9B = 1933.3 kg/h
F8B = 2833.3 kg/h F9P = 850.0 kg/h

Figure 4.3 Centrifuge (C-201)

Table 4.4 Mass Flowrate of the Component in Centrifuge

COMPONENT OUTLET STREAM 8 OUTLET STREAM 9

Mass Flowrate Mass Fraction Mass Flowrate Mass Fraction
(kg/h) (kg/h)
Glucose 0 0 0 0
Ammonium Nitrate 0 0 0 0
Biomass 2833.3 1 1983.3 0.7
Prochymosin 0 0 850.0 0.3
Water 0 0 0 0
Total 2833.3 1 2833.3 1

F9B = (0.7) F8B

= (0.7) (2833.3)

= 1983.3 kg/h
 21

F9P = (0.3) F8P

= (0.3) (2833.3)

= 850.0 kg/h

4.2.4 Rotary Filter 2 (R-102)

Rotary filter 2 (R-102) is used as phase separator as the product from centrifuge outlet
stream. Rotary filter is used to separate prochymosin that has already been extracted
from E. Coli from biomass. Stream 9 enters rotary filter with 30% of the biomass is
prochymosin while the rest is solid waste. Stream 11 is the top product of rotary filter
that consists of impure prochymosin. Stream 10 is the bottom product of rotary filter
which is the biomass, the side product from this process.

T9 = 310.15 K T11 = 310.15 K

F9B = 1933.3 kg/h F11P = 850.0 kg/h
F9P = 850.0 kg/h

T10 = 310.15 K
F10B = 1983.3 kg/h

Figure 4.4 Rotary Filter 2 (R-102)

 22

Table 4.5 Mass flowrate of the Component in Rotary Filter 2

COMPONENT INLET STREAM 9 INLET STREAM 10 INLET STREAM 11

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 0 0
Ammonium Nitrate 0 0 0 0 0 0
Biomass 1983.3 0.7 1983.3 1 0 0
Prochymosin 850.0 0.3 0 0 850.0 1
Water 0 0 0 0 0 0
Total 2833.3 1 1983.3 1 850.0 1

F9B = F10B = 1983.3 kg/h

F9P = F11P = 850.0 kg/h

4.2.5 Urea Tank (T-101)

Urea tank is used to activated the prochymosin to chymosin as final product. Stream 13
is contains 9M of urea is mix with Stream 12 that contains prochymosin to activate the
prochymosin. Stream 14 is product of centrifuge contains chymosin and waste product
which is urea, then be transfer to the chromatographer to separate our product and waste
product.

T12 = 310.15 K
F12P = 850.0 kg/h

T13 = 310.15 K T14 = 310.15 K

F13U = 914.0 kg/h F14U = 914.0 kg/h
F14C = 850.0 kg/h

Figure 4.5 Urea Tank (T-101)

 23

Table 4.6 Mass Flowrate of the Component in Urea Tank

COMPONENT INLET STREAM 12 INLET STREAM 13 INLET STREAM 14

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 0 0
Ammonium Nitrate 0 0 0 0 0 0
Biomass 0 0 0 0 0 0
Prochymosin 850.0 1 0 0 0 0
Urea 0 0 914.0 1 914.0 0.5181
Chymosin 0 0 0 0 850.0 0.4819
Total 850.0 1 914.0 1 1764.0 1

F12P = F14P = 850.0 kg/h

F13U = F14U = 914.0 kg/h

4.2.6 Chromatographer (C-101)

Chromatographer is used to separate chymosin from the urea. As stream 14 that consists
of prochymosin and waste urea enter the chromatographer, the mixture is separated into
its component parts. Stream 16 is the top product that consists of chymosin only. Stream
15 is the waste urea and is the bottom products of this process. The waste urea in bottom
product will be undergo hydrolytic pathway.
 24

T14 = 310.15 K
F14U = 914.0 kg/h
F14C = 850.0 kg/h

T16 = 310.15 K
F16C = 850.0 kg/h

T15 = 310.15 K
F15U = 914.0 kg/h

Figure 4.6 Chromatographer (C-101)

Table 4.7 Mass flowrate of the Component in Chromatographer

COMPONENT INLET STREAM 14 INLET STREAM 15 INLET STREAM 16

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 0 0
Ammonium Nitrate 0 0 0 0 0 0
Biomass 0 0 0 0 0 0
Prochymosin 0 0 0 0 0 0
Urea 914.0 0.5181 914.0 1 0 0
Chymosin 850.0 0.4819 0 0 850.0 1
Total 1764.0 1 914.0 1 850.0 1

F14C = F16C = 850.00 kg/h

F14U = F15U = 914.0 kg/h

 25

4.2.7 Overall Mass Balance

T1= 298.15 K T4 =310.15 K

F1O2 = 2096.8 kg/h F4CO2 = 4274.5 kg/h
F1N2 = 7887.9 kg/h F4N2 = 7887.9 kg/h

T7 = 310.15 K
T2 = 298.15 K F7W = 2303.0 kg/h
F2AN = 1099.2 kg/h
T10 = 310.15 K
F10B = 1983.3 kg/h
T3 = 298.15 K
F3G = 6214.1 T15 = 310.15 K

kg/h F15U = 914.0 kg/h

T16 = 310.15 K
F16C = 850.0 kg/h
T13 = 310.15 K
F13U = 914.0 kg/h

Figure 4.7 Overall Mass Balance

Table 4.8 Mass Flowrate of Overall Mass Balance I

COMPONENT INLET STREAM 1 INLET STREAM 2 INLET STREAM 3

Mass Mass Mass Mass Mass Mass
Flowrate Fraction Flowrate Fraction Flowrate Fraction
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 6214.1 1
Ammonium Nitrate 0 0 1099.2 1 0 0
Oxygen 2096.8 0.21 0 0 0 0
Nitrogen 7887.9 0.79 0 0 0 0
Biomass 0 0 0 0 0 0
Carbon Dioxide 0 0 0 0 0 0
Water 0 0 0 0 0 0
Total 9984.7 1 1099.2 1 6214.1 1
 26

Table 4.9 Mass Flowrate of Overall Mass Balance II

COMPONENT OUTLET STREAM 4 OUTLET STREAM 7 OUTLET STREAM 10

Mass Mass Mass Mass Mass Mass Fraction

Flowrate Fraction Flowrate Fraction Flowrate
(kg/h) (kg/h) (kg/h)
Glucose 0 0 0 0 0 0
Ammonium 0 0 0 0 0 0
Nitrate
Oxygen 0 0 0 0 0 0
Nitrogen 7887.9 0.3515 0 0 0 0
Biomass 0 0 0 0 1983.3 1
Carbon Dioxide 4274.5 0.6485 0 0 0 0
Water 0 0 2303.0 1 0 0
Total 12162.4 1 2303.0 1 1833.3 1

Table 4.10 Mass Flowrate of Overall Mass Balance III

COMPONENT OUTLET STREAM 15 OUTLET STREAM 16

Mass Flowrate Mass Fraction Mass Flowrate Mass Fraction
(kg/h) (kg/h)
Glucose 0 0 0 0
Ammonium Nitrate 0 0 0 0
Biomass 0 0 0 0
Prochymosin 0 0 0 0
Urea 914.0 1 0 0
Chymosin 0 0 850.0 1
Total 914.0 1 850.0 1

Check balance:

Fin = Fout

F1O2 + F1N2+ F2AN + F3G + F13U= F4CO2 + F4N2 + F7W + F10B + F15U + F16C

2096.8+7887.9 +1099.2+6214.1+914.0 = 4274.5+7887.9+2303.0+1983.3+914.0+ 850.0

18212.0 kg/h = 18212.7 kg/h (balanced!)

