TABLE OF CONTENT

ABSTRACT

PAGE

CHAPTER 1: INTRODUCTION
1.1

What is Liquid Nitrogen Bio-fertilizer?

1.2

4 Nitrogen Fixing Bacteria.

1.3

Biofertilizer Method of Application

CHAPTER 2: BIOCHEMICAL PRODUCT DESIGN

2.1

2.2

2.3

Identification of Needs
2.1.1

Increasing the Crop Yield

2.1.2

Reduce Harmful Effect of Chemical Fertilizer.

2.1.3

Demand of Fertilizer in Global Market

2.1.4

Demand Based On Agriculture in Malaysia

10

Identification of Ideas
2.2.1

Types of Fertilizer

12

2.2.2

Types of Biofertilizer (bacteria group)

14

2.2.3

Source of Substrate

16

Selection of the Best Ideas to Serves the Needs

(Concept screening and scoring)
2.3.1

Types of fertilizer

18

2.3.2

Types of biofertilizer (bacteria group)

19

2.3.3

Source of substrate

20

CHAPTER 3: PROCESS DESCRIPTION

3.1

Process Flow Chart

21

3.2

Detail Mass Production

22

3.3

Process Flow Diagram

26

3.4

Monthly Process Scheduling

28

3.5

Bacteria Growth Curve

29

3.6

Biochemical Reactions Involved

32

3.7

Kinetic control Fermentation

33

3.8

Biocatalyst Used

34

3.9

Excess of reactant used to increase product yield at equilibrium?

34

3.10

Issue on competing bioreaction and selectivity

34

3.11

Single Pass Conversion

35

CHAPTER 4: BIOREACTOR ENGINEERING DESIGN

4.1

Fed-batch Process

37

4.2

Input/Output Structure of Bioreactor

38

4.3

Material Balance in Bioreacting System

39

4.4

Chemical Engineering Design (sizing and scale-up) of Bioreactor

4.4.1

Introduction

43

4.4.2

Scale up Calculation

48

4.4.3

Bioreactor drawing and sizing

53

CHAPTER 5: DOWNSTREAM PROCESSING

5.1

Cross Flow Microfiltration

5.1.1

The Usage of Microfiltration (MF)

54

5.1.2

Type of Flow

54

5.1.3

System configuration

56

5.2

Material balance on seperating system

5.3

Waste Treatment

63

5.3.1

Wastewater from Associated Process

67

5.3.2

Characteristic and source of waste water

67

5.3.3

Wastewater Treatment Method

68

5.3.4

Pre-treatment of Downstream Processing Waste

70

5.3.5

Aerobic Treatment Unit

71

CHAPTER 6: PLANT DESIGN AND ECONOMIC ANALYSIS

6.1

Estimation of purchase cost at base condition and calculated Bare Module and Total
Module cost for each equipment.

72

6.2

Calculation for bare module cost (CBM) and total module cost (CTM).

73

6.3

Manufacturing Cost Estimation

6.4

6.3.1

Annual cost of raw material

77

6.3.2

Annual Cost of Waste Treatment

79

6.3.3

Annual cost of utilities

80

6.3.4

Annual cost of operating labour

82

Estimation of Total Direct Manufacturing Cost, Fixed Manufacturing Cost,General

Manufacturing Cost and Total Manufacturing Cost.

6.5

85

Profitability Analysis
6.5.1

Annual Revenue

88

6.4.2

NPV

90

CONCLUSION

94

APENDIX

95

REFERENCES

99

ABSTRACT

This report highlights the production of bio-fertilizer which includes product

development in term of bioreactor engineering, downstream processing, plant design and
economic analysis. The report started with identifying the needs, ideas and selection for the best
idea to serve the needs. This is further continued by selection the process route under Plant
design. Next is the development of product under bioreactor engineering which include material
balance in bioreacting system, justification of using fed batch process, determining the input and
output structure, designing bioreactor and material balance in bioreacting system. Then,
continued with the discussion under downstream processing which includes selection of
separation process, designing separation equipment, and material balance in separating system.
Lastly is the discussion under economic analysis which includes all the calculation needed to
estimate annual cost of raw material, waste treatment, utilities and operating labour. Besides, are
the estimation of total direct, fixed, general and total manufacturing cost and also profitability
analysis.

CHAPTER 1: INTRODUCTION

1.1

What is Liquid Nitrogen Bio-fertilizer?

Bio-fertilizer is microbial inoculants consisting of living cells of bacteria which may help in
increasing the crop productivity by way of helping in the biological nitrogen fixation. In other
word, this in not fertilizer which directly give nutrition to crop plants, but help the plants
indirectly to get nutrients through biological nitrogen fixation.

1.2

4 Nitrogen Fixing Bacteria.

This bio-fertilizer based on 4 selective strains of nitrogen-fixing beneficial bacteria. These

bacteria make nitrogen available to plant in an easily assimilable and utilizable form. The types
and role of the bacteria are as follow:

Nitrogen fixing bacteria Groups

Azotobecter
Free-living

Mode of action
It lives freely in soil and multiples by making use of the

chroococum

organic matter, as source of carbon (energy) for its

Rhizobium

Symbiotic

leguminosarum

growth and existence.

A nodulating type of micro-organism associating
symbiotically with the root of the legume plants. It
produces nodules and multiplies in it. By remaining

Azospirilum

inside the nodules it fixes atmospheric nitrogen.

Associative Capable of colonizing root surface of plant. By

brasilence

Symbiotic

establishing a symbiotic association ship, it helps plant

Acetobacter

Symbiotic

in getting nutrient 'N' (Nitrogen) from the atmosphere.

Capable of living inside the plant tissues. It can live in

diazotrophicus

1.3

high sugar levels that exists in sugarcane tissues

Biofertilizer Method of Application

Seed Treatment

Seed required for 1 hectare is to be treated with biofertilizer at the

(10 ml 1 kg of Seeds)

specified rate by adopting either seed dipping or seed coating

methods.
Treatment in Mechanical Biofertilizer at the recommended rate is to be mixed well with
Seed Treater

sufficient quantity of sticking agent/water. Seeds are then treated

(10ml/1kg of Seed)

with this culture mix in a seed treater in a manner so that all seeds
are coated with culture slurry properly. The seeds are then shade

Seedling Treatment

dried and sown immediately.

Biofertilizer is mixed with sufficient quantity of water and

(100 ml in 10 Litres of

organic fertilizer or field soil to form slurry and seedling roots are

water)

then immersed in this slurry for about 30 minutes before planting

Soil Application

so that the roots are well drenched with slurry.

Biofertilizer and organic fertilizer or field soil mixed at he

(3.0 liters in 1000 kg of recommended rate has to be applied uniformly in the soil and
organic fertilizer)
Drip System

watered well.
In places where drip irrigation system is in practice the liquid
formulation of biofertilizer can be used at 3 liters / ha in 500 liters

Tree Treatments

of water.
Biofertilizer at the specified rate is to be mixed with required
quantity of water and applied nearer feeder root zone / nearer tree
trunk following pan irrigation, drip irrigation and the like. The
treatment is to be done 2 or 3 times a year. The dosage of
biofertilizer varies according to varieties, age, and size of tree and
its canopy.

CHAPTER 2: BIOCHEMICAL PRODUCT DESIGN

2.1

Identification of Needs

2.1.1

Increasing the Crop Yield

Agriculture is the cultivation of varies plants developed mainly by farmer in good

condition area as source of food income. Eventually, these crops have been fully utilized for
many industrial sectors like energy, medicine, cosmetic, biofuel and many more. However,
global warming and climate change have resulted in unexpected disaster which affect the
production of crops. Those include drought, stormy rainfalls, extremely high temperature, cold
damage, hurricanes and tornadoes. Sometimes, the soil used for crops has reduced quality due to
repeated cycles of planting and harvesting where the plant has stripped the entire nutrient
available in soil. Eventually, the yield of crops is decreasing. Therefore, effective strategy must
be conducted to increase the crop yield by introducing fertilizer to provide a well-loamed and
nutrient-rich soil. In recent years, fertilizers were extensively applied to maintain high crop yield.

2.1.2

Reduce Harmful Effect of Chemical Fertilizer.

The excessive application of chemical based fertilizers has accelerated soil acidification
but also risked contaminating groundwater and the atmosphere. It also weakened roots of plants
that made them easy prey on unwanted diseases beside not good for human health. Hence, by the
ecofriendly approached such as the usage of Bio-fertilizers will helps to restore the soil fertility
and strength. Bio-fertilizers are microorganisms that assist plants to grow by increasing the
quantity of nutrients. The living microorganisms that co-exist with the plants will promote the
supply of important nutrients and, consequently are crucial for the overall productivity of the
soil. In addition, soil quality is also improved through the uptake of these environmental friendly
fertilizers.
2.1.3

Demand of Fertilizer in Global Market

Globally, agricultural sector plays an important role in the every country. This kind of industry
only can donate the biggest profit in economy chart of a country. Parallel to this proliferation
also increase in the use of fertilizer to enhance the crops yield. The diagram below shows the
usage of fertilizer from 1961 to 2007.

Figure 1: chart of global fertilizer usage through years.

From the chart above, there is significant increase (mostly in Asian countries) from 1961 to 1987
in the usage of fertilizer throughout the years. However there is slightly decreasing in usage
between 1987 and 1995. Then the value was rise again after 1995. These values are estimated to
be increase for the next few years. This shows the positive market available for biofertilizer. To
add, with the lower pricing of biofertilizer compared to the chemical fertilizer, the demand value
might shoot up in the following years.

Figure 2: global demand for different types of fertilizer

Among the most widely used biofertilizer is the nitrogen fixing bio fertilizer which is estimated
for 78 % for global demand in 2012 (Samani). The main applications for biofertilizers are for the
treatment of seed, root (plant surface) and soil (Gauraha). Seed treatment contributed the most in
application segment for biofertilizer which was valued at USD 316.5 million overall profit in
2012 (Samani).

India and China has becoming the largest producer of biofertilizer in Asia due to growing of
crops production there. In India, the increase in the agricultural business has open up the
opportunities for large scale production of biofertilizer. In fact, nowadays there are several
companies that making a profit by producing biofertilizer in India. The major crops that add to
the large segment of economy in India are paddy and vegetables. To enhance the production of
this crop, biofertilizer is used. Below is consumption chart for biofertilizer in India between 1992
and 1999 based on the production capacity.

2.1.4

Demand Based On Agriculture in Malaysia

Table 1: various crops land area for 2013 (hectare) (production statistic, crops area harvested in
Malaysia, 2015)

Agriculture plays an important role for the Malaysia's economy, contributing 12% to the
national Gross Domestic Products (GDP) and nearly 24% of Malaysia's land area is composed of
land dedicated to agriculture industries. From the data obtained from Department of Statistic
Malaysia, the total area harvested of selected crops for the year of 2013 as stated on the Table
above is 1,250,048 hectare. We assumed that our manufacturing will cover around 30% of total
crop area which is 375,014.4 hectare annually. Basically, 100 ml of biofertilizer able to be used
for seed treatment per acre of crop plant and hence 1 hectare requires 0.25 liter of bio-fertilizer.
Therefore approximately 94,000 liter of biofertilizer will be needed annually to meet the
demand. Further calculation is shown below.
Biofertilizer required per hectare =

Biofertilizer required per annum =

100 ml
x
acre

1 acre
0.4 hectare

0.25liter
hectare
0.25liter
x
hectare

375,014.4 hectare
1 year

= 93,753.6 liter
94,000 liter

Since our production is 4 batches monthly, therefore 1 year required 48 batches

1 year = 4 batch x 12 month = 48 batches.

So, 1 batch will produce 1958.3 liter of biofertilizer:

Biofertilizer produced per batch =

94,000liter
1 year
x
year
48 batches

= 1958.3 liter
2.2

Identification of Ideas

2.2.1

Types of Fertilizer

Consequently, type of fertilizer can be divided into five types which are inorganic, plant specific,
liquid, time release and fertilizer with pesticides. Each type of them brings a different function
with different advantage to the plant.

