Wastewater Full Report

INDUSTRIAL EFFLUENT TREATMENT SYSTEM
(IETS) DESIGN PROJECT
JASVINDER SINGH A/L TARA SINGH
UNIVERSITI KEBANGSAAN MALAYSIA

INDUSTRIAL EFFLUENT TREATMENT SYSTEM (IETS) DESIGN PROJECT
JASVINDER SINGH A/L TARA SINGH
WASTE AUDIT PROJECT SUBMITTED IN FULFILMENT FOR THE COURSE OF

KKKS6354 INDUSTRIAL EFFLUENT TREATMENT SYSTEM (IETS)
FACULTY SCIENCE AND TECHNOLOGY

UNIVERSITI KEBANGSAAN MALAYSIA
BANGI
2022
iii
DECLARATION
I hereby declare that the work in this project is my own except for quotations and
summaries which have been duly acknowledged.
28 January 2023 JASVINDER SINGH A/L

TARA SINGH
P115143
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TABLE OF CONTENTS
Page
DECLARATION iii
TABLE OF CONTENTS iv
LIST OF TABLES vii
LIST OF ABBREVIATIONS x
CHAPTER I INTRODUCTION
1.1 Introduction 1
1.2 History of Insulin 1
1.3 Insulin Global Market 2
1.3.1 Product Type Insight 4
1.3.2 Application Insight 5
1.3.3 Type Insight 5
1.3.4 Distribution Channel Insight 6
1.3.5 Regional Insight 7
1.3.6 Insulin Market Share Insight 7
1.4 Diabetic Condition in Malaysia 8
1.5 Insulin Production in Malaysia 9
1.6 Target and Addressable Market in Malaysia 11
1.6 Total Revenue of Insulin Manufacturing Industry 12
CHAPTER II PROCESS DESCRIPTION

2.0 Introduction 14
2.1 Process Description 17
2.2 Fermentation Section 19
2.2.1 Primary Recovery Section 21
2.3 Reaction Section 23
2.3.1 Inclusion Body Solubilization 23
2.3.2 CNBr Cleavage 23
2.3.3 Sulfitolysis 24
2.3.4 Refolding 25
2.4 Enzymatic Conversion 25
2.5 Final Purification System 26
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CHAPTER III MASS BALANCE

3.1 Fermentation 29
3.1.1 Mixing 29
3.1.2 Heat Sterilization 30
3.1.3 Air Filtration 30
3.1.4 Fermentation 31
3.1.5 Fermentation Air Release 31
3.1.6 Fermenter Output 32
3.2 Primary Recovery Section 32
3.2.1 Storage 32
3.2.2 Centrifugation 32
3.2.3 Blending 33
3.2.4 Homogenization 34
3.2.6 Blending 35
3.3 Reaction Section 36

3.3.1 IB Solubilization 36
3.3.2 Diafiltration 37
3.3.3 Dead End Filtration 37
3.3.4 CNBr Cleavage 38
3.3.5 Rotary/Evaporator 38
3.3.6 Sulfitolysis 39
3.3.8 S-Sepharose 40
3.3.9 Refolding 40
3.3.11 HIC Column 41
3.3.12 Enzyme Conversion 42
3.4 Final Purification Section 43
3.4.2 S-Sepharose 43
3.4.4 RP-HPLC 45
3.4.6 Gel Filtration 46
3.4.8 Crystallization 47
3.4.9 Basket Centrifugation 48
3.4.10 Freeze Drying 48
CHAPTER IV WASTEWATER DESIGN

4.1 Pharmaceutical Wastewater 50
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4.2 Wastewater Generated 51

4.2.1 Density of Liquid Waste 51
4.2.2 Primary Section Wastewater Generation 55
4.2.3 Reaction Section Wastewater Generation 55
4.2.4 Final Purification Section 56
4.2.5 Total Volume of Wastewater Generated 58
4.3 Wastewater Design 60

4.3.1 Sump Pit Design 60
4.3.2 Primary Clarifier 63
4.3.3 Calamity Tank 67
4.3.4 Equalization Tank 69
4.3.5 UASB 72
4.3.6 Aeration Tank 81
4.3.7 Final Clarifier 85
4.4 Waste Generated and Management 89
4.5 Sustainable Development Goal 92
CHAPTER V CONCLUSION
5.0 Conclusion 96
REFERENCES 97
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LIST OF TABLES
Table No. Page
Table 4.0 Table of Waste Generated for Primary Reaction 55
Table 4.1 Table of Waste Generated for Reaction Section 55
Table 4.2 Table of Waste Generated for Final Purification System 56
Table 4.3 Summary of Waste Generated 57
Table 4.4 Total Volume of Waste Generated 58
Table 4.5 Summary of Sump Pit Design 62
Table 4.6 Summary of Primary Clarifier Design 66
Table 4.7 Summary of Calamity Tank Design 68
Table 4.8 Summary of Equalization Tank Design 70
Table 4.9 Summary of UASB Tank Design 80
Table 4.10 CAS System Design 82
Table 4.11 Summary of Aeration Tank Design 84
Table 4.12 Summary of Final Clarifier Design 85
Table 4.13 Overall Efficiency of Wastewater 86

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LIST OF ILLUSTRATIONS
Figure No. Page
Figure 1.0 Insulin Market Size, by product type, 2020 to 2030 (USD
Million) 3
Figure 1.1 $ 20.4 billion of global insulin market share by

distribution channel 6
Figure 1.2 Fatality according to the type of incident for the year 2017
and 2018 9
Figure 1.3 Biocon Manufactured Insulin in Malaysia 10
Figure 1.4 Forecast Sales of Insulin in Malaysia 11
Figure 1.5 Total Revenue of Insulin Manufacturing Industry 12
Figure 2.0 Human Insulin from proinsulin fusion protein 15
Figure 2.1 Insulin Production PFD 17
Figure 2.2 Insulin Fermentation Vessel 20
Figure 2.3 Fermentation Vessel at Insulin Production Industry 20
Figure 2.4 Centrifugal Machine 22
Figure 2.5 Centrifugal Machine at Insulin Manufacturing Industry 22
Figure 4.0 Sump Pit 60
Figure 4.1 Primary Clarifier Design for Sludge Draw Off Pipe 64
Figure 4.2 Primary Clarifier Design 64
Figure 4.3 Equalization Tank 70
Figure 4.4 Conversion of Organic Pollutants to Biogas by Anaerobic 72

Microorganism
Figure 4.5 UASB Reactor 72
Figure 4.6 UASB Reactor with GLS Separator 73
Figure 4.7 Anaerobic Digestion Conversion Process of Organic Material 76

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Figure 4.8 Anaerobic Granular Sludge 77
Figure 4.9 Conventional Activated Sludge System 82
Figure 4.10 Overall Design of WWTP 88
Figure 4.11 Cenviro, Prescribed Premises Approved by DOE 90
Figure 4.12 Waste Management Hierarchy 92
Figure 4.11 SDG 6, clean Water and Sanitation 93
Figure 4.12 SDG 7, Affordable and Clean Energy 93
Figure 4.13 SDG 12, Responsible Consumption and Production 94
Figure 4.15 SDG 14, Life Below Water 95