 27

4.2.8 Superpro Result

Figure 4.8 SuperPro Result

Table 4.11 Comparison of The Composition in Material Balance and SuperPro

Result

Component Material Balance SuperPro Result Percentage Error

Glucose 0.0473 0.2248 78.96%
Ammnium Nitrate 0.0188 0.0188 0%
Oxygen 0.0896 0.0018 97.99%
Nitrogen 0.3856 0.3306 16.64%
Biomass 0.1506 0.1145 31.53%
Carbon Dioxide 0.1330 0.2011 33.86%
Water 0.1751 0.1084 61.53%

Percentage error is calculated by using equation:

𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒𝑠 𝑖𝑛 𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑀𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝐵𝑎𝑙𝑎𝑛𝑐𝑒 𝑎𝑛𝑑 𝑆𝑢𝑝𝑒𝑟𝑃𝑟𝑜 𝑅𝑒𝑠𝑢𝑙𝑡

×100%
𝐶𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑖𝑛 𝑆𝑢𝑝𝑒𝑟𝑃𝑟𝑜 𝑅𝑒𝑠𝑢𝑙𝑡
 28

4.3 ENERGY BALANCE

According to these laws, energy is never really created and it’s never really destroyed.
Rather, energy is transferred between entities. Energy balance is applied onto the system
in order to calculate the energy that is required to be input into the system, as well as
the energy that is released during the process.The biochemical process equation is as
shown:

C6H12O6 + 0.398NH4NO3 + 1.898O2 → 3.186CH1.94O0.52N0.25 + 2.814CO2 +3.706H2O (3.2)

For the calculation of energy balance, heat capacity, Cp, is required.

Table 4.12 Data for Calculation of Heat Capacity of Substances

Components A B C D
O2 25.48 1.520 x 10-2 -0.7155 x 10-5 1.312 x 10-9
CO2 22.26 5.981 x 10-2 -3.501 x 10-5 7.469 x 10-9
H 2O 32.24 -0.001924 1.055 x 10-5 -3.596 x 10-9

(Source: Lange’s Handbook of Chemistry)

Table 4.13 Calculation of Heat Capacity for Glucose

Elements RMM (g/mol) Cp (J/mol. K) Mole fraction Cp (J/mol. K)

C 12 85 0.4000 3.4000
H 1 20.8 0.0667 1.3874
O 16 21.9 0.5333 11.6793
Total 16.4667
 29

Table 4.14 Calculation of Heat Capacity for Biomass

Elements RMM (g/mol) Cp (J/mol. K) Mole fraction Cp (J/mol. K)

C 12 8.5 0.4658 3.9593
H 1 20.8 0.0753 1.5662
O 16 21.9 0.3230 7.0737
N 14 20.8 0.1359 2.8267
Total 15.4259

Table 4.15 Calculation of Heat Capacity for Ammonium Nitrate

Elements RMM (g/mol) Cp (J/mol.k) Mole fraction Cp (J/mol.k)

H 14 20.8 0.3500 7.28
N 1 20.8 0.0500 1.04
O 16 21.9 0.600 13.14
Total 21.46

Table 4.16 Calculation of Heat Capacity for Prochymosin

Elements RMM (g/mol) Cp (J/mol. K) Mole fraction Cp (J/mol. K)

C 12 8.5 0.6469 5.4987
H 1 20.8 0.0782 1.6266
O 16 21.9 0.0863 1.8890
N 14 20.8 0.1887 3.9250
Total 12.9393
 30

Table 4.17 Calculation of Heat Capacity for Urea

Elements RMM (g/mol) Cp (J/mol.k) Mole fraction Cp (J/mol.k)

C 12 8.5 0.2000 1.7000
H 1 20.8 0.0667 1.3874
O 16 21.9 0.2667 5.8407
N 14 20.8 0.4667 9.7074
Total 18.6355

4.3.1 Main Fermentor (F-101)

T1= 298.15 K
𝐹4𝑂2 = 2096.8 kg/h
T4 =310.15 K
𝐹1𝑁2 = 7887.9 kg/h 𝐹4𝐶𝑂2 = 4274.5 kg/h

T2 = 298.15 K 𝐹4𝑁2 = 7887.9 kg/h

F2AN = 1099.2 kg/h

T3 = 298.15 K
F3G = 6214.1 kg/h
T5 =310.15 K
F5B = 2833.3 kg/h
F5W = 2303.0 kg/h

Figure 4.1 Main Fermentor (F-102)

𝑇
The enthalpy of each component is calculated by using the formula ∆H = ∫𝑇 𝐶𝑝 𝑑𝑡
0

By using T0 = 298.15K
 31

Standard heat reaction, ∆𝐻𝑟 = -460𝑁𝑜

= -460 x (65.5250 x103)

= -30.14 x 106 kJ/kmol

𝑇
By using formula ∆𝐻 = ∫𝑇 2 𝐴 + 𝐵𝑇 + 𝐶𝑇 2 + 𝐷𝑇 3 𝑑𝑡 , the enthalpy for oxygen,
1

glucose, nitrogen and ammonium nitrate is calculated.

Feed enthalpy:

Stream 9:

298.15
∆𝐻𝑂2 = ∫298.15 25.48 + 0.0152𝑇 − 0.00007155𝑇 2 + 1.312 𝑥 10−9 𝑇 3 𝑑𝑇

= 0 kj/kmol

298.15
∆𝐻𝑁2 = ∫298.15 27.27 + 0.000593𝑇 + 4 𝑥 10−10 𝑇 3 𝑑𝑡

= 0 kJ/kmol

Stream 11:

298.15
∆𝐻𝐶6 𝐻12 𝑂6 = ∫298.15 16.4667 𝑑𝑇

= 0 kJ/kmol
 32

Stream 12:

298.15
∆𝐻𝑁𝐻4 𝑁𝑂3 = ∫298.15 21.46 𝑑𝑇

=0 kJ/kmol

(65.5250)(0)+(281.7107)(0)+(34.5228)(0)+(13.74)(0)
∆𝐻𝑖 = 395.4985

= 0 kJ/kmol

Product enthalpy:

Stream 13

310.15
∆𝐻𝐶𝑂2 = ∫298.15 22.26 + 0.05981𝑇 − 0.0003501𝑇 2 + 7.469 𝑥 10−9 𝑇 3

= 390.3561 kJ/kmol

310.15
∆𝐻𝑁2 = ∫298.15 27.27 + 0.000593𝑇 + 4 𝑥 10−10 𝑇 3 𝑑𝑇

= 185.1108 kJ/kmol

(97.1477)(390.3561)+(281.7107)(185.1108)
∆𝐻𝑂 = 378.8584

=237.7402 kJ/kmol
 33

Stream 14

310.15
∆𝐻𝐻2 𝑂 = ∫298.15 32.29 − 1.92 𝑥 10−3 𝑇 + 1.06 𝑥 10−5 𝑇 2 − 3.60 𝑥 10−9 𝑇 3 𝑑𝑇

= 329.5394 kJ/kmol

310.15
∆𝐻𝐵 = ∫298.15 15.4259 𝑑𝑇

= 449.0681 kJ/kmol

(109.9884)(449.0681)+(127.9444)(329.5394)
∆𝐻𝑂 = 237.9328

=384.7934 kJ/kmol

Q = Nout ∆𝐻𝑜𝑢𝑡 + Nout∆𝐻𝑜𝑢𝑡 − 𝑁𝑖𝑛 𝐻𝑖𝑛 + 𝑟∆𝐻𝑟

= (237.9328)(384.7934) + (378.8584)( 237.7402) – (395.4985)(0) + (34.52)

(-30.14 x 106 )

= -1.0403 x 109 kJ/h

The negative sign of Q indicates that the reaction is an exothermic reaction.

 34

4.3.2 Rotary Filter 1 (R-101)

T6 = 310.15 K
T8 = 310.15 K
F6B = 2833.3 kg/h
F8B = 2833.3 kg/h
F6W = 2303.0 kg/h

T7 = 310.15 K
F7W = 2303.0 kg/h

Figure 4.2 Rotary Filter 1 (R-101)

Feed Enthalpy:

Stream 15:

310.15
∆𝐻𝐵 = ∫298.15 15.4259 𝑑𝑇

= 185.1108 kJ/kmol

(109.9895)(185.1108)
∆𝐻𝑖 = 109.9895

= 185.1108 kJ/kmol
 35

Product Enthalpy:

Stream 16:

310.15
∆𝐻𝐵 = ∫298.15 15.4259 𝑑𝑇

= 185.1108 kJ/kmol

(76.9915)(185.1108)
∆𝐻0 = 76.9915

= 185.1108 kJ/kmol

Stream 17:

310.15
∆𝐻𝑃 = ∫298.15 12.9393 𝑑𝑇

= 155.2716 kJ/kmol

(2.2911)(155.2716)
∆𝐻0 = 2.2911

= 155.2716 kJ/kmol

Q = No ∆𝐻𝑜 + No∆𝐻𝑜 − 𝑁𝑖 𝐻𝑖

= (76.9915)( 185.1108) + (2.2911)( 155.2716) – (109.9895)( 185.1108)

= -5.7525 x 103 kJ/h

The negative sign of Q indicates that the reaction is an exothermic reaction.

