Final Compilation Design 2 PDF

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THE PRODUCTION OF RIBOFLAVIN
CHAPTER 1
INTRODUCTION
1.1 Project background
Vitamins are the nutrients which is needs by our body to function and fight off disease.
Shimizu (2011) reported that, vitamins are group of nutrients substances that necessary for
growth. The vitamins itself cannot be produced by our body, hence we must get them through
food or supplements. There are 13 vitamins that are important to our body. To know the
different types and understanding the purpose of these vitamins are quite important for good
health. Vitamins are divided into two types which are fat-soluble and water-soluble (Ricketts,
2013). Fat-soluble vitamins are stored in our fat cells, consequently requiring fat in order to be
absorbed. Meanwhile, water-soluble vitamins are not stored in our body, therefore, they need
to be replenished daily. Our body takes what it needs from the food we eat and then excretes
what is not needed as waste. Ricketts (2013) has listed all the vitamin types and some
common food sources as below:
1. The fat-soluble vitamins:

• Vitamin A - comes from orange colored fruits and vegetables; dark leafy greens, like kale
• Vitamin D - can be found in fortified milk and dairy products; cereals
• Vitamin E - is found in fortified cereals, leafy green vegetables, seeds, and nuts
• Vitamin K - can be found in dark green leafy vegetables and turnip/beet greens
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2. The water-soluble vitamins:

• Vitamin B1 (Thiamin) - come from whole grains, enriched grains; liver; nuts, and seeds
• Vitamin B2 (Riboflavin) - comes from whole grains, enriched grains, and dairy products
• Vitamin B3 (Niacin) - comes from meat, fish, poultry, and whole grains
• Vitamin B5 (Pantothenic Acid) - comes from meat, poultry, and whole grains
• Vitamin B6 (Pyridoxine) - comes from fortified cereals and soy products
• Vitamin B7 (Biotin) - is found in fruits and meats
• Vitamin B9 (Folate) - comes from leafy vegetables
• Vitamin B12 - comes from fish, poultry, meat, and dairy products
• Vitamin C - comes from citrus fruits and juices, such as oranges and grapefruits; red,
yellow and green peppers
Apart from that, this work is about to discuss specifically about vitamin B. Vitamin B
consists of eight types which are thiamin, riboflavin, niacin, biotin, pantothenic acid, pyridoxine,
folic acid and cobalamin. And those vitamins have their own function as shown in Table 1.1.
The vitamins B work together and have related roles in the body including their involvement in
the metabolism of carbohydrate, fat and protein and energy production (Carmenlita, 2014).
Carmenlita (2014) also said that, most of the vitamins B are water-soluble vitamins, which
means they aren’t stored in the body but instead any extra consumed is excreted through the
urine. Therefore, these vitamins need a continuous supply in our diet. B vitamins are found in a
variety of foods and deficiencies are not common. However, water-soluble vitamins can be
destroyed during food preparation, processing and storage. But, there are several ways to
prevent vitamins from being lost. For example;
• Avoid soaking produce in water.
• Cut produce in larger pieces – less exposed service area means fewer vitamins are
lost.
• Leave the skin on fruits and vegetables when possible, as most of the vitamins and
minerals are found just under the skin.
• Avoid overcooking vegetables.
• Steam or use the microwave vs. boiling vegetables in water.
• Keep milk in an opaque container when possible. If milk in a clear container is left out,
it will lose some of its riboflavin.
• Avoid rinsing grains before cooking – you’ll end up washing some of the nutrients right
down the sink.
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Table 1.1: All types of B vitamins and their functions

Vitamin Action in the body Good food sources Deficiency Excess
Vitamin B1 Helps convert carbohydrates into Dried milk, Egg, Enriched bread and Rare except in people who Excess vitamin B1 is excreted
(thiamin) energy; essential for functioning flour, Lean meats, Legumes, Nuts abuse alcohol. through the urine.
of the heart, muscles and and seeds, Organ meats, Peas,
nervous system. Whole grains
Vitamin B2 Growth, red blood cell production Dairy, Eggs, Green leafy vegetables, Rare except in those who Excess vitamin B2 is excreted
(riboflavin) and releasing energy from Lean meats, Legumes, Milk, Nuts, are severely through the urine.
carbohydrates. Fortified breads and cereals malnourished.
Vitamin B3 Helps the digestive system, skin Dairy, Eggs, Enriched breads and Rare; niacin is found in Excess niacin can result in
(niacin) and nerves to function; helps cereals, Fish, Lean meats, Legumes, protein rich foods. flushed skin, rashes, liver
convert food to energy. Nuts, Poultry damage.
Vitamin B4 Essential for growth and a Cereal, Chocolate, Egg yolk, Rare unless a person is Excess biotin is excreted
(biotin) component of enzymes that Legumes, Milk, Nuts, Organ meats eating raw egg whites through the urine.
break down fats & carbohydrate. (liver, kidney), Pork, Yeast frequently (a protein in raw
egg whites binds biotin
and prevents absorption).
Vitamin B5 Essential for growth and Avocado, Broccoli, kale, other Rare Large doses of pantothenic
(pantothenic metabolism; plays a role in the cabbage family veggies, Eggs, acid may cause diarrhea.
acid) production of hormones and Legumes and lentils, Milk,
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cholesterol. Mushrooms, Organ meats, Poultry,

White and sweet potatoes,
Whole-grain cereals, Yeast
Vitamin B6 Works as a coenzyme involved in Chickpeas, Beef liver, Yellowfin Rare Large doses of vitamin B6
(pyridoxine) over 100 enzymatic reactions, tuna, Salmon, Chicken breast, over time can cause nerve
particularly those concerned with Fortified breakfast cereals, Potatoes damage.
protein metabolism. Plays a role and other starchy vegetables,
in cognitive development, Turkey (meat only), Fruits (other
immune functioning, hemoglobin than citrus).
formation and metabolism.
Vitamin B9 Helps the body make and Fortified breakfast cereal, Beef liver, Though deficiency is rare, Excess folic acid may mask a
(folic acid/ maintain new cells. Pregnant Black-eyed peas, Spinach, women of child-bearing vitamin B12 deficiency. Some
folate) women need folic acid to help Asparagus, Enriched rice, Baked age have an increased medications may interfere with
prevent birth defects in babies. beans, Broccoli, Green peas, need for folate/ folic acid. the body’s absorption of
Enriched egg noodles folate.
Found in supplements as folic
acid. In food, it is found as folate.
Vitamin B12 Red blood cell formation, Animal products & some fortified Strict vegetarians and the Excess vitamin B12 is
(cobalamin) neurological function, DNA foods: Beef liver, Clams, Fortified elderly are at risk for excreted through the urine.
synthesis. breakfast cereals, Trout, Salmon, deficiency. Vegetarians
Haddock Yogurt, Beef, Tuna, Milk may not get it from their
food and the elderly may
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not be able to break

natural B12 from the
protein it is bound to be
able to use it.
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There is a large need for extra vitamins, and the quantities derived from plant and
animal food sources are not enough due to food shortage or disease (Chandra et al., 1996).
Added vitamins are now either prepared chemically or biotechnologically via fermentation
processes. Some vitamins, like riboflavin, are currently produced almost exclusively via
fermentation because several of the stages in the chemical process involve the use of toxic
reagents. Those processes shown Figure 1.1. The waste products, therefore, require
stringent environmental control and may need special forms of effluent treatment (Shimizu,
2001). Riboflavin, is one of the eight B vitamins that known as vitamin B2, is a water-soluble
vitamin that exist in most animal and plant tissues (Weil, 2016). According to Steven (2015),
all B vitamins help the body to convert carbohydrates (food) into glucose, which is used to
produced energy. Besides that, Carmenlita (2014) mentioned in her research that B vitamins
help form red blood cells, which means the cells is responsible for carrying oxygen to our
body’s tissues.
Chemical synthesis
Fermentation process
Glucose
Glucose
Molasses or soybean
Ribose
Xylidine
Fermentation
Ribamine
Aniline
Phenylazoribityl-amine
Barbitunic acid Riboflavin (in broth)
Raw riboflavin
Riboflavin (about 80% pure)
96% pure riboflavin
Figure 1.1: Chemical and fermentation processes for riboflavin synthesis (adapted from the
2001 Competition Commission report titled: BASF AG and Takeda Chemical Industries Ltd).
Steven (2015) reported that all these B vitamins, often referred to as B-complex
vitamins, which are necessary for healthy skin, hair, liver, eyes, and help the nervous system
function properly. Riboflavin is one of the essential B vitamins, known to help support adrenal
function, help calm and maintain a healthy nervous system, and facilitate key metabolic
processes, including helping to turn food into energy (Weil, 2016).
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CHAPTER 2
PROCESS BACKGROUND
2.1 Riboflavin Structure and Importance
The discovery of vitamin B2 and also known as riboflavin started with Alexander
Wynter Blyth member of Society for Public Analysts in 1872 (Burns, 2007). Wynter Blyth was
analyzing on the composition, structure and chemistry of dairy product where in the
observation of pigment in milk with yellow-green fluorescence can be traced (Clewes &
Thurnham, 2012). The research is continued by Frederick Gowland Hopkins where in the
early 1900, he investigates on the nutritional need by animal and human. Then follow by
Elmer McCollum where in 1916 he found water-soluble B in milk, egg yolk and wheat embryo,
and fat-soluble A. Then Paul György research on the important of vitamin B2 in 1932. György
reported that vitamin B2 may be essential for growth promoting and anti-pellagra factor
(Clewes & Thurnham, 2012). After that Harriette Chick and Margaret Honora Roscoe inspect
Elmer McCollum literature in 1938 (Clewes & Thurnham, 2012). They found that the
water-soluble B contains two type of vitamin which is vitamin B1 and vitamin B2. In 1939, a
research was conduct on 18 women conduct by William Henry Sebrell and Roy Edwin Butler.
A diet of low riboflavin was receiving by 13 women where they developed a reddened,
denuded lesion of the lips, maceration and fissuring of the angles of the mouth, and
seborrheic accumulations at the nasolabial folds. This finding proves that the important of
riboflavin to maintaining the human health. The colour of riboflavin is yellow or orange-yellow
where it can be found the food product which is milk, cheese, leafy green vegetables, liver,
yeast, almonds, and legumes such as mature soybeans.
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Moreover, it is used as a food colouring and to fortify some foods like baby foods,
breakfast cereals, and pastas, and processed cheese (Graham et al., 2005). To fuse pure
riboflavin in liquid, it is quite difficult due to the poor solubility in water. Therefore, the form of
soluble riboflavin is more expensive due to the riboflavin-5’-phosphate. It is the principal form
in which riboflavin is found in cells (Bacher et al., 2000).
Figure 2.1: Structure of Riboflavin.
Riboflavin was first isolated from milk in 1879 by Blyth and was called lactoflavin. It
has the empirical formula C17H20N4O6 and the structural formula is shown in Figure 2.1.
Riboflavin usually included in multivitamin and B-complex vitamins which come separately in
25mg, 50mg and 100mg tablet. Pure riboflavin has needle-shaped, practically odorless,
orange-yellow crystals, which begin to darken at about 240°C (464° F) and completely
decompose at about 80°C (536° F). Water solutions show a characteristic yellowish-green
fluorescence. Riboflavin is slightly soluble in water (12 milligrams in 100 milliliters at 27.5°C;
19 milligrams at 40°C.) and in several organic solvents. It is very soluble in alkali. Solutions are
relatively stable to acid, but riboflavin is readily destroyed by alkali and light. In neutral water
solutions, the compound exhibits a characteristic light absorption spectrum, with maxima at
445, 365, 265, and 220 millimicrons.
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Table 2.1: Recommended Amount of Riboflavin according age and gender

Life Stage Recommended amount
Birth to 6 months 0.3 mg
Infants 7–12 months 0.4 mg
Children 1–3 years 0.5 mg
Teen boys 14–18 years 1.3 mg
Teen girls 14–18 years 1.0 mg
Men 1.3 mg
Women 1.1 mg
Pregnant teens and women 1.4 mg
Breastfeeding teens and women 1.6 mg
In humans, signs and symptoms of riboflavin deficiency include cracked and red lips,
inflammation of the lining of mouth and tongue, mouth ulcers, cracks at the corners of the
mouth, and a sore throat (Graham et al., 2005). A deficiency may also cause dry and scaling
skin, fluid in the mucous membranes, and iron-deficiency anemia. The eyes may also
become bloodshot, itchy, watery and sensitive to bright light. In animals, riboflavin deficiency
results in lack of growth, failure to thrive, and eventual death. Other uses of riboflavin is to
increasing energy levels by boosting the immune system function, maintaining hair
health, skin, mucous membranes, and nails. Some research shows that it also may be
slowing down the aging process. In sport, it can boost up the athlete’s performance.
Riboflavin is important, but it needed the right amount to be consumed in daily life. The
amount also depend on the age and gender, below is the recommended amount for
consuming. Riboflavin also needs a proper storage due to the light sensitive and unstable in
alkaline solutions. The dry solid is stable to diffuse light, but is highly photolabile in solution,
especially in alkaline solutions. Neutral and acidic solutions are stable in the dark, but it will
decompose 3% per month at 27°C at pH 6.0.
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2.2 Process selection
2.2.1 Production of Riboflavin by Fermentation using Bacillus subtilis
This section describes the Bacillus subtilis bacterium. A major focus is on the
biosynthesis of riboflavin and on the strategies for obtaining strains of this bacterium with
up-regulated riboflavin productivity for the production of industrially relevant amounts of
vitamin B2. Bacillus subtilis is a gram positive, aerobically growing, rod-shaped bacterium, as
illustrated in Figure 2.2. The wild-type Bacillus is motile and able to form endospores for the
survival under hostile conditions (Priest, 1993). Species of the genus Bacillus play a
dominant role in industrial bioprocesses. They produce a variety of different products, such
as the fermented soybean product natto, several enzymes, insecticides, purine nucleotides,
vitamin B2 (riboflavin), and the flavor agent ribose.
Figure 2.2: Gram-positive Bacillus subtilis
The advantage of using Bacillus species for industrial purposes is that many of them
are generally regarded as safe (GRAS) by the US Food and Drug Administration and that
they grow rather fast which allows short production cycles (Schallmey et al., 2004). In
addition, the physiology and the genetics of Bacillus subtilis are well described, which allows
directed metabolic engineering of the producer strains. In order to produce riboflavin,
the Bacillus subtilis is cultured under a suitable condition.
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In detail, the Bacillus subtilis is inoculated onto a conventional medium containing a

suitable carbon source, nitrogen source, and inorganic compounds and cultured at a
predetermined temperature and pH under an aerobic condition. Examples of the carbon
source include glucose, molasses, lactose, sucrose, maltose, dextrin, starch, mannitol,
sorbitol, and glycerol. Preferably, glucose is chosen. Examples of the nitrogen source include
an inorganic source such as ammonia, ammonium chloride, and ammonium sulfate and an
organic source such as peptone, NZ-amine, beef extract, yeast extract, corn steep liquor,
casein hydrolysate, fish or fish meal, and defatted soybean cake or meal. Preferably, yeast
extract and corn steep liquor are chosen. Examples of the inorganic compounds include
potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate,
ferrous sulfate, manganese sulfate, and calcium carbonate. When needed, vitamins and
auxotrophic bases may be use (Eren & Mazor, 2016). For the culture, shaking culture or
stirring culture by aeration may be used. The culture temperature ranges from 30 to 45°C.,
and preferably 37 to 40°C. During the culture, the pH is adjusted to a fairly neutral level. The
culture period is 5 to 6 days.
The Bacillus subtilis is inoculated onto a seed medium and cultured at an aeration
flow rate of 1 vvm, 37°C., and 8,000 rpm for 20 hours. The seed culture is inoculated onto a
fermentation medium and subjected to shaking culture at an aeration flow rate of vvm, 40°C.,
800 rpm, and pH 7.0 for 60 to 70 hours. During the shaking culture, the fermentation culture
was supplied with a glucose supplement medium to maintain the residual glucose in the
culture to a level of 0.5 to 1% until the total content of glucose in the fermentation culture
reached 20%. Riboflavin was yielded at an increased level of about 18.4%, when compared
to the parent strain (Lee, 2006).
2.2.2 Production of Riboflavin by Fermentation using Candida famata
Riboflavin know as vitamin B2 can be producing using microorganism. There are 75

bacterial species been identified that can help the process. This are some bacterial that
commonly used in the process making of riboflavin which are Candida Famata, Ashbya
Gossypii, Clostridium Acetobutylicum, Eremothecium ashbyii and Mycobecterium
smegmatics. The riboflavin naturally contains in food source such as sugar cane, soybean,
egg, tempeh and many more with small amount. Within the technologies, the riboflavin can
be produce using chemical and biological techniques in the large volume (Seong,2001).
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Figure 2.3: The comparison of sensitivity for three type bacterial which is C.famate, D.
hansenii and Saccharomyces cerevisiae. (Kostyantyv, 2012)
The Candida Famata is belong to the group of Flavinogenic yeast which belong to the
families of yeast and fungi. Other name for Candida Famate is Candida flareri and it is most
flavinogenic yeast capable of overproduction during iron starvation. The reason for in
phonological role of riboflavin overproduction is unknown. The other reason for stimulation of
riboflavin production by iron reduction where the riboflavin act as an electron donor or iron
reduction act as a co-factor for the activity of intra and extracellular enzyme (Seong, 2001).
The osmotolerance for Candida Famate is high due to the yeast can adapt in high
concentration of NaCl up to 2.5M compared to Saccharomyces cerevisiae cannot present in
hypersaline condition compare to Candida Famate have been isolated from natural
hypersaline environment.
Hypersaline is a condition whereas contain high concentration of salt or sodium

chloride. The C.famate is one of species halotorent where the reason it can adapt hypersaline
condition. The advantage in biotechnology, the C.famate can be non-sterile production and
high product concentration. In this condition, can reduce the production cost (Kostyanty,
2012). The Figure 2.3 shows the experiment that done by the scientist where all three
bacterial were it be cultivate overnight on rich YPD medium.
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The sample been diluted at 1, 0.1, and 0.01. After that supply with 2m NaCl and
incubated 30oC for 4 and 8 days. Besides that, Figure 4 shows the process of C. famate by
using the glucose as the carbon source. The carbon will convert to ribulose-5-phosphate
(ribu-5P), precursor of L-3, 4-dihydroxy-2-butanone-4- phosphate (DBP). The
3-Phosphoglycerate, an intermediate of glycolysis, is also used for the synthesis of glycine
that is the precursor of GTP. The biosynthesis starts from GTP and end with product which is
riboflavin (Seong,2001).
Candida famate Glucose
Glucose
Ribu-5P
GTP
DRTP
ARP
DBP
DMRL
RIBOFLAVIN
Riboflavin
Figure 2.4: The process of Candida Famate to riboflavin (adapted from Enorch).
The Table 2.2 shows the three-major type of microorganism that use to produce
riboflavin with are bacteria, yeast and fungi. For the bacterial, it contains 2 type which are
Clostridum actobutylicum and Bacillus subrillis. Follow by yeast, Candida flareri (C.famate),
candida guilliermondii. For the fungi, 2 types that are commonly used which are
Eremothecium ashbyii and Asbhy Gassypii. For the Candida Flareri (C.famate), the riboflavin
that produce only 0.6 g/L by using glucose as carbon source. Compare to other the highest
produce riboflavin was Ashbya gossypii with is 5.5g/L. The less amount was Clostridum
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actobutylicum and Bacillus subrillis which is 0.1g/L. The media components that needed
producing riboflavin by using C.famate are glucose, urea, Monopotassium phosphate
(KH2PO4), magnesium sulfate (MgSo4) biotin, trace element, agar and distilled water. As for
the media fermentation process, the component need is the same as the media but only the
difference is the amount of components. For other chemicals that involve during the
fermentation are concentration ammonium sulphate solution, phenol crystals, diethyl ether
and lastly surfactanks- SLS and Triton 100x (Patil,2014).
Table 2.2: Three types of microorganism that use to produce riboflavin (Seong, 2001).
The C. famate must incubate at 28oC at 200rpm for 6-7 days. After the component
been mix, the fermentation broth was extraction. Next, the broth been heated at between 70
to 800oC for 39 minutes, the ammonium sulphate was added and centrifuge the broth at
between 5000 to7000rpm to separate precipitation. Add the phenol to the supernatant and
centrifugal once more at between 1000 to 2000rpm. The phenol is added constantly shaking
and centrifugal. Add water and ether shake and centrifugal. The riboflavin is detecting after
the separation water layer from ether layer (Patil, 2014).
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2.2.3 To check the riboflavin sample
Qualitative detection of riboflavin is using the thin layer chromatography (TLC). For
this technique, saturated solvent system consists of n-butanol, acetic acid and water
(40:10:50). Next, load with standard riboflavin and with the samples (water layer contains
riboflavin) for 6 to 8 hours. After the TLC is dry, the plate is observing for fluroscence at UV
short 254nm and UV long 365 nm in the ultraviolet fluroscence chamber. (Patil, 2014)
2.2.4 Improvement of Riboflavin Production Using Mineral Support in the Culture of