x
LIST OF ABBREVIATIONS
AD Anaerobic Digester
BOD Biological Oxygen Demand
CAGR Compound Annual Growth
CAS Conventional Activated Sludge
CH4 Methane Gas
CN Cyanide
CNBr Cyanogen Bromide
CO2 Carbon Dioxide
COD Chemical Oxygen Demand
CoE Centre Of Excellence
HCl Hydrochloric Acid
HRT Hydraulic Retention time
MrETOH Mercapethanol
NaCl Sodium chloride
NH4HCO3 Ammonium Bicarbonate
OLR Organic Loading Rate
IB Inclusion Bodies
TN Total Nitrogen
TP Total Phosphorus
TSS Total Suspended Solids
UASB Up Flow Anaerobic Sludge Blanket Reactor
WFI Water For Injection
WHO World Health Organisation
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CHAPTER I
INTRODUCTION
1.1 INTRODUCTION
Before insulin became accessible in the early 20th century, experts Allen and Joslin advocated
fasting and calorie-restricted diets for diabetes. This led to improved glucosuria and acidosis, a
decrease in a coma, and a delay in mortality among diabetic youngsters. In addition, all people
with diabetes were instructed to reduce their sugar and starch consumption, and those who were
overweight were told to lose weight (Mazur A., 2011).
1.2 HISTORY OF INSULIN
The discovery of insulin in 1922 heralded a significant medical and therapeutic advancement for
diabetic patients. Before the discovery of insulin, it was assumed that the pancreas released a
chemical that regulated the metabolism of carbohydrates. However, due to contaminants and
toxicity, attempts to prepare pancreatic extracts to decrease blood glucose have failed for years
(Bliss M., 1993).
Frederick Banting, an orthopedic surgeon, had the notion of isolating pancreatic islet extracts by
ligating the pancreatic duct of dogs, keeping them alive until the acini deteriorated, and separating
the islets. In September 1921, they demonstrated that the depancreatized dog developed diabetes
and that an intravenous infusion of their pancreatic extract, which they termed isletin, reduced
blood glucose. Late in 1921, J.B. Collip, a scientist, joined the group and helped refine isletin for
human use. Banting and Best administered the first pancreatic extract injection to a 14-year-old
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boy on January 11, 1922. The injection generated a sterile abscess, had little impact on ketosis,
and resulted in a slight drop in blood glucose. Collip's further injections of the refined extract had
encouraging results in the same year. Blood glucose and glucosuria declined, whereas ketonuria
vanished. Rosenfeld reported positive outcomes for six additional patients (Rosenfeld L., 2002).
Because the insulin mixture required many daily injections, researchers sought to extend its
duration of action. In the 1930s, Danish scientist H.C. Hagedorn added protamine to insulin to
develop its effect. In Toronto, Scott and Fisher added zinc to insulin to increase its duration of
action. These discoveries led to the launch of animal insulins with a longer term on the market
(Bliss M., 1993).
By using and combining the insulin A- and B- chains that have grown in Escherichia coli, David
Goeddel and his colleagues (of Genentech) created the first recombinant DNA human insulin in
1978. After that, Genentech and Lilly formed a contract to market rDNA insulin. As a result,
Humulin® R (rapid) and N (NPH, intermediate-acting), the first insulin made with rDNA
technology, were introduced to the market in 1982.
Chronic diabetes problems frequently increased as individuals with diabetes began to live longer.
However, the Diabetes Control and Complications Trial, conducted in 1993, demonstrated the
linear relationship between the level of glycemic control and complications for the first time (N
Engl J Med., n.d.).
1.3 INSULIN GLOBAL MARKET
The size of the worldwide insulin market was projected to be USD 20.35 billion in 2021 and is
anticipated to increase at a CAGR of 1.5% from 2022 to 2030. The insulin market is expanding
due to things like rising diabetes rates and improvements in the formulation. However, the rising
use of medications like GLP-1 RAs to treat type 2 diabetes may hinder market expansion.
Companies like Novo Nordisk A/S, Sanofi, and Eli Lilly and Company rule this oligopolistic
market. One of the chronic illnesses with the fastest global growth rates is diabetes. In recent
decades, its frequency has gradually risen. According to the World Health Organization (WHO),
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537 million individuals globally will have diabetes in 2021, with one in ten developing other forms
of the disease.
Figure 1.0: Insulin Market Size, by Product Type, 2020 to 2030 (USD Million)
Source: Statistica, 2021
Additionally, populations who are older and more fat are more prone to chronic illnesses. The
senior population worldwide was estimated by the World Bank Group to be over 727 million in
2020. Over the next three decades, this number is projected to quadruple, reaching almost 1.5
billion in 2050.
Another significant aspect influencing the expansion of space is developments in the formulation.
For instance, Fiasp (Novo Nordisk) is a mixture of niacinamide (Vitamin-B3) and insulin aspart
that can enhance the drug's early absorption. In addition, the only inhaled medication on the market
that does not require syringes or needles is Afrezza (Mannkind). Phase 3 clinical studies are also
being conducted for the oral insulin capsule made by Oramed Pharmaceuticals. By Q4 2023, the
business intends to submit the BLA. The successful introduction of this product may provide
patients with another choice (Hernandez et al., 2019).
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The COVID-19 epidemic caused the market to suffer. The tactics created to stop the spread of
SARS-CoV-2 during the epidemic caused a pause and interruption in the supply of medications.
In addition, fewer prescriptions for insulin have been written, according to hospitals. However, the
impact of COVID-19 began to wane in several nations after the second quarter of 2021, which
gave the market new life.
Insulin's high price can hamper the expansion of the market. Leading companies with patent
protection for their products include Eli Lilly and Company, Novo Nordisk A/S, and Sanofi. To
improve its position, Sanofi, for instance, submitted 74 patent applications for Admelog. These
corporations control more than 90% of the world market. Biosimilar players are, therefore,
insignificant in this market (Insulin Market Size, Share & Growth Analysis Report, 2030. (n.d.)).
1.3.1 Product Type Insight
In 2021, the sector for long-acting insulin provided a sizeable sales share of 56.53%. The segment
is anticipated to be driven by elements like patent protection and strong demand due to long-term
effects. The top goods in this category are Toujeo, Levemir, Xultophy, Insulatard, and Lantus. In
contrast, insulin with a rapid onset of action promotes quicker absorption. Aspart, Lispro, and
Glulisine are essential items in this market. Lispro stood out among them, having the most
significant sales in 2021 (Insulin Market Size, Share & Growth Analysis Report, 2030. (n.d.)).
The biosimilar insulin market is anticipated to grow at a promising CAGR of 18.3% during the
projected period. The category growth will be fuelled by the essential products' patents expiring
and the following release and adoption of biosimilar insulin products. For instance, the FDA
approved Semglee, a glargine biosimilar, in July 2021. It was the first interchangeable insulin
biosimilar of Lantus to receive approval in the United States.
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1.3.2 Application Insight
Due to the high level of insulin dependence among these patients, type 1 diabetes mellitus will
account for the most significant application category in 2021. The condition is more severe than
type 2 diabetes. Around 10% of people with diabetes, according to the International Diabetes
Federation, have type 1 diabetes (Insulin Market Size, Share & Growth Analysis Report, 2030.
(n.d.)).
The fastest-growing subgroup is anticipated to be type 2 diabetes mellitus. It is the kind that is
most prevalent worldwide. The main driver of the segment's expansion is its high prevalence. The
prevalence of type 2 diabetes, which the IDF 2021 study estimates to be at 405.6 million people,
is expected to rise to over 510.8 million people by 2030. Oral insulin is being researched and
developed by several businesses. However, only two oral insulin delivery systems have received
FDA approval thus far: Diasome's HDV-I and Oramed's ORMD-0801, which is now through a
phase 3 clinical study. Accepting this medication will provide patients with an alternative to
receiving insulin injections (Hernandez et al., 2019).
1.3.3 Type Insight
The analog insulin segment dominated the market with an industry share of 86.58% in 2021. The
reason for its supremacy is that it is more effective than traditional therapy and has fewer side
effects, such as fewer hypoglycemia spells and less weight gain. In addition, the majority of insulin
medications are long-acting. Companies like Eli Lilly, Novo Nordisk A/S, and Sanofi create long-
acting insulin analogues, including glargine, detemir, and degludec. These goods hold a sizable
portion of the market, and demand for them is anticipated to fuel growth.
On the other hand, human insulin is the standard kind of insulin. Therefore, products like Humulin,
Insuman, and Novolog are available on the market thanks to the top three producers. However, as
more potent insulin analogues become available, it is anticipated that these drugs' sales and
prescription rates will fall during the ensuing years (Cefalu et al., 2018).
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1.3.4 Distribution Channel Insight
The retail pharmacy category dominated the entire market in 2021 due to the increased illness load,
the viability of patients receiving home care, and the numerous discounts provided by retail
pharmacies. The market has seen chains of American retail pharmacies consolidate. In addition,
moderate growth is anticipated for the hospital pharmacy market. The growing number of diabetes
patients being admitted to hospitals is one of the reasons driving this market.
Online pharmacies are included in the others section, projected to have the most significant
increase throughout the projection period. The segment is primarily driven by online drug
purchases' convenience, comfort, and flexibility. Additionally, the incentives provided by online
pharmacies to customers to buy drugs online are anticipated to support industry expansion (Cefalu
et al., 2018).
Figure 1.1: $ 20.4 Billion of Global Insulin Market Share by Distribution Channel
Source: https://www.grandviewresearch.com/
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1.3.5 Regional Insight
In terms of revenue, North America led the market in 2021, primarily because of the dominance
of branded goods and the region's rising illness prevalence. In 2020, the CDC estimated that 37.3
million Americans had diabetes, of whom 28.7 million had been diagnosed, and 8.5 million had
not. Additionally, 96 million adults aged 18 and older have prediabetes. Novo Nordisk A/S, Eli
Lilly and Company, and Sanofi are a few leading companies presently active in this area. However,
Semglee and Rezvoglar, two new insulin biosimilars from Mylan/Biocon and Eli Lilly and
Company, respectively, have received FDA approval in the United States. Introducing these
biosimilars is anticipated to lower insulin's cost on the American market and increase
competitiveness (Jeganathan R et al., 2013).
Due to factors like growing geriatric and target populations, an increase in collaborations for the
development of biosimilars, geographic expansion of key players, and active involvement of the
government and nonprofit sectors in the market space, Asia Pacific is predicted to experience
significant growth during the forecast period. Additionally, regional market expansion may be
aided by health awareness campaigns and scientific conferences for the management of this
condition.
1.3.6 Insulin Market Share Insight
Through mergers and acquisitions, several organizations are increasingly choosing for
international growth, strategic alliances, and partnerships in growing and economically beneficial
locations. For instance, Biocon announced in February 2022 that it would acquire the biosimilar
portfolio of Viatris for USD 3.35 billion. This purchase expanded Biocon's line of biosimilar
products and increased its revenue-generating. Among the most notable essential companies in the
worldwide insulin market are:
• Novo Nordisk A/S
• Eli Lilly and Company
• Sanofi
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• Biocon Ltd
• Wockhardt
• Boehringer Ingelheim International GmbH
• Julphar
• United Laboratories International Holdings Limited
• Tonghua Dongbao Pharmaceutical Co. Ltd.
1.4 DIABETIC CONDITION IN MALAYSIA
Diabetes is a severe public health problem in Malaysia, where the incidence of type 2 diabetes
(T2D) has risen to 20.8% among adults older than 30 and affects 2.8 million people. Diabetes
management is the responsibility of primary and tertiary health care practitioners practicing in
various settings (Hussein et al., 2016).
Malaysia's diabetes incidence is the highest in the Western Pacific area and one of the highest in
the world, costing over $600 million every year. The prevalence of diabetes increased by 68.3%
between 2011 and 2019, from 11.2% to 18.3%. In Malaysia in 2019, a nationwide study found that
3,6 million persons (18 years and older) had diabetes, of which 49% (3.7 million cases) were
undiagnosed. Diabetes is anticipated to impact 7 million Malaysian individuals aged 18 or older
by 2025, providing a significant threat to public health with a prevalence of 31.3%. According to
published publications, the prevalence of diabetes in Malaysia ranges from 7.3% to 23.8%.
Multiple factors, including population growth, aging, urbanization, and increased obesity and
physical inactivity rates, have contributed to the upward trend (Jeganathan R et al., 2013).
Diabetes is a severe public health problem in Malaysia, associated with increased macro and
microvascular complications and early and unnecessary mortality. In Malaysia, the prevalence of
type 2 diabetes (T2D) among persons aged 30 has increased during the previous decade. In 2011,
according to the fourth Malaysian National Health and Morbidity Survey (NHMS IV), the
prevalence of T2D grew to 20.8%, impacting 2.8 million persons, compared to the third National
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Health and Morbidity Survey (NHMS III), which recorded a prevalence of 14.9% in 2006. Indians
had the most significant incidence of T2D (24.9% in 2011 and 19.9% in 2006) among the major
ethnic groups in Malaysia, followed by Malays (16.9% in 2011 and 11.4% in 2006) and Chinese
(13.8% in 2011 and 11.4% in 2006) (Hussein et al., 2016).
Figure 1.2: Fatality according to the type of incident for the year 2017 and 2018
Source: https://www.dosm.gov.my/
1.5 INSULIN PRODUCTION IN MALAYSIA
In Malaysia the insulin is only being manufactured at Biocon Sdn Bhd. Biocon Sdn Bhd is a
multinational company which is originated from India. Biocon Biologics has established in
Malaysia a Center of Excellence (CoE) for insulins with end-to-end manufacturing capabilities for
a comprehensive range of regular, basal, and fast insulins. It is the first and only biopharmaceutical
sterile injectables facility in Malaysia to get clearance from the U.S. Food and Drug Administration
(FDA) and the European Medicines Agency. In addition, Biocon's biosimilar insulin Glargine,
10
manufactured in Malaysia, got FDA clearance as the 'first interchangeable biosimilar' more
recently.
Biocon Biologics has established the most significant integrated insulin production and research
and development facility in Asia through Biocon Sdn Bhd in Malaysia at over USD 350 million.
This is Malaysia's most significant foreign investment in biotechnology, reflecting Biocon
Biologics' dedication to making cheap, life-changing insulins accessible to patients worldwide. In
2016, when Biocon Biologics' recombinant human insulin became the first locally made biosimilar
pharmaceutical licensed for sale in Malaysia, commercial activities began at the plant.
As the sole insulin manufacturer in Malaysia, Biocon Sdn Bhd has been able to ensure Malaysia's
insulin self-sufficiency, increase access to insulin, and save money for its partner, the Ministry of
Health (MoH), Malaysia. Since Biocon's 2011 arrival in Malaysia, human insulin costs have
decreased by almost forty percent, and insulinization has improved by thirty percent (Biocon,
2022).
Figure 1.3: Biocon Manufactured Insulin in Malaysia
Source: https://www.biocon.com/
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1.6 TARGET AND ADDRESSABLE MARKET IN MALAYSIA
For the year 2021, the total sales for insulin recorded were $16bn. The overall sales in 2022 are
expected to increase to $21bn. However, from the year 2022, the forecast sales are expected to
increase 3.3X overall, which are $25bn for the year 2023, $42bn for the year 2024, $62bn for the
year 2025, $69bn for the year 2026 and $73bn for the year 2027. The forecast sales are expected
to increase gradually as it aligns with the research that the number of diabetic patients will increase
to 7 million by 2025 (Biocon, 2022).
Figure 1.4: Forecast Sales of Insulin in Malaysia
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1.7 TOTAL REVENUE OF INSULIN MANUFACTURING INDUSTRY
Based on the annual report from the Biocon Ltd, it shows that the total income for the insulin
manufacturing companies continuously rises from the year of 2018 to 2022. However, profit only
raises slightly from the year 2018 to 2019 and from the year 2019 to 2022, the profit remains
constant.
The insulin production increased as the demand from the diabetic patient increases. However, the
profit of the industry remain same is due to the total expenses paid by the industry.
Figure 1.5: Total Revenue of Insulin Manufacturing Industry
In 2014, according to USC research, 70% of insulin money went to the manufacturers, and 30%
went to the middlemen, but by 2018, just 47% of insulin revenue went to the manufacturers. The
remaining % of insulin sales money was pocketed by the intermediaries, mostly PBMs and
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pharmacies (Insulin Prices Are Skyrocketing, but Just Who Is Driving the Rise in Costs? Hint: It’s
Not the Drug Manufacturers, 2022).
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CHAPTER II
PROCESS DESCRIPTION
2.0 INTRODUCTION
Insulin enhances glucose metabolism and is crucial for delivering energy to cells in the body.
Diabetes mellitus, the third leading cause of death in industrialized nations after cardiovascular
disease and cancer, is caused by impaired insulin production.
Insulin is a polypeptide composed of 51 amino acids organized in two chains, chain A containing
21 amino acids and chain B containing 30 amino acids. Two disulfide bonds link the A and B
chains together. The molecular weight of human insulin is 5808, and its isoelectric point is 5.4.
Human insulin can be manufactured using four distinct methods:
• Extraction from human pancreas