Inorganic fertilizers (chemical fertilizer)

They commonly consist of balanced amount of nitrogen, potassium and phosphorus that help in
fostering growth where they nourished plants roots, stems, shoots, leaves and blossoms in a
short time. (what is inorganic fertililzer) Inorganic fertilizer price normally way cheaper and it
does not need to decompose over time to supply nutrient to plants. The problem arises when the
inorganic substance absolutely will give some drawbacks to the soil and change the diversity as
long term effects. Synthetic fertilizer may destroy large percentage of soils naturally occurring
organism and earthworms causing a reduced root zone which means it need more water and
fertilizer to maintain a green plant. (Wilson)

Plant specific fertilizers

Particular plant may go under-fertilized or over-fertilized by using common fertilizer. Certain
plants cannot adapt with over-fertilizing condition because they have their own demand nutrient
with specific ratio precisely. Plant-specific fertilizer has been introduced to give an adequate
amount of nutrient needed by a particular plant to grow in optimum rate. The specificity is
commonly based on nitrogen-phosphorus-potassium ratio needed by plant whether plant in
active growth phase, stimulation of growth and specific plant to be fertilized. The difference
between these three types are, active growth phase type need high nitrogen while stimulation of

growth phase fertilizer need high phosphorus-potassium ratio and specific plant ratios may differ
depends on the plant species.
Liquid bio-fertilizers
This type of fertilizer is said to be a good choice in delivering nutrient to the plant where the
plant can immediately take up the nutrient because liquid form no need to be decomposed in soil.
Liquid fertilizer is also quick and easily to apply. The fertilizer is quite expensive comparing to
other can be disadvantage. Liquid fertilizer can be applied to large area by sprayers, seed
treatment or seedling treatment. It save time as much as water absorbed into the soil but
accompanied with benefits. Carrier based biofertilizer (granular) need time and water to deliver it
nutrient give advantages to liquid type. Liquid type easily taken care over granular where it does
not need fear of getting the fertilizer wet.

Time release fertilizer

This kind of fertilizer shows its popularity lately since it offers a slow release of nutrient over a
certain period. It can be between 2-6 months depends on the type, brand and also the weather.
(The Five types of Fertilizers) Plants have a lesser chance of getting burned by the excessive
used of fertilizer because the nutrient is not delivered in a bulk. The presence of water and high
moisture can speed up the nutrient release. Therefore, time release fertilizer performed its best
during warm weather. It is also quite expensive among fertilizers. But, the re-fertilize duration
can be easily predicted by checking the temperature and moisture of soil and surrounding.

Fertilizer with pesticide

Functioning in two ways where it can nourish the plant and controlling pests at the same time.
Pesticides can prevent crop failure, control invasive plants, or promote a uniformly green lawn.
Some pesticides reduce blemishes on fruit and vegetables, ensuring that a greater proportion of
the crop is marketable. Much way cheaper than buying these two separately give a good reason
to be chosen in planting industries. The pesticide and nutrient should be balance otherwise it may

kill the plants in addition to kill the pest. On the other hand, they both can cause water pollution
when erosion carries the chemicals off of farms along with eroded soils after each rainfall.
(Elliott, 2010)
2.2.2

Types of Biofertilizer (bacteria group)

There a few types of fertilizer that can be group according to types of bacteria used.

Nitrogen fixing bacteria

Nitrogen fixing bacteria can be further divided into 3 groups which is free-living, symbiotic and
associative symbiotic. (Organic farming :: Organic Inputs and Techniques, 2014) Most of the
plants required nitrogen is for growth. The plant is unable to grow without nitrogen. The
fertilizer works by the aid of the bacteria that will draw and convert nitrogen gas from the air and
stored it in the soil.
For example, free living nitrogen fixing bacteria not depending to each other but require all to
perform well. The bacteria used are Azotobacter, Beijerinkia, Clostridium, Klebsiella, Anabaena,
Nostoc. Meanwhile for symbiotic nitrogen fixing fertilizer, the bacteria depending to each other
without giving harm to others. The bacteria used are Rhibozium, Frankia and Anabae azollae.
Lastly, associative symbiotic nitrogen fixing fertilizer, the bacteria are intimately associated with
their host. There is only one bacteria that used this type of bacteria which is Azospirillum.

Phosphate solubilizing bacteria

Phosphate solubilizing biofertilizer have two groups which are bacteria and fungi. (Organic
farming :: Organic Inputs and Techniques, 2014) The microbial used can dissolve phosphate
available in the soil by using hormones, enzyme and organic acid and convert it into a form
which can be uptake by the plant. (Role of biofertilizer in soil fertility and agriculture, 2011) The
examples of bacteria that can be used for phosphate solubilizing biofertilizer are Bacillus

megaterium, Bacillus subtilis, Bacillus circulans and Pseudomonas striata. Meanwhile the
examples of fungi used are Penicillium sp. and Aspergillus awamori.

Phosphate mobilizing bacteria

The major example of phosphate mobilizing biofertilizer is Mycorrhiza which is generally
mutualistic association between a fungus and the roots of the plant. The fungus will colonize the
host plantss root by intracellularly or extracellularly. The fungus will be providing with access to
carbohydrates by this association. Therefore, the plant can gain the benefits of the myceliums
higher capacity for water and mineral. (Slideshare.net, 2015)

Plant growth promoting Rhizobacteria

Pseudomonas fluorescens can produce growth promoter called phytohormones which can
accelerate the growth of Rhizobacteria. They produce including indole-acetic acid, cytokinins, as
well as gibberallins. (Slideshare.net, 2015)

Biofertilizer for micronutrient

Bacillus sp. used in this fertilizer has the capability to degrading silicate and aluminium silicates
and can solubilize zinc in the solid for absorption of plant. (Slideshare.net, 2015)

2.2.3

Source of Substrate

Palm Oil Mill Effluent (POME)

POME is palm oil mill effluent produced during extraction of crude palm oil and cracked
mixture separation process. This waste water from the process can be used as carbon source for
fermentation since it contain cellulose. However, since the bacteria used unable to synthesis
enzyme to convert cellulose to glucose, certain upstream process has to carry out for recovery of
the cellulose. Although POME is cheap since it only cost in range US$ 250 to 300 (Rahim, 2014)
per Metric tonne but the operation cost increases to produce glucose from cellulose.
Composition of POME
Components
Range (% dry weight basic)
pH
4.00 5.00
Cellulose
0.25 8.00
Lignin
2.90 7.89
Crude protein
11.11 16.66
Crude fiber
14.44 16.66
Moisture content
8.00 - 12.00
Ash
18.88 22.22
Nitrogen free extract
28.88 53.33
Suspended solid
25.00 29.00
Table 2: approximate composition in 10 mL of palm oil mill effluent. (Mashitah, 2010)

Industrial Glucose powder.

The glucose is available in liquid state and solid state but not much different in term of price. 1
Metric tonne of either glucose powder and glucose syrup can be bought for US$ 480 to 600
(Han, 2014). This type of substrate is convenient since if plant is used as substrate, it has to
undergo a few process such as milling, pretreatment, liquification and saccharification to convert
starch or cellulose available into glucose since some of the bacteria unable to convert carbon
source available into substrate that can be consumed.

Algae
Nowadays, algae are used by human for many applications such as fertilizers, soil conditioners
and feedstock. Seaweed on types of algae contains unique polysaccharides and carbohydrates
that make them suitable as dried feed, human food, fertilizers and soil amendment agent.
Seaweed contains high carbohydrates content and could be efficiently saccharified to monosuga
(glucose). It could serve as good substrate for the fermentation process (Sung-Soo Jang, 2010) .
Sabah is the only state in Malaysia that is commercially producing seaweed.

Pineapple waste
The waste from pineapple can be used in fermentation process to produce biofertilizer. This agro
waste contains insoluble chemical constituents such as glucose and lignin and also contains
soluble constituents such as sugar, amino acid and organic acids (Subba Rao., 1993). A multitude
of different pretreatment technologies have been suggested during the last decades (Alvira et al.,
2010) in order to hydrolyze the cellulosic waste materials.

2.3

Selection of the Best Ideas to Serves the Needs

2.3.1

Types of fertilizer

CONCEPT SCREENING

Criterion
Immediate response
Effect to diversity
Handling way
Price
Total score
Rank

Inorganic
+
0
+
1
1

Types of fertilizer
Plant specific Liquid
Time release
0
+
0
0
0
0
+
0
0
0
1
-2
3
1
5

Pesticide
0
0
+
0
3

CONCEPT SCORING
Types of fertilizer
Criterion
Immediate response
Effect to diversity
Handling way
Price
Total score
Rank

Inorganic fertilizer
4
2
3
5
3.5
2

Liquid bio-fertilizer
5
3
5
2
3.75
1

By concept screening and scoring, liquid biofertilizer is the best idea since it is the top rank.

2.3.2

Types of biofertilizer (bacteria group)

CONCEPT SCREENING
Bacteria group
Criterion

More than 4 main role and

benefits
High rate of fertilizer
produce
(higher
than
100kg/ha per season)
Widely applicable to various
types of plant
Total score
Rank

Nitrogen
fixing

Phosphate
mobilizin
g
-

Plant
Growth

Micronutrien
t Biofertilizer

Phosphate
solubilizin
g
0

3
1

0
3

-1
4

2
2

0
3

CONCEPT SCORING
Bacteria group
Criterion
More than 4 main role and
benefits
High rate of fertilizer produce
(higher than 100kg/ha per
season)
Widely applicable to various
types of plant
Total score
Rank

Nitrogen fixing biofertilizers

5

Plant Growth biofertilizers

4

3
2

4
1

By concept screening and scoring, Nitrogen fixing bacteria is the best idea since it is the top
rank.
2.3.3

Source of substrate

CONCEPT SCREENING

Criterion
Availability
Price
Carbon source
content
Easy to handle
Total score
Rank

Palm Oil Mill

Effluent
(POME)
+
+

Source of substrate
Industrial
glucose
Algae
powder
+
0
+

Pineapple waste
0
0

0
1
2

+
2
1

-1
4

0
0
3

CONCEPT SCORING

Criterion
Availability
Price
Carbon source
content
Easy to pretreat
Total score
Rank

Weight
40%
20%
30%

Source of substrate
Palm Oil Mill Effluent (POME) Industrial glucose powder
4
5
5
2

10%

3
3.5
2

5
4.4
1

By concept screening and scoring, industrial glucose powder is the best idea since it is the top
rank.

CHAPTER 3: PROCESS DESCRIPTION

3.1

Process Flow Chart

The mass production of each microorganism is the same and illustrated as flow chart below:

Soil sample

Isolation
and Culture

Lab scale
fermentation
(4 liter)

Industrial Scale

Scale up

Pilot scale

(6000 liter)

(500 liter)

(50 liter)

Addition of
preservativ
e

Concentrati
on of broth

Addition of
preservativ
e
(recovery)

Packaging

The bio-fertilizer is produced separately for four different types of bacteria. The process flow is
the same for all microorganism but the timing as per unit operation is different according to the
growth curve for each microorganism.

3.2

Detail Mass Production

1.

Soil sample

The soil samples are collected from various fields and serial dilutions are done. The four bacteria
is

isolated each from the various type of microorganism

present by the analysis of the

characteristics according to the morphological and biochemical characteristics.

2.

Isolation and Starter Culture

Specific solid medium (agar) for each bacteria is used to grow the microorganism for the starter
culture. The culture is then used for preservation or mass production. The medium specifications
are as follow:
Bacteria
Azotobecter chroococum
Rhizobium leguminosarum
Azospirilum brasilence
Acetobacter diazotrophicus

3.

Medium
Mannitol Agar
Yeast extract mannitol agar
Dobereiner's malic acid agar
Mineral medium agar

Lab scale fermentation (4 liter)

For mass production, the bacterial strain from agar is inoculate into 400 ml liquid media of
Dextrose Medium in 4000 ml shake flask. The microorganism is grown and incubated in rotary
shaker until it reach half of exponential phase time before being used as inoculum for next pilot
scale fermenter. There are 10 flask needed to achieve 4 liter total volume of inoculum.
Volume: 4000 ml
Working volume (10% of flask volume) : 400 ml media used
10 units: 400 ml x 10 = 4 liter inoculum prepared.
The general liquid media used for all fermentation tanks for all types of Nitrogen Fixing bacteria
is Dextrose Medium. The prepared media needed for every single fermentation have to be

sterilized first using autoclave at 120

water bath for 20 minutes. Composition of required

components used to prepare 1 liter of Dextrose broth is as follow:

Component

4.