Ashbya gossypii
In this process for the production of riboflavin (vitamin B2), compared with other strain
that produce the same product, Ashbya gossypii give the highest yield of production. Ashbya
gossypii is a fungus that was first discover by Ashby and Nowell as a plant pathogen in 1926
that causes Stigmatomycosis which is a disease in a fungal form resulting in a wet, slimy
kernel or in fruit such as cotton (Gossypium hirsutum) or subtropical citrus fruits. The disease
itself is transmitted by insect mouthparts that penetrate the crops, however this disease is not
that severely a devastating plant pathogen because it is controllable where usually and
insecticides will be applied to control the pathogen.
An advance research shows that A. gossypii has an ability to produce a large quantity
of riboflavin which is responsible for its yellow colour (Demain, et.al, 1972; Stahman, et.al,
2000). Later then, riboflavin rank as one of the most successful commercial product as it can
be used not only as vitamin and food additive. Riboflavin also has a lot of contribution in food
colouring in soft drinks and dairy products. It is also found that, there were no negative cases
riboflavin are taken in an excess amount. Nowadays, more innovative research was done to
developed riboflavin production by using A. gossypii to increase the yield. The riboflavin itself
is soluble in water, other than used for animal’s consumptions, it also can be used for plants.
It plays an important role in live organisms because it is a precursor which are functioning as
coenzymes for wide variety of enzymes in metabolism. Usually in humans, riboflavin used to
avoid some deficiency symptoms like dermatitis (Demain, et.al, 1972; Stahman, et.al, 2000).
In this research of riboflavin production, according to Lim and his colleagues, they
found that by adding a mineral support, the concentration of riboflavin attained was 2.5 g/l in 4
days culture period. This result shows them that the production of riboflavin by using the
mineral support is 1.6 times higher than a culture without the addition of mineral support. In
this research, they enhanced their study by studying the effect of mineral support on the
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riboflavin production and mycelial morphology variation, intracellular oil droplets were
investigated by staining mycelia with Nile red. The A. gossypii were mixed with other material
in the culture medium along with soybean oil as the carbon source for its growing factor.
When soybean oil adsorbed on mineral support then added into the culture, the size of A.
gossypii getting thicker which then promote more riboflavin crystals, compared with a culture
that has no mineral support. The improvement of riboflavin production using mineral support
in the culture of ashbya gossypii research were done in a pilot scale production. In this
research, the production of riboflavin was done in a 5 L fermenter or usually called as
bioreactor. Table 2.3 are the chemicals material used in this production by using a. gossypii
strain. The strain used in this research which is A. gossypii grown in a 30 oC in a 1 litre of solid
medium containing of 10 g yeast extract, 3 g of glycine and 20 g of agar. The pH of the
medium was controlled and set to 6 which was then cultivated for two days and then stored in
a 4 oC environment.
Table 2.3: Material used in this production by using a. gossypii strain.

Materials
Glucose Cobalt (II) chloride
Soybean oil Magnesium sulfate heptahydrate
Gelatine Manganese (II) chloride tetrahydrate
Corn steep liquor Glycine
Potassium dyhydrogenphosphate Tween 80
Threonine Myo-inositol
Zinc sulfate heptahydrate Yeast
Potassium hydroxide Mineral support (AID-PLUS ML-50D)
The strain can be used then to produce inoculum or seed culture for flask culture first,
where in a litre of distilled water a total of 30 g corn steep liquor, 9 g yeast extract and 15 g of
soybean oil. The seed medium pH adjusted to 6.8 and then a total of 100 ml was taken from
the medium to be transfer into 500 ml shaking flasks. The seed was then cultivated for 40 hrs
with an agitation speed of 200 rpm at 28 oC. Generally, in riboflavin production process mostly
used a biological process which is usually can be called as a single step procedure. The
name of single step procedure usually came for a fermentation process as it only need a
short amount of step to get the desired product. In this research, the procedure does apply
the single step procedure, but it can be divide into much smaller step that involves in the
production of riboflavin. In fermentation, especially in this research process, after developing
the strain, then it need to be developed more or to be prepared first for the strain to reach it
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optimum reactivity. Therefore, a medium need to be prepare first. In medium preparation, the
preparation can be divided into two with the same types of medium. One medium to be used
for the inoculum to develop or for it to be active and the other medium was for the main
fermentation. The preparation of medium depends on the medium volume, where weight of
chemicals, volume of strains and also the volume of glucose and distilled water to be mixed.
Medium for inoculums usually prepared in a smaller quantity while for the main fermentation
the medium will be prepared in a larger quantity.
The next step comes after the medium preparation, are the sterilization process. In
sterilization process, all equipment, chemicals and medium need to be sterilized to minimize
the probability of contamination. Sterilization process also comes in different types of
sterilization depends on what types of material that need to be sterilize. For the medium and
equipment or apparatus, these materials usually sterilize by using wet steam or usually called
as autoclave in 121 oC for 15 minutes. While for the mineral support, the sterilization was
done by washing it first with phosphate buffer (pH = 7), then rinsed it two times and dried
overnight at 105 oC. After that, the soybean oil will be introduced with mineral support to be
adsorbed where after the adsorption between these two-material done it need to be
autoclave before adding it to the culture broth. The main fermentation usually divided
depends on the types of product to be produced. In this process, the main fermentation can
be divided into three where the first process was the Seed Culture 1, the second process was
the Seed Culture 2 where an amount of 1 ml taken from the seed culture 1 to be used in the
seed culture 2 that will then have cultivated in a rotary shaker at 200 rpm for 7 days at 28 o C,
and last but not least, the main fermentation or the production medium will run in a 5 L jar
fermenter. The condition of the fermenter need to be control especially for aeration, agitation
range, pH, temperature, and other parameters that can influenced the production process.
After some time, the product harvesting includes with filtering or centrifuging or
sometimes by decanting the broth. In this production, the broth will be filter then the filtrate
can be stored in -20 oC. Filtrate produce, or product produce need to be treated to reach it
highest purity. In analytical method or also called as the treatment of product, the product was
then washed. In the filter medium, there may be a lot of biomass collected in the filter where
this biomass usually called as mycelia. The determination of dry cell mas can be calculated
by subtracting the mineral support amount with the dried washed cell. The residual soybean
oil concentration was then extracted by using solvent extraction, mixed and centrifuged then
eliminate upper layer and dried it to determine the amount of oil extracted. In this part the
riboflavin was then measure and test with HPLC at a wavelength of 444nm. Lastly, the
product was dried for a better quality of product.
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CHAPTER 3
MARKET SURVEY
3.1 Market Study
Vitamin tonics help in strengthening and invigorating the body tone of an individual.
These tonics are used to prevent and treat several deficiency conditions such as rickets,
osteomalacia, angular stomatitis, and anemia. The tonics are available in several
formulations such as syrupy semisolids, liquids, capsules, tablets and pills, and multivitamin
injections. However, the liquid tonics are more popular compared to the others due to higher
patient compliance and acceptance amongst the children. The physicians prescribe these
products in vitamin deficiency anemia such as folate deficiency anemia, pernicious anemia
and vitamin C deficiency anemia. Another significant reason for increasing demand of these
products is hectic lifestyle of people across the globe. The global vitamin tonics market is
segmented into the following which are hospitals clinics, public clinics, private clinics, retail
pharmacies and drug stores and e-commerce (Shekhar, 2016).
In the global medical industry, market demand for vitamin B are mostly found at North
America, U.S, Europe, U.K, Asia Pacific, China, Japan, India, Latin America, Brazil, Middle
East and Africa. The U.S. market for the vitamin tonics is growing albeit with slowed growth
due to consumer questions about the actual benefits of the products and several studies
indicating that a large number of companies are simply selling placebos with minute
quantities of actual vitamins. Therefore, dominance of North America into global vitamins
market is being challenged by developing regions such as Asia, Latin America and Africa.
The market in emerging geographies is growing at a rapid pace owing to rising middle class
in countries such as China and India (Thomas, 2008).
19
Global demand for vitamins and provitamins is forecast to expand 2.6% annually
between 2014 and 2018. Belgium, China, Netherlands, Slovenia, and the United States are
considered the highest potential markets in the coming years. Based on global analysis in
2015, 97% of the U.S supplement users take vitamin and minerals. The highest demand is
multivitamin which is 77% followed by vitamin D 29%, calcium 26%, vitamin C 24% and the
least is vitamin B which is 20%. According to statistics by a leading international market
research company, Global Research & Data Services, the expansion of the global vitamin
and provitamin industry is forecast to reach 2.6% annually in the coming years. Between
2007 and 2013 the market increased with an average annual growth of 8.0%.
Currently, vitamin E accounts for 37.0% of the global demand while the remaining
market share is divided between vitamin C (17.4%), vitamin A (5.2%), vitamin B1 (4.4%),
vitamin B6 (4.0%), vitamin B12 (3.8%), vitamin B3 and B5 (3.0%), vitamin B2 (2.7%) and
other vitamins and provitamins (22.5%) (Sudeep, 2014). China, India, Japan, Singapore, and
the United States represent the largest vitamin and provitamin markets while the strongest
annual growth is forecast to occur in Slovenia (42.2%), Belgium (22.0%), Netherlands
(14.2%), Panama (11.3%), Slovakia (10.3%) (Sudeep, 2014).
There are two difference formula for riboflavin which are C17H20N406 (riboflavin) and
C17H20N4NaO9P (riboflavin-5-phosphate). For C17H20N406, the market price analysis is RM53,
040.00 per 200 kg. There are 11 country that produce this product which are China, United
States, Britain, Germany, India, Canada, Japan, France, Korea, Norway and Slovakia. The
package level come in difference weight which are milliliter and milligram up to ton. One of
the company that produce this product is Hubei Zhonglong Kangsheng Fine Chemical
Co.,Ltd located in Wuhan Hubei China. The product purity is 99.0% and the molecular weight
is 376.36400g/gmol. The company price for this product is RM 515.68 for one ton and one
cycle produce 20 ton. Besides, the minimum shipment of riboflavin is 2 days. Meanwhile, for
Shanghai Aladdin Bio-Chem Tehnology Co.,LTD company that located in Pudong New Area
Shanghai, China, the product purity is 98.0% with the same amount of molecular weight. This
company sells riboflavin in different weight which are 25g, 100g and 500g. The price for 25g
is RM78.90, 100g is RM264.54 and 500g is RM988.53. The shipment of riboflavin for
company Shanghai Aladdin Bio-Chem Technology Co,.LTD take 7 days and the purity is 98%
(Molbase 2013).
20
Figure 3.1: Vitamin and provitamin market size compared to market growth in different
countries. (Shekhar, 2016).
The market price analysis for C17H20N4NaO9P (riboflavin-5-phosphate) is RM2422.16

and the molecular weight is 478.326g/gmol. There are 6 country that produce riboflavin which
are China, United States, Britain, Germany, India and Korea. China is the largest supplier in
global market. Hubei Wei De Li Hua Xue Keji You Xi is one of the company that produce
riboflavin and located at Wuhan Hubei, China. The purity of this product is 76% and the price
is RM832. 42 for 25kg. Besides that, the duration for shipment take 7 days. Other than that,
Bide Pharmatech LTD is one of the company that produce riboflavin and supply to the global
market. This company sells riboflavin for RM 339.99 at 25kg. The purity of this product is
95.0+ % and the shipment duration is 3 days (Molbase, 2013).
21
3.2 Pricing
Pricing of materials used in this experiment comes in a different price depends on how
large is the quantities of materials to be purchased. All the market price shown in Table 3.1.
Table 3.1: Raw materials price

Raw Materials Price, RM
Glucose • 100 G  126.50

• 1 KG  242.50
• 5 KG  560.00
• 10 KG  752.50
• 25 KG  1375.00
Yeast [for microbial growth] • 250 G  305.00
• 1 KG  1157.50
Ashbya gossypii Strain • Not provided
• Produced by Mizusawa
Mineral support (AID-PLUS ML-50D)
Chemical CO., Niigata
Japan.
Others • An estimated price for all
other component to be
added during the
production 20 K (+ 6 K)
22
3.3 Site Survey
The site location for the production is selected based on several factors which involve
strategic location, raw material, technical, social and environmental impact and effluent
disposal. The geographical location of the production plant is a major factor that will
contribute to the development of selected site. The transportation of the product must
considerate based on the safety and the cost of expansion should be minimum. The final
selection for the site location should consider the advantages and disadvantages state
whereas different environment gives different impact. The factors will be explained as below:
1. Location
2. Raw materials
3. Technical
4. Social
5. Environment impact and disposal factor.
3.3.1 Location selected
The site location is the main factor for the success of a new company. Kawasan
Perindustrian Sri Gading 2 is selected for the production of the riboflavin due to the near
residential area. The product can be promoted and commercial to the nearby area which can
increase the sales of product thus give benefit to the company.
Figure 3.2: Chosen site; Chosen site; Kawasan Perindustrian Sri Gading, Batu Pahat, Johor
23
Based on the analyses location; “Kawasan Perindustrian Sri Gading 2” can be used to
manufacture riboflavin. The site also seems near with the residential area which can provide
labor. In addition, there have access with high amount of sugarcane, Johor seem to be the
ideal location to manufacture riboflavin which in Batu Pahat. Whereas the other two locaton
seem be disadvantages. In Negeri Sembilan the scale of the farm seems to be in medium
and to manufacture in Sarawak where there only one farm that produce sugarcane, if there
where shortage occur and the cost for the sugarcane transportation can be high.
3.4 Raw Materials
The raw materials are playing an important role in the production of riboflavin, this is
one of the major factor that influent in choosing site location. The main raw material for
production riboflavin is the glucose, where it can be obtained near the port or state of Johor.
Johor is the state that are popular with the development of industries, hence it is easier to get
the transportation and saving transportation cost. Malaysia Holding Berhad (MSM) is one of
the company that produce sugar (glucose, maltose and sucrose) in Tanjung Langsat, Johor.
So, this company can provide the raw material (glucose). The glucose is being chosen for the
production due to the cheaper price compare to sugar cane and it is commonly used in the
fermentation production which very suitable for riboflavin.
Table 3.2: Comparison prices of raw materials (Dewi, 2007)

Types Prices (RM)
Glucose 4.00 (per kg)
Sugar cane 15.00 (120g)
24
3.5 Technical.
For technical part, the equipment, machine and raw materials is influence by the
transportation. The transportation is very important to transport the raw materials, products,
equipment and machines; thus, the site location should have good facility. The example of
facility are road, airport and cargo. China is the higher demand of the riboflavin thus the
facility of road is importance. As the result, this production is forces on the oversea.
Besides, the expired period of the riboflavin is short because it is easily contaminant. For long
distance transport should reduce the delivery time by having a good condition facility and also
the safety of the products. Because the Batu Pahat is near to the airport and also seaport
(cargo) which make a good site location for this plant. The company such Shanghai Aladdin
Bio-Chem Technology Co, LTD is one of the supplier riboflavin and the period of the delivery
is 7 days. The location of the company is strategic due to the near area of shipping port
therefore, the cost of transportation can be reduced.
3.6 Environmental Impact and Disposal.
The environmental is a critical point to identify the location based on the safeness and
suitable disposal materials. The safety can be seen in terms land which describe a flat
ground, available of water, telecommunication capacity, possible environmental remediation,
sewer and solid waste disposal. As for the land, the government will interfere as the company
should know capital gain and losses, cost of compliance and time delay. Ethanol and pyruvic
acid is a certain example of waste production riboflavin. This product cannot dispose directly
to the land nor sea due to harm the environment and people near the resident area. This
waste is category as biological hazard therefore to discard the waste should follow the
description.
3.7 Social
With high population in the area will provide a job opportunity to the community and
most of the professional’s workers live at near city. To build a new company, a professional’s
worker should obtain to construct the production, therefore under skill worker can be train by
the professional to improve the quality of the products. Hence, the market of riboflavin is
increase due to the trusted of society by the products.
25
3.8 Comparison between the site locations
Factors Ladang tebu, Chuping, Perak Kawasan Perindustrian Sri

Gading 2
Raw materials The raw materials such as glucose can The glucose can obtain from
obtain from the MSM Perlis Sdn. Bhd MSM Johor Sdn. Bhd which
which located in Chuping Perlis. located in Johor
Locations near to The Chuping, Perlis is near the Thai Batu Phata is near to the port
the marketing area border. Therefore, it is a strategic site to which easy to obtain supplier
market the product to overseas such as and market to overseas. As a
Thailand. conclusion, the transportation is
one of the critical point for the
production of riboflavin.
Availability of The land is not commercial due to less This area is advanced with the
suitable land expose to technology, communication technology and transportation
and facility of the transportations are due to the nearby town.
lack. Therefore, the raw materials,
marketing products and obtain
the equipment is convenient.
Availability of This area is consist less residential due As the nearby town, the
labors to far from the city. population is large compare to
the Chuping, Pelis. As
conclusion, this provide a job to
the community
26
CHAPTER 4
EQUIPMENT DESCRIPTION
4.1 Process Flow Diagram
Figure 4.1: Process flow diagram of this plantation project of riboflavin production.
27
Figure 4.2: Process flow diagram (reference) from general studies (Waghmare et al., 2012).
As one said Vitamins are the nutrients needed by human body to be highly functional
as well as to fight off disease where it cannot be produce by human body itself other than by
taking it from other sources to be consume where then it will be divided into two types of
vitamins which is water soluble and also fat-soluble vitamins (Levine, 1935). Simply taking or
consuming any food and fruits for vitamins are not enough, this is because the concentration
of those vitamins content in fruits and foods much more lesser than we think and also the
amount may affect by the ways of handling and storing the food. Some vitamins can be
destroyed when exposed to bright light, heat and even can be washed away from too much of
washing (Anonymous A, 2017).
As technology keep on improving from time to time, Scientist were able to produce a
Pharmaceutical Vitamins product that can be consume just by itself in form of powder, drinks
an even tablets or pellets that has more high concentration of vitamins needed with a high
stability. In collaboration with engineering technology, each process of vitamins production
was monitored, studied and stabilized in order to get a high pharma-grade product. In this
design where it focuses more on the production of Riboflavin or also known as vitamin B2.
The production of riboflavin comes in two different method of production which is chemical
and also biological, but for safety purposes a biological methodology is more preferable.
In this design based on Figure 4.1 and 4.2, the production of riboflavin in a year gives
a total of 1000 tons/year of product. Figure 4.1 is the edited version process flow diagram
(PFD) in this project, while the Figure 4.2 is the reference PFD from general studies. The
process is divided into two major processes which is downstream processing and upstream
processing. In upstream processing, the preparation and sterilization of medium are needed,
where in preparation it involves with the development of the A. Gossyypii strain until the
28
development of inoculum. While preparing medium and other nutrients, be careful not to mix
the medium or biomass with the other nutrients during the sterilization as a reaction may be
happening and reduced the efficiency of production process. The sterilization usually will be
will take about 15 to 20 min in 121oC (Mohaghegh, 2015). Before entering the fermentation
phase, biomass will be mix first with other components such as the biomass itself, water and
nutrients needed, after mixing, as it is a simple step fermentation process, along the way to
the fermentation tank, the mixed medium will be sterilized one more time to minimize any
contamination then all materials will be mixed along with the inoculum of A. Gossyypii.
In the fermentation vessel, the process is being controlled with a few parameters that
will enhance the production process such as pH, temperature, agitation, dissolved oxygen
and other mechanical control such as fixed agitation (Wilkins et al., 2011). The fermentation
process period may vary depends on what types of product that need to be produced but
usually it will take about 8 to 9 days of fermentation excluded the strain and inoculum
development.
In downstream processing, the process usually involves with harnessing of product,