• Chemical synthesis via individual amino acids
• Conversion of pork insulin, or “semi synthesis”
• Fermentation of genetically engineered microorganisms
Due to the limited availability of raw materials, human pancreatic extraction cannot be performed.
Although technically feasible, total synthesis is not economically viable because of its low yield.
Production based on porcine insulin, commonly known as "semi-synthesis," converts the porcine
insulin molecule into a perfect clone of the human insulin molecule by swapping threonine for
alanine at position G-30. Novo Nordisk A/S has developed and implemented this technology
(Denmark). However, this technique is highly costly due to the collecting and processing vast
quantities of swine pancreas. In addition, the availability of porcine pancreas restricts the supply.
At least three alternative methods for generating human insulin using fermentation and
recombinant DNA technology have been proposed.
15
Two-Chain Procedure. The two-chain method was the first successful biosynthetic human insulin
(BHI) production methodology based on recombinant DNA technology. This method was created
by Genentech, Inc. (South San Francisco) and refined by Eli Lilly and Company (Indianapolis). In
Escherichia coli, each insulin chain is generated as an a-galactosidase fusion protein that forms
inclusion bodies. Two peptide chains are extracted from inclusion bodies (IBs), purified, and then
joined to produce human insulin. Eventually, the -galactosidase operon was replaced with the
tryptophan (Trp) locus, resulting in a significant increase in yield.
Proinsulin Technique. The so-called intracellular approach for producing proinsulin avoids the
requirement for separate fermentation and purification trains necessary for the two-chain method.
Instead, intact proinsulin is created. Eli Lilly has brought the proinsulin approach to the market.
Figure 2.0 depicts the essential transformation phases. E. coli bacteria overproduce Trp-LE'-Met-
proinsulin, which is recovered and solubilized as inclusion bodies (Trp-LE'-Met is a 121-amino
acid peptide signal sequence; proinsulin, with 82 amino acids, is an insulin precursor). CNBr
releases proinsulin by cleaving the methionine linker. The proinsulin chain undergoes a folding
process to form intermolecular disulfide bonds. The C peptide, which joins the A and B chains in
proinsulin, is cleaved by enzymes to produce human insulin. Several chromatography and
membrane filtration processes are necessary to purify the product.
16
Figure 2.0: Human insulin from proinsulin fusion protein.
Novo Nordisk A.S. created a second method (extracellular) for generating proinsulin. It is based
on yeast cells that release insulin as a precursor with a single chain. Secretion facilitates the
separation and purification of products. The precursor contains the necessary disulfide bridges,
making it identical to insulin. It is transformed into human insulin by transpeptidation in an organic
solvent containing a threonine ester and trypsin, followed by de-esterification. Utilizing a
continuous bioreactor–cell separator loop makes it possible to reuse the cells, which is another
advantage of the secreted proinsulin technique.
In this instance, a strategy based on intracellular proinsulin was selected for research.
17
2.1 PROCESS DESCRIPTION
The production of insulin is shown in Figure 2.1. It is divided into four sections: Fermentation,
Primary Recovery, Reactions, and Final Purification.
Figure 2.1 (a): Fermentation Section

18
Figure 2.1b: Reaction Section

19
Fig
Figure 2.1 (c): Final Purification Section
2.2 FERMENTATION SECTION
The fermentation medium is prepared in fermentation tank vessels made of stainless steel and
sanitized in a continuous heat sterilizer. The axial compressor and absolute filter supply the
fermenter with sterile air and ammonia at an average rate of 0.5 VVM. The fermenter will be
injected with 50 m3 of E. coli cells that have been modified. These cells create the insulin precursor
Trp-LE'-Met-proinsulin, which is maintained in the cellular biomass. The fermentation
temperature in the production fermenter is 37°C, and the fermentation time is around 18 hours.
The highest E. coli concentration in the manufacturing fermenter is around 30 g/L. (dry cell
weight). The Trp operon is activated when tryptophan is depleted during E. coli fermentation.
Intracellular accumulation of Trp-LE'-Met-proinsulin as insoluble aggregates (inclusion bodies)
20
reduces the rate at which the protein is destroyed by proteolytic enzymes. In the basic case,
inclusion bodies (IBs) were considered to account for 20% of total dry cell mass. After
fermentation, the broth is cooled to 10°C to prevent cell death. After each phase of processing in
the fermentation section (and subsequent sections), the equipment is cleaned in preparation for the
following product batch (Shuler & Kargi, 2002).
Figure 2.2: Insulin Fermentation Vessel
Figure 2.3: Fermentation Vessel at Insulin Production Industry

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2.2.1 Primary Recovery Section
After fermentation, the broth is transferred to a surge tank, separating the plant's upstream section
from the downstream section. Three parallel operating disk-stack centrifuges are utilized for cell
harvesting. The broth is concentrated from 37,000 L to 9,157 L during centrifugation, and most
extracellular impurities are removed. The rate of cell recovery is 98%. In a blending tank, the cell
sludge is diluted with an equal volume of buffer solution (composed of 96.4% by weight water for
injection (WFI), 0.7% EDTA, and 2.9% Tris-base). The buffer facilitates the separation of particles
of cell debris and inclusion bodies. Next, a high-pressure homogenizer is employed to rupture the
cells and liberate the inclusion bodies. The exit temperature is approximately 10 ℃. The identical
centrifuges as before are used to recover inclusion bodies. The IBs are recovered (with a yield of
98%) in the heavy phase, whereas most cell debris particles remain in the light phase. This is
possible because the IBs have a significantly greater density (1,3 g/cm3) and size (1 m in diameter)
than the cell debris particles. The inclusion body sludge, which contains approximately 20% solids
w/w, is washed with WFI containing 0.66% w/w Triton-X100 detergent (the volume of solution
is twice the volume of inclusion body sludge) and centrifuged again using the identical centrifuges
as before. The detergent solution aids in cleaning (dissociating debris and soluble proteins from
inclusion bodies). 10 degrees Celsius is maintained at the exit. The volume of slurry after the
primary recovery section is approximately 1,440 L (Harrison et al., 2015).
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Figure 2.4: Centrifugal Machine
Figure 2.5: Centrifugal Machine at Insulin Manufacturing Industry

23
2.3 REACTION SECTION
2.3.1 Inclusion Body Solubilization
The suspension of inclusion bodies is transferred to a reaction tank lined with glass and mixed
with urea and 2-mercaptoethanol to final concentrations of 5 M (300 g/L) and 40 g/L, respectively.
Urea is a chaotropic agent that dissolves the denatured protein in inclusion bodies, whereas 2-
mercaptoethanol is a reductant that breaks down disulfide bonds. To obtain a 95% solubilization
yield, a reaction time of 8 hours is necessary. The inclusion bodies are made up of 80% (w/w) Trp-
LE'-Met-proinsulin and the remaining 20% (w/w) other (contaminant) proteins. A diafiltration unit
is used to replace urea and 2-mercaptoethanol with WFI and concentrate the solution after the
solubilization reaction. This process takes 6 hours to complete and has a 98% recovery rate. A
polishing dead-end filter is utilized to remove all remaining fine particles (biomass, debris, and
inclusion bodies). This polishing filter safeguards the downstream chromatographic units.
Approximately 2200 L of the solution is present at this point (Shuler & Kargi, 2002).
2.3.2 CNBr Cleavage
In a glass-lined reactor, the chimeric protein is cleaved with CNBr (cyanogen bromide) into the
121-amino acid signal sequence Trp-LE'-Met and the denatured pro-insulin (82 amino acids). The
reaction is conducted in a 70% formic acid solution containing a 30-fold molar excess of CNBr
(one mole of CNBr is required per mole of Trp-LE'-Met-proinsulin, according to stoichiometry).
The reaction takes 12 hours at 20 degrees Celsius and yields 95%. The released pro-insulin has a
mass of approximately 30% of Trp-LE'-Met-proinsulin. The cleavage reaction produces a trace
amount of cyanide gas as a byproduct. By applying a vacuum and increasing the temperature to
approximately 35 °C, the formic acid, unreacted CNBr, and cyanide gas are eliminated (the boiling
point of CNBr). For one hour, this procedure is performed in a rotary vacuum evaporator (CSP-
101). Since cyanide gas is toxic, all air exhausted from the vessels is scrubbed with a locally
prepared and maintained hypochlorite solution (Harrison et al., 2015).
24
2.3.3 Sulfitolysis
Sulfitolysis of denatured pro-insulin occurs under alkaline conditions in a glass-lined reactor (pH
9–11). This step is intended to unfold pro-insulin, break any disulfide bonds, and add SO3 groups
to all cysteine sulfur residues. The desired product is human pro-insulin (S—SO3—)6 (protein–
S–sulfonate).
The sulfitolysis process is required for two reasons:
1. When expressed in pro-insulin is likely not folded in the correct configuration. E. coli as a
fusion protein component.
2. The treatment with cyanogen bromide tends to break existing disulfide bonds.
The final sulfitolysis mixture contains 50 wt % guanidine HCl (6 M), 0.35 wt % ammonium
bicarbonate (NH4HCO3), 3 wt % Na2SO3, and 1.5 wt % Na2S4O6. 12 hours of reaction time is
required to achieve a 95% yield. The denaturing reagent (guanidine HCl) prevents the refolding
and cross-folding of the same protein molecule onto itself or of two distinct protein molecules onto
one another. Additionally, urea may be used as a denaturing reagent. After the sulfitolysis reaction
is complete, the sulfitolysis solution is exchanged with WFI to achieve a final guanidine HCl
concentration of 20% by weight (Jiang & Chang, 2005). After IB solubilization, this procedure, P-
21, employs the DF-101 diafilter, which also handles buffer exchange. The human pro-insulin
(S—SO3—)6 is then chromatographically purified using three parallel ion exchange columns (C-
101) with four cycles per batch each. Each column has a bed height of 25 cm and a diameter of
140 cm. At pH 4.0, a cation exchange resin (SP Sepharose Fast Flow from Amersham-Pharmacia
Biotech) is utilized. The eluant solution consists of 69.5% (w/w) WFI, 29.0% (w/w) urea, and
1.5% (w/w) NaCl (Ser & Kentsis, 2020). Urea, a denaturing agent, prevents pro-insulin (S—SO—
)6 from improperly refolding and cross-folding. The following assumptions about operations were
made:
(1) The column is equilibrated for 30 minutes prior to loading.

(2) The total binding capacity of the resin is 20 mg/mL.
(3) The volume of eluant is equivalent to five column volumes (CVs),
(4) The total volume of column wash, regeneration, and storage solutions is 15 CVs.
(5) The target protein is recovered with a 90% recovery yield in 1.5 CVs of eluant buffer.
25
2.3.4 Refolding
This operation catalyzes the removal of the SO3 moiety and then allows disulfide bond formation
and correct refolding of the proinsulin to its native form. It takes place in a reaction tank. This
process step involves treatment with mercaptoethanol (MrEtOH), a reductant that facilitates the
disulfide interchange reaction. It is added at a ratio of 1.5 mol of mercaptoethanol to 1 mol of SO3.
Dilution to a proinsulin(S-SO3-)6 concentration of less than 1 g/L is required to prevent cross-
folding of proinsulin molecules. The reaction is carried out at 8°C for 12 h and reaches a yield of
85%. After completion of the refolding step, the refolding reagents are replaced with WFI and the
protein solution is concentrated using a diafiltration unit, which has a product recovery yield of
95% (5% of the protein denatures) (Kim et al., 2015). The volume of the solution at this point is
around 4500 L. Next, the human proinsulin is chromatographically purified in a hydrophobic
interaction chromatography (HIC) column. The following operating assumptions were made:
1. The column is equilibrated for 30 min prior to loading,
2. The total resin binding capacity is 20 mg/mL,
3. The eluant volume is equal to 6 CVs,
4. The total volume of the solutions for column wash, regeneration, and storage is 15 CVs,
5. The protein of interest is recovered in 1 CV of eluant buffer with a recovery yield of 90%,
and
6. The material of a batch is handled in three cycles.
2.4 ENZYMATIC CONVERSION
In a reaction vessel, the C-peptide is removed from human proinsulin enzymatically (using trypsin
and carboxypeptidase B). Trypsin cleaves internal lysine and arginine residues at their carboxyl
terminus, whereas carboxypeptidase B removes terminal amino acids. The amount of trypsin used
is rate-limiting and permits the formation of intact human insulin. The final concentration of
carboxypeptidase is 4 mg/L, while the final concentration of trypsin is 1 mg/L. The reaction occurs
at 30°C for 4 hours and yields a 95% conversion rate. At this point, the volume of the solution is
approximately 4300 L.
26
2.5 FINAL PURIFICATION SYSTEM
Biosynthetic human insulin is isolated using a purification sequence based on multimodal