 36

4.3.3 Chromatographer (C-101)

T19 = 310.15 K
F14P = 850.00 kg/h
F14WU = 914.0 kg/h

T16 = 310.15 K
F16C = 850.0 kg/h

T15 = 310.15 K
F15WU = 914.0 kg/h

Figure 4.6 Chromatography (C-101)

Feed Enthalpy:

Stream 19:

310.15
∆𝐻𝐶𝐻4 𝑁2 𝑂 = ∫298.15 18.6355 𝑑𝑇

= 223.626 kJ/kmol

310.15
∆𝐻𝑝 = ∫298.15 12.9393 𝑑𝑇

= 155.2716 kJ/kmol

(15.2333)(223.626)+(2.2911)(155.2716)
∆𝐻𝑖 = 17.5244
 37

= 214.6895 kJ/kmol

Product enthalpy:

Stream 20:

310.15
∆𝐻𝐶𝐻4 𝑁2 𝑂 = ∫298.15 18.6355 𝑑𝑇

= 223.626 kJ/kmol

(15.2333)(223.626)
∆𝐻0 = 15.2333

= 223.626 kJ/kmol

Stream 21:

310.15
∆𝐻𝐶 = ∫298.15 12.9393 𝑑𝑇

= 155.2716 kJ/kmol

(2.2911)(155.2716)
∆𝐻0 =
2.2911

= 155.2716 kJ/kmol

Q = No ∆𝐻𝑜 + No∆𝐻𝑜 − 𝑁𝑖 𝐻𝑖

= (15.2333) (223.626) + (2.2911) (155.2716) – (17.5244) (214.6895)

= 3.476 x 10-5 kJ/h

The positive sign of Q indicates that the reaction is an endothermic reaction

 38

CHAPTER V

SAFETY AND ECONOMIC ISSUES

5.1 INTRODUCTION

Chymosin is a part of the International Dairy Federation Group Experts work on the use
of enzymes in preparation for making dairy products. In the process of fermentation
production of chymosin, it goes through many stages where produce the main product
and waste product. Water and biomass are an environmental waste product of chymosin
during filtration and centrifugation. This waste product needs proper treatment to
prevent pollution to the environment.

The environmental issue for chymosin comes from the killing of young calf to
extract the enzyme from their stomach. Based on Malaysia Environmental Act it is that
stated no person allowed to kill any animal by shooting, unless during an emergency
case for disease control authorized by the veterinary authority. There is also an
environmental act for handling the waste product to prevent water pollution in Malaysia
water. This environmental act is important to sustain our environment for a future
generation.

Production of chymosin involves many chemical substances such as potassium

phosphate, ampicillin, formic acid etc. A wide range of health hazards such as
carcinogenicity, irritation, and allergic reaction can be caused by these substances.
Some however, like glucose, are non-hazardous. To ensure chemical safety, the
 39

information about hazardous and safety precautions of chemical substances is given to

avoid any safety issue. In order to grow the microorganism, there have first aid
measurement and handling storage methods of chemical substances to avoid health
issues to people. This kind of microorganism (E. coli) can cause digestive problems so
precautions need to be exercised when handling these bacteria.

5.2 SAFETY ISSUES

In the production process of fermentation produced Chymosin included many chemical

substances for difference purpose for example as a buffer solution, medium for the
growth of chymosin, medium of fermenter and others. Each substance needs to be
handled carefully as it might be harmful to human and environment. The information
below is referring to MSDS (LabChem Safety Data Sheet, 2012) of each substance:

Table 5.1 MSDS of Chemical Substances

Subst Hazard Safety Precaution First Aid Measurement Handling & Storage
ances
Gluco Non-hazardous - Eye contact: Flush eyes Handling: Wash
se with water. Call thoroughly after handling.
physician if irritation Storage: Protect from heat
develops. and keep away from
Skin contact: Wash skin oxidizers
with plenty of water.
Ingestion: Give large
quantities of water.

Amm May intensify fire, Wear protective Ingestion: Wash out Handling: Avoid skin and
onium harmful if gloves, mouth with water eye contact and breathing
Nitrat swallowed, causes protective provided person is in dust. Avoid handling
e serious eye clothing, eye conscious. which leads to dust
irritation, risk of protection and Eye contact: Immediately formation. Keep material
explosion if face protection irrigate with copious away from heat or ignition
heated under quantities of water, while sources.
confinement and holding eyelids open, for

liberates toxic gas at least 15 minutes. Seek Storage: Store in a cool,

when contact with medical attention if dry, well-ventilated place.
acids. irritation persists. Store away from sources of
Skin contact: Wash heat or ignition. Store away
affected areas with from combustible materials
copious amounts of
water. Remove all
contaminated clothing To be continued…
 40

…continuous
and launder before reuse.
Inhalation: Remove
affected person from
exposure to a well-
ventilated area. If the
affected person suffers
cardiac arrest commence
cardiopulmonary
resuscitation
immediately. Seek urgent
medical attention.
Urea May be harmful Wear protective Skin contact: Rinse with Handling: Keep container
by inhalation, gloves, plenty of water. closed when not in use.
ingest ion, or skin protective Eye contact: Rinse Storage: Avoid from
absorption. May clothing, eye immediately with plenty excess heat and dust
cause eye, skin, or protection and of water. formation. Protect from
respiratory system face protection. Ingestion: Rinse mouth. moisture.
irritation. Inhalation: Move to fresh
air. Get medical attention
immediately if symptoms
occur.
Sodiu May be harmful Wear protective Eye contact: Rinse Handling: Avoid
m by inhalation, gloves, cautiously with water for generating dust by
Chlori ingest ion, or skin protective several minutes. excessive or unnecessary
de absorption. May clothing, eye Skin contact: Wash movement.
cause eye, skin, or protection and thoroughly with water. Storage: Store in a dry
respiratory system face protection. Ingestion: A large bodily location. Avoid contact
irritation. load may cause vomiting, with aluminum or carbon
diarrheal, cramps, steel to minimize
tingling in hands and corrosion.
feet, weak pulse, and
circulatory disturbance.
Administer water to the
patient. Ingesting will
usually cause purging of
the stomach by vomiting.
Get medical attention.
Inhalation: Move to
fresh air area.
 41

5.3 ENVIRONMENTAL ISSUE

In the production of our product, there are a few wastes generated which are the
wastewater, waste gases and biomass. The wastewater is produces in stream 5 from the
reaction between glucose, ammonium nitrate and oxygen throughout the process of
producing recombinant chymosin in the main fermentor (F-102). The wastewater is then
being released at stream 7. Besides, waste gases are also produced in the main fermentor
and emits at stream 4. Next, for the solid biomass it is separated from wsatewater during
filtration process in the rotary filter I (R-101). After that, the solid biomass is separated
from prochymosin in the rotary filter II (R-102) where it is then discharged at stream
10. If these wastes are discharged into soil, rivers and lakes, they will be causing
pollution. Furthermore, if they are poured into a waterway, or sewer, they can deplete
the water oxygen levels, causing serious environmental damage.
 5.3.1
Waste Generation

Figure 5.1 PFD for Waste Generation

42
 43

Table 5.2 Type of Waste

Type Stream Component Quantity (kg/h)

Solid 10 Biomass 1983.3
Liquid 7 Wastewater 2303.0
Liquid 15 Waste urea 914.0
Gas 4 Waste gases 12162.4

Not every of the waste from the production of our product need to be treated.
The wastewater, biomass and waste urea need treatments but as for the waste gases
which consist of nitrogen gas, oxygen gas and carbon dioxide, they do not need to
undergo any treatment. So, the waste that need to be treated from our production plant
are the wastewater, biomass and waste urea.

5.3.2 Waste Treatment

As the recombinant chymosin produces wastewater, waste gases, biomass and waste
urea as the wastes, they have to be treated in a proper way to minimize their waste and
prevent the risk of pollution.