separation and also the sterilization of product. In harnessing usually, the product will be
stored first in a storage tanks then centrifuged to separate any large materials where only
biomass content is needed to be processed in the sterilization process. In this design, a total
of two dryer used to sterilize the product from any moisture content, the first dryer usually
used to dry the product up to a feed grade only, but then when it comes with a pharmaceutical
product that need a about 96% to 99% of purity a second downstream processing were done.
In pharma grade purification, the product will be mixed with active charcoal that will adsorb
any unwanted material that will affect the purity of product. the separation of active charcoal
that bring unwanted material will be washed away. A mixing of product was needed before
centrifuging the product since mixing will promote the efficiency of centrifugation of product to
be separated in a more purity product. Last but not least, the process of drying will be done
again as for pharma grade, the moisture content need to be lesser than in feed grade
riboflavin. The last form of product will be in powder form since it is easier to be transport,
dilute and absorbed by the body.
29
4.2 Description of The Equipments
4.2.1 Blending Storage Tank
The blending or mixing tank is commonly used to blend difference component

together through refined mixing. When the component is mixing, resulting a good of
mass production. During the mixing product of riboflavin, each of chemical need to put
individually in the tank to reduce hazardous substance reaction. The tank is made with
difference materials such as copper, plastic, stainless steel and rubber (James, 2014).
The most suitable for production of riboflavin is the stainless-steel due to create less
wastage compare to the manual mixing or other material. Besides, stainless steel
mostly used in the industry because the product can store in the tank such as chemical,
water and food.
The other reason using this type because the tank also durability of the tank and
can store outshine. It’s also resistant to corrosion in comparison to ordinary steel and
remains an attractive alternative for utilizing in any industry that uses corrosive
materials. Furthermore, stainless steel tanks highly stain resistant metal alloy, and is
considered an environment friendly material. It also is easy to clean, hygienic and
cost-saving storage alternatives. In comparison to other tanks, they are more
advantageous because of established physical and chemical attributes (Emanuel,
2017). The mixing tank is vertical cylindrical tanks which about 1000 gallons that have
baffle with the diameter of 25 m and height if 40 m.
This plant design project process flow diagram (as shown in Figure 1) consist
two units of mixing tank which has a diameter of 2.84 m and a height of 8.52 m with an
operating capacity of 750,000 L that runs usually about 4 to 5 days. The tank is
connected between the main source units and other tank (next stage) with pipeline. The
condition of pipeline should easy to clean and can be vacuum-operate with help of a
control system working at the main part of the mixing tank. The mixing system have
difference range which is from low to high-end performance (James, 2014).
Based on process flow diagram, the mixing tank (P-4/V-102) pipeline connected
to the main source are provided the component such as palmitic acid and yeast extract.
The component is in liquid form for an easier mixing process. Disadvantage of mixing
30
tank (stainless steel) are leaking, leaching and expensive. The leaking will only occur
after it has been used for ages where the corrosives compounds will contribute on this
disadvantages towards the tank. Next, a leaching phenomenon will occur when pores
form on the wall of the tank that will contributes on microorganism growth. Last but not
least, the building material of the tank itself is expensive as it is made from stainless
steel. However, stainless steel’s maintenance is easy to maintain due to its durability
when compared with other tank building materials.
4.2.2 Seed Fermenter
In seed fermenter the mixed liquor of palmitic and yeast extract from the
blending tank were then channeled into it to be mixed and inoculated with the selected
microorganism that will increase the production time by seeding it first before using it for
the biggest production of riboflavin in the fermenter. This is a closed tank equipped with
a jacket or coils by which the tank contents may be maintained at a uniform temperature
of 28°C. In the bottom of the tank are fine-porosity stones or perforated coils through
which compressed sterile air is supplied. A mechanical agitator assists in providing
adequate air distribution (Tanner, 1946). A total of 4 units of seed fermenter tank are
needed in this plant project where each tank has a total capacity of 74,350 L with a
diameter and height of 3.16 m and 7.482 respectively. The running time of these seed
fermenter took about 6 to 7 days before it is being used for the main fermentation
process.
4.2.3 Fermenter
In the fermenter, the fermented seed which has been inoculated with Ashbya
gossypii preferably operated at a temperature about 37°C. The fermentation process if
possible must be operated under a condition that will prevent the introduction of iron
and contaminating organisms. The fermenter itself has a capacity of 700,000 L with a
diameter and height of 5.79 m and 17.37 m respectively with a total of 6 units of
fermenter operating in this plant design project. A suitable inoculum may be prepared
from a stock culture by repeated transfers to a nutrient medium. An alkaline or acidic
reagent such as sodium hydroxide (NaOH) and hydrochloric acid (HCL) will be added to
the fermenter to maintain the pH of the medium to the desired pH which is around 6 to
7. Generally, a batch fermentation process can continue from 1 weeks to 2 weeks, but if
31
the production itself involved with a large amount of substrate, the fermentation time
also will increase. This plant project runs for about 31 to 32 days of fermentation which
is equivalent to a month of fermentation process. The gases formed during fermentation
can be vented. The solvents formed during fermentation were then undergoes for the
downstream processing which is the purification of product. The purification of the
product formed during the fermentation was done by separating the by-product with the
product by using several types of purification equipment such as decanter centrifuge,
ultrafiltration, ion-exchange chromatography and last but not least since the product
itself which is riboflavin need to be packed in powder form, therefore it need to undergo
a drying process where it will be dried by using a drum drier.
Besides that, there is an air compressor which has a function to force the air into
the reactor. The compressor need to generate sufficient pressure to force the air
through the filter, sparger holes and into the liquid. The air compressor used typically
produce air at 250kPa. The air should be dry and oil free so that the inlet air filter does
not contaminate the medium. The instrument air is generated at higher pressure but is
aspirated with oil and cannot be used in fermentation system (Kavitha, 2006).
4.2.4 Centrifuge (Decanter)
The centrifugation serves to separate the desired product which is the riboflavin
from the other by-products of the mixture product from the fermentation process where
the separation process are basically only separates large particles first by concentrating
the product in eliminating large amount of moisture. In this case, the separation process
will be separated by using decanter centrifuge (DC) which will separate Biomass +
Yeast extract + Water from the Riboflavin. The separation process by using DC will be
done twice where there will be two units of DC arrange one after another. The first DC
has a capacity of 2,773,920 L with a diameter and height of 10.56 m and 31.68 m
respectively. While for the second DC has a capacity of 562,840 L with a diameter and
height of 6.20 m and 18.6 m respectively. After each of centrifugation process, the
separated denser layer is richer in riboflavin than that of the previous centrifugation.
The undesired product will be separated as waste water. The temperature of the
mixture which consist of riboflavin, biomass and aqueous medium which is centrifuged
need to be maintain in the range 15°C to 80°C but for practical purposes, during the first
of several centrifugations, it is normally run in the range of 20°C to 40°C. During
32
centrifugation, the suitable relative centrifugal force (RCF) is in the range of about 2500
xg to 3000 xg and it is preferred from 2600 xg to 2800 xg (Hoffman, 1996). However, in
this plant design project the rotation speed of the DC is calculated in revolution per
minute (RPM) where for the first and second DC it gives a reading of 460.18 rpm and
600.56 rpm respectively.
The suitable centrifugation equipment which can be referred as decanter

centrifuge commercially available and well-suited to the purpose at hand and can be
used for equipment the parameters such as, the differential speed between bowl and
scroll, the weir diameter and the flux rate of the feed or liquid discharge can be varied in
relation to each other to achieve optimum results. It has been found that 2-4
centrifugations, particularly 3 centrifugations, suffice to afford, after separation, a
riboflavin material of sufficient purity to render it in a suitable condition for further
purification in the subsequent process steps (Hoffman, 1996).
4.2.5 Ultrafiltration
In ultrafiltration, the separation on purification process of product continued by

separating more fine materials. In decanter centrifuge (DC) the separation are basically
only separates large particles, but in ultrafiltration it is used to separates more fine
materials by using water as the washer or force to filter the product from passes from
the second DC. In this plant design project, the calculated capacity of the ultrafiltration
used is 3,861.54 L with a diameter and height of 4.46 m and 13.38 m respectively. As
riboflavin is known to be exist in a crystalline form when it losses most of its moisture,
an ultrafiltration provides an effective separation of the riboflavin from the smallest
soluble and suspended cellular moieties contained in the slurry, therefore giving a high
yield of riboflavin and also purer product. Separation process by using ultrafiltration is
preferred as it have the effectiveness of separation that resulting in highly pure product.
The washer uses which is water act as a dialysis where it is preferably conducted by
using soft water to gives a much more higher purity of riboflavin. The lower the solids
concentration in the water for dialysis, the higher the purity of final riboflavin product.
After the ultrafiltration process was done, further purification is done by using
ion-exchange chromatography to separate those unseparatable materials.
33
4.2.6 Ion Exchange
In Ion-exchange chromatography is a purification process where the working

principle for the separation to happen is done by separating the unwanted materials
with the riboflavin by using ionic charge. In this plant design project, the ion-exchange
column has a capacity of 176,910 L with a diameter and height of 4.22 m and 12.66 m
respectively. The ion-exchange operated in anion manner where it will bind with the
remaining by-product which in this plant design project it will bind wit glucose leaving
only water and riboflavin to pass freely and undergoes the last purification process. The
ammonium hydroxide will be used as the washer to wash the membrane after the
ion-exchange has done it job.
4.2.7 Drum Dryer
In riboflavin production, it is preferred to use drum dryer which is also called as a

roller dryer or film drum dryer. In this plant design project, the plant will require two units
of drum drier with a capacity of 246,350 L with a diameter and height of 4.7 m and 14.2
m respectively. The drum dryer construct from a hollow roller that have smoothly
polished external surface that can heated internally by steam. The roller rotates in
longitudinal axis. The placed known as feed pan is where the liquid dried. The roller
picked up the liquid as it rotates covering the surface in a thin film is removed
mechanically by a scrapper known as doctor knife. The construction of the drum dryer
theoretically consists of a hollow steel drum of 0.6 to 3 m diameter and 0.6 to 4.0 m
length which is horizontally mounted and its external surface is smoothly polished for
the easy removal of the dried cake. The drying of the riboflavin is done by the process
of steam when passed in to the drum which the temperature should be around 200 C to
35o C (Harned, 1967). Due to metallurgic nature of the drum the heat absorption is
more. The dried materials are scrapped by the knife and that falls in to the bin where the
purity of the riboflavin ican reach up to 99%. Therefore, leaving only water as waste to
be recycle. Saraswathi (2016) reported that the advantages of drum dryer it takes less
time to dry, where thermos sensitive’s drugs can also be dried, occupy less space. To
reduce the temperature of drying the drum can be enclosed in a vacuum chamber.
Rapid drying takes place due to rapid heat and mass transfer and the disadvantages
using drum dryer are the maintenance cost is high, need skilled operators are essential
to thickness control of the film and it is not suitable less solubility products.
34
4.2.8 Gas Compressor
Gas compressor act in a way to concentrate or compress the air first to

countable materials in a form of volume for an easier filtration and delivery. In this plant
design project, both ammonia and oxygen are being compressed and filter before being
used in the fermentation process. The working pressure of each gas compressor are 5
bar which is also equivalent to 500 kPa. There were four units of gas compressor used
in this plant design project, two in the seed fermenter and the rest in the fermenter.
4.2.9 Air Filtration
In this project, a total of six air filtration were used to filter compressed air before
entering and those air that being vented after fermentation process. Four of the air
filtration is used to filter compressed air (Ammonia and Oxygen), where two of them
placed in the seed fermenter and the other two in the fermenter. While the other two air
filtration placed on the venting of each seed fermenter and fermenter. The purpose of
filtering air before entering the medium is to eliminate microbes from relatively dry air
(Perkowski, 1983) while before releasing any gas vented the filtration once again
applied in order to lessen air odor from the fermentation process.
35
CHAPTER 5
MATERIAL BALANCE
5.1 Mass Balance
❖ Production per year = 1000 tone/year

❖ According to OSHA regulation; working days = 330 days
❖ Complete process;
• Preparation of medium = 4 days
• Fermentation process = 500 hours ×
1 𝑑𝑎𝑦
= 20.83 ≈ 21 𝑑𝑎𝑦𝑠
24 ℎ𝑜𝑢𝑟𝑠
• Cleaning process = 3 days

Total complete process = 28 days
330 𝑑𝑎𝑦𝑠 𝑏𝑎𝑡𝑐ℎ

❖ Total batch per year = = 11.79 ≈ 12
28 𝑑𝑎𝑦𝑠 𝑦𝑒𝑎𝑟
𝑡𝑜𝑛𝑒𝑠 𝐼 𝑘𝑔 1 𝑦𝑒𝑎𝑟 𝑘𝑔
❖ 1000 𝑦𝑒𝑎𝑟
=× 0.001 𝑡𝑜𝑛𝑒𝑠
× 12 𝑏𝑎𝑡𝑐ℎ
= 100,000 𝑏𝑎𝑡𝑐ℎ
𝑘𝑔
❖ 𝑅𝑖𝑏𝑜𝑓𝑙𝑎𝑣𝑖𝑛 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 100,000 𝑏𝑎𝑡𝑐ℎ
𝑘𝑔 1 𝑘𝑚𝑜𝑙 𝑘𝑚𝑜𝑙
❖ 𝑇𝑜𝑡𝑎𝑙 𝑚𝑜𝑙 𝑅𝑖𝑏𝑜𝑓𝑙𝑎𝑣𝑖𝑛 = 100,000 𝑏𝑎𝑡𝑐ℎ
× 376.36 𝑘𝑔
= 265.70 𝑏𝑎𝑡𝑐ℎ
36
5.2 Stoichiometry Balance
𝑂𝑖𝑙 + 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 + 𝐴𝑚𝑚𝑜𝑛𝑖𝑎 𝑠𝑎𝑙𝑡𝑠 → 𝑅𝑖𝑏𝑜𝑓𝑙𝑎𝑣𝑖𝑛 + 𝐶𝑂2 + 𝐻2 𝑂 + 𝐵𝑖𝑜𝑚𝑎𝑠𝑠
𝐶16 𝐻32 𝑂2 + 𝑎𝐶6 𝐻12 𝑂6 + 𝑏𝑂2 + 𝑐𝑁𝐻3 → 𝑑𝐶12 𝐻20 𝑁4 𝑂6 + 𝑒𝐶𝑂2 + 𝑓𝐻2 𝑂 + 𝑔𝐶17 𝐻1.8 𝑂0.5 𝑁0.2
𝑌𝑃 = 0.18
𝑆
𝑑(𝑀𝑊 𝑅𝑖𝑏𝑜𝑓𝑙𝑎𝑣𝑖𝑛)
=
1 (𝑀𝑊 𝑂𝑖𝑙)
𝑑(376.36 𝑘𝑔/𝑘𝑚𝑜𝑙)
=
1(256.43 𝑘𝑔/𝑘𝑚𝑜𝑙)
0.18(256.43)
𝑑= = 0.1226
376.36
𝑌𝑋 = 0.82
𝑆
𝑔(𝑀𝑊 𝐵𝑖𝑜𝑚𝑎𝑠𝑠)
=
1 (𝑀𝑊 𝑂𝑖𝑙)
𝑔(24.6)
=
1 (256.43)
0.82(256.43)
𝑔=
24.6
𝑔 = 8.5477
According to Papagianni (2007), given;
𝐶𝑂2 𝑒
𝑅𝑄 = = = 0.66
𝑂2 𝑏
𝐶16 𝐻32 𝑂2 + 𝑎𝐶6 𝐻12 𝑂6 + 𝑏𝑂2 + 𝑐𝑁𝐻3 → 𝑑𝐶12 𝐻20 𝑁4 𝑂6 + 𝑒𝐶𝑂2 + 𝑓𝐻2 𝑂 + 𝑔𝐶17 𝐻1.8 𝑂0.5 𝑁0.2
37
𝐶 𝑏𝑎𝑙𝑎𝑛𝑐𝑒 ∶ 16 + 6𝑎 = 17𝑑 + 𝑒 + 𝑔
𝐻 𝑏𝑎𝑙𝑎𝑛𝑐𝑒 ∶ 32 + 12𝑎 + 3𝑐 = 20𝑑 + 2𝑓 + 1.8𝑔
𝑂 𝑏𝑎𝑙𝑎𝑛𝑐𝑒 ∶ 2 + 6𝑎 + 2𝑏 = 6𝑑 + 2𝑒 + 𝑓 0.5𝑔
𝑁 𝑏𝑎𝑙𝑎𝑛𝑐𝑒 ∶ 𝑐 = 4𝑑 + 0.2𝑔
𝑑 = 0.1226
𝑔 = 8.5477
𝑅𝑄: 𝑒 = 0.66𝑏
5.2.1 C balance
16 + 6𝑎 = 17(0.1226) + 0.66𝑏 + 7.5053
16 + 6𝑎 = 2.0842 + 0.66𝑏 + 7.5053
16 − 2.0842 − 7.5053 + 6𝑎 = 0.66𝑏
6.4105 + 6𝑎 = 0.66𝑏
6.4105 + 6𝑎
𝑏=
0.66
𝑏 = 9.7128 + 9.0909𝑎
5.2.2 H balance
32 + 12𝑎 + 3𝑐 = 20𝑑 + 2𝑓 + 1.8𝑔
32 + 12𝑎 + 3𝑐 = 20(0.1226) + 2𝑓 + 1.8(8.5477)
32 + 12𝑎 + 3𝑐 = 2.452 + 2𝑓 + 15.3859
14.1621 + 12𝑎 + 3𝑐 = 2𝑓
𝑓 = 7.0811 + 6𝑎 + 1.5𝑐
𝑓 = 7.0811 + 6𝑎 + 1.5(1.9915)
𝑓 = 7.0811 + 6𝑎 + 2.9873
38
5.2.3 O balance
2 + 6𝑎 + 2𝑏 = 6𝑑 + 2𝑒 + 𝑓 + 0.5𝑔
2 + 6𝑎 + 2(9.7128 + 9.0909𝑎) = 6(0.1226) + 2(0.666) + 𝑓 + 0.5(8.5477)
2 + 6𝑎 + 19.4256 + 18.1818𝑎 = 0.7356 + 1.32𝑏 + 𝑓 + 4.2739
16.4161 + 24.1818𝑎 = 1.32𝑏 + 𝑓
16.4161 + 24.1818𝑎 = 1.32(9.7128 + 9.0909𝑎) + 𝑓
16.4161 + 24.1818𝑎 = 12.8209 + 11.9999𝑎 + 𝑓
3.5952 + 24.1818𝑎 = 𝑓
𝑓 = 3.5952 + 24.1818𝑎
Therefore, compare H balance with O balance;
7.0811 + 6𝑎 + 2.9873 = 3.5952 + 12.1819𝑎
6.4732 = 6.1819𝑎
𝑎 = 1.0471
5.2.4 N balance
𝑐 = 4𝑑 + 0.2𝑔
𝑐 = 4(0.1226) + 0.2(8.5477)
𝑐 = 2.1999
Therefore;
𝑏 = 9.1728 + 9.0909𝑎
𝑏 = 9.1728 + 9.0909(1.0471)
𝑏 = 19.2319
39
Therefore;
𝑑 = 0.1226
Therefore
𝑒 = 0.66𝑏
𝑒 = 0.66(19.2319)
𝑒 = 23.6931
Therefore;
𝑓 = 41164 + 12.1819𝑎
𝑓 = 4.1164 + 12.1819(10471)
𝑓 = 16.8721
Therefore;
𝑔 = 8.5477
5.3 Calculation of materials consumed
5.3.1 Glucose consumed
mol Glucose
= mol Riboflavin × total mol Riboflavin × MW Glucose
1.0471 kmol Glucose kmol Riboflavin kg Glucose

= 0.1226 kmol Riboflavin × 265.70 batch
× 180 kmol
kg Glucose
= 408,471.49 batch
40
5.3.2 Oil consumed
mol Oil
= mol Riboflavin × total mol Riboflavin × MW Oil
1 kmol Riboflavin kg Oil

= × 265.70 × 256.43
0.1226 batch kmol
kg Oil
= 555,737.77 batch
kg Glucose kg Oil kg total

∴ 408,471.49 batch
+ 555,737.77 batch
= 964,209.26 batch
5.3.3 Ammonium salt (NH3) consumed
mol NH3
= mol Riboflavin × total mol Riboflavin × MW NH3
2.1999 kmol Riboflavin kg NH3

= × 265.70 × 17.031
0.1226 batch kmol
kg NH3
= 81,197.78 batch
5.3.4 Pure O2 consumed
mol O
2
= mol Riboflavin × total mol Riboflavin × MW 02
19.2319 kmol Riboflavin kg O

= 0.1226
× 265.70 batch
× 32 kmol2
kg O2
= 1,333,746.38 batch
5.4 Calculation of materials produced
5.4.1 Biomass produced
g Oil consumed 964,209.26 kg Oil⁄batch

total mol substrate (oil) = MW Oil
= 256.43 kg Oil⁄kmol
total mol substrate (oil) = 3,760.13 kmol Oil⁄batch

41
Amount of Biomass produce
mol Biomass
= × total mol substrate × MW biomass
mol substrate
8.5477 kmol Oil kg Biomass
= 1
× 3,760.13 batch
× 24.6 kmol
kg Biomass
= 790,655.39
batch
5.4.2 Riboflavin produced
Amount Riboflavin produced
mol Riboflavin
= mol substrate
× total mol substrate × MW Riboflavin
0.1226 kmol Oil kg Riboflavin

= 1
× 3,760.13 batch
× 376.36 kmol
kg Riboflavin
= 173,498.93 batch
5.4.3 Water (H2O) produced
Amount H2O produce
mol H2 O
= × total mol substrate × MW H2 O
mol substrate
16.8721 kmol Oil kg H2 O

= 1
× 3,760.13 batch
× 18 kmol
𝑘𝑔 𝐻2 𝑂
= 1,141,943.21
𝑏𝑎𝑡𝑐ℎ
5.4.4 Carbon dioxide (CO2) produced
Amount CO2 produced

mol CO
2
= mol substrate × total mol substrate × MW CO2
12.6931 kmol Oil kg CO2
= 1
× 3,760.13 batch × 44 kmol
kg CO2
= 2,100,019.07
batch
42
5.5 MASS BALANCE
5.5.1 Mixing Tank 1
Figure 5.1: Mass balance for mixing tank.
Components Minput (kg/batch) Moutput (kg/batch)

Palmitic Acid (Oil), C16H32O2 60,351.51 60,351.51
Yeast Extract, C19 H14O2 36,210.91 36,210.91
Total 96,562.42 96,562.42
43
5.5.2 Seed Fermenter
Figure 5.2: Mass balance for seed fermenter

Inoculum 5,904.40 19,992.25
Glucose, C6H12O6 44,481.69 30,757.30
Palmitic Acid, C6H32O2 59,868.70 41,522.36
Riboflavin, C17H20N4O6 36,210.91 2,075.73
Yeast Extract, C19H14O2 36,210.90 38,446.63
Water, H2O - 19,992.25
Ammonia, NH3 8,817.73
Carbon Dioxide, CO2 - 39,130.37
Total 191,494.30 191,916.90
44
5.5.3 Fermenter
Figure 5.3: Mass balance for fermenter

Inoculum 19,992.25 498,911.01
Glucose, C6H12O6 475,574.20 42,763.80
Riboflavin, C17H20N4O6 2,075.73 114,036.80
Water, H2O 19,992.25 669,966.22
Ammonia, NH3 88,177.25 -
Carbon Dioxide, CO2 - 1,278,111.07
Total 191,494.30 1,425,460.00
45
5.5.4 Centrifugal 1
Figure 5.4: Mass balance for centrifuge 1.