chromatography, which exploits differences in molecular charge, size, and hydrophobicity.
Following is a description of all purification steps.
In a diafilter, the enzymatic conversion solution is exchanged for WFI and concentrated by a factor
of four. Using an ion exchange column, the insulin solution is purified. The following assumptions
about operations were made:
(1) Prior to loading, the column is equilibrated for 30 minutes.
(2) the total resin binding capacity is 20 mg/mL;
(3) the eluant volume is 8 CVs, and the eluant is an 11.5% w/w solution of NaCl in WFI;
(4) the total volume of the solutions for column wash, regeneration, and storage is 14 CVs;
(5) the protein of interest is recovered in 1.5 CV of eluant buffer with a 95% recovery yield; and
(6) the material from each batch is processed in four cycles.
The volume of the solution is approximately 1,780 L. In a diafilter, the ion exchange eluant
solution is then exchanged with WFI and concentrated by a factor of 2.0. For this step, a recovery
yield of 98% was assumed, and 2% was denatured. In order to purify the insulin solution, reversed-
phase high-performance liquid chromatography (RP-HPLC) is utilized.
The literature provides detailed information on the use of RP-HPLC for insulin purification.
Analytical studies utilizing various reversed-phase systems have demonstrated that an acidic
mobile phase can provide exceptional resolution of insulin from structurally similar insulin-like
components. Minor modifications to the insulin molecule, resulting in monodesamido formation
at the 21st amino acid of the A chain, or derivatization of amines via carbamoylation or
formylation, significantly increase the retention of insulin derivatives.
This is a typical example of the insulin-like derivatives found in the charge stream before reverse-
phase purification. Using an acidic mobile phase results in the elution of all derivatives after the
27
insulin peak, whereas using mildly alkaline pH results in the elution of derivatives on both sides
of the insulin peak.
The optimal pH range for insulin purification is 3.0 to 4.0, which is sufficiently below the
isoelectric pH of 5.4 to ensure insulin solubility. This operational criterion is satisfied by an eluant
buffer containing 0.25 M acetic acid because it is compatible with chromatography and provides
excellent insulin solubility. In the RP-HPLC step, a 90% insulin yield was suggested under the
following conditions:
1. The column is equilibrated for 30 minutes prior to loading.
2. The total resin binding capacity is 15 mg/ml.
3. The column height is 25 cm.
4. The eluant volume is 6 CVs, and its composition is 25% acetonitrile by weight, 1.5% acetic
acid by weight, and 73.5% WFI by weight.
5. The total volume of the solutions for column wash, equilibration, regeneration, and storage
is 6 CVs, and (5) 90% of the target protein is recovered from 1 CV of eluant buffer.
The RP-HPLC buffer is exchanged with WFI and concentrated by a factor of 2.0 in a diafilter with
a 98% (2% denatured) product recovery yield. A gel filtration chromatography column
accomplishes the purification. The following operational claims have been made:
1. The column is equilibrated for 30 minutes prior to loading.
2. The sample volume is 5% of the column volume.
3. The eluant volume is 4 CVs.
4. The total volume of the solutions for column wash, depyrogenation, stripping, and storage
is 6 CVs.
5. The protein of interest is recovered in 0.5 CV of eluant buffer with a 90% recovery yield.
The mobile phase is an acetic acid solution. Next, a diafilter is utilized to concentrate the purified
insulin solution tenfold. At this point, the solution volume is approximately 180 L and contains
approximately 12.8 kg of insulin. This substance is pumped into a jacketed, stirred tank. The
protein solution is treated with ammonium acetate and zinc chloride until each reaches a final
concentration of 0.02 M. Adjust the pH to between 5.4 and 6.2. The crystallization occurs at 5°C
28
for 12 hours. Insulin crystallizes with zinc according to the formula insulin6-Zn2. Step recovery
with insulin is approximately 90%.
The crystals are recovered with a recovery rate of 95% using a basket centrifuge. The crystals are
then freeze-dried. As determined by analytical high-performance liquid chromatography, the final
crystallized product has a purity between 99.5% and 99.9%. (HPLC). Per batch, approximately
11.5 kilograms of the product are recovered. The overall recovery rate is approximately 32%.
29
CHAPTER III
MASS BALANCE
3.1 FERMENTATION SECTION
3.1.1 Mixing
Media
Mixing Broth
Water
Media of 5335.42 kg/batch are inoculated inside the fermentation vessel. Carbon will be supplied
by glycerol and yeast, nitrogen by ammonium sulphate and thiamine, and inorganic nutrients by
potassium dihydrogen phosphate and dipotassium phosphate, which also serve as pH buffers. A
vitamin solution, sodium citrate, and magnesium sulphate will provide trace elements. E. coli will
receive all these elements for the fermentation process. The media compositions are 14% and the
remaining are added with water (Jing J.et al., 2018).
Media = 5335.42 kg/batch
Water composition calculation
Water = 100% - 14%
= 86%
Water = 33482.79 kg/batch
Media + Water = Broth
5335.42kg/batch + 33482.79 kg/batch = 38818.21 kg/batch

30
3.1.2 Heat Sterilization
In addition to sterilizing the media, heating glucose solutions promotes the production of many
glucose degradation products (GDPs) (Haybrard J. et al., 2017).
Broth Heat Sterilization Broth
In = Out
38818.21 kg/batch = 38818.21 kg/batch
3.1.3 Air Filtration
Air
Filtered Air
Air Filtration
Ammonia
Ammonia + Compressed gas = Air to fermenter
509.60 kg/batch + 24558.24 kg/batch = 25067.85 kg/batch

31
3.1.4 Fermentation
Broth CO2
Fermentation
Filtered Air Fermented Broth
Biomass = 11% of biomass will be inoculated inside the fermentation vessel.
= (11/100 X 38818.21 kg/batch)
= 44.41 kg/batch
Heat sterilization output + Air to fermenter + Biomass = Reaction in the fermenter
38818.21 kg/batch + 25067.85 kg/batch + 44.41 kg/batch = 63930.47 kg/batch
3.1.5 Fermentation Air Release (CO2)
Produced CO2 Air Filtration CO2 Emissions to air
40% gas will be produced from the insulin fermentation process.
(40/100) X 63930.47 kg/batch = 25920.30 kg/batch

32
3.1.6 Fermenter Output
Broth CO2
Fermentation
Filtered Air Fermented Broth
63930.47 kg/batch -25920.30 kg/batch = 38010.17 kg/batch
3.2 PRIMARY RECOVERY SECTION
3.2.1 Storage
Fermented Broth Storage Fermented Broth
Input = Output
Fermenter output = Storage Output
38010.17 kg/batch = 38010.17 kg/batch
3.2.2 Centrifugation
Fermented Broth Centrifuged Broth

Storage
Waste
Input = 38010.17 kg/batch

33
From the overall infeed, in centrifugation, 39% is wasted.
Output = 100% -39%
= 61%
Output = (61/100) X 38010.17 kg/batch
Output = 23265.75 kg/batch
To waste = 38010.75 kg/batch – 23265.75 kg/batch
= 14745 kg/batch
3.2.3 Blending
EDTA
Blending/Storage Broth
Centrifuged Broth
Input = 23265.75 kg/batch (from centrifugation)
Input (EDTA) = 40% of the EDTA in mass volume needs to be added.
= (40/100) X 23265.75 kg/batch
= 9300 kg/batch (EDTA solution)
Input = Output
23265.75 kg/batch + 9300 kg/batch = 32565.75 kg/batch

34
3.2.4 Homogenization
Broth Homogenization Broth
Input = Output
32565.75 kg/batch = 32565.75 kg/batch
Broth Centrifugation Centrifuged Broth
Liquid Waste 2
From centrifugation process, 5% of the product is recovered.
Product Recovery (Output) = (5/100) X 32565.75 kg/batch
Product Recovery (Output) = 1498.04 kg/batch
Waste from centrifugation = 32565.75 kg/batch – 1498.04 kg/batch
= 31067.71 kg/batch
35
3.2.6 Blending
Centrifuged Broth Blending/Storage Centrifuged Broth
Input (from centrifugation output) = 1498.04 kg/batch
65% of the product increment in the output.
Input (Triton X-100) = 2850 kg/batch
Input = Output
Input = 2850 kg/batch + 1498.04 kg/batch
Output = 4348.04 kg/batch
Centrifuged Broth Centrifugation Centrifuged Broth
Liquid Waste 3
Input = Output
33% of product recovery, while the remaining is discharged as liquid waste.
Product Recovery = (33/100) X 4348.04 kg/batch
= 1442.76 kg/batch
Input (from blending tank) = Product + Liquid waste
Liquid waste = 4348.04 kg/batch – 1442.76 kg/batch
= 2905.28 kg/batch
36
3.30 REACTION SECTION
3.3.1 IB Solubilization
MrETOH To Diafiltration
From Diafiltration IB Solubilization
To Dead End Filtration
From Centrifugation
Input:
MrETOH = 400 kg/batch
Urea = 2870 kg/batch
WFI = 6000 kg/batch
From Centrifugation = 1442.76 kg/batch
Input from Diafiltration = 2118.86 kg/batch
Output:
To Diafiltration = 10709.64 kg/batch
To dead end filtration = Input -Output
= 400 kg/batch + 2870 kg/batch + 6000 kg/batch + 1442.76 kg/batch +

2118.86 kg/batch – 10709.64 kg/batch
= 2121.98 kg/batch
37
3.3.2 Diafiltration
From IB Solubilization Diafiltration From IB Solubilization
Liquid Waste
Input:
WFI = 10004.21 kg/batch
From IB Solubilization tank = 10709.64 kg/batch
Output:
Recovery to IB Solubilization = 2118.86 kg/batch
Waste from diafiltration = 10004.21 kg/batch + 10709.64 kg/batch – 2118.86 kg/batch
= 18594.44 kg/batch
3.3.3 Dead-End Filtration
Output IB Solubilization Dead End Product Recovery

Filtration
Liquid Waste
38
Input (output IB Solubilization): 2118.86 kg/batch
Product Recovery from Dead End Filtration: 2093.86 kg/batch
Waste produced = 2118.86 kg/batch – 2093.86 kg/batch
= 25 kg/batch
3.3.4 CNBr Cleavage
CNBr/HCOOH
CNBr Cleavage CNBr Product Recovery
From Dead End Filtration
Input (Product Recovery from Dead End Filtration) = 2093.86 kg/batch
Input (CNBr/HCOOH) = 5460 kg/batch
Output = 2093.86 kg/batch + 5460 kg/batch
= 7553.86 kg/batch
3.3.5 Rotary/Evaporator
CNBr Product Rotary Evaporator Product
Input (output from CNBr Cleavage) = 7553.86 kg/batch
Recovery (product) = 1617.01 kg/batch
Waste (Evaporated) = 7553.86 kg/batch – 1617.01 kg/batch
= 5936.85 kg/batch
39
3.3.6 Sulfitolysis
From Diafiltration
From Rotary Evaporator Sulftolysis Product
Guan HCl
Input (Guan HCl) = 3151.00 kg/batch
Input (Output from Rotary/Evaporator) = 1617.01 kg/batch
Input (from Diafiltration) = 4761.96 kg/batch
Output (Product Recovered) = 4761.96 kg/batch
Output (to diafiltration) = 3151 kg/batch + 1617.01 kg/batch + 4761.96 kg/batch – 4761.96
kg/batch
=4768.01 kg/batch
3.3.7 Diafiltration
From Sulfolysis Diafiltration Product
Liquid Waste
Input (WFI) = 7135.57 kg/batch
Input (output from sulfitolysis) = 4768.01 kg/batch
Output (To sulfitolysis) = 4761.96 kg/batch
Output (liquid waste) = 7135.57 kg/batch + 4768.01 kg/batch – 4761.96 kg/batch

40
= 7141.62 kg/batch
3.3.8 S-Sepharose
Material Input
S-Sepharose Product to Refolding
Input from Sulfitolysis
Liquid Waste
Input 1 = 35728.56 kg/batch
Input from sulfitolysis = 4768.01 kg/batch
Product recovered = 6750.06 kg/batch
Output (liquid waste) = 35728.56 kg/batch + 22334.77 kg/batch + 8261.10 kg/batch + 12728.02
kg/batch + 8233.30 kg/batch + 4768.01 kg/batch – 6750.06 kg/batch
Output liquid waste = 8530 kg/batch
3.3.9 Refolding
Input from S-Sepharose

To Diafiltration
Input from Diafiltration Refolding
Product to HIC Column
MrETOH2
41
Input (MrEtOH 2) = 30000 kg/batch
Input (output from S-Sepharose) = 6750.06 kg/batch
From Diafiltration = 4537.25 kg/batch
Output from Diafiltration = 4537.25 kg/batch
3.3.10 Diafiltration
WFI
Diafiltration To Refolding
Input from Refolding
Liquid Waste
Input (WFI 3) = 18112.09 kg/batch
Input (from refolding) = 36750.06 kg/batch
Output (to refolding) = 4537.25 kg/batch
Output (waste) = 18112.09 kg/batch + 36750.06 kg/batch – 4537.25 kg/batch
= 50324.90 kg/batch
3.3.11 HIC Column
Material Input
Product Output to Enzyme
HIC Column Conversion Reaction
From Refolding
Liquid Waste
42
Input from refolding = 4537.25 kg/batch
Output (product recovery) = 4372.77 kg/batch
Output (Liquid waste) = 22573.68 kg/batch + 13022.22 kg/batch + 8681.48 kg/batch + 6644.93
kg/batch + 4298.37 kg/batch + 4537.25 kg/batch - 4372.77 kg/batch
= 55385.16 kg/batch
3.3.12 Enzyme Conversion
Product to Final Purification