Mass (g)

Glucose

39.2

Peptone

40.32

Beef extract

12.32

Sodium chloride

20.16

Pilot scale (50 liter)

The inoculum from lab scale fermentation is used to inoculate into 35 liter liquid media of
Dextrose Medium in 50 liter fermenter. The microorganism is grown in batch culture mode with
proper parameter and continuous agitation until it reach half of exponential phase time before
being used as inoculum for next scaled-up fermenter.
Volume: 50 liter
Working volume (70% of fermenter volume): 35 liter new media added
Inoculum (10% of working volume): 3.5 liter 4 liter inoculum from shake flask

5.

Scale up (500 liter)

The inoculum from 50 liter fermenter is used to inoculate into 350 liter liquid media of Dextrose
Medium in 500 liter fermenter. The microorganism is grown in batch culture mode with proper
parameter and continuous agitation until it reach half of exponential phase time before being
used as inoculum for next industrial scale fermenter.
Volume: 500 liter
Working volume (70% of fermenter volume): 350 liter new media added
Inoculum (10% of working volume): 35 liter inoculum from 50L fermenter
6.

Industrial Scale (6000 liter)

The inoculum from 500 liter fermenter is used to inoculate into 3600 liter liquid media of
Dextrose Medium in 6000 liter fermenter. The microorganism is grown in batch culture mode
with proper parameter and continuous agitation. The fermentation is fed batch by certain amount
of glucose (carbon source) until cell count reached to 1x109 colony forming unit per milliliter (
cfu/ml ) or concentration of 30g/L of total biomass dry weight per water.
Volume: 6000 liter
Working volume (65% of fermenter volume): 3900 liter new media added
Inoculum (10% of working volume): 390 liter inoculum from 500L fermenter

Stoichiometric equation: C6H12O6 + 3.45 O2 + 0.6 NH3 = 2.4 CH2N0.25O0.5 + 4.5 H2O + 3.6 CO2
Based on stoichiometric equation above, for growth, the microorganism required:
Carbon source = Glucose
Aerobic respiration = Oxygen
Nitrogen source = Ammonia
Product of fermentation= Biomass, carbon dioxide and water.

Parameter for each microorganism growth in every fermenter:

Bacteria
Azotobecter chroococum
Rhizobium leguminosarum
Azospirilum brasilence
Acetobacter diazotrophicus

7.

Temperature (C)
30
30
33
30

pH
6.5-7.5
6.5-7.5
6.5-7.5.
3.5

Pressure (bar)
1
1
1
1

Addition of preservative

The preservative is added (2% of total broth volume) and mix with the broth quickly to preserve
the microorganism in viable condition. The preservative also helps to prolong shelf life and

improve inoculant quality, such as including better adhesion to seed, stabilizing the product,
binding or inactivating soluble seed coat toxins. (Daniel, 2013) Composition for preservative in
2% of total broth volume are as follow:
Component

Composition

Polyvinyl pyrollidone

0.94

Carbomethylcellulose

0.04

Polysorbate 20

0.02

8.

Filtration

Cross-flow microfiltration is used to partially remove residual nutrients from the broth fostering
the microorganism into stationary phase. The broth is concentrated where 50% of water will be
permeated. The concentrate is further process, while the filtrate is further treated before
discharge.
9.

Addition of preservative (recovaery)

The preservative is added (2% of total broth) and mix with the broth to recover any preservatives
loss with water during previous filtration.
10.

Waste Treatment

Components permeated from the filtration will be channeled into waste treatment to be treated
before discharged into sewage system.
11.

Packaging

The broth is filled aseptically for 1 liter per bottle and stored as liquid biofertilizer. The
biofertilizer is stored in required temperature before distributed in market

3.3

Process Flow Diagram:

Bacteria
glucose

media

oxygen

broth 1

media

media

broth 3
6000L
FERMENTER

FERMENTE
biomass
R

35L
working
volume

3900L working
volume

350L
media
working

water

CO2

oxygen

broth
2
500L

50 L
FERMENT
biomassER

0.4 L
Working
volume

water
media

media

oxygen media

4L ROTARY
SHAKE
FLASK

biomass

oxygen

water

CO2

water

CO2

CO2

preservative

Biofertilizer MIXIN

STORAG
E

broth 4
biomass

media
water

biomass
media

preservative

CROSS

water
preservative

MICROFILTRATIO
biomass N

media

water

MIXIN
broth
5

Concentrate FLOW

biomass
media
water

preservative

preservative

filtrate

WASTE
TREATME
NT

media
water
preservative

3.4

Monthly Process Scheduling

All the time calculated for scheduling are based on different trend of growth curve for each Nitrogen fixing bacteria.
Time required for 1 batch of bacteria

A. Chroococum

R. Leguminosarum

A. Brasilence

A. Diazotrophicus

Shake flask

(hour)

12

25

25

50L fermenter

(hour)

12

25

25

500L fermenter

(hour)

12

25

25

6000L fermenter

(hour)

128

50

75

27

12

12

12

12

Preservative addition & filtration

(hour)

Packaging

(hour)

12

12

12

12

Total time

(days)

6.5

7.5

3.5

Gantt chart for 1 month production :

Days :
1 2 3 4 5 6 7 8 9 1

11 1

A. Chroococum

R. Leguminosarum

A. Brasilence

1 batch

1 batch

1 batch

A.
Diazotrop
1 batch

30

Maintenance

Total time for four batch

= 8 days + 6.5 days + 7.5 days + 3.5 days = 25.5 days 26 days.

3.5

Bacteria Growth Curve

Bacteria
Azotobecter

Growth curve

Time
Exponential

chroococum

16 hour

(Zhang, 2005)

Half

Optical Density
0.4 n.m

0.25 n.m

exponential
12 hour
Rhizobium

Exponential

leguminosaru

40 hour

1.4 n.m

m
Half
(Rhizobium sp,

0.75 n.m

exponential

2003)

25 hour

Azospirilum

Exponential

brasilence

35 hour

(Ona et al.,

Half

2005)

exponential

0.8 n.m

0.4 n.m

25 hour
Acetobacter

Exponential

diazotrophicus

18 hour

(Schrover,

Half

2015)

exponential
9 hour

Table X

1.2 n.m

0.7 n.m

Since the required cell concentration is 1x109 cfu/ml, the optical density of biomass need
to be produced during fermentation is 4.0. This is based on graph below which shows that optical
density is increase linearly with cell concentration.

2000000000
1500000000

Cell count (cfu/ml)

1000000000
500000000
0
0

Optical density (n.m)

Table3 : graph of cell count (cfu/ml) against optical density (n.m) (Gutenwik, 2003)

Therefore, by referring table X we can predict at what time the bacteria will reach optical density
of 4.0. Every bacteria will replicate when it grows during fermentation. To ensure the number of
replicated biomass is high, we have to prolong the exponential phase. This can be achieved by
maintaining the amount of carbon source using fed batch process. The amount of glucose to be
added during cell cultivation is calculated using formulae yield of biomass per substrate (X/S)
below.

Stoichiometric equation: C6H12O6 + 3.45 O2 + 0.6 NH3 = 2.4 CH2N0.25O0.5 + 4.5 H2O + 3.6 CO2

YX/S =

(stoichiometry )(molecular weight of biomass)

molecular weight of glucose
(2.4)(25.5)
180
0.34 gram dry weight biomass
1 gram glucose

The production of biofertilizer is 1958.3 liter or approximately 2000 liter per batch. To achieve
30 g/l of biomass concentration is therefore,

If 1 liter = 30 gram dry weight biomass

Then, 2000 liter = 60,000 gram dry weight biomass
If 0.34 gram dry weight biomass produced = 1 gram glucose consumed
Then, to produce 60,000 gram dry weight biomass = 176 kg of glucose needed.

During the scale up, glucose is fed for every fermenter in increasing amount. At the last
fermenter, where fed batch process is applied, the remaining glucose need to be fed into the
fermenter until it reach total of 176 kg of glucose in order to have cell number of 1x10 9 cfu/ml or
30 g dry weight biomass/liter.

3.6

Biochemical Reactions Involved

Since all bacteria are aerobic and consuming the same carbon source which is glucose, they will
have the same biochemical reaction used for their growth as listed below.
1.

Pentose phosphate pathway

This is reaction parallel to glycolysis that generates NADPH and pentoses (5-carbon sugars).
Glucose-6-phosphate + 2NADP+ + H2O
2.

ribose-5-phosphate + 2NADPH + 2H+ +CO2

Electron Transport Chain

Reaction involve oxidation of glucose, its primary role is anabolic rather than catabolic.
NADH + H+ + O2

3.
This

NAD+ + H2O &

FADH2 + O2

FAD + H2O

Tricarboxylic acid cycle.

reaction

generate

energy

carbohydrates, fats and proteins into carbon

through

the oxidation of acetate derived

dioxide and

chemical

energy

in

the

from
form

of adenosine triphosphate (ATP).

Acetyl CoA+3H2O +3NAD+FAD +ADP

HSCoA +2CO2 NADH+3H++FADH2+ATP

The biochemical reaction occurred when the bacteria is in its role for nitrogen fixation after
applied for plants is as follow
5.

Nitrogen fixation process

N2 + 8H+ 8e
4.

2NH3 + H2

Ammonium Assimilation

Formation of organic nitrogen compounds like amino acids from inorganic nitrogen compounds
present in the environment.
Glutamate + ATP + NH3

Glutamine + ADP + phosphate

3.7

Kinetic control Fermentation

All the bacteria are having kinetic control during the bioreaction because it has its own growth
curve during the fermentation. Here the rate of bacterial growth is depends on substrate
concentration where their relationship often assumes to form saturation of kinetics. The substrate
concentration is a growth limiting factor that its increases influence the growth rate.

Figure 3
Figure 3 shows that the increases of substrate concentration will increases the specific growth
rate until the concentration become the limiting factor. This kinetics can be described by the
Monod equation:
=

max S
Ks+ S

= specific growth rate

max = maximum specific growth rate
S = growth limiting substrate concentration
KS = saturation constant
max (maximum specific growth rate) is happen when the S >> K S. If endogeneous metabolism
is unimportant , then net = g. The constant K S is known as the saturation constant or half

velocity constant and is equal to the concentration of the rate-limiting substrate when the specific
rate of growth is equal to one-half of the maximum.
3.8

Biocatalyst Used

Since the biofertilizer consist of microbial inoculant, there is no need of biocatalyst. The
microorganism itself is catalyst to convert carbon source into products during its growth. In this
case the microbe converts glucose into biomass, carbon dioxide and water. The biomass is the
desired product without being purified to extract the intracellular or extracellular products. The
living biomass or microorganism dissolved in broth is the desired final product used as fertilizer
after mass production.

3.9

Excess of reactant used to increase product yield at equilibrium?

Since we have fixed our production to have concentration of biomass 30g/l, we have
calculated that we only needed 176 kg of glucose (reactant) for every batch. We increased our
volume of broth by scale up the fermentation tank but we increase the concentration of biomass
per liter by fed batch the calculated amount of glucose. Therefore, we do not need any excess of
reactant to increase the product yield.

3.10

Issue on competing bioreaction and selectivity

We do not have any issue on competing the bioreaction since our microorganism can be
easily found in the soil and several isolation techniques need to be done to capture the desired
bacteria. We also do not have any issue on competing the selectivity because we do not purify
our product. It is because our desired product is the biomass itself not the intercellular nor the
extracellular product it produced.

3.11

Single Pass Conversion.

Figure 4: Overview of single pass conversion and overall conversion

Single pass conversion gives the fraction of reactant converted on a single pass through the
reactor. In contrast, overall conversion gives the fraction of reactant converted by the process,
which may involve recycling reactant molecules many times through the reactor in order to
increase their conversion. To determine the single pass conversion, the equation use is as
follows:
reactant input
reactant input
single pass conversion= reactorreactant output reactor reactor 100

From the material balance calculation, the input and output value of the reactant is provided in
the table below. In this case, the limiting reactant is the glucose that acts as the carbon source for
the bacteria to grow.