Inoculum 498,911.01 499,620.22
C6H12O6 42,763.80 40,819.48
C6H32O2 57,018.40 57,018.40
C17H20N4O6 114,036.80 106,758.64
C19H14O2 42,763.80 548,311.73
H2O 669,966.22 31,633.37
Total 1,425,460.00 1,284,162.00
46
5.5.5 Centrifuge 2
Figure 5.5: Mass balance for centrifuge 2

Inoculum 499,620.22 24,210.43
Glucose, C6H12O6 40,819.48 39,532.48
Riboflavin, C17H20N4O6 106,758.64 105,419.95
Water, H2O 31,633.37 136,974.63
Total 1,284,162.00 313,996.00
47
5.5.6 Ultrafiltration
Figure 5.6: Mass balance for ultrafiltration

Inoculum 4,392.50 4,875.35
Glucose, C6H12O6 26,354.99 39,161.21
Riboflavin, C17H20N4O6 99,438.70 106,803.80
Water, H2O 68,083.72 69,714.14
Total 20,466.20 222,327.40
48
5.5.7 Ion Exchange
Figure 5.7: Mass balance for ion exchange.

Ammonium Hydroxide, NH4OH 13.92 47.32
Glucose, C6H12O6 39,161.21 39,995.73
Riboflavin, C17H20N4O6 106,803.30 39,995.73
Water, H2O 32,040.99 30,545.74
Total 178,019.40 110,584.50
49
5.5.8 Drum Drying
Figure 5.8: Mass balance for drum dying

Riboflavin, C17H20N4O6 104,133.22 107,428.23
Water, H2O 30,545.74 30,543.50
Total 134,679.00 137,971.70
50
5.6 ENERGY BALANCE
5.6.1 Mixing Tank 1
Figure 5.9: Energy balance for blending storage 1
Components ṁin (kg/batch) ṁout (kg/batch)

Total 96,592.42 96,592.42
Assumption:
• Temperature = 25 oC
• Time agitation = 15 minutes
• Power input = 22Kw.
ΔHrxn – MvΔhv – Q + W = 0
W=0
T = Constant = 25 oC
Ws = Power input x Mixing time
60 𝑠
= 22 kW x 15 min x 1 𝑚𝑖𝑛
Ws = 1.98 x 104 𝑘𝑊
𝑠
51
5.6.2 Seed Fermenter.
Figure 6.0: Energy balance for seed fermenter.
Components ṁin (kg/batch) ṁoutput (kg/batch)

Glucose, C6H12O6 44,481.69 31,137.18
Inoculum, Ashbya Gossypii 5,905.40 20,669.08
Riboflavin, C12H20N4O6 - 3,188.79
Water, H2O - 20,334.49
Ammonia, NH3 8,817.73 -
Total 101,451.24 153,786.50
Assumption:
• No evaporator
• Steady State
• Aerobic Fermentation
• Have Shaft Work
• Heat Reaction at 37oC = -460 kJ / gmol.
• Negligible sensible heat changes
• Fermentation day = 3 days
52
– Hrxn – MvΔhv – Q + W = 0
– Hrxn – Q + W = 0
Q = Ws + Hrxn
Hrxn = – 460 𝑘𝐽 𝑘𝑔 1000 𝑔 1 𝑔𝑚𝑜𝑙
x 153786.50 x x
𝑔𝑚𝑜𝑙 𝑏𝑎𝑡𝑐ℎ 𝑘𝑔 32 𝑔
𝑘𝐽
= – 2.21 x 10 9
𝑏𝑎𝑡𝑐ℎ
Assume Pconsume = 7.5 Fermentation Complete = 3 days

kW
So; Ws = Power x Production Fermentation
P = 7.5 kW x 3 days x 24 ℎ𝑟 3600 𝑠 𝑘𝑗/𝑠
𝑑𝑎𝑦
x ℎ𝑟
x 𝑘𝑊
= 1.94 x 10 6 kJ
Q = Ws + Hrxn
𝑘𝐽
= 1.94 x 10 6kJ + (– 2.21 x 10 9 )
𝑏𝑎𝑡𝑐ℎ
𝑘𝐽
= – 2.21 x 10 9 𝑏𝑎𝑡𝑐ℎ
53
5.6.3 Fermenter
Figure 6.1: Energy balance for fermenter.

Biomass, C17H1.8O0.5N0.2 20,669.08 502,949.17
Glucose, C6H12O6 75,954.08 40,033.78
Palmitic Acid, 16H32 O2 36,210.91 36,210.91
Water, H2O 20,334.49 684,594.00
Riboflavin, C12H20N4O6 3,188.79 107,355.75
Ammonia, NH3 88,177.25 -
Total 347,131.71 1,425,460.04
Assumption:
• No evaporator
• Steady State
• Aerobic Fermentation
• Have Shaft Work
• Heat Reaction at 37oC = -460 kJ / gmol.
• Negligible sensible heat changes
• Fermentation day = 15 days
54
– Hrxn – MvΔhv – Q + W = 0
– Hrxn – Q + W = 0
Q = Ws + Hrxn
Hrxn = – 460 𝑘𝐽 𝑘𝑔 1000 𝑔 1 𝑔𝑚𝑜𝑙

x 1424460.04 x x
𝑔𝑚𝑜𝑙 𝑏𝑎𝑡𝑐ℎ 𝑘𝑔 32 𝑔
𝑘𝐽
= – 2.048 x 10 10 𝑏𝑎𝑡𝑐ℎ
Assume Pconsume = 7.5 kW Fermentation Complete = 15 days
So; Ws = Power x Production Fermentation
P = 7.5 kW x 7 days x 24 ℎ𝑟 3600 𝑠 𝑘𝑗/𝑠

𝑑𝑎𝑦
x ℎ𝑟
x 𝑘𝑊
= 4.54 x 10 6 kJ
Q = Ws + Hrxn
𝑘𝐽
= 4.56 x 10 6 kJ + (– 2.048 x 10 10 )
𝑏𝑎𝑡𝑐ℎ
𝑘𝐽
= – 2.048 x 10 10
𝑏𝑎𝑡𝑐ℎ
55
5.6.4 Centrifuge 1
Figure 6.2: Energy balance for centrifuge (decanter) 1.

Biomass, C17H1.8O0.5N0.2 502,949.17 25,147.46
Glucose, C6H12O6 4,033.78 40,033.78
Yeast Extract, C19 H14O2 54,316.43 -
Riboflavin, C12H20N4O6 107,355.75 107,355.75
Water, H2O 684,584.00 136,918.80
Palmitic Acid, C16H32 O2 36,210.91 7,242.18
Total 1,389,450.03 316,697.97
Assumption:
• Power consume = 22.5 kW

• Centrifuge time = 240 minutes
ΔHrxn – MvΔhv – Q + Ws = 0
– Q + Ws = 0
Q = Ws
Ws = Power Input x Time
1 𝑘𝑗/𝑠 60 𝑠
= 22.5 kW x 240 min x 1 𝑘𝑊
x 𝑚𝑖𝑛
= 3.24 x 105 kJ
56
5.6.5 Centrifuge 2
S-108
Figure 6.3: Energy balance for centrifuge (decanter) 2.

Biomass, C17H1.8O0.5N0.2 25,147.46 5,029.49
Glucose, C6H12O6 40,033.78 40,033.78
Riboflavin, C12H20N4O6 107,355.75 107,355.75
Water, H2O 136,918.80 68,459.40
Palmitic Acid, C16H32 O2 7,242.18 1,448.44
Total 316,697.97 222,326.86
Assumption:
• Power consume = 22.5 kW
• Centrifuge time = 240 minutes
ΔHrxn – MvΔhv – Q + Ws = 0
– Q + Ws = 0
Q = Ws
Ws = Power Input x Time
1 𝑘𝑗/𝑠 60 𝑠
= 22.5 kW x 240 min x 1 𝑘𝑊
x 𝑚𝑖𝑛
= 3.24 x 105 kJ
57
5.6.6 Drum Drying
Figure 6.4: Energy balance for drum drying
Component ṁin ṁout ΔHin ΔHout

(kg/batch) (kg/batch) (kJ/batch) (kJ/batch)
Riboflavin, C17H20N4O6 104,133.22 107,428.23 7.0 × 108 4.84 X 1011
Water, H2O 30,545.74 30,543.50 1.54×109 2.10 × 109
Total 134,679.00 137,971.70 2.24×109 4.86 × 1011
T1 = 38.7 o C Heating Temp. = 152 o C T2 = 152 o C
Cp of Water;
35 OC 146.64 kJ/ kg
38.7 OC X
40.0OC 167.53 kJ /kg
Interpolate
40-35 167.53 − 146.64

=
40 -38.7 167.53 − 𝑋
837.65 -5 X = 27.16
kJ
= 162.10
kg
58
Cp Water 𝐤𝐉
= 162.10 𝐤.𝐤𝐠
Water Inlet Enthalpy, h = Cp(ΔT)

= [162.10 kJ/kg] × [ 311.75 K]
𝑘𝐽
= [50,534.68 𝑘𝑔
]
ΔHinlet = [50,534.68
𝑘𝐽
] × [30,545.74𝑏𝑎𝑡𝑐ℎ ]
𝑘𝑔
𝑘𝑔
𝑘𝐽
= 1.54×109 𝑏𝑎𝑡𝑐ℎ
Water Outlet Enthalpy, h = Cp(ΔT)

𝑘𝐽
= [162.10 𝑘.𝑘𝑔
] [ 425 k]
𝑘𝐽
= [ 1,487.024 ]
𝑘𝑔
ΔHoutlet = [68,892.50
𝑘𝐽
] [30,543.50𝑏𝑎𝑡𝑐ℎ ]
𝑘𝑔
𝑘𝑔
𝑘𝐽
= 2.10 × 109
𝑏𝑎𝑡𝑐ℎ
Cp Riboflavin, Inlet 𝒌𝑱
= [ 8116 𝒎𝒐𝒍
]
Riboflavin Inlet Enthalpy, h = Cp(ΔT)

𝑘𝐽 mol 1000 g
= [8,116 ][ ][ [311.75 k ]
𝑚𝑜𝑙 376.36 g kg
𝑘𝐽
= 6,722.72 𝑘𝑔
𝑘
ΔHinlet = [6,722.72
𝑘𝐽
𝑘 ] [104,133.22
𝑘𝑔
]
𝑘𝑔 𝑏𝑎𝑡𝑐ℎ
𝑘𝐽
= 7.0 × 108 𝑏𝑎𝑡𝑐ℎ
Cp Riboflavin, Outlet 𝑘𝐽
= [ 21,564.46 ]
𝑘𝑔
Riboflavin Outlet Enthalpy, = Cp(ΔT)

h 𝑘𝐽
= [21,564.46 𝑘𝑔
] [425 k]
𝑘𝐽
= [ 9.16 ×106 𝑘𝑔
𝑘 ]
ΔHoutlet = [ 9.16 ×106

𝑘𝐽
𝑘 ] [107,428.23𝑏𝑎𝑡𝑐ℎ ]
𝑘𝑔
𝑘𝑔
𝑘𝐽
= 4.84 X 1011 𝑏𝑎𝑡𝑐ℎ
58
59
Q = ∑ ΔHout − ∑ ΔHin
𝑘𝐽 𝑘𝐽
= [4.86 × 1011𝑏𝑎𝑡𝑐ℎ] – [2.24×109𝑏𝑎𝑡𝑐ℎ]
𝑘𝐽
= 4.84 X 1011 𝑏𝑎𝑡𝑐ℎ
59
60
CHAPTER 6
SIZING AND COSTING
6.1 Sizing and costing for blending storage
Figure 6.1: Blending storage
Table 6.1: List of the components flow out at blending storage

Components Density, ρ (kg/m3) Mass fraction, X
Palmitic Acid 853.00 0.625
Yeast Extract 990.35 0.375
60
61
6.1.1 Sizing
Step 1: Find volume of the vessel, V.

𝐹𝐿 𝜏
𝑉=2 [ ]
𝜌𝐿
Given;
1 1 𝑘𝑔
𝜌𝐿 = 𝑥 = 0.625 = 8, 99.80 ⁄ 3
∑ 0.375 𝑚
𝜌𝐿 [ 853 + 990.35]
𝑘𝑔⁄
𝐹𝐿 = 96, 562.42 𝑏𝑎𝑡𝑐ℎ
𝜏 = 𝑝𝑟𝑜𝑐𝑒𝑠𝑠 𝑡𝑖𝑚𝑒 = 80.47 ℎ𝑜𝑢𝑟𝑠
1 𝑏𝑎𝑡𝑐ℎ
96,562.42 𝑘𝑔 × × 80.47 ℎ𝑟
𝐹 𝜏 80.47 ℎ𝑟
So; 𝑉=2 [ 𝜌𝐿 ] = 2[ 𝑏𝑎𝑡𝑐ℎ
𝑘𝑔 ] = 107.32 m3
𝐿 8,99.80
𝑚3
Since there are 2 units of blending in the plant;

𝑉 107.32 𝑚3
𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑒 𝑢𝑛𝑖𝑡
= 2
= 53.66 𝑚3
Step 2: Find the diameter, D.
𝜋
𝑉 = [ ] × 𝐷2 × ℎ
4
Assume h = 3D;
𝜋
So; 53.66 𝑚3 = [ 4 ] × 𝐷 2 × 3𝐷
53.66 𝑚3 = 0.7854 × 3𝐷 3
53.66 𝑚3 = 2.3562 𝐷3
53.66 𝑚3
𝐷3 = = 22.77 𝑚3
2.3562
3
𝐷 = √22.77 𝑚3 = 2.84 𝑚
61
62
Step 3: Find height, h.

ℎ = 3𝐷
ℎ = 3 (2.84)
ℎ = 8.52 𝑚
6.1.2 Costing
Table 6.2: Details information of blending storage.

Details
Company Shanghai Joy Light Industry Machinery Co., Ltd.
Max. Capacity 50 000 L
Diameter -2.84 m
Height -8.52 m
Price RM 42,000.00
Calculation of the estimation purchased cost
𝑛
𝐶𝑎 = 𝐶𝑝 = 𝐶𝑏 [𝑉𝑎 ]
𝑉
𝑏
𝐶𝑝 53,660 0.6
= 𝑅𝑀 42,000 [ 50,000 ]
= 𝑅𝑀 43, 818.52
62
63
Table 6.3: Bare modules cost, CBM calculation for blending storage.
DIRECT
a) Equipment Cao = Cpo = RM 43, 818.52
b) Material Cm = αm Cpo
= 0.07 (RM 43, 818.52)
= RM 3, 067.30
c) Labor CL = αL (Cpo + Cm)
= 0.07 (RM 43, 818.52 + RM 3, 067.30)
= RM 3,282.01
d) Total Direct CDE = Cpo + Cm + CL
= RM 43, 818.52 + RM 3, 067.30 + RM 3, 282.01
= RM 50, 167.83
INDIRECT
a) Freight, etc CFIT = αFIT (Cpo + Cm)
= 0.07 (RM 43, 818.52 + RM 3, 067.30)
= RM 3, 282.01
b) Overhead CO = αO CL
= 0.10 (RM 3,282.01)
= RM 328.20
c) Engineering CE = αE (Cpo + Cm)
= 0.10 (RM 43, 818.52 + RM 3, 067.30)
= RM 4, 688.58
d) Total Indirect CIDE = CFIT + CO + CE
63
64
= RM 3, 282.01 + RM 328.20 + RM 4, 688.58
= RM 8, 298.79
Bare Module
CBM = CIDE + CDE
= RM 8, 298.79 + RM 50, 167.83
= RM 58,466.62 (1 unit)
Since blending storage in = RM 58,466.62 x 2 Units

plantation is 2 units, so;
= RM 116,933.24
64
65
6.2 Sizing and costing for seed fermenter
6.2.1 Sizing
Components Density (kg/m3) Mass Fractions
Biomass 1050.00 0.134
Glucose 1540.00 0.202
Riboflavin 1270.00 0.207
Water 1000.00 0.132
Flowrate 153780.50 kg/batch

Time 72 hours
Volume 7481.68 L
Diameter 3.160 m
Height 7.482 m
Impeller (axial types) 1.58 m
Baffle (8%) 0.2532 m
Headspace (7%) 52.045 m
65
66
6.2.2 Costing
Information from the company
Name Sentinain Inc

Prices RM 558, 527.00
Height 4.85 m
Diameter 2.2 m
Area 33.521 m2
Malaysia money currency on 11 Nov 2017 = 1 USD = RM 4.19.
Calculation from the superpro.

Area : π × D × h
= π × 3.160 m × 7.482 m
= 74.277 m.
74.277 𝑚3
Cp: [ 33.521 𝑚3 ] × 𝑅𝑀 558, 527.00
= RM 900, 257.20
Direct
Equipment CA = Cp RM 900, 257.20
Material Cm RM 63, 018.00
Labour CL RM 67, 429.25
Total direct RM 1,030, 704.45
Indirect
Freight, ect CFIT RM 67, 429.26
Overhead Co RM 4, 720.05
Engineering CE RM 96, 327.52
Total Indirect CIDE RM 168, 476.83
66
67
Bare module CBM = CIDE + CDE

= RM 168, 476 + RM 1,030,704.45
= RM 1, 199, 181.28
Overall total cost for seed fermenter.