For the wastewater generated, it needs to undergo the water treatment process.
This water treatment process can be conducted by the plants in Malaysia such as the
Sungai Semenyih Water Treatment Plant or by local companies for instance the Salcon
Engineering Berhad. Through the water treatment process, the wastewater will be
converted into bilge water that could be discharged back into the environment so that
the wastewater can be reused and hence conserved (BizCon, 2010).

Meanwhile for the biomass, it can be managed under the biomass waste-to-
energy plant. According to AltEnergyMag, the biomass wastes can be transformed into
clean energy by a variety of technologies. Besides recovery of substantial energy, these
technologies can lead to a substantial reduction in the overall waste quantities requiring
final disposal, which can be better managed for safe disposal in a controlled manner
while meeting the pollution control standards. Biomass waste-to-energy conversion
reduces greenhouse gas emissions in two ways. Heat and electrical energy are generated
 44

which reduces the dependence on power plants based on fossil fuels. The greenhouse
gas emissions are significantly reduced by preventing methane emissions from landfills.
Moreover, waste-to-energy plants are highly efficient in harnessing the untapped
sources of energy from wastes.

Lastly, for the urea waste, the most advanced and promising method of
decomposition of urea that can be used in industrial scale is the hydrolysis of urea
combined with the use of enzymes to increase its efficiency (W.J Appl Electrochem,
2016). This hydrolytic pathway can decompose urea into nitrogen, ammonium and
carbon dioxide before being released to the environment.

5.3.3 Regulation Act

According to Malaysia Environmental Act, it is stated that no person shall kill by way
of shooting any dog, cat or any other animals which may be prescribed by the Minister
by notification in the Gazette, unless it is authorized by the veterinary authority during
emergency or for the purposes of disease control specified under section 30.

In animal rennet, the active part of the enzyme used in the making of cheese is
chymosin. It is extracted from the stomach of calves that have not been weaned from
their mother’s milk. The calves are killed especially at a young age because if they get
older, the chymosin content in their body will start to decrease. Hence, an ethical issue
arose in slaughtering very young animals for protease sources. People for the ethical
treatment of animals (PETA) opposes the slaughtering of such animals.

Not with standing section 30, one of the exceptions for killing animals without
prohibition is unless the killing of animal is for human consumption. But in the long-
term effect, vegetarians might stop consuming cheese if they came to know that it
contains animal rennet. Due to this growing number of vegetarian diets, alternate
sources for animal rennet have been made available, allowing the supply of milk
coagulants to meet the demand for cheese production and providing alternative sources
 45

for vegetarians. Plant-based coagulants are also an appropriate alternative for animal
rennet for the vegetarian community as no controversial issues regarding animals will
be raised.

For gaseous released into the environment such as carbon dioxide and
nitrogen gas must comply with Environmental Quality (Clean Air) Regulations 1978 to
release the gas to atmosphere. This act required to control of air pollution according the
air emission standard. In the production of our product which is Chymosin, we must
follow the discharge limit based on Environment Quality (Industrial Effluent)
Regulations (3) 2009. This regulation applies only to industrial and services sectors’
wastewater and biomass discharges that conduct or perform activities regarded by the
law as processing, manufacturing, washing or servicing.
 46

CHAPTER VI

BIOCHEMICAL EQUILIBRIA

6.1 EQUILIBRIA CONSTANT

Equilibrium constant is defined as a number that expresses the relationship between the
amounts of products and reactants present at equilibrium in a reversible chemical
reaction at a given temperature. To determine the equilibrium constant, the following
steps is taken.

C6H12O6 + 0.398NH4NO3 + 1.898O2 → 3.186CH1.94O0.52N0.25 + 2.814CO2 +3.706H2O (3.2)

Table 6.1 Thermodynamic Data of each Component

Components Vi Gfø (kJ/mol) Hfø (kJ/mol)

Glucose -1 -917.22 -1264
Ammonium Nitrate -0.398 -184 -365.6
Oxygen -1.898 0 0
Biomass 3.186 -67 -91
Carbon dioxide 2.814 -394.4 -393.5
Water 3.706 -237.2 -285.8
Total 6.41 -1211.9148 -1046.901

(Source: Alberty, 1998)

 47

Table 6.2 Heat Capacity of each Component

Components A B C D
Glucose 1.9805 0 0 0
Ammonium Nitrate 2.5810 0 0 0
Oxygen 3.639 5.06 x10-4 0 -2.27 x104
Biomass 1.8553 0 0 0
Carbon Dioxide 5.457 1.05x10-3 0 -1.16x105
Water 3.47 1.45x10-3 0 1.21x104
Total 24.2123 7.35x10-3 0 -2.38 x105

(Source: Alberty, 1998)

The equation used to calculate heat capacity: Cp / R (J/mol. K) = A + BT + CT2 + DT-2

1 𝑇 ∆𝐶𝑝 1 310.15
∫ 𝑑𝑇 = 310.15 ∫298.15 24.2123 + 7.35 × 10−3 𝑇 − 2.38 × 105 𝑇 −2 𝑑𝑇
𝑇 𝑇∘ 𝑅

=0.9237

𝑇 ∆𝐶𝑝 𝑑𝑇 310.15
∫𝑇∘ = ∫298.15 24.2123𝑇 −1 + 7.35 × 10−3 − 2.38 × 105 𝑇 −3 𝑑𝑇 l
𝑅 𝑇

=0.9420

The temperature, T of biochemical reaction is 310.15K and operating at

atmospheric pressure 1 bar. We take the reference temperature, To = 298.15K.

By using the equation:

∆𝐺 𝑜 −∆𝐻 𝑜 ∆𝐻 𝑜 1 𝑇 ∆𝐶𝑝 𝑇 ∆𝐶𝑝 𝑑𝑇

− ln 𝐾 = + + 𝑇 ∫𝑇 𝑑𝑇 − ∫𝑇
𝑅𝑇𝑜 𝑅𝑇 𝑜 𝑅 𝑜 𝑅 𝑇

−1211.9148−(−1046.901) −1046.901
− ln 𝐾 = (8.3145)(298.15)
+ (8.3145)(310.15) + 0.9237 − 0.9420

𝐾 =1.6337
 48

The K value is larger than 1, the system favour the forwards reaction to form
products. Since it is an exothermic reaction in fermentor, the product formed can be
increased by decreasing the temperature, as a result, K also increased.

6.2 EQUILIBRIA COMPOSITION

Table 6.3 Composition of each Compenent

Glucose Ammonium Oxygen Nitrogen Biomass Carbon Water Total

Nitrate Dioxide
Vi -1 -0.398 -1.898 0 3.186 2.814 3.706 6.41
Nio 34.52 13.74 65.53 281.71 0 0 0 395.5

The composition of component shown as below:

34.52−ℰ 13.74−0.398ℰ 65.53−1.898ℰ

𝑥𝐺 = (395.5+6.41ℰ) ; 𝑥𝐴𝑁 = ( 395.5+6.41ℰ ) ; 𝑥𝑂2 = ( 395.5+6.41ℰ ) ;

281.71 3.186ℰ 2.814ℰ

𝑥𝑁2 = (395.5+6.41ℰ) ; 𝑥𝐵 = (395.5+6.41ℰ) ; 𝑥𝐶𝑂2 = (395.5+6.41ℰ)

3.706ℰ
; 𝑥𝐻2 𝑜 = (395.5+6.41ℰ)

Assume all components are in liquid phase and as an ideal solution. The extend of the
reaction,ℰ, can be determined by using equation:

∏(𝑥𝑖 )𝑣𝑖 = 𝐾

34.52−ℰ 13.74−0.398ℰ −0.398 65.53−1.898ℰ −1.898

(395.5+6.41ℰ) −1 × ( 395.5+6.41ℰ ) × ( 395.5+6.41ℰ ) ×
281.71 0
(395.5+6.41ℰ) ×

3.186 2.814 3.706

3.186ℰ 2.814ℰ 3.706ℰ
(395.5+6.41ℰ) × (395.5+6.41ℰ) × (395.5+6.41ℰ) = 1.6337
 49

By using trial and error method, substitute ℰ =0.9744

34.52−ℰ 34.52−(0.9744)
𝑥𝐺 = (395.5+6.41ℰ) = (395.5+6.41(0.9744))=0.0835

13.74−0.398ℰ 13.74−0.398(0.9744)
𝑥𝐴𝑁 = ( 395.5+6.41ℰ ) = ( 395.5+6.41(0.9744) )=0.0332