From HIC Column Enzyme Conversion
To Diafiltration
Input (from diafiltration) = 1087.74 kg/batch
Input from HIC column = 4372.77 kg/batch
Enzyme new = 11.07 kg/batch
Output (to diafiltration) = 1087.74 kg/batch + 4372.77 kg/batch + 11.07 kg/batch -1087.74
kg/batch
= 4383.84 kg/batch
43
3.4 FINAL PURIFICATION SECTION
3.4.1 Diafiltration
WFI
Diafiltration Product Recovered
From Enzyme Conversion
Process
Liquid Waste
In the final purification system, WFI are added
Input (from enzyme conversion) = 4383.84 kg/batch
From the overall input 12.5% of the product is recovered as product.
Product recovered = (12.5/100) X 8723.91kg/batch
Output (Product Recovered) = 1087.74 kg/batch
= 7636.17 kg/batch
3.4.2 S-Sepharose
Input
Product Recovered from
S-Sepharose Diafiltration
From Enzyme Conversion
Liquid Waste

44
7% of the product is recovered while the rest is waste.
Output (product recovery) = (7/100) X 24394.81
Output (Product recovery) = 1788.58 kg/batch
Output (liquid waste) = 6676.78 kg/batch + 9448.48 kg/batch + 2749.63 kg/batch + 4432.18 +
1087.74 kg/batch – 1788.58 kg/batch
Output (liquid waste) = 22606.23 kg/batch
3.4.3 Diafiltration
WFI
S-Sepharose Product Recovered
Input (from Sepharose)
Liquid Waste
Input (Sepharose) = 1788.58 kg/batch
24% of the product are recovered.
Output (Product recovered) = 847.24 kg/batch
= 2619.53 kg/batch
45
3.4.4 RP-HPLC
Input
RP-HPLC Product Recovered
Input From Diafiltration
Liquid Waste
Input 4 (product recovered from diafiltration) = 847.24 kg/batch
7% of the product are recovered from RP-HPLC.
93% is the waste.
Output (liquid waste) = 13170 kg/batch
Output (product recovered) = 1426.42 kg/batch + 6113.22 kg/batch + 5817.67 kg/batch + 847.24
kg/batch – 13170 kg/batch
= 1034.55 kg/batch
3.4.5 Diafiltration
WFI
Product Recovered to Gel
Diafiltration Filtration
From RP-HPLC
Liquid Waste
Input 1 (WFI) = 752.94 kg/batch

46
Input (from RP-HPLC) = 1034.55 kg/batch
90% of the material is discharged to the drain for the impurities recovery.
Output (liquid waste) = 1613.27 kg/batch
Output (product recovered) = 752.92 kg/batch + 1034.55 kg/batch - 1613.27 kg/batch
= 174.2 kg/batch
3.4.6 Gel Filtration
Input
Product Recovered to
Gel Filtration Diafiltration
Input from Diafiltration
Liquid Waste
96% of the waste is produced from the product recovery. This process is to obtain the purity of the
product.
Output (liquid waste) = (96/100) X 42191.72
Output (liquid waste) = 40430 kg/batch
Output (product recovered) = 7002.92 kg/batch + 14005.84 kg/batch + 21008.76 kg/batch + 174.2
kg/batch – 40430 kg/batch
= 1761.72 kg/batch
47
3.4.7 Diafiltration
Product Recovered from

Input from Gelfiltration Diafiltration Diafiltration
Liquid Waste
Input (from gelfiltration) = 1761.72 kg/batch
8% of the product are recovered from the final diafiltration process.
Output (liguid waste) = 100% - 8%
= 92%
= 92/100 X 2112.66
Output (liguid waste) = 1940 kg/batch
Output (product recovery) = 350.94 kg/batch + 1761.72 kg/batch – 1940 kg/batch
= 172.66 kg/batch
3.4.8 Crystallization
Input from Diafiltration

Crystallization Product Recovered
Input
Output (product recovery) = 0.76 kg/batch + 172.66 kg/batch

48
= 173.42 kg/batch
3.4.9 Basket Centrifugation
Input from Crystallization Basket Product Recovered

Centrifugation
Liquid Waste
Input = 64.11 kg/batch
Input (from crystallization) = 173.42 kg/batch
6.6% of product recovery for the basket centrifugation process for the insulin manufacturing
process.
Output (product recovery) = 6.6/100 X 237.53 kg/batch
= 221.74 kg/batch
3.4.10 Freeze Drying
Evaporation
Input from Basket Centrifugation Freeze Drying Product Recovery (Insulin)
Input (from basket centrifugation) = 15.79 kg/batch
Efficient Evaporation rate is 31% in the freeze drying.
Output (evaporation) = 31/100 X 15.79 kg/batch
Output (evaporation) = 4.86 kg/batch

49
Output (product final recovery) = 15.79 kg/batch – 4.86 kg/batch
= 10.93 kg/batch
50
CHAPTER IV
WASTEWATER DESIGN
4.1 PHARMACEUTICAL WASTEWATER
Diabetes is a metabolic disorder that causes an elevated blood sugar level. The cause is a lack of
or diminished efficacy of the body's insulin, a pancreatic hormone. The primary function of insulin
is to regulate blood glucose concentration. If this is not done correctly, insulin must be given
externally into the body. Previously, insulin was expensively harvested from the pancreas of pigs;
today, it is synthesized. The body accepts this (human) recombinant insulin. The insulin gene is
injected into the DNA of a bacterium cell to produce insulin. Typically, non-pathogenic strains of
Escherichia coli bacteria serve as hosts for producing required proteins. A non-ionic tenside, such
as Triton X-100, releases generated proteins from the cell membrane without altering their primary
structure. This form of tenside is hormonally active and resistant to degradation, necessitating its
removal during the subsequent wastewater treatment (Envirochemie GmbH, n.d.).
Insufficiently treated insulin manufacturing effluent threatens ecosystems and human health by
accumulating in the environment and the food chain due to the significant increase in insulin
product usage in recent years. Residue organic pollutant components in insulin production
wastewater, such as hazardous bacteria and chemicals, are typically challenging to eliminate or
mineralize. In contrast, pharmaceutical wastewater has elevated levels of chemical oxygen demand
(COD) and total organic carbon (TOC) (Deng et al., 2022).
In addition, insulin production effluent is one of the streams with the highest quantities of
hazardous and refractory organic contaminants. The pharmaceutical industry's wastewater consists
primarily of agents, solvents from equipment washing and cleaning operations, reactants, and
catalysts used in manufacturing. In many instances, effluents from these sectors have low
biodegradability due to high concentrations of refractory organics. Consequently, the pollutant
loads measured by biological oxygen demand (BOD) may be insignificant, although chemical
oxygen demand (COD) would be much higher than BOD. In contrast, the biological treatment
51
operations at sewage treatment plants only remove a portion of the insulin-producing chemicals,
which are then discharged into surface waters (Goodarzi et al., 2022).
It has always been challenging to treat insulin manufacturing wastewater to the appropriate effluent
requirements due to the enormous diversity of products produced in a medicine manufacturing
plant, resulting in variable wastewater composition, and fluctuating pollutant concentrations. The
pharmaceutical industry synthesizes structurally complex organic chemicals resistant to biological
breakdown. For this reason, traditional treatment technologies are typically unsuitable for insulin
manufacturing wastewater treatment (Vlyssides et al., 2008).
4.2 WASTEWATER GENERATED
4.2.1 Density of Liquid Waste
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟏 ∶ 𝑬. 𝒄𝒐𝒍𝒊

E.coli density 1.105 g/ml.
𝑚𝑎𝑠𝑠
𝜌=
𝑣𝑜𝑙𝑢𝑚𝑒
𝑔
𝑉𝑜𝑙𝑢𝑚𝑒 = 14745000 𝑔 𝑥 1.105
𝑚𝑙
𝑉𝑜𝑙𝑢𝑚𝑒 = 16293225 𝑚𝑙
𝑉𝑜𝑙𝑢𝑚𝑒 = 16.29 𝑚3
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟐 ∶ 𝑬. 𝒄𝒐𝒍𝒊

𝑘𝑔
𝐸𝐷𝑇𝐴 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 ∶ 860
𝑚3
31067.71𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 =
𝑘𝑔
860 3
𝑚
52
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟑 ∶ 𝑻𝒓𝒊𝒕𝒐𝒏 − 𝑿 − 𝟏𝟎𝟎

𝑘𝑔
𝑇𝑟𝑖𝑡𝑜𝑛 − 𝑋 − 100 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 ∶ 1070 3
𝑚
2905.28𝑘𝑔
𝑘𝑔
1070 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟒 ∶ 𝑾𝑭𝑰

𝑘𝑔
𝑊𝐹𝐼 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 = 998 3
𝑚
2121.98 𝑘𝑔
𝑘𝑔
998 3
𝑚
𝐿𝑖𝑞𝑢𝑖𝑑 𝑤𝑎𝑠𝑡𝑒 5 ∶ 𝑊𝐹𝐼

𝑘𝑔
𝑚
18594.44𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟔 ∶ 𝑾𝑭𝑰
𝑘𝑔
𝑊𝐹𝐼 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 = 998
𝑚3
25 𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑉𝑜𝑙𝑢𝑚𝑒 = 0.03𝑚3
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟕 ∶ 𝑾𝑭𝑰

𝑘𝑔
𝑚
7141.62𝑘𝑔
𝑉𝑜𝑙𝑢𝑚𝑒 ∶
𝑘𝑔
998 3
𝑚
𝑘𝑔
𝑉𝑜𝑙𝑢𝑚𝑒 = 7.16 3
𝑚
53
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟖 = 𝑾𝑭𝑰
𝑘𝑔
𝑊𝐹𝐼 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 = 998
𝑚3
8530𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟗 = 𝑾𝑭𝑰

50324𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝒘𝒂𝒔𝒕𝒆 𝟏𝟎 = 𝑨𝒎𝒎𝒐𝒏𝒊𝒖𝒎 𝑨𝒄𝒆𝒕𝒂𝒕𝒆
𝑘𝑔
𝐴𝑚𝑚𝑜𝑛𝑖𝑢𝑚 𝐴𝑐𝑒𝑡𝑎𝑡𝑒 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 = 1170
𝑚3
55385.16𝑘𝑔
1170𝑘𝑔
𝑚3
55385.16𝑘𝑔
1170𝑘𝑔
𝑚3
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟏 ∶ 𝟕𝟔𝟑𝟔. 𝟏𝟕 𝒌𝒈

998𝑘𝑔
𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑊𝐹𝐼 =
𝑚3
7636.17𝑘𝑔
𝐿𝑖𝑞𝑢𝑖𝑑 𝑤𝑎𝑠𝑡𝑒 11 =
998𝑘𝑔
𝑚3
3
𝑉𝑜𝑙𝑢𝑚𝑒 = 7.65𝑚
54
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟐 ∶ 𝟐𝟐𝟔𝟎𝟔. 𝟐𝟑 𝒌𝒈
22606.23𝑘𝑔
𝐿𝑖𝑞𝑢𝑖𝑑 𝑊𝑎𝑠𝑡𝑒 12 =
998𝑘𝑔
𝑚3
𝐿𝑖𝑞𝑢𝑖𝑑 𝑤𝑎𝑠𝑡𝑒 12 = 22.65 𝑚3
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟑 ∶ 𝑾𝑭𝑰
998𝑘𝑔
𝑚3
2619.53𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟒 ∶ 𝑾𝑭𝑰

13170𝑘𝑔
𝑘𝑔
998 3
𝑚
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟓 ∶ 𝑨𝒄𝒆𝒕𝒊𝒄 𝑨𝒄𝒊𝒅

𝑘𝑔
𝐷𝑒𝑛𝑠𝑖𝑡𝑦 = 1050 3
𝑚
1613.27𝑘𝑔
1050𝑘𝑔
𝑚3
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟔 ∶ 𝑾𝑭𝑰