CBM × Multiple unit
= RM 1, 199, 181.28 × 4
= RM 4, 796, 725.12
67
68
6.3 Fermenter
Figure 6.2: Fermenter
Table 6.4: List of the components flow out at fermenter

Glucose 1540.0 0.028
Biomass 1050.0 0.353
Water 1000.0 0.480
68
69
6.3.1 Sizing

𝐹𝐿 𝜏
𝑉=2 [ ]
𝜌𝐿
Given;
1 1
𝜌𝐿 = 𝑥 = 0.353 0.028 0.038 0.075
∑ 0.48 0.025
𝜌𝐿 [ 1050 + 1540 + 853 + 1270 + 1000 + 990.35]
𝑘𝑔
= 1038.18 ⁄ 3
𝑚
𝑘𝑔⁄
𝐹𝐿 = 8, 484.88 𝑏𝑎𝑡𝑐ℎ
𝜏 = 𝑝𝑟𝑜𝑐𝑒𝑠𝑠 𝑡𝑖𝑚𝑒 = 168 ℎ𝑜𝑢𝑟𝑠
8,484.88 𝑘𝑔 × × 168 ℎ𝑟
𝐹 𝜏 168 ℎ𝑟
𝑘𝑔 ] = 2,746.07 m3
𝐿 1038.18
𝑚3
Since there are 6 units of fermenter in the plant;

𝑉 2,746.07 𝑚3
= 6
= 457.68 𝑚3
𝜋
𝑉 = [ ] × 𝐷2 × ℎ
4
Assume h = 3D;
𝜋
So; 457.68 𝑚3 = [ 4 ] × 𝐷 2 × 3𝐷
457.68 𝑚3 = 0.7854 × 3𝐷 3
457.68 𝑚3 = 2.3562 𝐷3
457.68 𝑚3
𝐷3 = = 194.25 𝑚3
2.3562
3
𝐷 = √194.25 𝑚3 = 5.79 𝑚
69
70

ℎ = 3𝐷
ℎ = 3(5.79)
ℎ = 17.37 𝑚
Step 4: Working volume.

75% from working volume can be used; since volume calculated = 457.68 m3
1000 𝐿
𝑉 = 457.68 𝑚3 × 1 𝑚3
= 457, 679.10 𝐿
75
So; working volume = 100
× 700, 000 𝐿 = 525, 000 𝐿
Step 5: Diameter of impeller (Using axial impeller).

50% from diameter of the fermenter vessel; since diameter calculated = 5.79 m
50
= 100
× 5.79 𝑚 = 2.895 𝑚
Step 6: Baffle width (8% - 10% of the tank diameter).

8
= 100
× 5.79 𝑚 = 0.463 𝑚
10
= × 5.79 𝑚 = 0.579 𝑚
100
= in range of 0.491 𝑚 − 0.614 𝑚
Step 7: Headspace (70% - 80% from tank volume).

70
= × 457.68 𝑚3 = 320.38 𝑚3
100
80
= × 457.68 𝑚3 = 366.14 𝑚3
100
= in range of 320.38 𝑚3 − 366.14 𝑚3
70
71
6.3.2 Costing
Table 6.5: Details information of fermenter from Ahmedabad, India.

Details
Company Shree Bio Care,Vatva Industrial Estate, Ahmedabad
Max. Capacity 500 000 L
Diameter 4.3 m (r = 2.15 m)
Height 15.0 m
Price RM 831, 547.40
Calculation of area (based on company’s information)

Area = 𝜋𝐷ℎ
= 𝜋 (5.79)(17.37)
= 315.96 m2
Calculation of area (based on plantation)

Area = 𝜋𝐷ℎ
= 𝜋 (4.3)(15.0)
= 202.63 m2

𝑛
𝐶𝑎 𝐴
= 𝐶𝑝 = 𝐶𝑏 [𝐴𝑎]
𝑏
𝐶𝑝 315.96 0.6
= 𝑅𝑀 831,547.40 [ 202.63 ]
= 𝑅𝑀 1, 085, 535.49
71
72
Table 6.6: Bare modules cost, CBM calculation for fermenter.

DIRECT
a) Equipment Cao = Cpo = RM 1, 085, 535.49
= 0.07 (RM 1, 085, 535.49)
= RM 75, 987.48
= 0.07 (RM 1, 085, 535.49 + RM 75, 987.48)
= RM 1,090,854.61
= RM 1,085,535.49 + RM 75,987.48 + RM 1,090,854.61
= RM 2,252,377.58
INDIRECT
e) Freight, etc CFIT = αFIT (Cpo + Cm)
= 0.07 (RM 1,085,535.49 + RM 75,987.48)
= RM 1,090,854.61
f) Overhead CO = αO CL
= 0.10 (RM 1,090,854.61)
= RM 109,085.46
g) Engineering CE = αE (Cpo + Cm)
= 0.10 (RM 1,085,535.49 + RM 75,987.48)
= RM 116,152.30
h) Total Indirect CIDE = CFIT + CO + CE
= RM 1,090,854.61 + RM 109,085.46 + RM 116,152.30
= RM 1,316,092.37
Bare Module
CBM = CIDE + CDE
= RM 1,316,092.37 + RM 2,252,377.58
= RM 3,568,469.95 (1 unit)
Since Fermenter in = RM 3,568,469.95 x 6 Units
plantation is 6 units, so; = RM 21,410,819.70
72
73
6.4 Centrifuge
Figure 6.3: Centrifuge (Decanter)
Table 6.7: List of the components flow out at centrifuge (decanter).

Centrifuge 1 Centrifuge 2
Biomass 1050 0.35 0.08
Glucose 1540 0.03 0.13
Palmitic Acid 853 0.04
Riboflavin 1270 0.07 0.34
Water 1000 0.48 0.43
Yeast 990.35 0.03 0.03
73
74
6.4.1 Sizing
6.4.1.1 Centrifuge 1

FL τ
V=2 [ ]
ρL
Given;
1 1 kg
ρL = x = 0.35 = 1027.76 ⁄ 3
∑ 0.03 0.04 0.08 0.48 0.03 m
ρL [1050 + 1540 + 853 + 1270 + 1000 + 990.35]
kg⁄
FL = 1,425,460.039 batch
τ = process time = 4.0 hours
1 batch
1,425,460.039 kg × × 4.0 hr
F τ 4.0 hr
So; V=2 [ ρL ] = 2[ batch
kg ] = 2773.92 m3
L 1027.76
m3
π
V = [ ] × D2 × h
4
Assume h = 3D;
π
So; 2773.92 m3 = [ 4 ] × D2 × 3D
3531.86 m3
D= 3 = 10.56 m
3

h = 3D
= 3(10.56m)
= 31.68 m
74
75
Step 4: Find W.
1⁄
Gt. g 2
W= [ ]
R. t
Given;
Gt bacteria = 18 x 106 s
g = 9.81 m/s2
R = 5.28 m
t = 4 hrs × 60 min 60 s
1 hr
× 1 min
= 14,400 s
1⁄
(18×106 s).(9.81 m/s2 ) 2
So; W = [ (5.28 m).(14,400 s)
] = 48.19 rad/s
rad rev 60 s
= 48.19 s
× 2π rad × min = 460.18 rpm
Step 5: Find Q
Q  Vg  ------------------------------ 1


L Ro 2  R12 w2  ------------------------------ 2
R 
g ln o 
 R1 
 2a 2 (    o ) g 
Vg    ------------------------------ 3
 9 
Where;
R = Distance from centre of rotation to the top packed column
Ro = Distance from centre of rotation to top of the liquid in centrifuge tube
a = For bacterial cell, 0.5 μm
ρo = For bacterial cell density, 1.10 g/cm3
μ = Viscosity, 0.01 g/cm.s
75
76
  2
3 
  2  2.5m X 1 X 10 m   1.1 g  1.0 g   1 X 10 cm  
6 6
  m    cm 3 cm 3   m3 
   
Vg 
  0.01 g 100cm  
 9 X  
  cm.s m  
 
= 5.45 X 10-8 m/s
  
 g ln R  
  R 
Vg    o
2  ------------------------------ 4
 wt 
 
 
Step 6: Calculation of w2 and constant value [2a2& (ρ – ρo)]
  6

2

2a   2 0.5 mX  
2 1 X 10 m
  m  

= 5 X 10 -13m2
 g g   1 X 106 cm 3 
(    o )  1.1 3  1.0 3   
 cm cm   m3 
g
= 100000
m3
2
 rev min 2rad 
w   460.18
2
X X 
 min 60 s rev 
= 2322.27 s
Therefore; 34
76
77
  
 g ln R  
  R 
Vg    o
2 
 wt 
 
 
  m   5.28 m  
  9.81 2  ln 
m   s   Ro  
5.45 X 10-8  
s    
2322.27 s  2 14400s  
 
 
 m
 
  5.45 X 10-8  2322.27 s  2 14400s   
 s   ln 5.28 m 
  m   R 
  9.81 2    o 
  s  
  
 
   5.45 X 10-8  2322.27 s  2 14400s 
m
  

Exponent  
s   ln 5.28 m  
  m   R 
   9.81 2    o 
 s  
  
 5.28 m 
1.20   
 Ro 
 5.28 m 
Ro   = 4.4 m
 1.20 
All calculated value into 2

 
 31.68m  4.4m 2  5.28m 2 2322.27 s 
------------------------- 5
 m   4.4m 
 9.81 2  ln 
 s   5.28m 
= 1100775.52 m2
77
78
3 & 5 into 1
Q  Vg 
 m

Q   5.45 X 108  1100775.52 m2 
 s
m3 L 60 s
= 0.06 X 3
X
s 0.001m min
L
= 3600
min

FL τ
V=2 [ ]
ρL
Given;
1 1 kg
ρL = x = 0.08 = 1125.35 ⁄ 3
∑ 0.13 0.34 0.43 0.03 m
ρL [1050 + 1540 + 1270 + 1000 + 990.35]
kg⁄
FL = 316,697.967 batch
τ = process time = 4.0 hours
1 batch
316,697.967 kg × × 4.0 hr
F τ 4.0 hr
So; V=2 [ ρL ] = 2[ batch
kg ] = 562.84 m3
L 1125.35
m3
π
V = [ ] × D2 × h
4
78
79
Assume h = 3D;
π
So; 562.84 m3 = [ 4 ] × D2 × 3D
716.63 m 3
D= 3 = 6.20 m
3

h = 3D
= 3(6.20m)
= 18.6 m
Step 4: Find W.
1⁄
Gt. g 2
W= [ ]
R. t
Given;
Gt bacteria = 18 x 106 s
g = 9.81 m/s2
R = 5.28 m
t = 4 hrs × 60 min 60 s
1 hr
× 1 min
= 14,400 s
1⁄
(18×106 s).(9.81 m/s2 ) 2
So; W = [ (3.1 m).(14,400 s) ] = 62.89 rad/s
rad rev 60 s
= 62.89 s
× 2π rad × min = 600.56 rpm
Step 5: Find Q
Q  Vg  ------------------------------ 1


L Ro 2  R12 w2  ------------------------------ 2
R 
g ln o 
 R1 
 2a 2 (    o ) g 
Vg    ------------------------------ 3
 9 
79
80
Where;
R = Distance from centre of rotation to the top packed column
Ro = Distance from centre of rotation to top of the liquid in centrifuge tube
a = For bacterial cell, 0.5 μm
ρo = For bacterial cell density, 1.10 g/cm3
μ = Viscosity, 0.01 g/cm.s
  2
3 
  2  2.5m X 1 X 10 m   1.1 g  1.0 g   1 X 10 cm  
6 6
  m    cm 3 cm 3   m3 
   
Vg 
  0.01 g 100cm  
 9 X  
  cm.s m  
 
= 5.45 X 10-8 m/s
  
 g ln R  
  R 
Vg    o
2  ------------------------------ 4
 wt 
 
 
Step 6: Calculation of w2 and constant value [2a2& (ρ – ρo)]
  6

2

2a   2 0.5 mX  
2 1 X 10 m
  m  

= 5 X 10 -13m2
 g g   1 X 106 cm 3 
(    o )  1.1 3  1.0 3   
 cm cm   m3 
g
= 100000
m3
80
81
2
 rev min 2rad 
w   600.56
2
X X 
 min 60 s rev 
= 3955.21 s
Therefore; 34
  
 g ln R  
  R 
Vg    o
2 
 w t 
 
 
  m   3.1m  
  9.81 2  ln 
-8 m   s   Ro  

5.45 X 10
s    
3955.21s  2 14400s  
 
 
 m

  5.45 X 10-8  3955.21s  2 14400s    
 s   ln 3.1 m 
  m   R 
  9.81 2    o 
  s  
  

   5.45 X 10-8  3955.21s  2 14400s 
m
   

Exponent  
s   ln 3.1 m  
  m   R 
   9.81 2    o 
 s  
  
 3.1 m 
1.37   
 o 
R
 3.1m 
R o  = 2.26 m
 1.37 
81
82
All calculated value into 2

 
 18.6m  2.26m 2  3.1m 2 3955.21s 
------------------------- 5
 m   2.26m 
 9.81 2  ln 
 s   3.1m 
= 335636.43 m2
3 & 5 into 1
Q  Vg 
 m

Q   5.45 X 108  335636.43. m2 
 s
m3 L 60 s
= 0.02 X 3
X
s 0.001m min
L
= 1200
min
82
83
6.4.2 Costing
Table 6.8: Details information of centrifuge (decanter).

Details
Company KES Energy Equipment Manufacturing Hebei Co., Ltd.
Max. Capacity 57 m3/h
Diameter 1.09 m
Height 3.27 m
Price RM 125,706.00

Area = 2πrh + 2πr 2
= 2π (0.545 m)(3.27 m) + 2π(0.545 m)2
= 13.06 m2

= 2π (5.28 m)(31.68 m) + 2π(5.28 m)2
= 1226.16 m2

n
Ca A
= Cp = Cb [ A a ]
b
Cp 1226.16 0.6
= RM 125,706 [ ]
13.06
= RM 1,918,308.22
83
84
Table 6.9: Bare modules cost, CBM calculation for centrifuge (decanter).
DIRECT
a) Equipment Cao = Cpo = RM 1,918,308.22
= 0.07 × RM 1,918,308.22
= RM 134,281.58
= 0.07 (RM 1,918,308.22 + RM 134,281.58)
= RM 143,681.29
= RM 2,196,271.09
INDIRECT
= 0.07 (RM 1,918,308.22 + RM 134,281.58)
= RM 143,681.29
= 0.10 (RM 143,681.29)
= RM 14,368.13
= 0.10 (RM 1,918,308.22 + RM 134,281.58)
= RM 2,052,588.8
= RM 2,210,638.22
Bare Module
CBM = CIDE + CDE
= RM 2,196,271.09+ RM 2,210,638.22
= RM 4,406,909.31
84
85

= 2π (0.545 m)(3.27 m) + 2π(0.545 m)2
= 13.06 m2

= 2π (3.1 m)(18.6 m) + 2π(3.1 m)2
= 422.67 m2

n
Ca A
= Cp = Cb [ A a ]
b
Cp 422.67 0.6
= RM 125,706 [ ]
13.06
= RM 1,012,490.50
85
86
Table 6.10: Bare modules cost, CBM calculation for centrifuge (decanter).
DIRECT
e) Equipment Cao = Cpo =RM 1,012,490.50
f) Material Cm = αm Cpo
= 0.07 × RM 1,012,490.50
= RM 70,874.34
g) Labor CL = αL (Cpo + Cm)

= 0.07 (RM 1,012,490.50 + RM 70,874.34)
= RM 75,835.54
h) Total Direct CDE = Cpo + Cm + CL
= RM 1,159,200.38
INDIRECT
= 0.07 (RM 1,012,490.50 + RM 70,874.34)
= RM 75,835.54
= 0.10 (RM 75,835.54)
= RM 7,583.55
= 0.10 (RM 1,012,490.50 + RM 70,874.34)
= RM 108,336.48
= RM 191,755.57
Bare Module
CBM = CIDE + CDE
= RM 1,159,200.38 + RM 191,755.57
= RM 1,350,955.95
Total Bare module
ΣCBM = Σ(CBM1 + CBM2)
= RM 4,406,909.31 + RM 1,350,955.95
= 5,757,865.26
86
87
6.5 Ultrafiltration
Figure 6.4: Ultrafiltration
Table 6.11: List of the components flow out at ultrafiltration.

Biomass 1050.0 0.023
Water 1000.0 0.308
Glucose 1540.0 0.180
87
88
6.5.1 Sizing

𝐹𝐿 𝜏
𝑉=2 [ ]
𝜌𝐿
Given;
1 1 𝑘𝑔
𝜌𝐿 = 𝑥 = 0.023 0.308 0.180 = 1,062.00 ⁄ 3
∑ 0.483 0.006 𝑚
𝜌𝐿 [ 1050 + 1000 + 1540 + 9893.97 + 990.35]
𝑘𝑔⁄
𝐹𝐿 = 222,326.85 𝑏𝑎𝑡𝑐ℎ
222,326.85 𝑘𝑔 × × 24 ℎ𝑟
𝑘𝑔 ] = 209.15 m3
𝐿 1,062.00
𝑚3
Since there are 1 units of fermenter in the plant;

𝑉 209.15 𝑚3
= 1
= 209.15 𝑚3
𝜋
𝑉 = [ ] × 𝐷2 × ℎ
4
Assume h = 3D;
𝜋
So; 209.15 𝑚3 = [ 4 ] × 𝐷 2 × 3𝐷
209.15 𝑚3 = 0.7854 × 3𝐷 3
209.18 𝑚3 = 2.3562 𝐷3
209.15 𝑚3
𝐷3 = = 88.77 𝑚3
2.3562
3
𝐷 = √88.77 𝑚3 = 4.46 𝑚

𝜋
209.15 𝑚3 = [ ] × (4.46)2 × ℎ
4
209.15 𝑚3 = 15.62 × ℎ
ℎ = 13.39 𝑚
88
89
6.5.2 Costing
Table 6.12: Details information of ultrafiltration.

Details
Company Xi’an Xinshengtai (XST) Water Treatment Technology Co., Ltd.
Max. Capacity 6000L/H
Diameter
Height
Price USD 1,250 ̴ RM 5,237.50

𝑛
𝐶𝑎 𝑉
= 𝐶𝑝 = 𝐶𝑏 [ 𝑎 ]
𝑉 𝑏
𝐶𝑝 3,861.55 𝐿/ℎ 0.6

= 𝐶𝑎 = 𝑅𝑀 5,237.50 ( )
6,000 𝐿/ℎ
= RM 4,020.59
89
90
Table 6.13: Bare modules cost, CBM calculation for ultrafiltration.

DIRECT
a) Equipment Cao = Cpo = RM 4,020.59
= 0.07 × RM 4,020.59
= RM 281.44
= 0.07 (RM 4,020.59+ RM 281.44)
= RM 301.14
= RM 4,020.59 + RM 281.44 + RM 301.14
= RM 4,603.17
INDIRECT
= 0.22 (RM4,020.59 + RM 281.44)
= RM 946.45
= 0.1 (RM 301.10)
= RM 30.11
= 0.1 (RM 4,020.59 + RM 281.44)
= RM 430.20
= RM 946.45 + RM 30.11 + RM 430.20
= RM 1,406.76
Bare Module
CBM = CIDE + CDE
= RM 4,603.17+ RM 1,406.76
= RM 6,009.88 (1 unit)
Since blending storage in = RM 6,009.88x 1 Units
plantation is 1 units, so; = RM 6,009.88
90
91
6.6 Ion Exchange
Components Density, (kg/m3) Mass fraction

Water 1000 0.17
Riboflavin 1270 0.6
Glucose 1540 0.23
Ammonium Hydroxide 989.97 0.1
6.6.1 Sizing
F t
Vessel Volume, V  2 L 
 pL 
Given;
FL = 178005.5 kg/m3 t = 12.5 hr
1 1 kg
pL    1006.22 3
x  0.23 0.6 0.1 
p
0.17 0.1 m
     
L  1540 1270 1000 989.97 989.97 
 kg batch 
 178005.5 X X 12.5h 
V  2 batch 12.5h 
 kg 
 1006.22 3 
 m 
= 176.91 m3
Find D
 
V    X D2 X h
4
Assume h = 3D
 
So; 176.91 m3 =   X D X 3D
2
4
 
 176.91m3 
3D3 =  
  
 
 4 
225.25 m 3
D= 3 = 4.22 m
3
91
92
H = 3D r = (4.22 m / 2) = 2.11 m
H = 3(4.22 m)
H = 12.66 m
Vf 
Bed Volume, VB  FD   (EBCT )
 t 
Where;
FD = Overdesign factor of Equipment = 1.0
VF = Total feed volume per procedure cycle = 0.23 cycle/week
T = breakthrough time (process time) = 12.5 h
EBCT = Empty bed contact time = 3 min
 185,200 L 10 batch yr week 

VF   X X X  = 167,753.32 L.Cycle-1
 batch yr 48 week 0.23 cycle 
 167,753.62 L 
VB  1.0  (3 min)
 12.5 hr 
 13,420 L hr 0.001m3 
VB  1.0 X X  (3 min) = 0.671 m3 ≈ 671 L
 hr 60 min L 
To find D and H of Bed
 
V    X D2 X h …………………………1
4
H:D ratio = 3:2
3H : 2D
2
H= D = 0.6667 D ……………..….2
3
Therefore, 2  1
 
0.671m3    X D 2 X 0.6667 D
4
92
93
0.671m3
D= 3 = 1.09 m
0.524
2
H= (1.09 m) = 0.7267 m
3
Bed to column height ratio = 0.5

V 
Column volume, VC   B 
 0.5 
 0.671 m3 
=  
 0. 5 
= 1.34 m3
To find D and H of column
 
VC    X D 2 X h
4
Assume h = 2D
1.34 m3 = 1.57 D3
1.34 m3
D= 3 = 0.95 m
1.57
H = 2(0.95 m) = 1.9 m
93
94
6.6.2 Costing
Table 6.14: Details information of ion exchange.