65.53−1.898ℰ 65.53−1.898(0.9744)
𝑥𝑂2 = ( 395.5+6.41ℰ ) = ( 395.5+6.41(0.9744) ) =0.1585

281.71 281.71
𝑥𝑁2 = (395.5+6.41ℰ) = (395.5+6.41(0.9744)) =0.7012

3.186ℰ 3.186(0.9744)
𝑥𝐵 = (395.5+6.41ℰ) = (395.5+6.41(0.9744)) =0.0067

2.814ℰ 2.814(0.9744)
𝑥𝐶𝑂2 = (395.5+6.41ℰ) = (395.5+6.41(0.9744))= 0.0079

3.706ℰ 3.706(0.9744)
𝑥𝐻2 𝑜 = (395.5+6.41ℰ) = 395.5+6.41(0.9744) =0.0090

∑ 𝑥 =0.0835+0.0332+0.1585+0.7012+0.0067+0.0079+0.0090=1

Table 6.4 Comparison of The Composition in Biochemical Equilibria, Material

Balance and SuperPro Result

Component Biochemical Material SuperPro Percentage Percentage

Equilibria Balance Result Error 1 Error 2
Glucose 0.0835 0.0473 0.2248 76.53% 62.86%
Ammnium 0.0332 0.0188 0.0188 76.60% 76.60%
Nitrate
Oxygen 0.1585 0.0896 0.0018 76.90% 98.86%
Nitrogen 0.7012 0.3856 0.3306 81.85% 112.10%
Biomass 0.0067 0.1506 0.1145 95.55% 94.15%
Carbon Dioxide 0.0079 0.1330 0.2011 94.06% 96.07%
Water 0.0090 0.1751 0.1084 94.86% 91.70%
 50

Percentage error 1 is calculated by using equation:

𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒𝑠 𝑖𝑛 𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝐵𝑖𝑜𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑎 𝑎𝑛𝑑 𝑀𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝐵𝑎𝑙𝑎𝑛𝑐𝑒

× 100%
𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑖𝑛 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑏𝑎𝑙𝑎𝑛𝑐𝑒

Percentage error 1 is calculated by using equation:

𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒𝑠 𝑖𝑛 𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝐵𝑖𝑜𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑎 𝑎𝑛𝑑 𝑆𝑢𝑝𝑒𝑟𝑃𝑟𝑜 𝑟𝑒𝑠𝑢𝑙𝑡

×100%
𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 𝑖𝑛 𝑆𝑢𝑝𝑒𝑟𝑝𝑟𝑜 𝑟𝑒𝑠𝑢𝑙𝑡

Based on the Table 6.4, shown percentage error 1 is differences in composition

between Biochemical equilibria and material balance whereas percentage error 2 is
difference between Biochemical equilibria and SuperPro result. Then, we can conclude
that the composition calculated in material balance by using Superpro and manual are
different than the composition calculated in chapter biochemical equilibria. Three
different method is used in calculating composition. In biochemical equilibrium, we
need to determine the equilibrium constant, K, in order to get the composition, the Gibbs
energy, enthalpy of formation and heat capacity is taken into considered. Besides, there
is some assumption to be made. We assuming all the component is liquid phase at ideal
condition. Since it is an endothermic reaction in fermentor, the product formed can be
increased by increasing the temperature, as a result, K, also increased.
 51

CHAPTER VII

BIOLOGICAL INFORMATION OF CHYMOSIN BIOCHEMICAL

7.1 INTRODUCTION

Chymosin is the principal milk-coagulating enzyme present in rennet. It is a protein that

consists of a single polypeptide chain of 323 amino acids with intramolecular disulfude
linkages. Commercial synthesis of calf rennet contains two form of chymosin which is
Chymosin A and B. The only difference between the two is that the amino acid at
position 286 in their polypeptide chain. Chymosin A has an aspartic acid residue while
Chymosin B has glycine residue on that position. Chymosin A slightly more active in
terms of proleotic activity compared to Chymosin B (El-Sohaimy et al., 2010). It is
suggested that the enhanced binding affinity of κ-casein through the stronger
electrostatic interactions between the substrate and the chymosin A is the reason for the
higher activity. Chymosin A is less stable than the B form, though the difference in
stability is marginal (Kumar, 2010).

7.2 CHEMICAL AND BIOCHEMICAL PROPERTIES

Escherichia coli is a facultative anaerobic, Gram-negative organism which is capable of

using a wide spectrum of organic carbon sources for heterotrophic growth. The presence
of electron acceptors provides energy production for fermentation or respiration.
Glucose is mainly imported and phosphorylated to glucose-6-phosphate using
 52

phosphotransferase system (Hunter and Kornberg, 1979; Escalante et al., 2012). The
glycolysis pathway processes the two molecules of pyruvate and the release of two ATP
and two NADH molecules. Under toxic conditions, pyruvate is then converted to acetyl-
CoA and carbon dioxide. Under these conditions, acetyl-CoA is further processed
within citric cycle to produce more ATP.

7.3 SELECTION OF PRODUCER CELL

The producer cell that will be used in this plant will be Escherichia coli K-12 HB101.
E.coli of this strain are facultative anaerobic, it can produce energy through both aerobic
and anaerobic respiration depending on the presence of oxygen. It is Gram negative,
rod shaped, and is nonmotile. On average, it is 2.85 μm long and 0.65 μm wide
(Rathgeber et al., 2005). E.coli cannot sporulate, hence simple boiling or basic
sterilization would eradicate them. E. coli K-12 HB101 is a laboratory strain, in which
it is grown in a laboratory environment, though its wild variant can be found naturally
in soil or aqueous environment.

E.coli is classified in the domain Bacteria due to the fact that it does not have
nuclei in which their DNA is stored. E.coli has a cell wall that contains peptidoglycan,
hence the classification of its kingdom in Eubacteria. Phylum Proteobacteria is one of
the major phylum for Gram-negative bacteria and E.coli is under the class
Gammaproteobacteria. E.coli is calssified under the order of Enterobacteriales, which
is a monotypic order and contains only one family that is Enterobacteriaceae. Members
of this family are rod-shaped. While most of them have many flagella for motility,there
are few genera that are nonmotile. Genus Escherichia is one of it (Castellani and
Chalmers, 1919).
 53

The classification of E. coli is as stated below:

Table 7.1 Taxonomic Classification

Domain Bacteria
Kingdom Eubacteria
Phylum Proteobacteria
Class Gammaproteobacteria
Order Enterobacteriales
Family Enterobacteriaceae
Genus Escherichia
Species E. coli

(Source: BacDive, 2019)

Besides E. coli, other possible cells that can be used to manufacture chymosin
are Kluyveromyces lactis and Aspergillus niger variant awamori.

Table 7.2 Producer Cells Comparison

Producer cell Eschericia coli Kluyveromyces lactis Aspergillus niger

variant awamori
Kingdom Bacteria Fungi Fungi
Optimal growth ̊
37 C 30 ̊C 35-37ºC.
temperature
Optimal growth pH 7 7-7.5 4-6.5
Type of chymosin Chymosin A Chymosin B Chymosin B
produced
Type of fermentation Aerobic fermentation Aerobic fermentation Aerobic fermentation
Location of chymosin Intracellular Extracellular Extracellular
produced
Acceptable Daily Intake Not specified Not specified Not specified

(Source: Pfizer Central Research, 1988)

 54

Fermentation process of E. coli starts by growing it in aqueous solution that

consists of suitable source of carbohydrates, nitrogen, mineral salts and miscellaneous
inorganic and organic compounds (Pfizer Central Research, 1988). The prochymosin
produced is an intracellular product, hence cell disruption is done to harvest the
inclusion bodies. The harvested inclusion bodies are washed with phosphate buffer
solution containing 1-4 M urea. The mixture is then being held for at least one hour in
a condition of pH under 2.0 to inactivate any residual E. coli cells. Addition of urea is
done next to dissolve the inclusion bodies followed by dilution with a buffer. The
solution is being held for 2 hours at pH 8.5-9.5 to allow renaturing of prochymosin
which is then activated into chymosin by holding it for one hour under pH 1.8-2.2. After
readjustment of pH to 5.5-6.0, the chymosin is absorbed on a suitable anion exchange
resin to purify it followed by elution.