998𝑘𝑔
𝑚3
40430𝑘𝑔
998𝑘𝑔
𝑚3
55
𝑳𝒊𝒒𝒖𝒊𝒅 𝑾𝒂𝒔𝒕𝒆 𝟏𝟕: 𝒁𝒊𝒏𝒄 𝒄𝒉𝒍𝒐𝒓𝒊𝒅𝒆

𝑘𝑔
𝑍𝑖𝑛𝑐 𝑐ℎ𝑙𝑜𝑟𝑖𝑑𝑒 = 2 910 000 3
𝑚
𝑉𝑜𝑙𝑢𝑚𝑒 = 221.74 𝑘𝑔
221.74𝑘𝑔
2910000𝑘𝑔
𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 = 0.0000762𝑚3
4.2.2 Primary Section Wastewater Generation
Table 4.0: Table of Waste Generated for Primary Reaction

Primary Reaction
Stream Waste Output (kg/batch) Output COD TN TP

(m3/batch) (mg/L) (mg/L) (mg/L)
6 Liquid waste 1 14745 16.29 13412 451 112
8 Liquid Waste 2 31067.71 36.12 2345 78.9 6.4
9 Liquid Waste 3 2905.28 2.72 41021 0.2 0.1
Total 48717.99 55.13 20000 32 1.5
The total volume of wastewater generated from the primary section are 55.13 m3/batch. The
wastewater generated are from the centrifugation process.
4.2.3 Reaction Section Wastewater Generation
Table 4.1: Table of Waste Generated for Reaction Section
Reaction Section
Stream Waste Output (kg/batch) Output (m3/batch) COD TN TP

(mg/L) (mg/L) (mg/L)
15 Liquid Waste 4 2121.98 2.126 2348 13 -
16 Liquid Waste 5 18594.44 18.63 424 0.2 -
21 Liquid Waste 6 25 0.03 4061 38.6 0.1
23 Liquid Waste 7 7141.62 7.16 41.9 0.1 12
26 Liquid Waste 8 8530 8.55 25.1 24.5 2.5

56
28 Liquid Waste 9 50324.9 50.42 2134 12.3 3
Total 86737.94 86.916 2120 450 0.7
The total volume of wastewater generated from the reaction section are 86.916 m3/batch. The
wastewater generated are from the diafiltration, dead end filtration, and ion exchanger.
4.2.4 Final Purification Section
Table 4.2: Table of Waste Generated for Final Purification System

Final Purification Section
No Waste Output (kg/batch) Output (m3/batch) COD TN TP
31 Liquid Waste 10 55385.16 47.34 6094 504 2
33 Liquid Waste 11 7636.17 7.65 476 45 6
35 Liquid Waste 12 22606.23 22.65 752 0.1 0.1
37 Liquid Waste 13 2619.53 2.63 163 27 2.6
39 Liquid Waste 14 13170 14.2 108 0.4 -
40 Liquid Waste 15 1613.27 1.54 642 0.2 0.1
42 Liquid Waste 16 40430 40.51 108 0.05 -
45 Liquid Waste 17 1940 0.0000762 893 0.14 0.03
Total 82379.03 81.5300762 980 18 20
The total volume of wastewater generated from the final purification section is 81.53m 3. The
wastewater generated are from the diafiltration, ion exchanger, and centrifugation.
57
Summary of Overall Waste Generated
Table 4.3 : Summary of Waste Generated

Stream Waste COD TN TP
6 Liquid waste 1 (E.coli) 13412 451 112
8 Liquid Waste 2 (E.coli) 2345 78.9 6.4
9 Liquid Waste 3 (Triton X-100) 41021 0.2 0.1
15 Liquid Waste 4 (WFI + Residue) 2348 13 -
16 Liquid Waste 5 (WFI + Residue) 424 0.2 -
21 Liquid Waste 6 (WFI + Residue) 4061 38.6 0.1
23 Liquid Waste 7 (WFI + Residue) 41.9 0.1 12
26 Liquid Waste 8 (WFI + Residue) 25.1 24.5 2.5
28 Liquid Waste 10 (Ammonium acetate) 6094 504 2
31 Liquid Waste 11 (WFI) 476 45 6
33 Liquid Waste 12 (WFI) 752 0.1 0.1
35 Liquid Waste 13 ( WFI) 163 27 2.6
37 Liquid Waste 14 (WFI) 108 0.4 -
39 Liquid Waste 15 (Acetic Acid) 642 0.2 0.1
42 Liquid Waste 16 (WFI) 108 0.05 -
45 Liquid Waste 17 (Heavy metal) 893 0.14 0.03

58
4.2.5 Total Volume of Waste Generated
Table 4.4: Total Volume of Waste Generated

Waste Output (kg/batch) Output (m3/batch)
Liquid waste 1 14745 16.29
Liquid Waste 2 31067.71 36.12
Liquid Waste 3 2905.28 2.72
Liquid Waste 4 2121.98 2.126
Liquid Waste 5 18594.44 18.63
Liquid Waste 6 25 0.03
Liquid Waste 7 7141.62 7.16
Liquid Waste 9 50324.9 50.42
Liquid Waste 10 55385.16 47.34
Liquid Waste 11 7636.17 7.65
Liquid Waste 12 22606.23 22.65
Liquid Waste 13 2619.53 2.63
Liquid Waste 15 1613.27 1.54
Liquid Waste 17 1940 0.0000762
Total 281078.03 278.5660762
Total volume of wastewater generated from the insulin manufacturing industry is 278.57m3.
59
Table 4.5 (a): Summary of Mixed Wastewater and Isolated Stream 45

Parameters Values Average
pH 5.5-7.0 mg/L 6.3 mg/L
COD 5388-6292 mg/L 5840 mg/L
BOD5 2713-5345 mg/L 4029 mg/L
BOD5/COD 0.21-0.35 mg/L 0.68 mg/L
TP 2.26–2.72 mg/L 2.49 mg/L
TN 396–422 mg/L 409 mg/L
pH 4.69–4.86 mg/L 4.77 mg/L

60
4.3 WASTEWATER DESIGN
4.3.1 Sump Pit Design
The main function of the sump pit is act as holding tank to store and hold the wastewater. The
sump pit is designed to collect all the insulin manufacturing industry wastewater and also function
as to homogenize all the wastewater and store. Sump pit is designed equipped with 2 submersible
pumps. The main function of submersible pump is to transfer the wastewater to the primary
clarifier.
Figure 4.0: Sump Pit
The wastewater generated are 278.57m3 from the insulin manufacturing industry for 8 hours.
61
278.57m3 = 280m3
Time = 8 hours
BOD & COD removal efficiency is 40%.

𝑚𝑔
5840
𝐶𝑂𝐷𝑡 = 𝑙
0.6
𝑚𝑔
𝐶𝑂𝐷𝑡 = 9733
𝑙
𝑚𝑔
1945
𝐵𝑂𝐷𝑡 = 𝑙
0.6
𝑚𝑔
𝐵𝑂𝐷𝑡 = 3242
𝑙
TSS Removal Efficiency is 60%.
𝑚𝑔
309
𝑇𝑆𝑆 = 𝑙
0.4
𝑚𝑔
𝑇𝑆𝑆 = 515
𝑙
Sump pit designed to collect for 500m3 of wastewater, to collect and hold the wastewater.
Sump pit layout:
Length: 6.1 m
Width: 5.5 m
Height: 14.9m
62
Height: 14.9m
Length: 5.5m
280𝑚3
𝑃𝑢𝑚𝑝 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑚𝑒𝑛𝑡 =
8ℎ𝑟
35𝑚3
𝑃𝑢𝑚𝑝 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑚𝑒𝑛𝑡 =
ℎ𝑟
The influent pump sump with 3 pumps will be used for a peak flow of 50 m3/h. Before the primary
clarifier, a screen press (self-cleaning) will be installed, to prevent large solid from entering the
plant.
Flow: 35 m3/hr Flow: 35 m3/hr

COD:9733 mg/L COD:9733 mg/L
TSS: 515 mg/L TSS: 515 mg/L
TN: 409 mg/L TN: 409 mg/L
TP : 2.49 mg/L TP : 2.49 mg/L
Sump Pit
Table 4.5: Summary of Sump Pit Design
63
Description Unit Design Criteria
Length (L) m 6.1
Height (H) m 5.5
Width (W) m 14.9
Volume (V) m3 280
Pump Quantity unit 2 (1 run, 1 standby)

• (0.35kW/0.5HP)
• Head up – 10 meters
Flow Rate m3/hr 35
4.3.2 Primary Clarifier
Primary clarification, also known as sedimentation, is the first step in the water treatment process for
removing suspended solids (TSS), oil and grease. During this step, solids floating at the surface and
other large particles from the water or wastewater flow are removed before biological treatment. The
primary clarifiers are used to separate settle able solids from the raw incoming wastewater. These
are located on the downstream of the plant. Sludge is settled to the bottom of the clarifier basins and
collected by a rake and removed by a sludge removal system. Meanwhile, oil and grease float to the
surface and is skimmed off. A typical primary clarifier removes 60 percent of suspended solids and
30 to 40 percent of Biological Oxygen Demand (BOD).
Circular clarifiers are round facilities consisting of an inlet structure, a cylindrical clarification
zone, a conical sludge accumulation zone, and effluent weirs (Figure 2). The effluent weirs are
placed near the facility perimeter to create a radially directed flow pattern from the tank centre
towards the walls. The slope of the bottom conical floor is usually 1:10 to 1:12 and depends on the
type of the sludge collection mechanism. The tank diameter ranges from 3 meters (10 ft) to over
100 meters (300 ft). Circular clarifiers are typically built-in pairs of 2 or 4 to simplify the influent
flow distribution between the individual units. Circular tank side water depth varies from 2.5 to 5
meters (8 to 16 feet).
64
Figure 4.1: Primary Clarifier Design for Sludge Draw Off Pipe
Fig 4.2: Primary Clarifier Design

65
Design Volume: 500 m3/batch
Flow: Design for continuous
500𝑚3
𝐹𝑙𝑜𝑤𝑟𝑎𝑡𝑒 =
24ℎ𝑟
𝑚3
𝐹𝑙𝑜𝑤𝑟𝑎𝑡𝑒 = 20.8
ℎ𝑟
Hydraulic load: 1m/h
Active Surface Area:
𝐹𝑙𝑜𝑤𝑟𝑎𝑡𝑒
𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 =
𝐻𝑦𝑑𝑟𝑎𝑢𝑙𝑖𝑐 𝐿𝑜𝑎𝑑
20.8𝑚3
𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 = ℎ𝑟
1𝑚
ℎ
𝑚
𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 = 20.8
ℎ
Surface Area:
𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 = 𝜋𝑟 2
20.8 = (3.142)(𝑟 2 )
𝑟 = 2.6𝑚
Volume of clarifier:
𝑉𝑜𝑙𝑢𝑚𝑒𝑐𝑦𝑙𝑖𝑛𝑑𝑒𝑟 = 𝜋𝑟 2 ℎ
42 𝑚3 = 𝜋(2.6)2 ℎ
ℎ = 1.98𝑚
𝜋𝑟 2 ℎ
𝑉𝑜𝑙𝑢𝑚𝑒𝑐𝑜𝑛𝑒 =
3
𝜋(2.6)2 (ℎ)
10 =
3
ℎ = 1.42 𝑚
66
h=1.98 m
h=1.42m

TSS: 515 mg/L TSS: 309mg/L
TP : 2.49 mg/L TP : 2.49 mg/L
Primary Clarifier
Sludge: 3893 mg/L

67
Table 4.6: Summary of Primary Clarifier Design

Type of Primary Clarifier - Circular
Radius m 2.6
Surface Area m 20.8

Hydraulic load m/h 1m/h
Volume (V) m3 52

Mono pump:
• 10m3/hr
• 2.2 kW
Flow Rate m3/hr 35
4.3.3 Calamity Tank
Calamity tanks act an emergency tank to store the water that found critical to be treated or to collect
and store the accidental discharge effluent from the insulin manufacturing company.
Retention time = 8 hours
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 =
𝑉𝑜𝑙𝑢𝑚𝑒
𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 = 8 ℎ𝑟 𝑥 20.8
ℎ𝑟
Calamity tank dimension to store 167m3.
Length = 5.96m
Width = 7m
Height = 4m
68
Calamity Tank
4m
5.96m