Details
Company Xian Landau Mechanical & Electrical Co., Ltd.
Max. Capacity 10,000 m3
Diameter 16.1906 m
Height 48.5718 m
Price RM 83, 832.00

= 2π (8.0953 m)(48.5718 m) + 2π(8.0953 m)2
= 2882.33 m2

= 2π (2.11 m)(12.66 m) + 2π(2.11 m)2
= 195.81 m2

n
Ca A
= Cp = Cb [ A a ]
b
Cp  2882.33 m 2 
0.6
= RM 83,832  2 

 195.81m 
= RM 420,876.94
94
95
Table 6.15: Bare modules cost, CBM calculation for ion exchange.
DIRECT
a) Equipment Cao = Cpo = RM 420,876.94
= 0.07 × RM 420,876.94
= RM 29,461.39
= 0.07 (RM 420,876.94+ RM 29,461.39)
= RM 31,523.68
= RM 481,862.01
INDIRECT
= 0.07 (RM 420,876.94+ RM 29,461.39)
= RM 31,523.68
= 0.10 (RM 31,523.68)
= RM 3,152.37
= 0.10 (RM 420,876.94+ RM 29,461.39)
= RM 45,033.83
= RM 79,709.88
Bare Module
CBM = CIDE + CDE
= RM 481,862.01+ RM 79,709.88
= RM 561,571.89 (1 unit)
95
96
6.7 Drum Drying
Figure 6.5: Drum dryer
Table 6.16: List of the components flow out at drum dryer.

Water 1000.0 1.0000
Water 1000.0 0.0160
6.7.1 Sizing

𝐹𝐿 𝜏
𝑉=2 [ ]
𝜌𝐿
Given;
1 1 𝑘𝑔
𝜌𝐿 = 𝑥 = = 559.39 ⁄ 3
∑ 0.98 0.016 1.0 𝑚
𝜌𝐿 [1270 + 1000 + 1000]
𝑘𝑔⁄
𝐹𝐿 = 741, 575.52 𝑏𝑎𝑡𝑐ℎ
741,575.52 𝑘𝑔 × × 24 ℎ𝑟
𝑘𝑔 ] = 279.96 m3
𝐿 559.39
𝑚3
Since there are 3 units of drum dryer in the plant;

𝑉 279.96 𝑚3
= 3
= 93.32 𝑚3
96
97
𝜋
𝑉 = [ ] × 𝐷2 × ℎ
4
Assume h = 3D;
𝜋
So; 205.46 𝑚3 = [ ] × 𝐷 2 × 3𝐷
4
93.32 𝑚3 = 0.7854 × 3𝐷 3
93.32 𝑚3 = 2.3562 𝐷3
3
93.32 𝑚3
𝐷 = = 39.61 𝑚3
2.3562
3
𝐷 = √39.61 𝑚3 = 4.43 𝑚

𝜋
205.46 𝑚3 = [ ] × (4.43)2 × ℎ
4
205.46 𝑚3 = 15.41 × ℎ
ℎ = 13.30 𝑚
97
98
6.7.2 Costing
Table 6.17: Details information of drum dryer.

Details
Company Gongyi Xiaoxi Mingyang Machinery Plant., China
Max. Capacity 1500-2000 kg/h
Diameter 1.5 m (r = 0.75 m)
Height / Length 12 m
Price USD 20150.32 @ RM 84, 450.00

𝑛
𝐶𝑎 𝐴
= 𝐶𝑝 = 𝐶𝑏 [𝐴𝑎]
𝑏
𝐶𝑝 209.67 0.6
= 𝑅𝑀 84, 450 [ ]
56.55
= 𝑅𝑀 185, 382.20
98
99
Table 6.18: Bare modules cost, CBM calculation for ion exchange.
DIRECT
a) Equipment Cao = Cpo = RM 185, 382.20
= 0.07 × RM 185, 382.20
= RM 12, 976.75
= 0.07 (RM 185, 382.20+ RM 12, 976.75)
= RM 13, 885.13
= RM 185, 382.20 + RM 12, 976.75 + RM 13, 885.13
= RM 212, 244.08
INDIRECT
= 0.07 (RM 185, 382.20+ RM 12, 976.75)
= RM 13, 885.13
= 0.10 (RM 13, 885.13)
= RM 1,388.51
= 0.10 (RM 185, 382.20+ RM 12, 976.75)
= RM 19, 835.90
= RM 13, 885.13 + RM 1,388.51 + RM 19, 835.90
= RM 35, 109.54
Bare Module
CBM = CIDE + CDE
= RM 35, 109.54 + RM 212, 244.08
= RM 247, 353.62 (1 unit)
Since Drum Dryer in = RM 247, 353.62 x 2 units
plantation is 2 units, so; = RM 494, 707.24
99
100
CHAPTER 7
PROCESS CONTROL
7.1 Introduction
Process control of the plantation is very importance in the industry. It is also related to
the method of convert the raw material to become products by certain process, for example
chemical process, biological process or physical process (Bequette, 2006). The phase of the
raw materials that are used can be liquid, gaseous or slurry which either remain or pass
through during the process, transferred, measured, mixing, heating or cooling, filter, stored or
handled in produce the product at end of the process (Bequette, 2006; Teschke, 1998). In the
process industry include chemical industry, oil and gas industry, food and beverage industry,
the pharmaceutical industry, waste treatment industry and lastly power industry. As for the
process control is refer to the method that are used to control process variable during
manufacturing the product (Bequette, 2006).
There are three main importance in the manufacturing control which are reduce
variability, increase efficiency and ensure the safe. To ensure high quality of the product and
save money, it is state in the reduce variability (Anonymous A, 2017). The explanation of
saving money for the production can reduce by reducing need for product padding to meet
required product specifications. Padding is forces on the process of making a product of
higher-quality compare to the specification required. Pad is importance for the manufactured
which lead to the specifications that need to be match and also involve the cost production
(Levitt, 1983).
100
101
Levitt (1983) also mentioned that, if the production is accurately which depend on the
set point resulting to the aim of product specification thus save manufacturer money, and also
increase the efficiency which some processes need to be maintained at specific point to
maximize efficiency. Furthermore, ensure the safety can be interpreting as an out-of-control
(run-away process) of chemical reaction that interfere the manufacture production due to the
uncontrolled all the process variable (Levitt, 1983). The process variable can be divided into
several which depend on the equipment such as temperature, pH, pressure and others
(Bequette, 2006). This can be concluding that; the process control gives huge impact to the
process which can lead to the result of the product and also the safety. With the benefit of
process control tools, enable the process of runs smoothly.
101
102
7.2 Process control of Seed Fermenter
Figure 7.1: Piping and instrumentation diagram (P&ID) for seed fermenter.
Table 7.1: P&ID symbol and abbreviation for seed fermenter.

Symbol Abbreviation
FIC Flow indicator controller
TI Temperature indicator
pH pH probe
PI Pressure indicator
LI Level indicator
Foam Anti-Foam probe
102
103
Table 7.2: Process control description for seed fermenter
Controller Measured variable Manipulated variable Disturbances Set Point

variable
Level indicator Level meter in the Inlet flowrate Too much volume will The maximum level is 75% from
seed fermenter result in poor agitation, the seed fermenter volume. The
dissolved oxygen and flow will manually adjust
slows down the rate according to the maximum
operating fermenter capacity.
Temperature Heat produced Water jacketed cooling Cell growth will be Living cells are very sensitive to
during the system slower as the temperature. Therefore,
metabolic reaction temperature are not in temperature probes should be
of the living cell the optimum condition. steam-serializable in place and
detected by The temperature is set stable over several weeks, so
thermocouple or up that they can be used for in-situ
RTD. The sign will measurement in fed-batch and
display perfusion cultures.
pH The pH reading pH loop system Cell needs suitable Initially, a three-point calibration
range by pH probe. environment (pH) in is typically performed. One
The probe pH will order to grow. standard for the adjustment of
detect change of Outbound pH range will the transmitter output, typically
temperature and result in growth at pH 7, a second one for the
display to the restriction slope adjustment, and the third
computer. one for a check on linearity.
This calibration is needed to
ensure the probe are valid
throughout the process. The pH
is set up at 6.0±0.1 in the
process. Any natural changes
103
104
due to cell growth reaction, the

loop system will automatically
adjust to the set pH.
Foam formation Antifoam sensor at Antifoam and air are supply The excesses of The antifoam occurs to low the
the seed fermenter into the seed fermenter formation antifoam may formation if it is excesses.
(tank). This affect the production
and also cause the
contamination
104
105
7.3 Process control of fermenter
Figure 7.2: Piping and instrumentation diagram (P&ID) for fermenter
Table 7.3: P&ID symbol and abbreviation for seed fermenter.

Symbol Abbreviation
FIC Foam Indicator Controller
PI Pressure Indicator
TI Temperature Indicator
LI Level Indicator
pH pH probe
Foam Anti-foam probe
105
106
Table 7.4: Process control description for fermenter
Control Measured Manipulated Disturbance Set Point

Variable Variable Variable
Temperature of Heat produced Water jacketed Cell growth will be slower as the Living cells are very sensitive to
fermenter during the cooling system temperature are not in the temperature. Therefore, temperature
metabolic optimum condition probes should be steam-sterilizable in
reaction of the place and stable over several weeks, so
living cell that they can be used for in-situ
detected by measurement in fed-batch and perfusion
thermocouple or cultures.
RTD
pH of mixture The pH reading pH loop system Cell needs suitable environment Initially, a three-point calibration is
range by pH probe (pH) in order to grow. Outbound typically performed. One standard for
pH range will result in growth the adjustment of the transmitter output,
restriction typically at pH 7, a second one for the
slope adjustment, and the third one for a
check on linearity. This calibration is
needed to ensure the probe are valid
throughout the process. The pH is set up
at 6.0±0.1 in the process. Any natural
changes due to cell growth reaction, the
loop system will automatically adjust to
the set pH.
Pressure Pressure meter Venting system Affecting the dissolved oxygen It is naturally measured as a gauge
indicator in the detection level in cell and the aeration in pressure, as a relative to atmospheric
tank the process conditions. The vent will be
automatically adjusted to transfer out air
just enough to maintain 101.325±10 kPa.
106
107
Level indicator Level meter in the Inlet Flowrate Too much volume will result in The level is set to be 75% from the total
tank poor agitation, dissolved oxygen volume of the fermenter as it involved
and slows down the rate. with living organisms. The flow will
manually adjusted according to the
maximum operating fermenter capacity.
107
108
7.3 Process control of centrifuge (decanter).
Figure 7.3: Piping and instrumentation diagram (P&ID) for centrifuge (decanter).
Table 7.5: Symbol and abbreviation for centrifuge (decanter).

Symbol Abbreviation
FI Flow Indicator
FC Flow Control
Table 7.6: Process control description for centrifuge (decanter).

Control Measured Manipulated Disturbances Set point
variable variable Variable
Flow of Outlet Inlet flowrate Decanter Specified flow of liquid into the
liquid flowrate overflowed decanter centrifuge should not exceed
into the with liquid, 1.5 Million L for decanter centrifuge 1
decanter poor separation and 312,321.977 L for decanter
centrifuge process centrifuge 2 or else the flow indicator
alarm will be activated.
108
109
7.4 Process control of ion exchange
Figure 7.4: Piping and instrumentation diagram (P&ID) for ion exchange.
Table 7.7: Symbol and abbreviation for ion exchange.

Symbol Abbreviation
FC & FI Flow Controller and Flow Indicator
pHC & pHI pH Controller and pH Indicator
LC & LI Level Controller and Level Indicator
pHA, LIA, FIA pH, Level and Flow Indicator Alarm
109
110
Table 7.8: Process control description for ion exchange.

Control Measured Manipulated Disturbances Set point
variable variable Variable
Liquid level in Outlet flowrate Inlet flowrate Overflow of liquid Specified level in the
the column exceed optimum column to prevent
capacity of column column from flooding.
Does not exceed
185,200 L at one time.
If exceed the level
indicator alarm will be
activated
pH of solute Outlet flowrate Inlet flowrate pH of column pH of column exceeds
exceeding optimum + 0.5 of the optimum
pH of product pH then the pH alarm
will be activated.
110
111
CHAPTER 8
PROCESS SAFETY
8.1 Introduction
A good safety in an organization give benefits to all parties either employees,

management or customer. It provides a good working environment in the sector. A good safety
system lead to decrease in expenses for insurance cover as the risk of injury is small. The quality
of the product produced also maintained. As an organization, the brand or reputation is important
as it reflect the management system to the customer and gain interest among people to work
there. A good safety system is one of the factor contribute to it. In order to create good safety
system, several factors must be identified and considered for example hazard, risk, risk
assessment and control measures.
Hazard is the risk to the occurring of injury. Generally, in biochemical industry, hazard
can be identified as physical, electrical, mechanical and chemical (Richardson, 2012). A hazard
and operability study (HAZOP) is a structured and systematic examination of a complex planned
or existing process or operation in order to identify and evaluate problems that may represent
risks to personnel or equipment (Rausand, 2005).
HAZOP are the most widely used in the process industries today. It is one of the method
of risk assessment. It can be used to identify problems even during the early stages of project
development, as well as identifying potential hazards in existing systems. A HAZOP
systematically investigates each element in a process. The goal is to find potential situations that
111
112
would cause that element to pose a hazard or limit the operability of the process as a whole
(Rausand, 2005). There are four basic steps to the process:
1. Forming a HAZOP team
2. Identifying the elements of the systems
3. Considering possible variations in operating parameters
4. Identifying any hazards or failure points
The task of analysing hazards in a workplace or system can be daunting. However, without
an effective analysis, potential hazards may not be discovered before they result in injuries and
loss. The cost of an accident is often many times greater than the cost of the analysis that could
have stopped it (Rausand, 2005).
112
113
8.2 Process safety for seed fermenter.
Table 8.1: HAZOP study on seed fermenter.

Plant / Operation: Seed Fermenter Review Date: 21/10/2017
Line / Vessel / Node: P-1 / SFR-101
Design Intention: Growth of microbes
Guide Deviations Possible Causes Possible Action Required Recommendation
Word Consequences
LESS Level 1. Rupture of vessel Backflow of Install level & flowrate Maintenance by Technician
wall material indicator, new gasket &
2. Outlet valve fails to Level decreased in alarm
close the tank
3. Leak from vessel Routinely inspect water tight
door seal before each use
MORE Level 1. Inlet valve does not Overflow of material

close Install a flowrate &
2. Cooling jacket emergency shut off indicator
ruptures into vessel and alarm
LESS Pressure 1. Rapid drop in Pressure decrease Vent should be open and Isolation must be placed on
temperature in tank could lead to pressure controlled at all valves to ensure not opening
2. Liquid drawn from failure times without safeguards in place
tank without second
opening
MORE Pressure 1. Pump run for longer Pressure increase High pressure alarm and
than required in tank could lead to level indicators on tanks
failure
113
114
LESS Temperature 1. Refrigeration unit set Fermentation Unit must be switched off to Ensure set point is set
at wrong set point stopped and need change preset correctly
2. Control valve fails to to restart
open
MORE Temperature 1. Refrigeration unit Fermentation out of Control manually

malfunction control, spoiled
2. Refrigeration side product.
pump failure
LESS Flow 1. Pipe was blocked Slow time moving to Maintenance of pipes to Do not break an isolation to
2. Cooling system next process unit ensure clean open a valve
pump fails
MORE Flow 1. Pump speed was set Over pressure of Ensure all equipment is
up too high downstreams unit operating correctly before
starting work
114
115
8.3 Process safety for fermenter
Table 8.2: HAZOP study for fermenter

Plant / Operation: Fermenter Review Date: 21/10/2017
Line / Vessel / Node: P-7 / FR-101
Design Intention: Growth of microorganisms
Guide Deviations Possible Causes Possible Action Required Recommendation
Word Consequences
LESS Level 4. Rupture of vessel Backflow of Install level & flowrate Maintenance by Technician
wall material indicator, new gasket &
5. Outlet valve fails to Level decreased in alarm
close the tank
6. Leak from vessel Routinely inspect water
MORE Level door tight seal before each use
3. Inlet valve does not Overflow of

close material Install a flowrate &
4. Cooling jacket emergency shut off indicator
ruptures into vessel and alarm
LESS Pressure 3. Rapid drop in Pressure decrease in Vent should be open and Isolation must be placed on
temperature tank could lead to pressure controlled at all valves to ensure not opening
4. Liquid drawn from failure times without safeguards in place
tank without second
opening
MORE Pressure increase in High pressure alarm and
Pressure 1. Pump run for longer tank could lead to level indicators on tanks
than required failure
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LESS Temperature 3. Refrigeration unit set Fermentation Unit must be switched off to Ensure set point is set
at wrong set point stopped and need to change preset correctly
4. Control valve fails to restart
open
MORE Temperature 3. Refrigeration unit Fermentation out of Control manually

malfunction control, spoiled
4. Refrigeration side product.
pump failure
LESS Flow 3. Pipe was blocked Slow time moving Maintenance of pipes to Do not break an isolation to
4. Cooling system to next process unit ensure clean open a valve
pump fails
MORE Flow 2. Pump speed was set Over pressure of Ensure all equipment is
up too high downstreams unit operating correctly before
starting work
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8.3 Process safety for centrifuge (decanter)
Table 8.3: HAZOP study for centrifuge (decanter)

Process Deviation Possible Causes Possible Consequences Action Required
Parameter (Guide Words)
Flow & Level NO 1. Control valve 1. No product separation. 1. Select valve to open.
fails open. 2. No product separation 2. Install filter with maintenance procedure.
2. Plugged inlet Install flow meter and flow alarm.
pipes.
HIGH 1. Control valve 1. Poor separation of product, 1. Select valve to close.

fails close. high moisture in concentrate. 2. Install flow meter and flow alarm.
1. Increase operation time

LOW 1. Control valve and low separated product. 1. Install filter with maintenance procedure.
partially Poor separation of Install flow meter and flow alarm.
open/plugged. concentrate and liquid. 2. Install flow meter and flow alarm.
2. Increase operation time

2. Control valve fail and low separated product.
to respond. Poor separation of
concentrate and liquid.
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8.5 Process safety for ultrafiltration
Table 8.4: HAZOP study for ultrafiltration

Process Guide Words Deviation Possible Possible Action
Parameter Cause Consequences
Flow MORE Excess of Valve fully -Overflowing of the -Connect the system with
material onto open. materials. flow indicator.
the -Equipment efficiency
ultrafiltration. decrease.
LESS Less amount of -Valve is partly -Equipment efficiency -Set the minimum flowrate.
material enters open. decrease. -Connect the flow indicator
the -Leaking in the to alarm system.
ultrafiltration. pipe.
Pressure MORE High pressure. Pressure Explosion of the -Installed pressure indicator
control failure equipment -Connect the pressure
indicator with alarm system,
LESS Low pressure. -Pressure Lack of biomass. -Installed pressure indicator
control failure.
-Vacuum pump
malfunction.
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8.6 Process safety for ion exchange
Table 8.5: HAZOP study for ion exchange

Process Deviation Possible Causes Possible Consequences Action Required
Parameter (Guide Words)
Flow & NO 1. Control valve fails 1. No product separation. 1. Select valve to open.
Level open. 2. No product separation 2. Install filter with maintenance procedure.
2. Plugged inlet pipes. Install flow meter and flow alarm.
HIGH 1. Control valve fails 1. Poor separation of 1. Select valve to close.