Fermentation process for chymosin production using K. lactis consists of 3 steps

which laboratory propagation, inoculum fermentation and primary fermentation.
Prochymosin production occurs during primary fermentation where, in a fermentor
equipped with suitable devices for pH, oxygen and temperature controls, the production
strain is grown aerobically under aseptic condition. The process is stopped by adding
sulfuric acid and sodium benzoate after around 100 hours of fermentation. After the
yeast cells is killed, low pH level will activate the prochymosin into chymosin. Several
cell filtrations is done to purify it before being concentrated by ultrafiltration. The final
product is formulated with sodium chloride and sodium benzoate and may also be
formulated with glycerol, propyleneglycol or sorbitol (Gist-brocades, 1990).

For large scale production, Aspergillus niger var awamori cell is grown in
several stages to build up the inoculum. Cells are then grown aerobically under the
suitable condition of pH, temperature, nutrient composition, etc. The fermentation is
stopped after chymosin level reaches a desired amount. The fungal cells are inactivated
and separated from the liquid. The chymosin is recovered by filtering the broth followed
by chromatographic purification and concentration(Hansen, 1989).
 55

It is determined that E. coli is used in this work due to several reasons:

1. E. coli is one of the most studied microorganism with its gene sequence
have been discovered.
2. E. coli K-12 HB101 belongs to the Risk Group 1, which means it
represents little to no risk to an individual or towards a community
(Leibniz Institute).
3. Can be grown easily and inexpensively in laboratory settings due to its
ability to use various metabolite as its carbon source with positive
growth (Rathgeber et al., 2005).
4. Chymosin that is produce by E. coli K-12 HB101 has been evaluated as
safe for consumption (Pfizer Central Research, 1988).
5. Enzymatic properties of the chymosin produced by E. coli K-12 are
indistinguishable from native calf chymosin (Meisel & Frister, 1988)

7.4 CULTIVATION AND PROPAGATION OF CELLS

E. coli K-12 HB101 is used in the production of chymosin. The producer cells will
first be introduced with prochymosin A gene from bovine . E. coli K-12 strain HB101
growth in M9 medium which supplemented with glucose and ammonium nitrate
(Emtage et al, 1983). The temperature range for growth of this certain strain of E. coli
is as of mesophilic bacteria. Its growth temperature range is 4-37 ̊C and is at optimum
at 30 ̊C. The strain can grow at pH 6-11 and will grow at optimum pace at pH 8.5
(Rathgeber et.al, 2005). Hence, the productivity or yield product is at highest at30 ̊C
and pH 8.5.

7.5 TYPE OF CARBON SOURCE

Glucose is the best carbon source for E. coli because it provides the fastest growth than
any other sugars and is consumed first in sugar mixtures. The enzymes to metabolize
glucose are made constantly by E. coli.
 56

CHAPTER VIII

FLUID MECHANICS

8.1 FRICTION LOSS

Friction loss occurs in the pipe due to the effect of fluid’s viscosity near the surface of
the pipe and the movement of fluid molecules against each other and against the wall
of pipe. From the process flow diagram, pump between main fermenter (F-102) and
rotary filter 1 (R-101) is chose to be calculated. The inlet pressure of the pump is 1 bar
and the outlet pressure are 2 bar. The pump that we use is centrifugal pump. The reason
why we choose this pump is due to the low initial cost, low maintenance costs, simple
in operation, ability to operate under a wide variety of conditions and how it gives a
smooth, continuous flow that is free from pulsation. The temperature at both stream 5
and 6 are 310.15K. The flowrate of water is the greatest in the inlet stream, thus it is the
major component of the stream. So, the calculations are made based on the properties
of water
 57

Figure 8.1 Process Flow Diagram from Main Fermenter to Rotary Filter 1

8.1.1 Friction Loss in Pipe

The type of pipe that we use is commercial steel, schedule 40, 4-inch nominal diameter
(Appendix A.5, Geankoplis, 2014).

Mass flowrate of water, ṁw = 2303.00 kg/h = 0.64 kg/s

Density of water at 310.15K, ρw = 993.28 kg/m3 (Appendix A.2, Geankoplis, 2014).

Viscosity of water at 310.15 K, μw = 6.95 × 10−4kg/ms (Appendix A.2, Geankoplis,

2014).
 58

Internal diameter, D = 0.1023m

Total Length, ∆L = 11m

πD2
Area of pipe, A = 4

π(0.1023m)2
=
4

= 8.22 × 10−3 m2

ṁ𝑤
Velocity of the flow, v =
ρ𝑤 A

0.64kg/s
=
993.28kg/m3 (8.22×10−3 m2 )

= 0.08m/s

ρ𝑤 𝑣𝐷
Reynolds Number, NRe = 𝜇𝑤

(993.28kg/m.s)(0.08m/s)(0.1023m)
=
6.95×10−4 kg/ms

= 11696.41

≈ Since NRe ˃ 104, hence it is turbulent flow.

≈ Therefore, α = 1.0
 59

Assumption:

1. Size of pipe is the same from Main Fermenter to Rotary Filter 1, therefore
the velocity is constant from Main Fermenter to Rotary Filter 1, ⃗⃗⃗⃗
𝑣1 = ⃗⃗⃗⃗
𝑣2

2. Friction, ∑ 𝐹 in pump is negligible.

The total friction loss, ∑ 𝐹 include:

i. Contraction loss at the exit of Main Fermenter, hc

𝑨𝟏
For contraction from A1 to A2 cross-sectional area, = 𝟎, A1 of the Rotary
𝑨𝟐

Filter is very large compared to A2.

𝐴1
Kc = 0.55(1 − ) = 0.55(1-0) = 0.55
𝐴2

𝑣2 0.08m2
hc = Kc2𝛼 = 0.55( 2(1.0) ) = 1.76 x 10-3 J/kg

ii. Friction loss in the 4-inch straight pipe, Ff

For commercial steel pipe, 𝜺 = 𝟒. 𝟔 × 𝟏𝟎−𝟓 𝒎
Using the formula for rough pipes relative roughness, fanning friction factor is
find using the formula below,

𝟏 𝟔.𝟗 𝜺
= -3.6log10 [𝑵 + (𝟑.𝟕𝑫)𝟏𝟎/𝟗 ]
√𝐟 𝑹𝒆

𝟏 𝟔.𝟗 𝜺 𝟏𝟎/𝟗
= -3.6log10 [𝟏𝟏𝟔𝟗𝟔.𝟒𝟏 + (𝟑.𝟕(𝟎.𝟏𝟎𝟐𝟑𝒎) ]
√𝐟

1
= 11.51
√f
 60

√f = 0.087

f = 7.55 x 10-3

∆L = 2 + 3 + 1 + 4 + 1 = 11m

∆𝐿 𝑣 2 11m (0.08m/s)2
Ff = 4𝑓 = 4(7.55 × 10−3 ) ( ) = 0.01J/kg
𝐷 2 0.1023m 2

iii. Friction loss in 3, 90° elbows

From table 2.10-1 of Geankoplis, 2014, for turbulent flow, Kf for elbow = 0.75

𝑣2 (0.08m/s)2
hf = 3 Kf = 3(0.75) = 7.20 x 10-3 J/kg
2 2

iv. Expansion loss at the entrance of rotary filter 1, hex

𝐴3
For contraction from A3 to A4 cross-sectional area, = 0, A3 of the tank 1 is
𝐴4

very large compared to A4.

𝐴 2
Kex = (1 − 𝐴3 ) = (1-0)2 = 1
4

𝑣2 (0.08𝑚/𝑠)2
hex = Kex 2 = 1 = 3.2 x 10-3 J/kg
2

Therefore, the total friction loss, ∑ 𝐹:

∑ 𝐹 = ℎ𝑐 + 𝐹𝑓 + ℎ𝑓 +ℎ𝑒𝑥
 61

= 1.76 x 10-3 + 0.01 + 7.20 x 10-3 + 3.2 x 10-3

= 0.02 J/kg

8.1.2 Mechanical Energy Balance

By substituting value of ∑ 𝐹 from 8.1.1 into mechanical energy balance equation

(Geankoplis, 2014).