0.01 kmol glucose

0.006 kmol glucose

Variables

Input reactant
Stream 1
Stream 2
1.88674
50.0000
4.97196
1173.6591
0.010
0.278
Fermenter

Flow rate (kg/batch)

Concentration (g/L)
Number of mol (n) (kmol)

Output reactant
1.10095
0.262842
0.006

0.278 kmol glucose

The single pass conversion in the fermenter can be calculated as follow:

reactant input
reactant input
single pass conversion= reactorreactant output reactor ractor 100

single pass conversion=

0.288 kmol0.006 kmol

100
0.288 kmol

single pass conversion=97.92 98

In a nutshell, the single pass conversion in the reactor is approximately 98%. The glucose at the
output stream will be existed in small concentration that can be negligible. Since that the glucose
concentration present in the output stream is just in small quantity, the requirement of the recycle
stream can be terminated. This is because, considering the cost of separating the glucose in the

mixture of broth is expensive and since that glucose concentration in the output stream is very
small, the recycle stream seems not economically for this process.
CHAPTER 4: BIOREACTOR ENGINEERING DESIGN

4.1

Fed-batch Process

The bioreactor that will be used in liquid biofertilizer production is a fed-batch reactor.
Fed batch is the intermediary model of bioreactor operation that characterised by predetermined
or controlled addition of nutrients into the bioreactor at certain times of fermenter operations.
This mode of feeding nutrients into the fed batch bioreactor will allow temporal variations in the
supply of nutrients. The good thing about fed batch operation of a bioreactor it allows us a
degree of control on the process and operations of the fed batch bioreactor. We can control the
rate of growth of the microorganisms or the concentration of the biomass by controlling
parameters such as frequency of feeds, hydraulic loadings and concentrations of feed. Fed batch
is mainly used in industry to maintain high concentration of microorganisms and reduce
feedback inhibition especially catabolite repression. Fed batch is the most popular mode in
fermentation industries.

In this production, the very crucial point is to maintain a high concentration of

microorganism, because in the end the final product of the liquid biofertilizer is the amount of
concentrated microorganism that is desired for the plantation. Since the fed batch mode can
control the rate of growth of the microorganism in order to keep it at the exponential phase by
controlling the fermentation parameters, it is the most suitable mode of operation that can be
used in this industry.

4.2

Input/Output Structure of Bioreactor

Figure above show the input and the output structure of bioreactor for biofertilizer
production. After the inoculation of Azospirilium, Azotobactor, Acetobactor, and Rhizobium the
microorganism is being transferred into the bioreactor for further growth in the large scale
production. Inside the bioreactor, the bacteria will increase in mass as the fermentation is
occurred. This mass growth of bacteria is vital for biofertilizer production. Thus, by creating the
suitable condition for the bacterial to growth will ensure the higher yield of production.

In the input stream, glucose is added as the carbon source for the bacteria in fed batch
process. Bacteria will utilize the glucose for survival. This process simulates the fermentation
process as the glucose will be converted to certain products. In this case, the product from the
glucose fermentation is the undesired product. The desired product from this process is the
bacteria itself as the bacteria increase in mass. The growth and the mass doubling time of the
bacteria are monitored until it about to reaches the stationary phase.

As the bacteria at the end of exponential phase, the product from the fermentation will be
channel out into the output stream. The fermentation product will be consisting of bacteria and
by-product from the fermentation such as biomass, water and carbon dioxide. The reason why

the bacteria are extracted during at the end of exponential phase is to make sure the bacteria fully
utilized all the carbon source.
4.3

Material Balance in Bioreacting System (fed batch)

The stoichiometry equation in the reaction is obtained as follows:

C6H12O6 + 3.45 O2 + 0.6 NH3 2.4 CH2N0.25O0.5 + 4.5 H2O + 3.6 CO2

2100 mol O2
7900 mol N2

100 mol C6H12O6

Fed batch
fermenter
n mol C6H12O6
n mol O2
n mol N2
n mol CH2N0.25O0.5
n mol H2O
100 mol NH3

n mol CO2
n mol NH3

In this reaction, the single pass conversion of limiting reactant is 98%, Nitrogen gas is an inert in
the reaction, and the desired output for the bacteria is 60000 g.
The molecular weight for the bacteria (CH2N0.25O0.5) is 25.5 g/mol. Convert the cell dry weight of
the bacteria into mol;
n=

m
MW

n=

60 000 g
=2352.94 mol biomass generated
25 kg /mol

Nitrogen balance
input=output

n=7900mol of N 2 generated

Glucose balance
Since 98% of the glucose is reacted then the 2% remain unconverted
n=0.02 ( 100 mol )=2 mol of glucose generated

Ammonia balance
By using stoichiometry ratio;
2.4 mol CH 2 N 0.25 O0.5 generated
0.6 mol NH 3 consumed

n=2.35 mol CH 2 N 0.25 O0.5 generated

0.6 mol NH 3 consumed

=0.59mol NH 3 consumed
2.4 mol CH 2 N 0.25 O0.5 generated

n=100 mol0.59 mol=99.41 mol NH 3 generated

Water balance
Using stoichiometry ratio;
1 mol C 6 H 12 O6 consumed
4.5 mol H 2 O generated

n=0.98 (100 mol ) glucose consumed

4.5 mol H 2 O generated

=441mol H 2 O generated
1 mol C 6 H 12 O6 consumed

Carbon dioxide balance

Using stoichiometry ratio;
1 mol C 6 H 12 O6 consumed
3.6 mol C O 2 generated
n=0.98 (100 mol ) glucose consumed

3.6 mol C O2 generated

=352.2 mol C O2 generated
1 mol C 6 H 12 O6 consumed

Oxygen balance
input=output + consumption

2 mol of glucose generated+ 352.2mol C O2 generated+ 441 mol H 2 O generated

2100 mol O2=n mol O2 generated +
n=1304.8mol O2 generated

The material balance in fed batch fermenter;

2100 mol O2
7900 mol N2

100 mol C6H12O6

Fed batch
fermenter
(6000 L)

2 mol C6H12O6

1308.8 mol O2
7900 mol N2
2352.94 mol CH2N0.25O0.5
441 mol H2O
100 mol NH3

352.2 mol CO2

99.41 mol NH3

Table 4: The summary table of material balance in fed batch fermenter

INPUT STREAM
Gluocse (C6H12O6)
Ammonia (NH3)
Oxygen gas (O2)
Nitrogen gas (N2)

100 mol
100 mol
2100 mol
7900 mol

OUTPUT STREAM
Gluocse (C6H12O6)
Ammonia (NH3)
Oxygen gas (O2)
Nitrogen gas (N2)
Biomass
(CH2N0.25O0.5)

2 mol
99.41 mol
1308.8 mol
7900 mol
2352.94 mol

Water (H2O)
441 mol
Carbon dioxide (CO2) 352.2 mol

4.4

Chemical Engineering Design (sizing and scale-up) of Bioreactor

4.4.1

Introduction

Scale up of bioreactor is crucial in order to obtain same performance of large scale

production with the small scale production. Through many experiments and research, engineers
have been developed several methods in scaling up bioreactor from the lab scale until industrial
scale. Obviously, the performance of animal culture is so different at 6000 L to 50 L. This is
because it is hard to maintain homogeneity in large system, changes in surface to volume ratios
and changes in the cultures itself due to increase in length of culture time. There are three basic
types of bioreactor designs which are reactors with internal mechanical agitation for example,
stirred reactor (STR), bubble columns and loop reactors. In this design of bioreactor, we are
designing STR for our production of liquid bio-fertilizer.

We are using standard geometry ratios of stirred tank reactor for single agitator to do the
scale up calculation from benchtop fermenter which is 50 L to the industrial fermenter which is
6000 L. The type of agitator we are using is Rushton turbine for the lab scale and pilot scale and
pitched blade impeller for the industrial scale. Pitched blade impeller is often used in batch or fed
batch cultures and it also commonly used for continuous and perfusion processes. Selecting the
right impeller is very crucial to increase yields, avoid damages to the cells and to homogenized
mix cells, gases and nutrients throughout the culture vessel.

The mixing purposes is to evenly distributes oxygen and nutrients to cells for healthy
growth, keeps them from settling to the bottom of the vessel and helps to maintain a uniform
culture temperature. Many impellers proposed different types of flow regime which are radial
flow, axial flow or combination of both flows. The axial and radial flows are illustrated as in
Figure 1 below. Radial flows happen when he fluid is pushed away from the impellers axis
toward the vessel wall while the axial flow occurs when the fluid is being pushed up or down
along the axis or shaft of the impeller. The direction of the agitation of the axial flow will
influence the movement of the fluid whether it is upward or downward move. If the agitation is
clockwise, the fluid will push in upward direction and if the counterclockwise agitation will push
the fluid downward toward the bottom of the vessel. It is important to decide where to place
blades of the impeller shaft and to set direction of the impeller (clockwise or counterclockwise)
in order to increase the mixing performance. (Rich Mirro and Kevin Voll, 2009)

Figure 5: The axial and radial flow

To select the right type of impeller for a process, one require to consider all aspects including
shear sensitivity, type of flow regime, suitability of the impeller for fluid viscosity and many
other aspects. The summary of the impeller selection can be summarized as in the table below.

Type of
impeller
Flow
direction
Flow regime
Preferred
field of
application

Rushton turbine

Pitched blade impeller

Radial

Axial and radial

Turbulent
Turbulent/transitional
Preferable
use
in Most often used with mammalian or other shearfermentation of cell line sensitive cell line growing and widely used in
that are not considered fermentation processes involved highly viscous
shear sensitive such as cultures, such as filamentous bacteria and fungi
yeast, bacteria and some and in some anaerobic biofuels processes.
fungi.
Table 5: differences between Rushton turbine and pitched blade impeller

Both of these two types of agitator have its own advantages and for this process, since we are
dealing fermentation of cell cultures which are not consider as very sensitive to the shear.
However, pitched-blade impeller is also suitable to be used in the fed-batch fermentation. (Rich
Mirro and Kevin Voll, 2009). With this information, we decided to use Rushton turbine in the 50
L and 500 L bioreactor and pitched-blade impeller for 6000 L bioreactor because the industrial
scale production is a fed-batch process. Therefore, for both fermenter in the lab scale (50 L),
pilot scale (500 L), and for industrial fermenter (6000 L), the vessels will be designed with single
agitator.

a)

Rushton turbine

b)

Pitched Blade impeller

Standard Geometry of Stirred Tank

a)

Figure 1: Standard geometry of STR for single agitator

(POWER CONSUMPTION AND EFFICIENCY IN LIQUID
MIXING (MIX))

b)

Figure 2: STR with 3 impellers, 6 blades and

4 baffles

According to (Michael L.Shuler and Fikret Kargi, 2014), fermenter maintains a height to
diameter ratio of 2 to 1 or 3 to 1. If the height to diameter ratio remains constant, thus the surface
to volume ratio decreases dramatically during scale up. Common scale up rules are maintenance
of constant power to volume ratios (P/V), constant K La, constant tips speed, a combination of
mixing time and Reynolds number and the maintenance of constant substrate to product level.