close. product. 2. Install flow meter and flow alarm.
Column overflow.
LOW 1. Control valve partially 1. Increase operation time 1. Install filter with maintenance procedure.
open/plugged. and low separated product. Install flow meter and flow alarm.
2. Install flow meter and flow alarm.
2. Control valve fail to 2. increase operation time
respond. and low separated product.
pH MORE 1. Liquid pH entering 1. Poor Separation of 1. Add acid to lowered pH according to set point.
column. product. Install pH meter and pH alarm.
LESS 1. Liquid pH entering 1. Poor Separation of 1. Add base to lowered pH according to set
column. product. point. Install pH meter and pH alarm.
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CHAPTER 9
WASTE MANAGEMENT
9.1 Introduction
Waste or rubbish are trash, junk, garbage that depend on the type of material or the
regional terminology which is unwanted or undesired material or substance. It may consist of
unwanted material left over from a manufacturing process (industrial, commercial, mine or
agriculture operations) or from community and household activities (Devendra, 2017; Marshall
and Farahbakhsh, 2013). The material may be described or accumulated, stored or treated
(physical, chemically or biologically), prior to being discard or recycled. It is also used to
describe something we use inefficiently or inappropriately (Marshall and Farahbakhsh, 2013).
Most contemporary waste management efforts are focused at local government level
and based on high tech or high energy waste disposal by methods such as landfill and
incineration (Zhang et al., 2010). However, these methods are becoming increasingly
expensive and energy inefficient. The financial cost of managing the long-term environmental
impacts of waste disposal are many times what is actually charged for this service and in many
cases corrective action is not remotely feasible. The purely environmental costs such as
negative effects on habitat, wildlife and biodiversity are also recognized (Fahrig, 2003). In other
words, waste disposal is not sustainable and will have negative implications for future
generations.
Waste treatment is a biological, chemical or mechanical method that is used to remove

pollutant from industrial or municipal wastes. It is the method to change the character and
composition of medical waste. It is also can help to reduce or eliminate its potential for harm to
living beings and the environment (Sharholy et al., 2008; Tchobanoglous et al., 2002). Waste
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management and treatment comes into two types of management which is the one that used
conventional method and also the recently used one which is the integrated system of waste
management (Chifari et al., 2017). In conventional, the cost on energy used, technology and
also all possible cost are higher than usual. While for the integrated system, it applies a much
better system where it promotes on preventing or reducing those sources of waste first then if
those sources is less than all other criteria will be decrease automatically. Figure 9.1 and 9.2
shows the differences of the conventional and integrate waste management system.
Figure 9.1: Conventional Figure 9.2: Integrated
Figure 9.1 and 9.2 above shows a clear difference of conventional and integrated waste
management system where the integrated system is much more preferable than the
conventional because it saves cost and much more environmental free. These systems need
to be apply both in industrial and municipal waste management system. Waste to be managed
including all forms of matter such as gaseous, liquid, solid and radioactive matter. The issue of
untreated waste being release to the environment which will contaminate the environment that
can pose a long-term health risk and also pollution. However, when it comes to plant design
the raw materials waste can be used for another production of produce. The process itself will
undergoes several processes or method by including other materials to produce the desired
product.
Production plant does not only produce product because it is impossible to reach a
100% conversion of reactant to product where by-products also produces whether it is a new
form of by-products or residue of the production process. Those by-products usually denoted
as waste or unwanted materials derived from the process. Any waste should be treated first
before being discarded to the open environment. Therefore, those waste from this project need
to be treated first before being discarded where each waste has a different methodology on
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how to treat them. The methodology treatment itself divided into three which is physical or
mechanical, chemical and biological treatment. The treatment process is a method to change
the properties of the waste turning it into less or non-hazardous substances first before being
discarded into open environment. Therefore, it will reduce or eliminate the potential of those
materials to harm the environment. The Environmental Protection Regulation 2009, they
intended to recognize the resources potential of industrial wastes. In order to keep the
environment free from pollution and good management, there has to be a level of
consciousness with regard to the importance of the environment. It means that awareness
building regarding the environmental conservation and development are to be made
immediately (Clarke et al., 1994).
9.2 Waste identification and treatment
9.2.1 Carbon dioxide
Carbon dioxide (CO2) is one of the waste derive from the production plant during the
fermentation process. Excessive carbon dioxide in the air may cause headache, sweating,
rapid breathing, increased heartbeat, shortness of breath and dizziness. The number of impact
or symptoms may vary between individuals where high concentration or levels of CO 2 in air
may even cause coma, asphyxia, convulsions, unconsciousness and even death. All of these
problems may arise if the emitted carbon dioxide is trapped in a closed area without a proper
ventilation. This problem may be settle by enlarging the working area or make sure the
ventilation of the room is good by supplying more air into the room or by exhausting or circulate
the air inside the room with the outer air. Another solution can be used is by installing pipes on
the venting of the gas, but usually this method only applied when the carbon dioxide gas need
to be used gain or recover.
9.2.2 Wastewater
Wastewater is one of the by-product derived from the production plant. In this project,
some wastewater derived from rotary vacuum filter while the other one is from the decanter.
Both wastewater may contain different content of materials that they bring together while being
excreted such as they may possess sludge from the decanter, nitrogen content, carbon dioxide
and other properties. However, they will undergo the same treatment process. It was done by
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collecting those wastewater in one tank before treating it. The treatment process may or may
not undergoes through all these four steps in wastewater treatment process which is the
Screening, the Primary treatment, secondary treatment and final treatment. However, in this
production plant, the wastewater will not possibly go through all those four treatment steps.
The wastewater in this production plant may undergoes the screening process to remove any
large particles, after that it need to be tested for their COD, BOD or other wastewater criteria
content before being treated further. If the wastewater exceeds any limits of wastewater
criteria, then it will be treated. Since this is a wastewater produce from a pharmaceutical plant,
therefore the treatment process would be done after adjusting those limits of wastewater into
an allowable release range. Before releasing the wastewater, it needs to meet the water quality
standards as mention in table 5.1.
Table 9.1: National Water Quality Standards for Malaysia

Parameter Unit Class
I IIA IIB III IV V
Ammoniacal Nitrogen mg/l 0.1 0.3 0.3 0.9 2.7 > 2.7
Biochemical Oxygen mg/l 1 3 3 6 12 > 12
Demand
Chemical Oxygen mg/l 10 25 25 50 100 > 100
Demand
Dissolved Oxygen mg/l 7 5-7 5-7 3-5 <3 <1
pH - 6.5 - 6-9 6-9 5-9 5–9 -
8.5
Colour TCU 15 150 150 - - -
Electrical µS/cm 1000 1000 - - 6000 -
Conductivity*
Floatables - N N N - - -
Odour - N N N - - -
Salinity % 0.5 1 - - 2 -
Taste - N N N - - -
Total Dissolved Solid mg/l 500 1000 - - 4000 -
Total Suspended mg/l 25 50 50 150 300 300

Solid
Temperature °C - Normal + - Normal + 2 - -
2 °C °C
Turbidity NTU 5 50 50 - - -
Faecal Coliform** count/100 ml 10 100 400 5000 5000 -
(20000) a (20000) a
Total Coliform count/100 ml 100 5000 5000 50000 50000 > 50000
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9.2.3 Decanter cake and biomass
This form of waste usually comes in a form of solid. Both decanter cake and Biomass
can be treated in the same way where it can be dried first before releasing it. Other than that, it
can be treated by combusting it and turn it into less weight and less hazardous solid.
9.2.4 Summarize of waste of treatment process
Table 9.2: Treatment process of waste

Waste form Pollution control/ treatment process
Carbon Dioxide, CO2 • Enlarging working space.
• Supply more air into the room.
• Good air ventilation circulation
• Install recovery pipes on the venting
Waste water • Filtration
• Test for COD, BOD, Nitrogen
• Treat the waste content into allowable range
• Release wastewater meet the water quality standard
Decanter cake • Combustion process
Hydrochloric Acid, HCL • Stored in bottles before treating.
• Diluting it into neutral pH
Biomass • Combustion process by cofiring with other fuels or through
gasification
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9.2.5 Type of waste generated from the equipments
Table 9.3: Amount of waste generated from all the equipments

Equipment Stream Waste Component Amount Waste Generated Total
Blending/ Storage None None None None
Ammonia = 6,170.28 kg/batch =146,715.17 kg/batch
Seed Fermenter S-102 Carbon dioxide = 39,130.38 kg/ batch
Nitrogen = 175.62 kg/batch
Oxygen = 101,238.88 kg/batch
Ammonia = 1,762.73 kg/batch = 1,310,861.40 kg/batch
Carbon dioxide = 127,811.07 kg/batch
Fermenter S-107 Nitrogen = 1,596.06 kg/batch
Oxygen = 2,939.53 kg/batch
Palmitic Acid 3 Palmitic Acid = 54,316.43 kg/batch = 54,316.43 kg/batch
Biomass = 477,801.71 kg/batch = 1,054,445.64 kg/batch
Decanter 1 B+Y+W (1) Water = 547,675.20 kg/batch
Yeast Extract = 28,968.73 kg/batch
Biomass + Biomass = 20,117.97 kg/batch = 94,371.11 kg/batch
Yeast + Water
Decanter 2 Water = 68,459.40 kg/batch
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Biomass = 5,029.49 kg/batch = 443,321.35 kg/batch
Ultrafiltration S-123 Water = 37,843.43 kg/batch
Ammonia NH4OH = 14.32 kg/batch = 14.32 kg/batch

Hydroxide out
Ion Exchange
NH4OH & NH4OH = 1.99 kg/batch = 40,035.77 kg/batch
Glucose
Glucose = 40,033.78 kg/batch
Drum Dryer Stream Steam Water = 30,003.66 kg/batch = 30,003.66 kg/batch
Total waste generated = 3,174,084.85 kg/batch
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CHAPTER 10
PROFITABILITY ANALYSIS
10.1 Raw Materials Cost
Table 10.1: Price list of raw materials for riboflavin production

No. Material Mass used (kg/year) Price/kg Price (RM)
1. Glucose 4,892,985.9 RM 19.50 RM 95,413,225.05
2. Palmitic Acid 6,638,665.7 RM 192.72 RM 1,279,403,654.00
3. Yeast Extract 362,109.1 RM 678.60 RM 245,727,235.30
4. Ashbya gossypii 59,054.0 RM 436.76 RM 25,792,425.04
Price/m3
5. Oxygen 15,906,080.1 RM 14.82 RM 235,728,107.10
6. Ammonia 969,949.8 RM 2.35 RM 2,279,382.03
7. Ammonia Hydroxide 163.17 RM 22.00 RM 3,589.74
Total RM 1,884,347,618.26
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10.2 Labor Cost
The table below shows all the salary employee for all position in the production of riboflavin
(Vitamin B2).
Table 10.2: Salary of employee per month.
Position Salary No of Person Total Salary of Each Workers

Manager RM8,000.00 1 RM 8,000.00
Safety officer RM6,300.00 1 RM 6,300.00
Assistant Safety RM3,500.00 1 RM 3,500.00
Finance Executive RM5,850.00 1 RM 5,850.00
Assistant Finance RM2,500.00 1 RM 2,500.00
Senior Engineer RM6,900.00 1 RM 6,900.00
Engineer RM2,600.00 2 RM 5,200.00
Administration RM1,600.00 2 RM 3,200.00
Executive
Technician RM1,800.00 3 RM 5,400.00
Operator RM1,700.00 20 RM 34,000.00
TOTAL RM 80,850.00
Table10.3: Salary of employee per year.
Type of Occupation Total Salary of Each Workers Salary of Year

Manager RM 8,000.00 RM96,000.00
Safety officer RM 6,300.00 RM 75,600.00
Assistant Safety RM 3,500.00 RM 42,000.00
Finance Executive RM 5,850.00 RM 70,200.00
assistant Finance RM 2,500.00 RM 30,000.00
Senior Engineer RM 6,900.00 RM 82,800.00
Engineer RM 5,200.00 RM 62,400.00
Administration Executive RM 3,200.00 RM 38,400.00
Technician RM 5,400.00 RM 64,800.00
Operator RM 34,000.00 RM 408,000.00
TOTAL RM 80,850.00 RM970,200.00
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Table 10.4: Summarization of no. of workers and no. of equipment units for all the equipment.
Types equipment Multiple units No. of workers

Seed Fermenter 4 5
Fermenter 6 7
Mixing 2 2
Ultrafiltration 1 1
Centrifuge 2 2
Ion Exchange 1 1
Drum Drying 2 2
TOTAL 28 20
NOL 6.57
No of shift per years 990 shifts / day
No of operates for an operator 245 shifts / years
No of operator needed 4.05 operators
Operating Labours 540 persons
Labours cost in Malaysia (2017) RM 11,578,200.00
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10.3 Waste Management and Treatment Cost
10.3.1 Cost for waste transportation.
Figure 10.1: Schedule A (Transportation Cost for Quality Alam)
Notes: The pallet size 120 cm x 120 cm that crates for four (4) 200 liters Drums per units.
Table 10.5: Type of waste produce in every equipment

Equipment: Type of Waste:
Blending/Storage None.
Seed Fermenter Ammonia and carbon dioxide (Gasses).
Fermenter Ammonia and carbon dioxide (Gasses).
Centrifuge 1 Biomass, palmitic acid, water and yeast extract.
Centrifuge 2 Biomass, water and yeast extract.
Ultrafiltration Biomass and yeast extract.
Ion Exchange Ammonia hydroxide and glucose.
Drum Drying Water (Steam).
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10.3.1.1 Total liquid/solid waste produce:
a) Centrifuge 1: 109,9930.53 L/Batch

Therefore
109,9930.53 L
=
𝐿
200 𝐷𝑟𝑢𝑚
= 5,499.65 ≈ 5,500 𝐷𝑟𝑢𝑚𝑠
For 1 pallet equal 4 drums

5,500 𝐷𝑟𝑢𝑚𝑠
=
𝐷𝑟𝑢𝑚𝑠
4
𝑃𝑎𝑙𝑙𝑒𝑡
= 1,375 𝑃𝑎𝑙𝑙𝑒𝑡
b) Centrifuge 2: 94,174.90 L/Batch

Therefore
94,174.90 L
=
𝐿
200
𝐷𝑟𝑢𝑚
= 470.88 ≈ 471 𝐷𝑟𝑢𝑚𝑠

471 𝐷𝑟𝑢𝑚𝑠
=
4
= 117.75 ≈ 118 𝑃𝑎𝑙𝑙𝑒𝑡
c) Ultrafiltration: 44,490.61 L/Batch

Therefore
44,490.61 L
=
𝐿
= 222.45 ≈ 223 𝐷𝑟𝑢𝑚𝑠

=
4
= 55.75 ≈ 56 𝑃𝑎𝑙𝑙𝑒𝑡
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d) Ion exchange: 35,857.55 L/Batch

Therefore
35,857.55 L
=
𝐿
= 179.29 ≈ 180 𝐷𝑟𝑢𝑚𝑠

=
4
= 45 𝑃𝑎𝑙𝑙𝑒𝑡
Total waste on pallet: 1594 Pallet

Since the location of the company in Johor and pallet for the rate transportation fee fall under
2nd tier.
Per Pallet (RM) = 1594 x 41.13

= RM 63,710.37
Per MT (RM) = 1594 x 102.82
= RM 168,562.18
Total transportation = RM 63,710.37 + RM 168,562.18
= RM 232,272.55
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10.3.2 Cost for waste treatment.
Figure 10.2: Schedule B (Organic Waste for Incineration)
Notes: Packaged Waste refers to waste packed in standard 200 liters drums.
Total Drums for organic waste incineration (Waste Group A):

= 5500 + 471 + 223
= 6194 Drums
Therefore:
= 6194 x 810
= RM 5,017,140.00
Figure 10.3: Schedule B (Inorganic Waste for Physical/Chemical Treatment)
Total Drums for inorganic waste:

= 180 Drums
Therefore:
= 180 x 1620
= RM 291,600.00
Total waste treatment: RM 5,017,140.00 + RM 291,600.00

= RM 5,308,740.00
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10.4 Land Cost
The industrial site as proposed by the team which has the criteria to enhance the
probability of the company to grow much faster and build more relationship with other party
since the site itself is located nearer to SILC Iskandar Nusajaya which is in Gelang Patah which
known to be an industrial hub in Johor that rounded with several amenities. The land has a total
area of 43776.82 square feet with RM 95.94 per square feet which gives a total of RM
4,199,948.11. The land itself has a lot of advantages since it is located near to public facilities,
Universities, residential homes, ready infrastructure, levelled land plot, got a lot of central
utilities and most importantly it is a strategic location which is near to Singapore factory
relocation to Johor Bahru that can enhance more International cooperation not only with
Singapore but other country as well. The location of the proposed site will provide working
opportunity to the residential around and also will grow much faster since the location itself is
connected to all Major Highway and town which also closer to speed train station from
Singapore to KL travel. The price of the land may be quite expensive since it has a lot of benefit
around the site itself. However, the cost of plant operation can be cut down since all utilities
needed for the plant to operate already provided surrounding Gelang Patah. Figure 10.4 and
Figure 10.5 below shows the surrounding of the proposed site location.
Figure 10.4: Gelang Patah Location in Johor.
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Figure 10.5: Nusajaya, Johor
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10.5 Utility Cost
10.5.1 Electricity
Based on TARIFF E3 - HIGH VOLTAGE PEAK/OFF-PEAK INDUSTRIAL TARIFF (For all

kWh during the peak period: 33.70 sen/kWh)
Steam factor = number of days plant operates per year / number of days per year (OSHA)
= 310.82 days/yr / 330 days/yr
= 0.94
Equipment Power used Period Steam Price/batch Price/yr (10 batch)

(kW/batch) (hr) factor
Blending 10.53 80.47 0.94 RM 268.42 RM 2,684.20
Seed 288.99 72 0.94 RM 6,591.33 RM 65,913.30
Fermenter
Fermenter 2425.17 168 0.94 RM 129,065.22 RM 1,290,652.30
Centrifuge 214.26 4 0.94 RM 271.49 RM 2,714.90

Centrifuge 117.17 4 0.94 RM 148.47 RM 1,484.70
Ultrafiltration 18.54 24 0.94 RM 140.95 RM 1,409.50
Drum Drying 3.75 24 0.94 RM 28.51 RM 285.10
TOTAL 3,078.41 RM 136,514.39 RM 1,365,143.90
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10.5.2 Water
10.5.2.1 Stream
The annual overall production consists of 10 batch which,

Water 1 (Stream-110) = 92,677.01 L/batch x 10 batch/yr
= 926,770.10 L/yr
Total water consumption = 926,770.10 L/yr
= 926.77 m3/yr
Water consumption costs is based on: Tariff rates for Non-domestic: >35m3 = RM 3.30/m3
Price for water consumption = 926.77 m3/yr x RM 3.30/m3

= RM 3,058.34/yr
10.5.2.2 Washing
Annual usage (L/yr) = (N x W) x (1 - R)
Where:
N = Number of annual units washed, processed or manufactured
W = Liter consumed per unit
R = Percentage recycled or reused (in decimal)
18 𝑢𝑛𝑖𝑡𝑠 10 𝑏𝑎𝑡𝑐ℎ
(( × ) × 100𝐿) × (1 − 0.6)
𝑏𝑎𝑡𝑐ℎ 𝑦𝑟
𝐿 1𝑚3
= 7200 ×
𝑦𝑟 1000𝐿
7.2 𝑚3 𝑅𝑀 3.30
= ×
𝑦𝑟 𝑚3
𝑅𝑀 23.76
=
𝑦𝑟
Total water consumption = RM 3,058.34/yr + RM 23.76/yr
= RM 3,082.10/yr
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10.5.3 Internet
Criteria Details
Internet service provider Telekom Malaysia Berhad
Broadband plan UniFi Pro
Package download speed 100Mbps
Monthly price RM 239.00
Annually price RM 2,868.00
10.5.4 Total Utility Costs
Utility Annually Price

Electric RM 1,365,143.90
Water RM 3,082.1
Internet RM 2,868.00
TOTAL RM 1,371,094.00
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10.6 Capital Cost
10.6.1 Grass Root Capital (GRC)
The equipment costing part was done in previous chapter which is in Chapter 6. This part
is done to determine the grass root capital for Ribo Tech Ent Plant. Grass root capital cost is
the largest part of total fixed capital cost. Cost of equipment installed in a plant is referred as
grass root capital cost. To estimate the cost for every equipment in the plant, Bare Module
method is used. Next, to calculate the GRC, contingency and fees (18% of bare module cost),
auxiliary facilities (30% of bare module cost) is added to the initial bare module cost.
Total Bare Module Cost (CBM) = MYR 11,646,272.12
• Contingency and Fees:

Contingency = 0.15 x CBM = MYR 1,746,940.12
Fees = 0.03 x CBM = MYR 349,388.16
Capital Cost = Contingency + Fees + CBM

= MYR 2,096,328.28
• Auxiliary Facilities:
Site Development = 0.06 x CBM = MYR 698,776.33
Auxiliary Building = 0.04 x CBM = MYR 465,850.88
Offsite Facilities = 0.2 x CBM = MYR 2,329,254.42
Total Grass Root Capital (GRC) = Auxiliary Facilities + Capital Cost

= MYR 5,590,209.91
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10.6.2 Total Capital Investment (TCI)
The Total Capital Investment (TCI) is a one-time expense for the design, construction and
start-up of a new plant. It can be represented by the equation below:
Total Capital Investment = Fixed Capital Investment + Working Capital + Start-up
Fixed Capital Investment (FCI) is a capital that is needed to install the process equipment with
all auxiliaries to complete the process operation. The direct and indirect cost is included for
every equipment. The working capital funds are needed to cover operating costs required for
the early operation of the plant, including the cost of the inventory and funds to cover accounts
receivable because they involve the costs of the raw materials and the values of the
intermediates products and by-product. Usually, the working capital is 12% of the fixed capital
investment whereby start-up cost is 8% of the fixed capital cost.
Table 10.6: The direct cost and indirect cost for all equipment.
No. Equipment Direct Cost (RM) Indirect Cost (RM) Bare Module, CBM (RM)
1 Blending Tank 50,167.83 8,298.79 58,466.62
2 Seed Fermenter 1,030,704.45 168,476.83 1,199,181.28
3 Fermenter 2,252,377.58 1,316,092.37 3,568,469.95
4 Centrifuge 1,159,200.38 191,755.57 5,757,865.26
5 Centrifuge 2 2,196,271.09 2,210,638.22
6 Ultrafiltration 4,603.17 1,406.76 6,009.88
7 Ion Exchange 481,862.01 79,709.88 561,571.98
8 Drum Drying 212,244.08 35,109.54 494,707.24
TOTAL 7,387,430.59 4,011,487.96 11,646,272.12
Cost of Land (Gelang Patah, Johor):

Price per square feet = RM 95.94
Total area of land = 43,776.82 square feet
Total price of land = RM 4,199,948.11
Fixed Capital Investment (FCI) = Total Direct Cost + Total Indirect Cost + GRC
= RM 87,631,572.91
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Working Capital = 0.12 x FCI = RM 10,515,788.75

Star-up Cost = 0.08 x FCI = RM 7,010,525.83
Total Capital Investment (TCI) = FCI + Working Capital + Start-up Cost

= RM 17,526,314.58
10.6.3 Direct Manufacturing Cost (DMC)
Range Specification Costs (RM)

Raw material 1,884,347,618.26
Utilities 142,440.73
Waste treatment 5,308,740
Operating labour 970,200
Maintenance 10% 87,631,572.91 8,763,157.29
Supervision 10% 970,200 97,020
Laboratory charge 7% 970,200 67,914
Pattern and loyalties 1% 87,631,572.91 876,315.73
Total 1,900,573,406.01
141
142
10.6.4 Fixed Manufacturing Cost

Local text 1% 87,631,572.91 876,315.73
Insurance 1% 87,631,572.91 876,315.73
Plant overhead 25% 970,200 242,550
Depreciation 10% 87,631,572.91 8,763,157.29
Total 10,758,338.75
Total Manufacturing Expenses (AME) = Direct + Fixed Manufacturing Cost

= RM 1,911,331,745.00
10.6.5 General Expenses (AGE)
Range Specification Cost (RM)

Administration 10% 9,830,377 983,037.73
Distribution and selling expensive 5% 87,631,572.91 4,381,578.65
Research and development 4% 87,631,572.91 3,505,262.92
Total 8,869,879.30
Total Cost of Manufacturing (COM) = AME + AGE

= RM 1,920,201,624.00
10.7 Revenue Sales
• Selling price of purified riboflavin per kg = RM 1,120.00

• Production rate per year = 1,073,557.50kg/year
Total selling price per year = RM 1,202,384,400.00
142
143
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APPENDIX
150
151
APPENDIX FOR LABOUR
NOL = [6.29 + 31.7 (1)2 + 0.23 (no. of operators)]0.5

NOL = [6.29 + 31.7 (1)2 + 0.23 (20)]0.5
= 6.53
No of shift per year:
3 𝑠ℎ𝑖𝑓𝑡 330 𝑑𝑎𝑦𝑠
= ×
𝑑𝑎𝑦𝑠 𝑦𝑒𝑎𝑟𝑠
𝑠ℎ𝑖𝑓𝑡
= 990
𝑦𝑒𝑎𝑟𝑠
No of operates for an operator:
1 𝑠ℎ𝑖𝑓𝑡 5 𝑑𝑎𝑦𝑠 49 𝑤𝑒𝑒𝑘𝑠
= 𝑑𝑎𝑦𝑠
× 𝑤𝑒𝑒𝑘𝑠
× 𝑦𝑒𝑎𝑟𝑠
𝑠ℎ𝑖𝑓𝑡
= 245 𝑦𝑒𝑎𝑟𝑠
No of operators needed:
𝑠ℎ𝑖𝑓𝑡 𝑦𝑒𝑎𝑟𝑠
= 990 ×
𝑦𝑒𝑎𝑟𝑠 245
= 4.05 𝑜𝑝𝑒𝑟𝑎𝑡𝑜𝑟𝑠
Operating Labours:
= 4.05 × 6.53
= 26.43 ≈ 26.00
= 26.0 × 20 (no of operator)
= 520 persons.
Labours cost in Malaysia (2017)
= RM 1,700.00 × 12 months
= RM 20,400.00
Labour cost, COL
= 540 persons × RM 20,400.00
= RM 10, 608,000.00 + RM 562,200.00
= RM 11,170,200.00
151
152
APPENDIX FOR PROFITABILITY ANALYSIS
Direct manufacturing cost

Raw material 1,884,347,618.26
Utilities 142,440.73
Waste treatment 5,308,740
Operating labour 11,170,200.00
Maintenance 10% 87,631,572.91 8,763,157.29
Supervision 10% 11,170,200.00 1,117,020.00
Laboratory charge 7% 11,170,200.00 781,914.00
Patents and loyalties 1% 87,631,572.91 876,315.73
Total 1,913,736,059.28
Fixed Manfacturing Costs

Local text 1% 87,631,572.91 876,315.73
Insurance 1% 87,631,572.91 876,315.73
Plant overhead 25% 11,170,200.00 2,792,550.00
Depreciation 10% 87,631,572.91 8,763,157.29
Total 13,308,338.75
152
153
General Expensive (AGE)

Range Specification Cost (RM)
Administration 10% 9,830,377 983,037.73
Distribution and selling expensive 5% 87,631,572.91 4,381,578.65
Research and development 4% 87,631,572.91 3,505,262.92
Total 8,869,879.30
AGE = General expensive 8,869,879.30

AME = Direct manufacturing cost + Fixed manufacturing 1,927,044,398.03
Total cost of manufacturing = AME + AGE 1,935,914,277.33
153
154
APPENDIX UNDISCOUNTED CASH FLOW.
Capital Sales Income Depreciation Annual Expenses Cash Income Net Profit Net Cash Cummulative
Year Investment(TCI) (AS) (AD) (APC) (AS - APC) (AS-APC)-AD Income Cash Flow CD
0 102,528,940.30 -20,016,330.27 -20.02
1 71,770,258.21 -91,786,588.48 -111,802,918.75 -111.80
2 20,505,788.06 -40,522,118.33 -152,325,037.08 -152.33
3 10,252,894.03 -30,269,224.30 -182,594,261.38 -182.59
4 1,907,760,400.00 8,763,157.29 1,911,331,745.00 -3,571,345.00 -12,334,502.29 -3,571,345.00 -186,165,606.38 -186.17
5 1,926,838,004.00 876,315.73 1,911,331,745.00 15,506,259.00 14,629,943.27 15,506,259.00 -170,659,347.38 -170.66
6 1,946,106,384.04 87,631.57 1,911,331,745.00 34,774,639.04 34,687,007.47 34,774,639.04 -135,884,708.34 -135.88
7 1,965,567,447.88 8,763.16 1,911,331,745.00 54,235,702.88 54,226,939.72 54,235,702.88 -81,649,005.46 -81.65
8 1,985,223,122.36 876.32 1,911,331,745.00 73,891,377.36 73,890,501.04 73,891,377.36 -7,757,628.10 -7.76
9 2,005,075,353.58 87.63 1,911,331,745.00 93,743,608.58 93,743,520.95 93,743,608.58 85,985,980.48 85.99
10 2,025,126,107.12 8.76 1,911,331,745.00 113,794,362.12 113,794,353.36 113,794,362.12 199,780,342.60 199.78
11 2,045,377,368.19 0.88 1,911,331,745.00 134,045,623.19 134,045,622.31 134,045,623.19 333,825,965.79 333.83
12 2,065,831,141.87 0.09 1,911,331,745.00 154,499,396.87 154,499,396.78 154,499,396.87 488,325,362.66 488.33
13 2,086,489,453.29 0.01 1,911,331,745.00 175,157,708.29 175,157,708.28 175,157,708.29 663,483,070.95 663.48
14 2,107,354,347.82 0.00 1,911,331,745.00 196,022,602.82 196,022,602.82 196,022,602.82 859,505,673.78 859.51
15 2,128,427,891.30 0.00 1,911,331,745.00 217,096,146.30 217,096,146.30 217,096,146.30 1,076,601,820.08 1,076.60
16 2,149,712,170.21 0.00 1,911,331,745.00 238,380,425.21 238,380,425.21 238,380,425.21 1,314,982,245.29 1,314.98
17 2,171,209,291.92 0.00 1,911,331,745.00 259,877,546.92 259,877,546.92 259,877,546.92 1,574,859,792.21 1,574.86
18 2,192,921,384.84 0.00 1,911,331,745.00 281,589,639.84 281,589,639.84 281,589,639.84 1,856,449,432.05 1,856.45
19 2,214,850,598.68 0.00 1,911,331,745.00 303,518,853.68 303,518,853.68 303,518,853.68 2,159,968,285.73 2,159.97
20 2,236,999,104.67 0.00 1,911,331,745.00 325,667,359.67 325,667,359.67 325,667,359.67 2,485,635,645.40 2,485.64
154
155
RM3,000,000,000.00
RM2,500,000,000.00
Cumulative Cash Flow RM2,000,000,000.00
RM1,500,000,000.00
RM1,000,000,000.00
RM500,000,000.00
RM0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
-RM500,000,000.00
Year
Figure 10.6: Cumulative Cash Flow (Undiscounted Cash Flow).
155
156
APPENDIX DISCOUNTED CASH FLOW.

Discounted Cash Flow for 10%
Year Net Cash Income Discount Factor (fd) Discounted cash flow CD cash flow CD
1/(1+0.1)^n (i=10%) net cash income x fd
0 0.00 0.00 0.00 0.00 0.00
1 -91,786,588.48 0.91 -83,442,353.16 -83,442,353.16 -83.44
2 -40,522,118.33 0.83 -33,489,353.99 -116,931,707.16 -116.93
3 -30,269,224.30 0.75 -22,741,716.23 -139,673,423.38 -139.67
4 -3,571,345.00 0.68 -2,439,276.69 -142,112,700.07 -142.11
5 15,506,259.00 0.62 9,628,166.85 -132,484,533.22 -132.48
6 34,774,639.04 0.56 19,629,377.17 -112,855,156.05 -112.86
7 54,235,702.88 0.51 27,831,491.23 -85,023,664.82 -85.02
8 73,891,377.36 0.47 34,470,872.87 -50,552,791.95 -50.55
9 93,743,608.58 0.42 39,756,441.14 -10,796,350.81 -10.80
10 113,794,362.12 0.39 43,872,652.69 33,076,301.88 33.08
11 134,045,623.19 0.35 46,982,173.18 80,058,475.06 80.06
12 154,499,396.87 0.32 49,228,269.16 129,286,744.22 129.29
13 175,157,708.29 0.29 50,736,948.93 180,023,693.15 180.02
14 196,022,602.82 0.26 51,618,877.87 231,642,571.02 231.64
15 217,096,146.30 0.24 51,971,091.37 283,613,662.39 283.61
16 238,380,425.21 0.22 51,878,525.93 335,492,188.32 335.49
17 259,877,546.92 0.20 51,415,387.22 386,907,575.55 386.91
18 281,589,639.84 0.18 50,646,371.87 437,553,947.42 437.55
19 303,518,853.68 0.16 49,627,757.94 487,181,705.36 487.18
20 325,667,359.67 0.15 48,408,377.87 535,590,083.23 535.59
156
157
0 0.00 0.00 0.00 0.00 0.00
1 -91,786,588.48 0.83 -76,488,823.73 -76,488,823.73 -76.49
2 -40,522,118.33 0.69 -28,140,359.95 -104,629,183.68 -104.63
3 -30,269,224.30 0.58 -17,516,912.21 -122,146,095.90 -122.15
4 -3,571,345.00 0.48 -1,722,292.15 -123,868,388.04 -123.87
5 15,506,259.00 0.40 6,231,617.72 -117,636,770.33 -117.64
6 34,774,639.04 0.33 11,645,956.25 -105,990,814.07 -105.99
7 54,235,702.88 0.28 15,136,189.30 -90,854,624.77 -90.85
8 73,891,377.36 0.23 17,184,772.76 -73,669,852.02 -73.67
9 93,743,608.58 0.19 18,168,139.38 -55,501,712.64 -55.50
10 113,794,362.12 0.16 18,378,424.78 -37,123,287.86 -37.12
11 134,045,623.19 0.13 18,040,930.42 -19,082,357.43 -19.08
12 154,499,396.87 0.11 17,328,135.52 -1,754,221.91 -1.75
13 175,157,708.29 0.09 16,370,918.85 14,616,696.94 14.62
14 196,022,602.82 0.08 15,267,527.36 29,884,224.29 29.88
15 217,096,146.30 0.06 14,090,727.74 43,974,952.04 43.97
16 238,380,425.21 0.05 12,893,494.92 56,868,446.95 56.87
17 259,877,546.92 0.05 11,713,524.11 68,581,971.06 68.58
18 281,589,639.84 0.04 10,576,798.81 79,158,769.88 79.16
19 303,518,853.68 0.03 9,500,402.35 88,659,172.23 88.66
20 325,667,359.67 0.03 8,494,724.77 97,153,896.99 97.15
157
158
0 0.00 0.00 0.00 0.00 0.00
1 -91,786,588.48 0.77 -70,605,068.06 -70,605,068.06 -70.61
2 -40,522,118.33 0.59 -23,977,584.81 -94,582,652.87 -94.58
3 -30,269,224.30 0.46 -13,777,525.85 -108,360,178.73 -108.36
4 -3,571,345.00 0.35 -1,250,427.16 -109,610,605.88 -109.61
5 15,506,259.00 0.27 4,176,286.38 -105,434,319.50 -105.43
6 34,774,639.04 0.21 7,204,477.96 -98,229,841.54 -98.23
7 54,235,702.88 0.16 8,643,344.17 -89,586,497.37 -89.59
8 73,891,377.36 0.12 9,058,305.08 -80,528,192.29 -80.53
9 93,743,608.58 0.09 8,839,984.36 -71,688,207.93 -71.69
10 113,794,362.12 0.07 8,254,432.54 -63,433,775.39 -63.43
11 134,045,623.19 0.06 7,479,555.05 -55,954,220.34 -55.95
12 154,499,396.87 0.04 6,631,420.40 -49,322,799.94 -49.32
13 175,157,708.29 0.03 5,783,166.21 -43,539,633.73 -43.54
14 196,022,602.82 0.03 4,978,508.11 -38,561,125.62 -38.56
15 217,096,146.30 0.02 4,241,327.71 -34,319,797.91 -34.32
16 238,380,425.21 0.02 3,582,423.73 -30,737,374.18 -30.74
17 259,877,546.92 0.01 3,004,220.25 -27,733,153.93 -27.73
18 281,589,639.84 0.01 2,504,011.57 -25,229,142.36 -25.23
19 303,518,853.68 0.01 2,076,165.55 -23,152,976.81 -23.15
20 325,667,359.67 0.01 1,713,591.06 -21,439,385.76 -21.44
158
159
600.00
500.00
Cumulative Cash Flow, x10^6 (RM)
400.00
300.00
10%
200.00
20%
30%
100.00
0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
-100.00
-200.00
Year
Figure10.7: Cumulative Cash Flow (Discounted Cash Flow).
159
160
Table 10.7: Total area of all the equipment in riboflavin plantation.

No Equipment Area (m2)
1 Mixing 76.02
2 Seed Fermenter 74.28
3 Fermenter 202.63
4 Centrifuge 1,226.16
5 Centrifuge 2 422.67
6 Ultrafiltration 187.61
7 Ion Exchange 195.81
8 Drum Drying 56.55
Total 2,441.73
160

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1

THE PRODUCTION OF RIBOFLAVIN

1.1 Project background

1. The fat-soluble vitamins:

2. The water-soluble vitamins:

THE PRODUCTION OF RIBOFLAVIN

Table 1.1: All types of B vitamins and their functions

THE PRODUCTION OF RIBOFLAVIN

cholesterol. Mushrooms, Organ meats, Poultry,

THE PRODUCTION OF RIBOFLAVIN

not be able to break

Barbitunic acid Riboflavin (in broth)

Riboflavin (about 80% pure)

96% pure riboflavin

2.1 Riboflavin Structure and Importance

Figure 2.1: Structure of Riboflavin.

Table 2.1: Recommended Amount of Riboflavin according age and gender

2.2 Process selection

2.2.1 Production of Riboflavin by Fermentation using Bacillus subtilis

Figure 2.2: Gram-positive Bacillus subtilis

In detail, the Bacillus subtilis is inoculated onto a conventional medium containing a

2.2.2 Production of Riboflavin by Fermentation using Candida famata

Riboflavin know as vitamin B2 can be producing using microorganism. There are 75

Hypersaline is a condition whereas contain high concentration of salt or sodium

Candida famate Glucose

2.2.3 To check the riboflavin sample

2.2.4 Improvement of Riboflavin Production Using Mineral Support in the Culture of

Table 2.3: Material used in this production by using a. gossypii strain.

3.1 Market Study

The market price analysis for C17H20N4NaO9P (riboflavin-5-phosphate) is RM2422.16

Table 3.1: Raw materials price

Glucose • 100 G  126.50

3.3 Site Survey

3.3.1 Location selected

3.4 Raw Materials

Table 3.2: Comparison prices of raw materials (Dewi, 2007)

3.6 Environmental Impact and Disposal.

3.8 Comparison between the site locations

Factors Ladang tebu, Chuping, Perak Kawasan Perindustrian Sri

4.1 Process Flow Diagram

In downstream processing, the process usually involves with harnessing of product,

4.2 Description of The Equipments

4.2.1 Blending Storage Tank

The blending or mixing tank is commonly used to blend difference component

4.2.2 Seed Fermenter

4.2.4 Centrifuge (Decanter)

The suitable centrifugation equipment which can be referred as decanter

In ultrafiltration, the separation on purification process of product continued by

4.2.6 Ion Exchange

In Ion-exchange chromatography is a purification process where the working

4.2.7 Drum Dryer

In riboflavin production, it is preferred to use drum dryer which is also called as a

4.2.8 Gas Compressor

Gas compressor act in a way to concentrate or compress the air first to

4.2.9 Air Filtration

5.1 Mass Balance

❖ Production per year = 1000 tone/year

• Cleaning process = 3 days

330 𝑑𝑎𝑦𝑠 𝑏𝑎𝑡𝑐ℎ

5.2 Stoichiometry Balance

𝑂𝑖𝑙 + 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 + 𝐴𝑚𝑚𝑜𝑛𝑖𝑎 𝑠𝑎𝑙𝑡𝑠 → 𝑅𝑖𝑏𝑜𝑓𝑙𝑎𝑣𝑖𝑛 + 𝐶𝑂2 + 𝐻2 𝑂 + 𝐵𝑖𝑜𝑚𝑎𝑠𝑠

According to Papagianni (2007), given;

𝐻 𝑏𝑎𝑙𝑎𝑛𝑐𝑒 ∶ 32 + 12𝑎 + 3𝑐 = 20𝑑 + 2𝑓 + 1.8𝑔