1 𝑃2 − 𝑃1
(𝑣22 − 𝑣12 ) + 𝑔(𝑧2 − 𝑧1 ) + + ∑ 𝐹 + 𝑊𝑆 = 0
2𝛼 𝜌𝑤

𝑤ℎ𝑒𝑟𝑒 𝑣22 − 𝑣12 = 0

1 (2 × 105 − 1 × 105 )Pa

(0) + 9.81m/s2 (4 − 2)m + + 0.02J/kg + WS = 0
2(1.0) 993.28kg/m3

WS = -120.32 J/kg

8.2 PERFORMANCE RATING OF PUMP

Assumption:

1. The reference point is pump position to the Main Fermenter and Rotary Filter

2. Z1 is the reference height of main fermenter from reference point.

3. Z2 is the reference height of rotary filter 1 from reference point.

 62

8.2.1 Suction Head Calculation

The suction head, H1 is determined using the formula

𝑉12 𝑃1
H1 = + 𝑔𝑧1 + + ∑ 𝐹1
2 𝜌𝑤

The total friction loss, ∑ 𝐹 include:

i. Contraction loss at main fermenter, hc

𝐴
Kc = 0.55(1 − 𝐴1 ) = 0.55(1-0) = 0.55
2

𝑣2 (0.08m/s)2
hc = Kc2𝛼 = 0.55 = 1.76 × 10−3 J/kg
2

ii. Friction loss in the 4-inch straight pipe, Ff

∆𝐿 = 2 + 3 = 5m

∆L v2 5m (0.08m/s)2
Ff = 4f = 4(7.55 × 10−3 ) (0.1023m) = 4.72 x 10-3 J/kg
D 2 2

iii. Friction loss in 1, 90° elbow, hf

From table 20-1 (Geankoplis,2014), Kf =0.75

𝑣2 (0.08m/s)2
hf = Kf = (0.75) = 2.4 x 10-3 J/kg
2 2
 63

Therefore, the total friction loss, ∑ 𝐹1 :

∑ F1 = hc + Ff + hf

= 1.76 × 10−3+ 4.72 x 10-3+ 2.4 x 10-3

= 8.88 x 10-3 J/kg

𝑉12 𝑃
Suction head, H1 = + 𝑔𝑧1 + 𝜌 1 + ∑ 𝐹1
2 𝑤

(0.08m/s)2 1×105 m
= 2
+ 9.81 m/𝑠 2 (2m) + 993.28 kg/𝑚3 +8.88 x 10-3 J/kg

= 120.31 m2/s2

8.2.2 Discharge Head Calculation

The discharge head, H2 is determined using the formula

𝑉22 𝑃
H2 = + 𝑔𝑧2 + 𝜌 2 + ∑ 𝐹2
2 𝑤

The total friction loss, ∑ 𝐹 include:

i. Friction loss in straight pipe, Ff

∆𝐿 = 1 + 4 + 1 = 6m

∆𝐿 𝑣 2 6m (0.08m/s)2
Ff = 4𝑓 = 4(7.55 × 10−3 ) (0.1023m) = 5.67 x 10-3 J/kg
𝐷 2 2
 64

ii. Friction loss in 2, 90° elbows, hf

From table 2.10-1 (Geankoplis,2014), Kf =0.75

𝑣2
hf = 2 Kf
2

(0.08m/s)2
= 2(0.75) 2

= 4.8 x 10-3 J/kg

iii. Expansion loss at the entrance of rotary filter 1, hex

𝐴 2
Kex = (1 − 𝐴3 ) = (1-0)2 = 1
4

𝑣2 (0.08m/s)2
hex = Kex 2 = 1 = 3.2 x 10-3 J/kg
2

Therefore, the total friction loss, ∑ 𝐹2 :

∑ 𝐹2 = 𝐹𝑓 + ℎ𝑐 + ℎ𝑒𝑥

= 5.67 x 10-3+ 4.8 x 10-3 + 3.2 x 10-3

= 0.01J/kg

𝑉22 𝑃
Discharge head, H2 = + 𝑔𝑧2 + 𝜌 2 + ∑ 𝐹2
2 𝑤

2 5
= (0.08m/s)
2
+ 9.81m/s 2 (4m) +
2×10 Pa
993.28 kg/m3
+ 0.01J/kg
 65

= 240.61 m2/s2

8.2.3 Pump Fluid Power Calculation

Pump head, ∆𝐻 = 𝐻2 − 𝐻1

= 240.61 – 120.31

= 120.30 m2/s2

∆𝐻 120.30m2 /s2
Pump head in unit length, =
𝑔 9.81m/s2

= 12.26m

Pump head calculation is 120.30 J/kg and 12.25m in unit length.

8.2.4 Pump Efficiency

Based on Geankoplis, 2014, efficiency of Centrifugal Pump, 𝜂 are given according to

their volumetric flowrate, Q (m/s3).

Volumetric flowrate that enters the pump are calculated using formula:

ṁ𝑤
Q= 𝜌

0.64𝑘𝑔/𝑠
= 993.28𝑘𝑔/𝑚3

= 6.44× 10−4 m3 /s

m3 60s
= 6.44 × 10−4 × 1 min
s
 66

= 0.04 m3/min

The efficiency for this pump, 𝜂 is determined by extrapolation based on the given range
of data below where our volumetric flowrate, Q is 0.04 m3/min,

Table 8.1 Efficiency of Pump according to Volumetric Flow Rate

Volumetric Flow Rate (m3/min) Efficiency (%)

0.075 50
0.19 62
0.38 68
0.76 75
1.89 82
3.80 85

Hence, the pump efficiency extrapolated from the data above is 46.35% (0.46) at 0.04
m3/min.

Calculation for the shaft work delivered the pump:

𝑊𝑃 = −𝑊𝑆 . 𝜂

= -(-120.32)(0.46)

= 55.34 J/kg

Pump power = 𝑚̇. 𝑊𝑃

= (0.64kg/s)(55.34J/kg)

= 35.42 W
 67

8.3 NET POSITIVE SUCTION HEAD (NPSH)A

The (NPSH)A by the pump in order to prevent cavitation for safe and reliable operation
of the pump. The available (NPSH)A of the system should always exceed the required
(NPSH)r to avoid vaporization and cavitation of the pump.

Total friction loss in suction head, ∑ 𝐹1 = 8.88 x 10-3 J/kg

Vapour pressure of water, Pvp = 6.31 kPa (Appendix 2, Geankoplis, 2014)

𝑃1 −Pvp 𝑉2
g(NPSH)A = + 𝑔𝑧1 − − ∑ 𝐹1
𝜌𝑤 2

(1×105 − 6.31 ×103 )Pa (0.08m/s)2

9.81(NPSH)A = + 9.81m/s2 (2m) − − 8.88 x 10-3 J/kg
993.28kg/𝑚3 2

(NPSH)A = 10.61m

Table 8.2 Comparison of Manual Calculation and iCON calculation

Manual Calculation iCON Calculation Percentage Error (%)

Total head, (m) 12.26 10.28 16.15
Efficiency, (%) 46.35 107.64 56.94
Pump Power, (W) 35.42 59.90 40.87
 68

8.4 PUMP PERFORMANCE CURVE

Volumetric Flowrate against Efficiency, Head loss

and (NPSH)A
90.00
Efficiency (%), Head loss(m), (NPSH)A(m)

80.00

70.00

60.00

50.00

40.00

30.00

20.00

10.00

0.00
0.000 0.500 1.000 1.500 2.000 2.500 3.000 3.500 4.000
Volumentric Flowrate,Q (m³/min)

Efficiency(%) Head loss (m) (NPSH)A (m)

Figure 8.2 Graph of Efficiency, Head Loss and NPSH against Volumetric
Flowrate
 69

8.5 iCON RESULT

Figure 8.3 Calculation on iCON for inlet Stream 5

Figure 8.4 Calculation on iCON for the value of Pump Head, Efficiency
and Power in the Pump
 70

Figure 8.5 Calculation on iCON for outlet Stream 6

 71

CONCLUSION

The major component in rennet is chymosin, it is a complex compound, a protein

formed of a single chain of 323 amino acids with intramolecular disulfide linkages. It
achieved its optimum activities at pH 5.3-6.3 and temperature between 44-55°C.
Chymosin is generally is used in dairy industry as a milk-clothing agent.