TSS: 309mg/L TSS: 309mg/L
TP : 2.49 mg/L TP : 2.49 mg/L
Calamity Tank
In the calamity tank, there is no reaction, and the calamity tank is only utilized for the temporary
storage for the emergency tank. In the insulin manufacturing industry the Triton X-100 chemical
are dangerous for the biological process especially when it is discharged in large amount in which
indicate the high concentration of toxic material in the wastewater. Thus, the presence of the
calamity tank have a capacity to hold the wastewater that is contaminated with higher
concentration of chemical which can be harmful to biological process and cause toxic shock.
The capacity of the calamity tank to hold the wastewater is equivalent to 1 cycle of production
which are equal to 8 hours for the insulin manufacturing industry. The calamity tank is designed
to store 166m3 of wastewater.
69
Table 4.7: Summary of Calamity Tank Design
Length (L) m 6
Height (H) m 4
Width (W) m 7
Volume (V) m3 166

• (0.35kW/0.5HP)
Mixer unit 1
• 1.3 kW/1.7hP
• Mixing flow 0.83 m3/s
Flow Rate m3/hr 35
4.3.4 Equalization Tank
Providing consistent flow and loading to a biological process is important to maintain optimal
treatment. Equalization (EQ) Basins are designed to provide consistent influent flow to
downstream processes by retaining high flow fluctuations. Due to the additional retention time,
aeration and mixing are required to prevent the raw wastewater from becoming septic and to
maintain solids in suspension. Besides of that, pre hydrolysis process are carried out in the
equalization tank. In addition, the wastewater pH also will be adjusted at equalization tank by
dosing sodium hydroxide (NaOH) or hydrochloric acid (HCl).
70
Figure 4.3: Equalization Tank
Effluent is collected for equalization, which is designed for a normal storage minimum 8 hours.
Here civil water is mixed with primary treated water. Coarse bubble aeration is provided by means
of an air blower and distribution system to achieve a uniform and homogenous mixture of
concentration of the discharge. The raw waste from the main plant is first collected in the
equalization tank through a bar screen. The equalization tank is designed for a hydraulic retention
time of around 10 hours and is provided with air grids connected to air blowers for maintaining
the solids in suspension (Belal et al., 2015).
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 =
𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 = 10 ℎ𝑟 𝑥 20.8
ℎ𝑟
𝑉𝑜𝑙𝑢𝑚𝑒 = 208𝑚3
Equalization Tank 4m
7.43m
71
Equalization Tank dimension to store 208m3.
Length = 7.43 m
Width = 7 m
Height = 4 m
Table 4.8: Summary of Equalization Tank Design
Length (L) m 7.4
Height (H) m 4
Width (W) m 7
Volume (V) m3 208 m3

• (0.35kW/0.5HP)
Mixer unit 1
• 1.8 kW
• Mixing flow 0.92 m3/s
Flow Rate m3/hr 35
72
4.3.5 Up flow-Anaerobic Sludge Blanket Reactor (UASB)
Anaerobic Wastewater Treatment is a biological system that treats wastewater without utilizing
air or oxygen. The objective was to eliminate organic contamination from wastewater, slurries,
and sludge. Anaerobic microbes convert organic pollutants into "biogas" containing methane and
carbon dioxide.
Fig 4.4: Conversion of Organic Pollutants to Biogas by Anaerobic Microorganisms
Up-flow anaerobic sludge blanket technology, sometimes referred to as UASB reactor, is a type
of anaerobic digester utilized in wastewater treatment. The UASB reactor is a methane-producing
digester that forms a blanket of granular sludge and is digested by anaerobic bacteria.
Figure 4.5: UASB reactor

73
The UASB reactor is based on a so-called three-phase separator, which allows the reactor to
separate gas, water, and sludge mixtures under circumstances of intense turbulence. This enables
smaller, less expensive designs. The reactor is equipped with numerous gas hoods for biogas
separation. Due to the extensive gas/water interfaces, relatively high loading rates of 10 to 15
kg/m3.d are feasible. Separation in the UASB reactor needs just 1 meter of height, preventing
flotation effects and, as a result, floating layers.
During typical UASB reactor treatment, the substrate initially travels through an enlarged sludge
bed with a high biomass percentage. The remaining portion of the substrate next travels through a
less thick biomass known as the sludge blanket.
Figure 4.6: UASB Reactor with GLS Separator
The influent is pushed from the bottom of the UASB reactor using a Peristaltic pump. The influent
moves higher and comes into contact with the biomass in the sludge bed. The influent then
continues to travel upwards, and the remaining substrates interact with the biomass in the sludge
blanket, which has a lower biomass concentration than the sludge bed below.
The volume of the sludge blanket must be adequate to treat wastewater channeled around the
sludge bed's lowest layer. In addition, it will aid in maintaining consistent effluent quality. A 3
phases (Gas-Liquid-Solid or GLS) separator is positioned above the sludge blanket to separate
74
solid particles from the combination (gas, liquid, and solid) after treatment, enabling liquid and
gas to exit the UASB reactor.
The effluent collection system will collect the treated wastewater via a number of launders
dispersed across the whole area discharging to the reactor's main launder. And the biogases
produced will be collected for use as fuel or disposal. The UASB's typical full-scale design loading
is 10 kg COD/m3.d.
4.3.5.1 UASB Reactor Height and Area
To lower the plan area and land cost, the reactor's GLS separator and influent distribution
arrangement must be as high as feasible. Furthermore, the height of the sludge bed must be
adequate to avoid channelling and maintain the liquid upflow velocity within the maximum
allowable limits (1.2 – 1.5 m/h). In order to accommodate the sludge bed, sludge blanket, and 3-
phase separator, the height of the reactor should be limited to 4 meters, and the height of the sludge
bed should be between 1.5 and 2.5 meters. As specified by the standard, the maximum height of a
reactor is approximately 8 meters; however, the average height ranges from 4.5 to 6 meters.
In addition, the sludge bed occupies 30 to 60% of the whole reactor volume, the sludge blanket
occupies 20 to 30%, and the GLS separator fills the remaining 15 to 30%.
4.3.5.2 Gas, Liquid and Solid Separator
The primary purpose of gas, liquid and solid (GLS) design is to ease the return of sludge without
the assistance of an external energy source or control mechanism. The function of the GLS
separator is to provide sufficient gas-water interfaces inside the gas dome and an excellent settling
area outside the dome to control the surface overflow rate. The sufficient aperture was opened at
the bottom to avoid the turbulence caused by the high inlet velocity of liquid in the settler so that
the solid could be appropriately returned to the reactor. To guarantee that the GLS separator
functions correctly, it is necessary to pay close attention to the unit's geometry and hydraulics.
75
4.3.5.3 Operation Description And Principle
Anaerobic digestion mainly involves four main processes which are hydrolysis, acidogenesis,
acetogenesis and methanogenesis. Hydrolysis is a process of large organic compound are
converted into simple monomeric compound. Usually, the process of hydrolysis is accomplished
by the extracellular enzyme.
In acidogenesis process, the soluble monomeric compound formed by hydrolysis will undergo
fermentation process. The products obtained from the acidogenesis fermentation are propionic acid
and butyric acid which are the intermediaries and other such as acetic acid, ethanol, hydrogen and
carbon dioxide. Acidogenesis fermentation is usually carried out by the Lactobacillus Sp and in
the acidogenesis fermentation the pH will reduced.
Acetogenesis is process where some of the acetate is produced through mixed acid fermentation.
Rest of the acetate is produced through secondary fermentation of products obtained in previous
stage. Acetate formation is important for formation of methane. The acetogenic bacteria are
Acetibacter sp.
Besides of that, methanogenesis is the final metabolic stage in an anaerobic digestion. Methane
(CH4) is formed either from acetate or carbon dioxide (CO2) and hydrogen. The bacteria
responsible for carrying out methanogenesis are called methanogens and are classified as
Litotrophic or Hydrogenotrophic which produce methane from CO2 and H2. Acetotrophs or
Acetoclastic microorganism are responsible to convert acetate to methane. The whole process is
shown in the Figure 4.7.
76
Figure 4.7: Anaerobic digestion conversion process of organic material
Source: An Introduction to Anaerobic Digestion of Organic Wastes, 2003.
Temperature, pH, nutrients, mixing and seeding are the main factor that affecting the anaerobic
digester.
4.3.5.4 Temperature
Most methane forming bacteria are active in two temperature range. Which are in mesophilic range
from 30 to 38oC. and thermophilic range from 50 to 60oC. While the temperature range between
40 to 50oC the process will be inhibited. The optimum temperature range is between 35 to 38oC
(Lohani et al., 2017).
77
4.3.5.5 pH
When the digester pH is 7.2 or lower, the presence of ammonium (NH4+) is favored. When the
digester pH is greater than 7.2, the presence of ammonia (NH3) is favored. Dissolved ammonia gas
or NH3 is toxic to bacteria, especially methane forming. The optimum pH value for the anaerobic
digester is 6.8 to 7.2 (Lohani et al., 2017).
4.3.5.6 Nutrients
Macronutrients and micronutrients are an essential element in the anaerobic digester. For
macronutrients the two main nutrients are phosphorus and nitrogen. The C: N:P ratio should be
maintained between C:N:P = 100:3:1. Micronutrients are elements that also essential for anaerobic
digester such as cobalt, nickel, iron and sulfide (Lohani et al., 2017).
4.3.5.7 Mixing
Mixing enhances the digestion process by distributing bacteria, substrate, and nutrients throughout
the digester as well as equalizing temperature. Methane forming bacteria are very sensitive to rapid
mixing which can washes out the bacteria. The figure 4.8 represent wash out anaerobic granular
sludge (Lohani et al., 2017).
Figure 4.8: Anaerobic Granular Sludge
Source: Environmental XPRT

78
4.3.5.8 Seeding
To seed an anerobic digester with an adequate population off facultative anaerobes and anaerobes
including methane forming bacteria, a ratio of 1:10 of secondary sludge to primary sludge may be
used. Without seeding it may take long time (up to few month) to get the anaerobic digester to
work. Proper seeding will attain a quick balance of reaction (Lohani et al., 2017).
1945𝑚𝑔
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ∶
𝐿
𝐵𝑂𝐷𝑡
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛: 1.945 𝑘𝑔
𝑚3
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝐿𝑜𝑎𝑑
𝑘𝑔𝐵𝑜𝐷𝑡
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 500𝑚3 𝑥 1.945
𝑚3
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐵𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 972.5 𝑘𝑔 𝐵𝑂𝐷𝑡
Effective loading rate

Design Organic loading rate
Substrate Feeding Rate 0.6.
VSS in the UASB reactor: 10 kg
𝐵𝑂𝐷𝑡 𝐿𝑜𝑎𝑑
𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝐹𝑒𝑒𝑑𝑖𝑛𝑔 𝑅𝑎𝑡𝑒 ∶
𝐾𝑔 𝑉𝑆𝑆 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒
972.5 𝑘𝑔 𝐵𝑂𝐷𝑡
0.6 𝑥 10
𝑉𝑜𝑙𝑢𝑚𝑒 = 162.08 𝑚3
UASB Reactor Tank Dimension:
Length = 4m
Width = 5.1m
Height = 8m
79
Volume =163m3
UASB Tank Reactor

8m
4m
A minimum of 80% of COD removal is expected from the UASB reactor.
Anaerobic Effluent = 100% -80%
= 20%
20 𝑚𝑔
𝐴𝑛𝑎𝑒𝑟𝑜𝑏𝑖𝑐 𝐸𝑓𝑓𝑙𝑢𝑒𝑛𝑡 (𝐶𝑂𝐷) = 𝑥 5840
100 𝑙
𝑚𝑔
𝐴𝑛𝑎𝑒𝑟𝑜𝑏𝑖𝑐 𝐸𝑓𝑓𝑙𝑢𝑒𝑛𝑡 (𝐶𝑂𝐷) = 1168
𝐿
Biogas: 25.62 kg/hr