4.4.2

Scale up Calculation

The calculation below performed for scaling up 50 L to 500 L volume of bioreactor. The
geometry similarity ratios of height to diameter used is 3 to 1. The type of impeller use is
Rushton turbine. Since that H/Dt=3/1, then H=3Dt and Da=0.33 for Rushton turbine with
agitator speed of 500 rpm=8.33 rps
Reynolds number
=

N D2

Since that the fermentation broth fulfilled the criteria for a Newtonian fluid throughout the
fermentation with consist of 72% of water and the rest are glucose, nutrients, biomass and side
product, then the viscosity and dynamic viscosity are simply assumed as properties of water.
=1000

kg
kg
=0.03
N=500 rpm=8.33rps
3
m. s
m

(1000
=

kg
)(8.33 rps )(0.276 m)2
3
m
=2.115 10 4 >104
kg
0.03
m. s

Since that the Reynolds number falls into turbulent flows region, the power number can be
determined based on power curve of flat-blade turbine. Based on the graph, when the Reynolds
4
number is 2.115 10 , the power number approximately equal to 9.1. By using Power number

formula,
N p=

P
N 3 D5

Rearrange the equation, we got:

8.33 rps

P=N p N 3 D5 =(9.1)(1000

kg
)
m3

Figure 6: Example of Power curve for flat blade impeller

The volume formula;

V=

2
3 3
Dt H =
Dt
4
4

V1=50 L=50,000 cm3 and V2=500 L=500,000 cm3

V =50,000 cm 3=
Dt=27.69 cm

3 3
Dt
4

H=83.06 cm
Da=9.14 cm

The scale up factor to maintain the geometry similarity;

V 2 Dt , 2
=
V 1 Dt , 1

Dt , 2 V 2 1/ 3
=(
) =(500 L/50 L)3 =2.15
Dt , 1 V 1
Then;
Dt=59.53 cm

H=178.58 cm
Da=19.65 cm

The dimension for the large fermenter and agitator speed is calculated as follows for:
1. P/V
P=N 3 D5V =D2
( P/V )1 ( N 3 D5 )1
=
( P/V )2 ( N 3 D5 )2

N 1 3 Dt , 1
)=
N2
Dt , 2

N 2=N 1

Dt ,1 3 (
1 3
= 500 rpm )
=300.15 rpm
Dt ,2
2.15

2. Constant tip speed

( )

N 2=N 1

( DtDt ,1,2 )=( 500 rpm ) ( 2.151 )

N 2=232.56 rpm

3. Constant Reynolds number

N 2=N 1

Dt ,1
1
=( 500 rpm )
Dt ,2
2.15

( )

N 2=108.17 rpm

The calculation is repeated for scaling up fermenter 6000 L. The calculation is summarized as in
the table below and since that we are growing cell lines cultures that are not very sensitive to
shear, scale up on the basis of constant P/V would preferably choose.
Fermenter volume
Diameter of tank
Height of the vessel
Diameter impeller
Agitator speed based on

50 L
27.69 cm
83.06 cm
9.14 cm
500.00 rpm

500 L
59.53 cm
178.58 cm
19.65 cm
232.56 rpm

6000 L
233.23 cm
699.66 cm
76.95 cm
12,094 rpm

P/V
Table 6: Summary table for scaling up of bioreactor

In conclusion, the fermentation is started with seed culture in the 4000 mL shake flask
with 350 rpm. The agitator speed for the seed culture was selected based on the common used
agitation value in the lab scale. Then, the fermentation is continued in the benchtop fermenter
with 50 L in size and next to the industrial scale fermenter with volume of 6000 L. The
revolution per minutes was set at 500 rpm. From this value, the scale up of bioreactor is
calculated and the values are shown in table above. The table above indicates the data of the
reactor dimension as well as the agitator speed that is needed to be designed for the production of
liquid bio-fertilizer. Notice that, the agitator speed decreases with increasing volume. Note that,

P/V=N3D2 thus, 1/V is inversely proportional to the N. This resulted increase in volume will
decrease the agitator speed. Then, the type of impeller is also crucial in the designing of a
bioreactor. Different type of impeller give different type of flow thus affects the mixing
performance and yield of the desire product. The impeller type that is used for the bioreactor of
50 L and 500 L is Rushton turbine while for the 6000 L bioreactor, pitched-blade impeller is
chooses.

4.4.3

Bioreactor drawing and sizing

CHAPTER 5: DOWNSTREAM PROCESSING

5.1

Cross Flow Microfiltration

5.1.1

The Usage of Microfiltration (MF)

Microfiltration is part of important process in production of liquid biofertilizer, where the

end product, the biomass solution need to be concentrated by removing 50% of water from the
broth. The separated water will carry some of dissolved components like glucose, media and
preservatives. Hence, half of the glucose, beef extract, sodium chloride, peptone,
carboxymethylcellulose, polysorbate 20 and polyvinylpyrolidone are separated from the broth.
This partial removal of residual nutrients in the medium makes cell biomass unavailable to
reproduce and grow, thereby fostering the transformation of the cell biomass into the dormant
phase. (NAVIN PROCESS SYSTEM)
. Cross-flow microfiltration is ideal for this process, since they ensure that a majority of
cells remain viable. It also separating solid component of biomass from broth solution according
to microorganism size by flowing it under a pressure difference condition in a porous medium.

(G.Harrison, Todd, R.Rudge, & P.Petrides, 2003) . It also heatless system since need to separate
the cell biomass without involving heat. This is because the components of the biomass consist
of protein which is a heat sensitive substance. Overheat can destroy the structure of the protein
and also its function which risking the viability of cell.

5.1.2

Type of Flow
Type of flow in MF can be divided into two which is cross-flow filtration (CFF) and

dead-end flow. CFF preferred rather than dead-end flow since it prevent the formation cake
layer on the filter membrane surface. CFF apply with tangential flow filtration where the flow
direction is parallel to the filter. Therefore, the flow itself will wash away any filter cake present
on filter membrane. The flow can be illustrated as follow.

Principal of cross flow filtration (Cross Flow Filtration Method Handbook, 2014)
By referring to figure above, fluid that is flow into the membrane called as feed while
fluid that cannot passes through the membrane surface and back to the feed reservoir called
retentate. Solutions that can pass through the membrane called as permeate. Retentate normally
pump back to feed reservoir and recirculated to get a high concentration of biomass. Flow
direction for dead-end is oppositely perpendicular to the filter. It causes the retained particles to
coagulate and forms a cake eventually reduce the filtration performance (CrossFlow-Filtration vs
Dead-End, n.d.). It also suitable selected for feeds containing high proportion of small particle
size solids compared to dead-end flow (Cross-flow filtration, 2015). Table below is given to
summarize the choosing of CFF over dead-end filtration. (Bhave, 1996)

Process goal
Ability to handle wide
variation in particle size
Ability to handle wide
variations in solids
concentration
Continuous concentration
with recycle
Waste minimization

CFF
Excellent

Dead-end filtration
Generally poor

Excellent

Poor or unacceptable

Excellent

Poor or unacceptable

Superior

Can minimize waste if handling

low solids feed where cartridge
disposal is infrequent
High product purity or
Excellent but may require Performance
is
generally
yield
diafiltration to overcome acceptable except in situations
excessive fluc loss at higher involving high solids or
recovery
adsorptive fouling
5.1.3 System configuration

System configuration of CCF consists of pump, valves, pressure sensors, flow sensors, reservoir
level sensors, air sensors, additional sensor and illustrated below.

Figure 7: Basic configuration of a CCF system (Cross Flow Filtration Method Handbook, 2014)
Pump (Cross Flow Filtration Method Handbook, 2014)

Feed pump
Retentate pump

maintain the flow of feed into the filter

maintaining and controlling the flow of retentate back into the feed

(pressure gauge)
Permeate pump
Transfer pump
. Sensors

reservoir
control the permeate side of the filter to not has a negative pressure
washing and diafiltration application

Pressure sensors
Flow sensors
Reservoir sensor
.

monitor and control the pressure in the feed, retentate and permeate
sense the fluid flow
controls amount of liquid in the reservoir

Form of filter

Figure 8: configuration of hollow fiber cartridges and flat sheet cassettes. (Cross Flow
Filtration Method Handbook, 2014)

Referring to the figure above, there are two types or form of filter which are cartridge
filters and cassette filters.
Cartridge filters. (hollow fiber filters)
Cassette filters (spiral-wound filters)
a form of membrane that created by a set of composed of several flat sheets of membrane
parallel hollow fibers
that held apart and housing by support screens
feed would enter passes through the lumen of feed flow into the space between two
fibers and permeate collected at the outside of membrane sheets while permeate collected at
the fibers
other side of sheets
characterized in terms of fiber length, lumen Characterized by the flow path length, channel

diameter and number of fibers as well as pore height and pore size.
size

Hollow cartridge filters preferable since it has high surface area and provides a high product
recovery with low shear denaturation (Beauchemin). Other than that, cartridge often use for cell
clarification or harvesting while cassette commonly used for protein concentration in
ultrafiltration. (UniFlux system (GE Healthcare Bio-Sciences AB), 2006)
A common hollow fiber may consist of several hundred to 10,000 fibers. The typical dimension
of the membrane may vary by manufacturer and approximate ranges are given as table below.
Dimensions
Outside diameter
Inside diameter
Fiber wall thickness
Fiber length

Range
0.5 2.0 mm
0.3-1.0 mm
0.1-0.6 mm
1-2 meters

Pore size
MF is therefore defined as a membrane separation process using membrane with pore
size of approximately 0.03 to 10 microns, a MWCO of greater than 100000 Daltons and
relatively low feedwater operating pressure of approximately 100 to 400kPa. (MF/UF system,
2002) Rule of thumb in designing a MF is to have a membrane with pore sizes that are the same
of slightly smaller than the particle size. Particle size to pore size ratio of 10 is preferable which
means to has membrane with pores that one-tenth the particle size. (Cheryan, 1998)
Bacteria
Rhizobium
Acetobacter
Azotobacter
azospirillum

Ratio

Size
1 to 2 m long and 0.1 m diameter (Umar, 2001)
1 to 4 m long and 0.6-0.8 m diameter (Maal & Shafiee, 2010)
2-10 m long and 1-2 m diameter (Pea, Reyes, & Larralde-Corona,
February 2002)
2-30 m long and 1-1.5 m diameter (Eckert, et al., 2001)
Table7

In different case, during harvesting of Erwinia carotovora cells, the 0.45m membrane gave
almost double the flux of a 0.6 m membrane, but the 0.2 m membrane gave lower flux than
0.6 m membrane. (Cheryan, 1998) This shows that, the one-tenth ratio is not a must in
modelling a MF but can be as a guidelines.
Material membrane and size
Thus, the most suitable type of membrane to use is Modified Polyethersulfone (mPES)
which has pore size of 0.65 m which would trap those biomass in table 1. mPES is an advanced
hydrophilic membrane filtration that provides higher flux rates for faster processing times and
excellent selectivity for separation applications. Hydrophilic membrane is a water attracting
membrane and such materials readily adsorb water. This water-loving membrane also has high
surface tension values which allowed the material to be wetted by forming a water film or
coating on membranes surface (Hydrophilicity and Hydrophobicity, n.d.).
Spectrum's new modified PES hollow fiber filters provide all the benefits of modified
PES chemistry in fully encapsulated filter modules that are priced for Single-Use. With
Spectrum's Single-Use mPES hollow fiber filters, total development and production costs are
decreased through the combination of faster processing times, decreased labor costs associated
with filter cleaning for re-use and a reduction in the use of costly WFI water. Single-Use
eliminates the need for costly stainless steel systems and increases manufacturing flexibility
((mPES) Hollow Filter Filter Modules Product Number Key, n.d.). The membrane parameters
such as fiber channel internal diameter of 0.75mm, membrane internal diameter of 108mm
has been choose based on manufacturer from SPECTRUMSLAB which has the pore rating of
0.65m.