The global market of chymosin is expected to grow at CAGR of 4.3% from 2019
to 2025 which achieved 6.4% defficiency to the global demand. Our plant capacity is
850 kg/h as we planning to cater 0.0025% of the world’s demand of Chymosin.

E. coli K-12 HB101 is chosen as producer cell in our plant. E.coli of this strain
are facultative anaerobic, it can produce energy through both aerobic and anaerobic
respiration depending on the presence of oxygen. It is one of the most studied
microorganisms with its gene sequence have been discovered.

In terms of sustainability, waste products produced from our plant are waste
water, biomass, urea, and waste gaseous. Waste water, urea, and biomass is being
treated before expelled to the environment while the waste gaseous can released to
environment directly without treatment.

From the calculation of energy balance, 1.0403 x 109 kJ/h heat is being released
in the main fermenter. Apart from this, the process in rotary filter is exothermic process
which produced -5.7525 x 103 kJ/h of heat, thus, cooling jacket should be equipped at
the outer of rotary filter as well as the main fermenter to dissipate heat that being
released. For the chromatographer, it is an endothermic process which 3.476 x 10-5 kJ/h
amount of heat is being absorbed.

The equilibrium constant, 𝐾, calculated is 1.6337. Assuming all species are in

liquid phase and in ideal condition, by using trial and error method, the extend of
reaction, ℰ, calculated is 0.9744.
 72

LIST OF REFERENCE

Andreas H.Forster and Johannes Gescher. Front. Bioeng. Biotechnol., 23 May 2014 |
Metabolic engineering of Escherichia coli for production of mixed-acid fermentation
end products, from, https://doi.org/10.3389/fbioe.2014.00016

Anon. (2015). No Title. Retrieved September 19, 2019, from

https://gentleworld.org/what-is-rennet/

Anon. (2016). OUR PRODUCTS. Retrieved September 19, 2019, from

http://www.renconz.com/products/natural-enzyme-products/rennet/

Anon. (2017). Chymosin. Retrieved September 21, 2019, from

https://www.chemistryworld.com/podcasts/chymosin/3007682.article

Anon. (2018). Rennet Market (2018-2023): Size: Analysis: Forecasts: Industry Trends.
Retrieved October 2, 2019, from https://www.mordorintelligence.com/industry-
reports/rennet-market

Anon. (2019). Animal-Derived Chymosin Market 2019 With Top Countries Data:
Market Size, Industry Trends, Growth Insight, Share, Competitive Analysis, Statistics,
Regional, And Global Industry Forecast To 2025. Retrieved September 30, 2019, from
https://www.marketwatch.com/press-release/animal-derived-chymosin-market-2019-
with-top-countries-data-market-size-industry-trends-growth-insight-share-
competitive-analysis-statistics-regional-and-global-industry-forecast-to-2025-2019-
09-10

Anon. (2019). CarlinaTM Animal Rennet: For traditional cheese making - DuPont:
Danisco. Retrieved September 20, 2019, from
https://www.dupontnutritionandbiosciences.com/products/carlina.htm

Anon. (2019). Chymosin. Retrieved November 16, 2019, from

https://www.uniprot.org/uniprot/Q9GK11

Anon. (2019). CLAL, Rennet and Acid Casein, from

https://www.clal.it/en/?section=caseine [6 October 2019]

Anon. (2019). Maxiren®. Retrieved September 19, 2019, from

https://www.dsm.com/markets/food-specialties/en/products/dairy/maxiren.html

Anon. (5304). Calf Rennet - REN-NA. Retrieved September 18, 2019, from
https://www.ingredientsnetwork.com/calf-rennet-renna-

Anon. (n.d.). Centrifugal pumps.

Anon. (n.d.). Escherichia coli.

 73

Anon. (n.d.). Inorganic Compounds: Physical and Thermochemical Data. Retrieved

November 23, 2019, from
http://www2.ucdsb.on.ca/tiss/stretton/database/inorganic_thermo.htm

Anon. (n.d.). VIVO Pathophysiology. Retrieved September 20, 2019, from

http://www.vivo.colostate.edu/hbooks/pathphys/digestion/stomach/rennin.html

Apelblat, A., & Korin, E. (1998). The vapour pressure of saturated aqueous solutions
of D (H) -glucose, D (H) -galactose, and ␤ -lactose at temperatures from T s 278 K to
T s 318 K, 1263–1269.

Barbosa et al. (2007). Temperature and Concentration Dependence of Density of Model

Liquid Foods, 2912. https://doi.org/10.1081/JFP-120017815

Boyer H.W. & Roulland-Dussoix D. 1969. A Complementation Analysis of the

Restriction and Modification of DNA in Escherichia coli. J. Mol. Biol. 41(5): 459-
472.https://(doi.org/10.1016/0022-2836(69)90288-5)

Dickson et al. 1978. Purification and Properties of an Inducible, B-Galactoside Isolated

from the Yeast Kluyvermyces Iactic. Journal of Bacteriology 137(1): 51-61.

El-Sohaimy et al. 2010. Cloning and in vitro transcription of chymosin gene in E. coli.
Open Nutraceuticals 3(3): 63-68.

Emtage et al. (1983). Synthesis of calf prochymosin (prorennin) in Escherichia coli,

80(June), 3671–3675.

F.S.D Lin. (n.d.). CHYMOSINS A AND B FROM GENETICALLY MODIFIED

MICROORGANISMS. Retrieved November 22, 2019, from
http://www.inchem.org/documents/jecfa/jecmono/v28je08.htm

Geankoplis, C. J. (2014). Transport processes & separation process principles

principles (includes unir operations) (Fourth Edi). United State of America:
PEARSON.

Hammes, G. G. (2015). Standard Gibbs Energies and Enthalpies of Formation at 298 K

1 atm pH 7 and 0.25 M Ionic Strength, 465–466.

Hellmuth et al. (2019). Learn more about Rennet Microbial production of enzymes used
in food applications Enzymes Used in the Food Industry: Friends or Foes?

Kawaguchi et al. (1987). Production of Chymosin in Escherichia coli Cells and Its
Enzymatic Properties, 51(7), 1871–1877.

Kingston et al. (2014). Use of grape pomaces to produce biomass of a Komagataella

pastoris strain expressing a bovine chymosin activity. https://doi.org/10.1002/fsn3.128
 74

Kumar et al. (2006). Purification and characterization of milk clotting enzyme from
goat (Capra hircus), 145, 108–113. https://doi.org/10.1016/j.cbpb.2006.06.010

Kumar et al. (2010). Chymosin and other milk coagulants: sources and biotechnological
interventions. Retrieved November 23, 2019, from https://www.brenda-
enzymes.org/literature.php?e=3.4.23.4&r=708236

Meisel H. & Frister H. 1998. Chemical Characterization of a Caseinophosphopeptide

Isolated from in vivo Digests of a Casein Diet. Biological Chemistry Hoppe-Seyler
369(2): 1275-1280.

M.J.C. Crabbe. (2001). Rennets: General and Molecular Aspects, 1, 3652.

Montica Sawant. (2016). Industrial Production of Microbial Enzyms. Retrieved October

23, 2019, from https://www.slideshare.net/monticasawant/production-of-enzymes

Ozdemir et al. (n.d.). Collaborative Publishing. Retrieved from

https://www.wikigenes.org/e/gene/e/529879.html

Patent et al. (1994). United States Patent (19), (19).

Percival, S. L. and Williams, D. W. Microbiology of Waterbourne Diseases (Second

Edition), chapter 6- Escherichia coli., from, https://doi.org/10.1016/B978-0-12-
415846-7.00006-8

Rathgeber et al. 2005. Roseicyclus mahoneyensis gen. nov., sp. nov., an aerobic
phototrophic bacterium isolated from a meromictic lake. International Journal of
Systematic and Evolutionary Microbiology 55(4): 1597-
1603.https://doi.org/10.1099/ijs.0.63195-0

van den Berg et al. 1990. Kluveromyces as a host for heterologous gene expression:
expression and secretion of prochymosin. Nature Publishing Group 8(2): 135-
139.https://doi.org/10.1038/nbt0290-135
 75

APPENDIX

View publication stats