Positive-pressure
Particularly, MF was design to harvest the biomass. The MF estimated in the SuperPro to
be operated 4227L volume of per batch. Thus, feed has to be inside-out flow with positive
pressure instead of negative pressure since the glucose and water need to be push out of the
system. (MF/UF system, 2002) Positive pressure give the meaning of having a relatively high

pressure transmembrane compared to the outside of the membrane. Pressure drop or

Transmembrane pressure, TMP is the change in the pressure of fluid that passes through the
membrane. In addition, the difference in pressure will sieve out small particle that pass through
the pore membrane and leave larger particle in the membrane.
Thus, certain pressure is applied to feed which in the range of 100 to 400kPa. Same
pressure between inside membrane and outside the membrane will not help in sieving out the
water and salt while too high pressure will cause damage the membrane and rise problem to flux.
(Membrane Filtration, 1999). Therefore, it is decided to choose the pressure of 200kPa by
considering the electricity cost and efficiency of the membrane. General estimation of TMP in
a cartridge has been made by referring to journal which typically in the range of 5-20psig that
equivalent to 2-10kPa at time 180s to 1800s (SHERMAN+, 1992).
There are few ways to increase the filtration rate such as increase the filtration area,
reduce the pressure drop, reduce the cake mass, reduce the liquid viscosity and reduce the
specific cake resistance by increase the porosity and reduce the particle size. Therefore, some
calculation has been made with a few parameter held to be constant. Thus, in order to find the
optimum filtration rate, certain equation has been involved.
Flux
Flux term commonly use in transport phenomena where it is specifically expressed in
terms of gallon of flow per square foot of membrane filter surface area per day(gpd/ft 2) (Bird,
1960). In this case, the feed flow has to be monitored in order to control the flux. Using the
concept of Bernoulli, a high pressure region will has a low fluid velocity or flux. Equally
important to note, the pressure supplied by the pump would effects the flux. High pressure in
membrane will reduce the flux and effect the performance such as membrane fouling which
cause by the accumulation of constituents called as cake formation and clogged pores. (Field,
WU, Howell, & Gupta, 1995) Therefore, optimum pressure must be use to prevent reduction in
flux and its performance. A reference state that normal flux rates range from 20 to 100gpd/ft 2
which equivalent to 34 to 170L/m2h depending on the application (Baruth, 2005).
The process time of the CFF to completes its filtration process, may vary widely depends
on the application, requirements, and system characteristic, economics and stability of the

product. It stated that the total time required for a typical filtration ranges from 3 to 8 hours.
This range may including the preparing the filter and system for processing up to 2 hours,
conducting the filtration process and backwashing the system up to 2 hours (Cross Flow
Filtration Method Handbook, 2014). Therefore, the estimation of filtration time has been
decided up to 3 hours or 10800s.

Therefore, the volumetric flowrate can be calculated by dividing the volume and filtration time.
Assumptions are listed as below.
1. Volume of feed need to be filtrated, v= 4227L/batch
2. Filtration time, t=3hours=10800s
Q(m3 /s)=( 4.227 m /batch) x( batch/3 h)
1.409 m /h

3.914 x 10 m /s
The volumetric flowrate of the filtration is solved. Next, calculating the membrane surface area
is vital in order to find the flux and length of the membrane. Specific cake resistance was assume
at pressure drop of 10kPa at 30minutes. Mass and viscosity of the system was referred to the
properties of cell culture. Assumptions listed below.
1. Viscosity of cell culture , =1.00cp or 0.001kg/m.s (Christopher R. Jacobs, 2012)
2. Specific cake resistance, =171x106m/kg (SHERMAN+, 1992)
3. Mass of dry cake solids per volume of filtrate, c= 1000kg/m3 (Martine Meireles, 2003)
4. Volume at 10kPa and 30minutes, V= 3.914x10-4m3/s x 1800s=0.7043m3
5. Volumetric flowrate of feed need to be filtrated, Q=3.914x10-4m3/s
6. Pressure drop, p=10kPa (SHERMAN+, 1992)

7. Cake formation time, tc=30min=1800s (SHERMAN+, 1992)

8. Filtration time, t=3h=10800s (Cross Flow Filtration Method Handbook, 2014)

A 2=

V
2 pt c

1 /2
0.001 kg
171 x 10 m 1000 kg (
)
.s
0.7043m
m
kg
m
]
= [
1000
N
m
2 ( 10 kPa ) ( 1800 s )
. m kg . . s
kPa
N

)(

)(

)(

1.829 m

A =10800/1800 x 1.828 m
10.97 m 11 m2

Length of the membrane can be easily divide the volume of the MF by area of the surface area.
3

L ( m)=

V 4.227 m
=
A
11 m2

0.39 m
39 cm 40 cm

Calculation of the flux has been made by dividing volumetric flowrate in L/h by area of surface
membrane.

J ( L/m 2 . h)=Q/ A
( 4227 L/batch)(batch/3 h)/11 m2
3

((3.914 x 10(4 m ) )/s )/(11m 2)

128 L /m2 . h

Next, velocity of the fluid find to determine the type flow in the hollow tube. After that,
Reynolds number was calculated. The range value of Reynolds is less that 2100 is laminar flow
while over than 2100 are turbulence flow. The results shown that the type of flow is in turbulence
flow, laminar flow will result in a high pressure in the hollow membrane which is not suitable.
Turbulence flow will lead to a low pressure condition which is normally use.
u(m/ s)=Q/ A
3

((3.914 x 10(4 m ) )/s )/(11 m 2)

0.0427 m/s

R= ud/
(1000 kg /m3)(0.0427 m/s )(0.108 m)/((0.001 kg /m)/s)

4611.6 >2100

Finding volume of wash liquid per volume of liquid in unwashed cake is crucial step to find the
washing time.
Assumptions
1. Weight of fraction of solute remaining in the cake after washing, R=0.04% (Bhave,
1996)
2. Percentage wash efficiency, E=60% (G.Harrison, Todd, R.Rudge, & P.Petrides, 2003)
R =(1E /100)
0.04=(160/100)

log 0.04=nlog 0.4

n=3.51

Next, estimate the weight of dry cake solid filtered.

Vf =V

( 1000m kg )( 0.70434 m )
3

704.34 kg

Weight of water in filter cake by assuming a typical filtration to have 70% water in the filter cake
V=(70 /30)(704.34 kg)/(1000 kg /m3 )

 1.643 m

Ratio of volume of the residual liquid in cake to volume filtrate.

f=

V
Vf

1.643
0.70434

0.0233

Therefore the washing time,

tw=2 nft
2 ( 3.51 ) ( 0.0233 ) ( 1800 s )
294.4 s 295 s
4.9 min

Accumulation of the biomass on the membrane surface lead to reduction in flux that
results into low recoveries of the product or incomplete removal of impurities. Thus,
backwashing commonly designed to remove contaminant in polymer membrane but in this case,
backwashing used to collect the biomass from the fermentation tank. (Bhave, 1996) Direction of
flow is reverse for 30 seconds to 3minutes and occur in relative frequent interval such as 5min to
several hours. (Team, 2010) The backward flow and force dislodge the contaminants at
membrane surface and wash accumulated solids out through the discharge line. By generating a

high backward pressure, another pump was used to provide pressure up to 3bar for 295s as the
washing time calculated above. Commonly 1 to 3% volume of permeate consumed but in this
case 50% volume of permeate will be used in the backwashing purposely did. (Bhave, 1996)

5.2

Material Balance in Seperating System

The material balance in microfilter;

Beef extract

0.0002

Biomass

0.0007

Carboxyl

0.0016

peptone

0.0048

polysorbate

0.0003

Polyvinyl

0.0017

Sodium chloride0.0048
Water

0.960

Glucose

0.0048

Cross flow

microfilter
Beef extract

0.0028

Beef extract 0.0028

Biomass

0.0354

Biomass

0.0162

Carboxyl

0.0007

Carboxyl

0.0074

Peptone

0.0047

Glucose

0.0089

Polysorbate

0.0036

Peptone

0.0095

Polyviny

0.0170

Polysorbate

0.0003

Sodium chloride0.0046

Polyvinyl

0.174

Water

0.9200

Sodium chloride0.0047

Glucose

0.0047

Water

5.3

Waste Treatment

5.3.1

Wastewater from Associated Process

0.94

Most of the water consumed in the liquid fertilizers processing plant is used in associated
process such as washing of related laboratory apparatus, cleaning in-place (CIP) of factory

equipment and tanks. The CIP system will consist of several steps such as a pre-rinse step to
remove any loose raw materials or product remains usually incorporated with soap, a cold rinse
to remove remaining traces of soap and application of hot steam to sterilize the bioreactor.

5.3.2

Characteristic and source of waste water

The volume, concentration and composition of the effluent arising from the plants greatly
depend on the type of the product being processed, production program and operating method.
The liquid wastewater may be divided into categories:
1

Wastewater from downstream processes which is the microfiltration. Filtrate contains

high concentration of liquid salts and water.

Processing water, which include water used in the cooling and heating processes. These
effluents are usually free of pollutant and can be recycled to be used with minimum
treatment. It can also be discharged into sewage water system.

Cleaning wastewater which rises from the cleaning of equipment that has been in contact
with the products or raw materials, CIP cleaning procedure, and other related cleaning
process contains minute amount of raw materials or fermentation broth.

5.3.3

Wastewater Treatment Method

The nature of the wastewater varies in terms of volume and in terms of pH and nutrient
contents. Thus, this mainly resulted in the choice for wastewater treatment to be employed.
Because the production wastewater is highly biodegradable and should be treated accordingly
since it still possesses a potential harm to the environment. There are several options for
wastewater treatment which are:
1

Directly discharge to and subsequent treatment by the nearby sewage treatment plant.

Treatment of wastewater by waste disposal contractors.

Removal and treatment of significant amount of wastewater until permitted concentration

level stated by the Department of Environment (DOE) in on site wastewater.

Since minute amount of water is used for sterilization and cleaning processes and the
contaminant concentration is completely negligible, the waste water from these processes can be
directly discharged into the sewage system. Whereas the wastewater generated from the
downstream process should be undergoes pre-treatment process first to reduce the chemical
oxygen demand (COD) and biological oxygen demand (BOD), the pH should also be adjusted
until allowable value as directed by DOE before being discharge main sewage system. Refers
table 1 below.

Table 8: Wastewater Discharge Standards and Raw Water Quality Criteria for Malaysia.

5.2.4

Pre-treatment of Downstream Processing Waste

Microfiltration is the only equipment involved in downstream process of liquid bio-fertilizer

where biomass containing living cells is isolated. The filtrate of each fermentation broth will
contains numerous amounts of salts and water, whereas the values of COD, BOD and pH of each
filtrate varies due to fermentation of different bacteria used as shown of table below.

Process area

COD
(mg/L)
Negligible

Temperature
(C)
120

pH

Autoclave sterilization water

BOD
(mg/L)
Negligible

Fermenter Cleaning

Negligible

Negligible

25

Filtrate of Rhizobium
leguminosarum fermentation
broth
Filtrate of Azotobacter
chroococum fermentation broth

30

6.5-7.5

30

6.5-7.5

Filtrate of Acetobacter
diazotrophicus fermentation
broth
Filtrate of Azospirilum
brasilense fermentation broth

30

3.5

33

6.5-7.5

Table 9: Chemical characteristic of production wastewaters

The liquid waste water derived from the downstream processing thus not contains any solid
residue and mainly composed of water soluble salts, impurities and very high amount of water.
Hence, simple waste water treatment should be incorporated, since it has a very low organic load
and can be treated by on site aerobic treatment unit.

5.2.5

Aerobic Treatment Unit

Figure 9: Proposed liquid waste treatment

The filtrated liquid effluent will enters activated sludge reactor where air is constantly blown into
the liquid to provide oxygen for mixing and to promote the growth of micro-organisms. The
active biomass will use the oxygen and consumes organic pollutants and nutrients in the
wastewater to grow and reproduce. From the aeration tanks, the mixture of wastewater and
micro-organisms passes into a secondary sedimentation tank (also known as a clarifier) where
the biomass settles under gravity to the bottom of the tank and is concentrated as sludge. Some of
this sludge is recycled to the inlet of the aeration tank to maintain the biomass, hence the name
for the process activated sludge. The remaining is pumped to anaerobic digesters for further
treatment. The treated wastewater is discharged into main sewage system for further treatment
since the in site wastewater treatment will only reduce the COD, BOD and pH values until
acceptable values stated by DOE.

CHAPTER 6: PLANT DESIGN AND ECONOMIC ANALYSIS

6.1

Estimation of purchase cost at base condition and calculated Bare Module and Total
Module cost for each equipment.

No.
1

Equipments

2
3
4
5
6
7
8
9
10
11
12

Centrifugal Compressor (G101)

Air Filter (AF-101)
Shake Flask Rack (SFR-101)
Autoclave (ST-101)
Fermenter (FR 101)
Fermenter (FR 102)
Fermenter (FR 103)
Microfilter (MF 101)
Mixer (MX 101)
Mixer (MX 102)
Clarifier (CL 101)
Aeration basin (AB 101)

13

Sludge Dryer (SLDR-101)

14

Filler (FL-101)
Total

Purchase cost
(RM/unit)
157 250

Bare module cost

(Cbm) (RM)
440 300

Total module
cost (Ctm) (RM)
519 554

37 000
15 799
22 200
1 850
16 650
22 200
42 550
138 754
27 750
7 400
2 867.50

66 600
63 196
62 160
7 400
66 600
88 800
140 032.05
19 147.50
38 295
24 353.40
9 436.95

78 588
74 571.28
73 348.80
8 732
78 588
104 784
165 237.80
22 594.05
45 188.10
28 737.10
11 135.60

37 000

59 200

69 856

18 500
568 120.50

60 883.50

72 451.36

6.2

Calculation for bare module cost

(CBM) and total module cost (CTM).

15% x RM 7 400 = RM 1 110

3% x RM 7 400 = RM 222
CTM = RM 7 400 + RM 1 110 +
RM 222

Shake Flask Rack (SFR-101)

= RM 8 732

Cp = RM 15 799 (Placeholder5)
From Table A.7, FBM = 4.00
Bare Module Cost, CBM = Cp FBM
C BM = RM 15 799 x
4.00

Fermenter (FR-102)
Cp = RM 16 650 (alibaba website, 2015)
FBM = 4.0

= RM 63 196

CBM = RM 16 650 x 4.0

Total Module Cost, CTM = CBM + CCONT +

CFEE
CCONT = 15% from CBM
CFEE = 3% from CBM
15% x RM 63 196 =RM 9 479.40

= RM 66 600
15% x RM 66 600 = RM 9 990
3% x RM 66 600 = RM 1 998
CTM = RM 66 600 + RM 9 990 + RM
1 998

3% x RM 63 196 = RM 1 895.88

= RM 78 588

CTM = RM 63 196 + RM 9 479.40 +

RM 1 895.88
= RM 74 571.28

Fermenter (FR-103)
Cp = RM 22 200 (website Alibaba, 2015)

Fermenter (FR-101)
Cp = RM 1 850 (website Alibaba, 2015)
From table A.7 in appendix, we got F BM =
4.0
CBM = Cp FBM
= RM 1 850 x 4.0
= RM 7 400

FBM = 4.0
CBM = RM 22 200 x 4.0
= RM 88 800
15% x RM 88 800 = RM 13 320
3% x RM 88 800 = RM 2 664
CTM = RM 88 800 + RM 13 320 +
RM 2 664
= RM 104 784

Microfilter ( MF-101)

Cp = RM 42 550 (Alibaba website, 2015)

= RM 19 147.50
15% x RM 19 147.50 = RM 2 872.13

Items

Percentage
of
equipment
cost (%)
100.0
71.4
63.0
8.0
63.4
23.3

Equipment
Materials
Labor
Freight
Overhead
Engineerin
g
(Richard Turton, 2013)

Cost
multi
pliers
1-0
M
L
FIT
O
E

Value of
multipliers

3% x RM 19 147.50 = RM 574.43
CTM = RM 19 147.50 + RM 2 872.13
+ RM 574.42
= RM 22 594.05

0.714
0.368
0.047
1.005
0.136

FBM = ( 1 + L + FIT + LO + E) ( 1 + M)
= [ 1 + 0.368 + 0.047 + (1.005 x 0.368)
+ 0.136 ][ 1 + 0.714]

Centrifugal Compressor (G-101)

Cp = RM 157 250 (Alibaba, 2015)
From Table A.6, CS centrifugal compressor
identification number = 1
From Figure A.9, FBM = 2.80 in appendices,
CBM = CpFBM
CBM = RM 157 250 x 2.80

= 3.291

= RM 440 300
CBM = RM 42 550 x 3.291
= RM 140 032.05
15% x RM 140 032.05 = RM 21 004.80
3% x RM 140 032.05 = RM 4 200.96

15% x RM 440 300 =RM 66 045

3% x RM 440 300 = RM 13 209
CTM = RM 440 300 + RM 66 045 +
RM 13 209
= RM 519 554

CTM = RM 140 032.05 + RM 21

004.80 + RM 4 200.96
= RM 165 237.80

Mixer (MX-101)
Cp = RM 13 875 (Alibaba, 2015)
From Table A.7, FBM = 1.38
CBM = RM 13 875 x 1.38

Air Filter (AF-101)

Cp = RM 37 000 (Source: SuperPro
Designer)
From Table A.7, FBM = 1.8
CBM = RM 37 000 x 1.8

= RM 66 600

= 60 883.50

15% x RM 66 600 =RM 9 990

15% x RM 60 883.50 =RM 9 741.35

3% x RM 66 600 = RM 1 998

3% x RM 60 883.50 = RM 1 826.50

CTM = RM 18 000 + RM 9 990 + RM

1 998
= RM 78 588

CTM = RM 60 883.50 + RM 9 741.35

+ RM 1 826.50
= RM 72 451.36

Aeration basin (AB-101)

Mixer (MX-102)

Cp = RM 2 867.50 (Kompareit website)

Cp = RM 27 750 (Alibaba, 2015)

FBM = 3.291

From Table A.7, FBM = 1.38

CBM = Cp FBM

CBM = RM 27 750x 1.38

= RM 2 867.50 x 3.291

= RM 38 295

= RM 9 436.95

15% x RM 38 295=RM 5 744.25

15% x RM 9 436.95 = RM 1 415.54

3% x RM 38 295= RM 1 148.85

3% x RM 9 436.95 = RM 283.10

CTM = RM 38 295 + RM 5 744.25 +

RM 1 148.85
= RM 45 188.10

CTM = RM 9 436.95 + RM 1 415.54

+ RM 283.10
= RM 11 135.60

Filler (FL-101)

Clarifier (CL-101)

Cp = RM 18 500 (Source : SuperPro

Designer)

Cp = RM 7 400 (Source : SuperPro

Designer)

FBM = 3.291

FBM = 3.291

CBM = RM 18 500 x 3.291

CBM = RM 7 400 x 3.291

= RM 24 353.40

Fp from equation A.3 in appendices,

15% x RM 24 353.40 =RM3 653.10

Log10 FP = C1 + C2 log10 P +

3% x RM 24 353.40 = RM 730.60

C3(log10 P)2

CTM = RM 24 353.40 + RM 3 653.10

+ RM 730.60
= RM 28 737.10

Where value for C1, C2 and C3 = 0, take from

table A.2 in appendices.
So,

Log10 FP = 0 +0 log 10 1 + 0(log10 1)2

FP = 1

Thus, CBM = RM 22 200 x 2.8 x 1 x 1

Sludge Dryer (SLDR-101)

= RM 62 160

Cp = RM 37 000 (Alibaba, 2015)

15% x RM 62 160 = RM 9 324

From Table A.7, FBM = 1.6

3% x RM 62 160 = RM 1 864.80

CBM = RM 37 000 x 1.6

= RM 59 200
15% x RM 59 200 =RM 8 880

CTM = RM 62 160 + RM 9 324 + RM

1 864.80
= RM 73 348.80

3% x RM 59 200 = RM 1 776
CTM = RM 59 200 + RM 8 880 + RM
1 776
= RM 69 856

The grassroots cost can be calculated from

this equation :
CGR = CTM + O.50
Where,CTM =

TM

C
= 1.18

BM

Autoclave (ST-101)
Cp = RM 22 200 (Alibaba website, 2015)
From table A.5 in appendices,
CBM = Cp FBM FP FT
Where FT = 1
FBM from figure A.19 in appendices is, 2.8

= 1.18 x RM 541 065.90

= RM 638 457.76
Thus, CGR = RM 638 457.76 +
0.50(RM 686 738.50)
= RM 981,827.01

BM

6.3

Manufacturing Cost Estimation

The manufacturing cost has to be estimated and tracked from day to day before the economic
feasibility can be determined. Process flow diagram, estimation of fixed capital investment and
estimation on number of operator required to operate the plant is needed to estimate the
manufacturing cost. The cost of manufacturing can denote by RM per unit time or simply
denoted by RM in contrast of capital cost. (Turton, 2014)

6.3.1

Annual cost of raw material

Raw Material
Price
Powdered sodium chloride (NaCl)
RM 159.1/ metric tonne
Peptone powder
RM 47.73/ kilogram
Beef extract
RM 19092/ metric tonne
Industrial grade glucose powder
RM 1527.36/ metric tonne
Water
RM 2.07 / 35m3
Anhydrous ammonia
RM 2227/ metric tonne
Polysorbate 20
RM 6523/ metric tonne
Carboxylmethyl cellulose
RM 7636.80/ metric tonne
Polyvinylpyrrolidone
RM 31.82/ metric tonne
Table 10: The market price of raw materials used in the production of liquid biofertilizer.

Chemical Required
Sodium Chloride
Peptone
Beef extract
Glucose
Water

Amount required per batch Cost for amount required per

(kg)
batch (RM)
39.44
6.27
59.35
2832.76
31.47
4101.73
214.84
328.14
8,866.73
18.34

Total cost
7287.24
Table 11: Composition of 4200L of Dextrose Medium with price of each component.

Chemical required

Amount required per batch Cost for amount required per

(kg)
batch (RM)
Ammonia
16.69
37.17
Carboxymethyl cellulose
23.67
180.76
Polysorbate 20
21.59
140.83
Polyvinylpyrrolidone
117.04
3.72
Total cost
362.48
Table 12 : Composition of preservatives and nitrogen sources used with its respective price per
batch.

Annual cost of raw materials: (RM 7287.24+RM 362.48)(4 productions per month)(12 months)
: RM 367,186.56

Since all production involving different bacteria using the same medium, the cost raw material
per annum can be determine by multiply the cost of raw material per batch with total batches per
year. Since one batch of four different bacteria per month using the same raw material it can be
consider four batches per week multiplying with 12 months which the cost of raw material per
annum is RM 367,186.56

6.3.2

Annual Cost of Waste Treatment

Waste water costing per year.

The formula for calculating the water in the liquid biofertilizer production is shown below
(Assistance, April 2015):

Wastewater cost = raw material (water) cost + treatment & disposal cost

Amount of waste water enter the bioOxidation reactor is 1997.88 kg/batch.

Cost of raw material (water) per year = 1997.88 m3 x RM 2.07 x 12 month
= RM 4549.01
Treatment and disposal coasting (in bioOxidation reactor, sludge draying and clarification) is RM
24,448.87. This value is based on the report of the super pro designer.

Therefore, waste costing per year = RM 4549.01 + RM 24,448.87= RM 28,997.88

6.3.3

Annual cost of utilities

a. Electric consumption

These productions required 807,340 kWh per year. 276,660 kWh used during peak hour and
538,208 kWh used during off-peak hour. The cost of electricity per year can be calculated
according to the Tenaga Nasional Berhad Electricity Tariff as state at table 13. Since operating
hour for each bacterial is different, the power consumption for each production also different as
stated below.

Power consumption for acetobacter sp. 263,572 kW-h

Power consumption for azospirilium sp. 165,921 kW-h

Power consumption for azotobacter sp. 163.637 kW-h

Power consumption for rhizobium sp. 214,210 kW-h

Electric consumed per hour =

807,340 kWh
1 year
1 day

year
318 days 24 hours

= 105.78 kW
Peak hour electricity consumption = 8 hours x 105.78 kW x RM 0.337
= RM 285.19 per day
Off-peak hour electricity consumption = 16 hours x 105.78 kW x RM 0.202
= RM 341.88 per day
Total Cost per year = (RM 285.19 + RM 341.88)(318 days per year)
= RM 199,408.26 per annum

b. Water consumption

Water used for Cleaning-in-place (CIP) utilized 76,601m3/year. The cost of water consumption is
calculated based on the Syarikat Air Syabas Berhad tariff as stated in table 13. The cost of water
per annum calculated as below.
Amount of water used per year

Water consumption for acetobacter sp per year. 20,883 m3

Power consumption for azospirilium sp. per year 18,119 m3

Power consumption for azotobacter sp.per year 17,969 m3

Power consumption for rhizobium sp. per year 19,630 m3

Cost of water per year

35 m3 x RM 2.07 x 12 months = RM 869.40 per year

[(20,883+18,119+17,969+19,630)m3 - (35m3 x 12 months)] x RM 2.28 = RM 173,692.68

per year

Thus, the total cost of water consumption is RM 173,692.68

Type of Utilities

Cost per annum

(RM/year)
Electric
Peak period (8 am to 5 pm) = RM 199,408.26
0.337/kW-h
Off-peak period = 0.202/kW-h
Water
223,683
35m3 = 2.07
RM 173,692.68
3
3
m /year
35m and above = 2.28
Steam
21 MT/year
RM 44.44/MT
RM 933.24
Total
RM 374,034.18
Table 13: Estimation of the cost of utilities for all production per year.

6.3.4

Consumption
per annum
2,848,390.49
kW-h/year

Cost per Unit (RM)

Annual cost of operating labour

According to Alkayat and Gerard, the number of operators per equipment per shift, N OL, can be
estimated using equation as follows:

NOL = (6.29 + 31.7P2 + 0.23 Nnp) 0.5

where: NOL = the number of operators per shift
P

= the number of processing steps involving the handling of particulate solids

Nnp = the number of non-particulate processing steps & includes compression, heating &
cooling mixing, & rotation.
=Equipment
Nnp only includes the compressor, tower, heaters, reactors, and exchangers.

Assumption that has been made during the calculation of the operating labour:
1

Average working weeks annually allocated for a single operator are 48 weeks a year
(Quota of 3 weeks for vacation and sick leave).

An operator works for five 8-hour shifts a week which required 245 shifts per operator
per year.

This plant is set to operate 24 hours per day for 318 days per year. The remaining 47 days
are allocated for plant shut-down and maintenance.

For one operators shift in a year:

= (48 weeks/year)(5 shifts/weeks)
= 240 operating shifts per year
Total shifts per year:
= (330 days/year)(3 shifts/day)
= 990 total operating shifts per year
Number of operator needed per shift:
= (990 operating shifts/year)/(240 operating shifts/year)
= 4.125 4 operators needed per shift.
Equipment types

Nnp

Compressor

Exchanger

Heater/ furnace

Reactors

Tower

Total

Table 14 : List of equipment in non-particulate processing steps

Based on table above, Nnp = 24 and P = 6, thus

NOL = (6.29 + 31.7P2 + 0.23 Nnp)0.5

= [6.29 +31.7(0)2 + 0.23(22)]0.5
= 2.77
Thus, the operating labor can be calculated by:

Operating labor = 4(NOL)

= 4(2.77)
= 11.08 11 operators

Under Federal Government Gazette of Malaysia, minimum wages order according to Act
732 is released in 2012 required the employers in private sector to pay up to RM900/month to
employees in peninsular region while RM800/month for Sabah, Sarawak and Labuan Federal
Territory. This order took effect starting on 1 January 2014. However, current movement is
underway in implementing an increase in monthly wages up to RM 1200/month. However, based
on the Salary Guides released by the Kelly Service in Malaysia, the average salary for the
Operation managers with experinces of 5 7 years, the minimum salary is RM7,500. By
assuming that this new regulation may be well enforced before the plant operated and comparing
it with the average salary for 5 7 years experienced operators, the Plant is targeted with 1 2
years experienced operators during the early plant startup with a salary to be given RM2,500.
The labor cost (COL) thus,

COL = (Salary/month) x (12 months/year) x operating labor

= (RM 2,500/month) x (12 months/year) x 11

= RM 330,000 /year

6.4

Estimation of Total Direct Manufacturing Cost, Fixed Manufacturing Cost, General

Manufacturing Cost and Total Manufacturing Cost.

Cost Item

Typical range of
multiplying factors
Direct manufacturing cost

Raw Materials
Waste Treatment
Utilities
Operating Labour
Direct supervisory and
Clerical labour
Maintenance and repair

CRm
CWT
CUT
COL
0.18COL
0.06FCI

Operating Supplies

0.009FCI

Pattern and royalties

0.03COM

Item Value (RM)

367,186.56
28,997.88
374,034.18
330,000.00
= 0.18(330,000)
=59400
=0.06(981,827.01)
=58,909.62
=0.009(981,827.01)
=8,836.44
=0.03(2,052,488.92)

= 61,574.67
Fixed Manufacturing Costs
Depreciation
Local taxes and insurance
Plant Overhead Costs

Administration costs

0.1FCI

=0.1(981,827.01)
=98,182.70
0.032FCI
=0.032(981,827.01)
=31,418.46
0.708COL + 0.036FCI =0.708(330,000)+0.03(981,827.01)
=263,094.81
General Expenses
0.117COL + 0.009FCI

=0.117(330,000)+0.009(981,827.01)
=47,446.44
Distribution and Selling
0.11COM
= 0.11(2,052,488.92)
Cost
=225,773.78
Table 15: Summary of general expenses, fixes cost and manufacturing cost.

Cost of Manufacturing (COM) with depreciation

= 0.208FCI + 2.73COL+1.23(CUT+CWT+CRM)
= 0.208(981,827.01) + 2.73(330,000) + 1.23(374,034.18+28,997.88+ 367,186.56)
= RM 2,052,488.92

Cost of Manufacturing (COMs) without depreciation

= 0.180FCI + 2.73COL+1.23(CUT+CWT+CRM)
= 0.180(981,827.01) + 2.73(330,000) + 1.23(374,034.18+ 28,997.88 + 367,186.56)
= RM 2,024,997.78

Total Direct Manufacturing Cost

= CRM + CWT + CUT + 1.33COL + 0.03COM + 0.069FCI
= 367,186.56 +28,997.88+ 374,034.18+ 1.33(330,000) + 0.03(2,052,488.92) +
0.069(981,827.01)

=RM 1,338,439.35

Total Fixed Manufacturing Costs

= 0.708COL+0.068FCI+depreciation
= 0.708(330,000) + 0.068(981,827.01) + 98,182.70
= RM 5, 6,412,320.66

Total General Manufacturing Costs

= 0.177COL + 0.009FCI + 0.16COM
= 0.177(330,000) + 0.009(981,827.01) + 0.16(2,052,488.92)
= RM 948.332.17

Total Costs
= CRM + CWT + CUT + 2.215COL + 0.190COM + 0.146FCI + depreciation
= 367,186.56+ 28,997.88+ 374,034.18+ 2.215(330,000) +0.190(2,052,488.92)
+ 0.146(981,827.01) + 98,182.70
= RM 2,131,020.96

6.5

Profitability Analysis

6.5.1

Annual Revenue

Based on the data collected from the simulation of biofertilizer production on SuperPro , the
yield of biofertilizer for one batch is 1958.3 L/batch. Every month 4 batches of bacteria is
produced. Annually, the production of this biofertilizer undergoes 48 batches only. Thus:
1958.3 L x 48 batches = 93,725.856 L biofertilizer produce annually

Standardize biofertilizer price for this plant is RM20 for every 250ml (IBG,2015). Therefore, the
estimated annual revenue for this plant will be:
93,725.856 L

1000 ml
250 ml RM 20 = RM 7,498,068.48 /year
1l

Profit Margin
Profit margin = Product sold cost of raw material

Based on the price of the raw material used for biofertilizer production the profit margin can be
calculated:

Total cost of raw material for one batch:RM 7287.24 + RM 362.48 = RM 7649.72/ batch

Total product sold for one batch:

1958.3 L

1000 ml
250 ml RM 20 = RM 156,209.76 /batch
1l

Hence ,
Profit margin(per batch) = Product sold(per batch) cost of raw material(per batch)
= RM 156,209.76 - RM 7649.72
= RM 148,560.04 (per batch)
Annual profit margin = RM 148,560.04 x 48 batches = RM 7,130,881.92

Net Annual profit

Working Capital, WC
Item
Raw material
Operating labor
Waste treatment
Utilities
Direct supervisory and clerical labor
Maintenance and repair
Operating supplies
Pattern and royalties
TOTAL

Value(RM/yr)
367,186.56
330,000.00
28,997.88
374,034.18
59400
58,909.62
8836.45
61,574.67
1,288,939.35

Fixed Capital Investment, FCI

RM 981,827.01

nd of
ar (k)
0
1
2
3

6.4.2

NPV

NPV is the cumulative discounted cash position at the end of the project. In other words,
the greater the value of NPV, the more profitable the business is. In order to determine the NPV
value, there are certain factors needed to be considered carefully such as fixed capital investment
and so on. Cumulative cash flow for both non-discounted and discounted are needed to be
calculate first.

For the non-discounted, depreciation value is calculated using straight line

method which is:

dkSL = (FCIL s)/n where n is plant life span
All the data can be shown as below:
Items
Land cost
FCIL
FCIL during year 1
FCIL during year 2
WC
R
Interest
COMd
Taxation rate
Salvage value
Depreciation
Plant life span

Value (RM)
1000000.00
981827.01
589096.21
392730.80
1288939.35
7498068.48
0.15
2024997.78
25% (KPMG International, 2015)
0
5 years straight line method
10 years

investment

dk

FCI-dk

COM

1000000
589096.21
1681670.15
-

196365.402

981827.01
981827.01
981827.01
785461.608

7498068.48

2024997.78

(R-COMdk)*(1-t)+dk
4153894.376

cash flow
-1000000
-589096.21
-1681670.15
4153894.37
6

cumulat
cash flo
-100000
-1589096
-3270766
883128.0

196365.402

589096.206

7498068.48

2024997.78

4153894.376

196365.402

392730.804

7498068.48

2024997.78

4153894.376

196365.402

196365.402

7498068.48

2024997.78

4153894.376

196365.402

7498068.48

2024997.78

4153894.376

7498068.48

2024997.78

4104803.025

7498068.48

2024997.78

4104803.025

10

7498068.48

2024997.78

4104803.025

11

7498068.48

2024997.78

4104803.025

12

2288939.35

8498068.48

2024997.78

4854803.025

4153894.37
6
4153894.37
6
4153894.37
6
4153894.37
6
4104803.02
5
4104803.02
5
4104803.02
5
4104803.02
5
7143742.37
5

5037022.

9190916.

13344811

17498705

21603508

25708311

29813114

33917917

41061659

Table 16. Non-discounted cash flows

end of year
0
1
2
3
4
5

nondiscounted cash flow

-1000000
-589096.21
-1681670.15
4153894.376
4153894.376
4153894.376

discounted cash flow

-1000000
-512257.5739
-1271584.234
2731252.98
2375002.591
2065219.644

cumulative discounted cash flow

-1000000
-1512257.574
-2783841.808
-52588.82862
2322413.762
4387633.407

6
7
8
9
10
11
12

4153894.376
4153894.376
4104803.025
4104803.025
4104803.025
4104803.025
7143742.375

1795843.169
1561602.756
1341867.39
1166841.209
1014644.529
882299.5908
1335216.529
Table 17. discounted cash flows

6183476.576
7745079.332
9086946.722
10253787.93
11268432.46
12150732.05
13485948.58

50000000
40000000
30000000
cumulative cash flows (RM)

20000000
10000000
0

0
-10000000

3
2

5
4

7
6

9 11 13
8 10 12

time (years)

Figure 30. cumulative cash flow graph for non-discounted after-tax cash flows

16000000

14000000

12000000

10000000

8000000

cumulative discounted cash flows

6000000

4000000

2000000

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
-2000000

-4000000
time (years)

Figure 11. cumulative cash flows graph for discounted after-tax cashs flows

Based on the Table 2, the NPV value is RM13485948.58

CONCLUSION

It can be concluded that, liquid biofertilizer has high demand either globally or locally. It
is a good choice of manufacturing product since it could give high profit in long term. Form the
starting until the end of production, it cost an acceptable amount. By selling the product in 10
years of plant life span, it manages to give payback period at second year of manufacturing.
Based on agriculture sector in Malaysia which is very active, it is believed that this product can
go further by having a good promotion and do some testimony to gain customer trust. To have
further good strategies in competing with other company, every worker has to do their job
accordingly. This include every worker in their department like, chief technologist, sales and
marketing director, finance director, intellectual property director and of course the chief
executive officer.

APPENDIX

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