Other : 71.556 kg/hr
Anaerobic
Reactor
Flow: 20.8 m3/hr Flow: 20.8 m3/hr

TSS: 309mg/L TSS: 74 mg/L
TP : 2.49 mg/L TP : 2.49 mg/L
80
Biogas Production:
5840𝑚𝑔
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ∶
𝐿
𝐶𝑂𝐷𝑡
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛: 5.840 𝑘𝑔
𝑚3
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝐿𝑜𝑎𝑑
𝑘𝑔𝐶𝑂𝐷𝑡
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 500𝑚3 𝑥 5.840
𝑚3
𝐼𝑛𝑓𝑙𝑢𝑒𝑛𝑡 𝐶𝑂𝐷𝑡 𝐿𝑜𝑎𝑑 = 2920 𝑘𝑔 𝐶𝑂𝐷𝑡
𝐶𝑂𝐷𝑡
(2920 𝑘𝑔 𝑥 0.8 𝑥 0.35 𝑁𝑚3 )
𝐵𝑖𝑜𝑔𝑎𝑠 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 𝑑
0.75
𝑁𝑚3
𝐵𝑖𝑜𝑔𝑎𝑠 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 1090
d
1090Nm3
𝐵𝑖𝑜𝑔𝑎𝑠 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = d
1.489
𝑙
𝐵𝑖𝑜𝑔𝑎𝑠 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 732 𝑑𝑎𝑦
1 𝑙𝑖𝑡𝑟𝑒 = 0.84 𝑘𝑔
𝑘𝑔 732 𝑙 𝑘𝑔
𝐵𝑖𝑜𝑔𝑎𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 = 𝑥 0.84
𝑑𝑎𝑦 𝑑𝑎𝑦 1𝑙
𝑘𝑔 𝑘𝑔
𝐵𝑖𝑜𝑔𝑎𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 = 614.88
𝑑𝑎𝑦 𝑑𝑎𝑦
𝑘𝑔 𝑘𝑔 1𝑑𝑎𝑦
𝐵𝑖𝑜𝑔𝑎𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 = 614.88 𝑥
ℎ𝑟 𝑑𝑎𝑦 24ℎ𝑟
𝑘𝑔 𝑘𝑔
𝐵𝑖𝑜𝑔𝑎𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 = 25.62
ℎ𝑟 ℎ𝑟
1090 Nm3/day
81
Table 4.9: Summary of UASB Tank Design

Design Criteria Ranges Designed
Influent COD 4500 – 7500 mg/L
Influent Inflow 10 – 35 m3/hr
Length (L) 4m
Height (H) 8m
Volume 163m3
Organic Loading Rate (OLR) 5.840 kg COD/m3.day
Up-flow Velocity 0.18 m/h
Hydraulic Retention Time (HRT) 8 hr
Solid Loading Rate (SLR) 0.6 kgCOD/KgVSS.d
Biogas Production (CH4) 1090 Nm3/day
Valve 8 Valve (4 run, 4 circulation)
4.3.6 Aeration Tank
As seen in figure 4.9, a Conventional Activated Sludge (CAS) system typically consists of an
aeration tank used for biological degradation and a secondary clarifier (sedimentation tank) where
the sludge is separated from the treated wastewater. In the first phase of a CAS system, wastewater
is combined with air to activate microorganisms in the aeration tank. As the organisms digest the
wastewater, they collide and create flocs, which have a more significant potential to destroy the
biological components of the wastewater.
Following the aeration basin is a secondary clarifier or settling tank. During this process,
microorganisms, and the organic substance they have absorbed settle. Water is carried from the
82
clarifier to facilities for disinfection and ultimate discharge or to additional tertiary treatment units
for further purification.
The surplus microorganisms may be readily routed to any of the sludge treatment options, where
energy can be extracted from biosolids. This extra stage completes the wastewater treatment plant's
energy cycle, allowing it to operate without fossil fuel.
A portion of the microorganisms is fed into the aeration tank to maintain a sufficient quantity of
microorganisms for the biological degradation processes to continue.
CAS involve a biological floccule that oxidizes the sewage's organics. This material (known as
mixed liquid suspended solids or MLSS) consists primarily of saprophytic bacteria and protozoa,
along with rotifers and filamentous bacteria. In the aeration basin, the MLSS is combined with the
entering wastes and aerated. The MLSS is dumped into settling tanks whenever the treated effluent
overflows into weirs and is run off for further treatment prior to disposal. A portion of the settled
solids, the sludge, is returned to the head of the aeration basin in order to reseed the incoming
wastewater. This portion of floc is referred to be Return Activated Sludge (R.A.S.). Waste
Activated Sludge is excessive sludge (WAS). WAS is eliminated from the treatment technique to
maintain a balanced ratio of biomass to food within the wastewater, and is then digested either
under anaerobic or aerobic conditions before to disposal.
Figure 4.9: Conventional activated sludge system

83
Table 4.10: CAS System Design
Design Condition Description

Minimum no. aeration tanks 2
F/M ratio 0.2-0.6
Hydraulic Retention Time (HRT) 6-16 (for a system where only ammonia removal
required) 12-16 (for total nitrogen removal)
O2 requirements (for BOD and NH3 removal) 2.0
Mixed Liquor Suspended Solids (MLSS) 1500-3000
Aeration Device Rating Continuous
Sludge age 5-10
CAS System
MLVSS = 4000 mg/L
Effluent COD = 1168 mg/L
CAS System F/M ratio should be maintained at 0.2 to 0.6.
F/M ratio = 0.6
𝐹 𝑊𝑎𝑠𝑡𝑒𝑤𝑎𝑡𝑒𝑟 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑒𝑑 𝑝𝑒𝑟 𝑑𝑎𝑦 𝑥 𝐶𝑂𝐷

𝑅𝑎𝑡𝑖𝑜 =
𝑀 𝑀𝐿𝑉𝑆𝑆 𝑥 𝑇𝑎𝑛𝑘 𝑉𝑜𝑙𝑢𝑚𝑒
20.8𝑚3 𝑚𝑔
𝑥 24ℎ𝑟 𝑥 1168
𝑇𝑎𝑛𝑘 𝑉𝑜𝑙𝑢𝑚𝑒 = ℎ𝑟 𝑙
4000 𝑥 0.6
𝑇𝑎𝑛𝑘 𝑉𝑜𝑙𝑢𝑚𝑒 = 243 𝑚3
𝐻𝑦𝑑𝑟𝑎𝑢𝑙𝑖𝑐 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒 =
243𝑚3
𝐻𝑦𝑑𝑟𝑎𝑢𝑙𝑖𝑐 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒 =
20.8𝑚3
ℎ𝑟
𝐻𝑦𝑑𝑟𝑎𝑢𝑙𝑖𝑐 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒 = 11.7 ℎ𝑟
84
Tank Design for Aeration Tank:
Length = 5.51 m
Width = 5.51 m
Height = 8 m
8m
Aeration Tank
5.51m
90 𝑚𝑔
𝐶𝐴𝑆 𝑠𝑦𝑠𝑡𝑒𝑚 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝑥 1168
100 𝑙
𝑚𝑔
𝐶𝐴𝑆 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 1051 𝑟𝑒𝑚𝑜𝑣𝑒𝑑
𝑙
𝑚𝑔 𝑚𝑔
𝐹𝑖𝑛𝑎𝑙 𝐶𝑂𝐷 = 1198 − 1051
𝑙 𝑙
𝑚𝑔
𝐹𝑖𝑛𝑎𝑙 𝐶𝑂𝐷 = 147
𝑙
Flow: 5.2 m3/hr

MLSS: 4800 mg/L
Secondary
Aeration Tank
Clarifier
Flow: 20.8 m3/hr Flow: 20.8 m3/hr Flow: 20.8 m3/hr

COD:1168 mg/L COD:147 mg/L COD:147 mg/L
TSS: 4000 mg/L TSS: 4000 mg/L TSS: 18 mg/L
TN: 89 mg/L TN: 31 mg/L TN: 31 mg/L
TP : 2.49 mg/L TP : 0.3 mg/L TP : 0.3 mg/L
85
Table 4.11: Summary of Aeration Tank Design

Design Criteria Ranges Designed
Influent COD 800 – 1500mg/L
Influent Inflow 10 – 35 m3/hr
Length (L) 5.51 m
Height (H) 8m
Volume 243m3
MLSS Value 4000 mg/L
Sludge Retention Time 6 Days
Hydraulic Retention Time (HRT) 11.7 hr
F/M ratio 0.6
Aerator 2 unit of surface aerator. (1 controlled by VFD)

• 35kW of surface aerator enough to provide oxygen
level up to 6 mg/L.
4.3.7 Final Clarifier
Clarifiers (also referred to as sedimentation tanks or settlers) are an integral part of every wastewater
treatment plant. At these treatment facilities, solids are removed from the wastewater by using gravity
sedimentation in quiescent conditions. All clarifiers have two functional zones – a clarification zone,
where the process of gravity sedimentation occurs, and a thickening zone, where the settled solids are
accumulated forming a dense layer of sludge (sludge blanket). Clarifier effluent of low solids
concentration is collected from the top of the clarification zone over overflow weirs and into collection
channels where it is conveyed to the tank outlet. The sludge collected at the bottom of the clarifier is
removed for further treatment at the wastewater treatment plant’s solids handling facilities. The depth
of the clarification zone is commonly referred to as the clear water zone (CWZ) depth, while the depth
86
of the zone of sludge accumulation is called the sludge blanket depth (SBD). The sum of the CWZ
depth and the SBD is defined as the side water depth (SWD).
Secondary tanks are located downstream of the biological (secondary) treatment facilities of the
wastewater treatment plant (such as activated sludge aeration basins or trickling filters) and are used
to separate the biomass generated during the secondary treatment process from the treated plant
effluent.
Secondary
Clarifier
Flow: 20.8 m3/hr Flow: 20.8 m3/hr

TSS: 4000 mg/L TSS: 18 mg/L
TP : 0.3 mg/L TP : 0.3 mg/L
Table 4.12: Summary of Final Clarifier Design

Type of Primary Clarifier - Circular
Radius m 2.6
Surface Area m 20.8
Hydraulic load m/h 1m/h
Volume (V) m3 52

Mono pump:
• 10m3/hr
• 2.2 kW
Flow Rate m3/hr 35
87
Table 4.13: Overall Efficiency of Wastewater
Parameter Sump Pit Primary Equalization Anaerobic Aeration Tank Final Multimedia Standard A
(influent) Clarifier Tank Reactor (CAS) Discharge Sand Filter
(effluent) (effluent) (UASB) (effluent) (effluent)
(effluent)
(effluent)
COD (mg/L) 9733 5840 5840 1168 147 147 30 100 mg/L
BOD (mg/L) 6781 4029 4029 311 31 31 20 mg/L
TSS (mg/L) 515 309 309 74 18 18 2 50 mg/L
TN (mg/L) - 409 409 89 31 31 6 10 mg/L
TP (mg/L) - 2.49 2.49 0.3 0.3 0.1 3 mg/L
COD & BOD - 40% - 80% 90% 80% -

Removal
Efficiency
TSS Removal - 60% - 76% 75% 88% -

Efficiency
TN Removal - - 78% 62% 77% -

Efficiency
TP Removal - - 90% 67% -

Efficiency
88
Figure 4.10: Overall Design of WWTP

89
4.4 WASTE GENERATED AND MANAGEMENT
Fermentation, reaction, and purification stage in the insulin manufacturing industry will generate
the waste which are in the liquid form and will be discharged to the wastewater treatment plant.
a) Stream 45 – Zinc Chloride (ZnCl2)
Streams which contain zinc chloride (ZnCl2) is isolated and treated separately. The reason for this
stream is separated is to prevent any contamination of the of the wastewater due to presence of
heavy metal which can cause the other process to be toxic. The waste generated from this stream
is isolated and treated in prescribed premises which is approved by the Department of Environment
(DOE) under the Environmental Quality (Schedule Waste) Regulation 2005.
Figure 4.11: Cenviro, Prescribed Premises Approved by DOE
b) Primary Treatment Sludge from Wastewater
The sedimentation process from the primary clarifier will sediment with the sludge for 8 hours.
The sludge is treated with a decanter unit. The primary function of the decanter is to reduce the
moisture content from the sludge. Based on the designed model for Aldec 45 (Alfa Laval), the
solid, the sludge, will be produced with 30% moisture content. The produced sludge can be used
90
as compost because the sludge is rich in nitrogen content; the sludge is mainly produced from the
slurry E. coli cells, which are released from the reaction stage.
c) Anaerobic Reactor – Biogas
The high-strength wastewater will be treated to the low strength of wastewater using an anaerobic
reactor. The UASB reactor has been chosen due to its 80 to 90% efficiency in treating wastewater.
The high-strength wastewater will be treated, and the by-product is methane gas (CH4). The CH
can be used as a fuel to generate electricity through the steam turbine and can be used to feed as
fuel to the boiler system. This can reduce electricity consumption, and this approach is towards
cradle to cradle, which promotes the circular economy.
d) Secondary Treatment Sludge
The conventional activated sludge (CAS) system is a biological treatment system that generates
sludge from the aerobic tank. Over time, the sludge will be produced excessively, and as the mean
cell residence time (MCRT) exceeds the setpoint, which is 6 to 10 days or exceeds the MLSS value
of 4000mg/L, the sludge must be wasted through the decanter. The cake or solids produced from
the decanter can be used as a fertilizer via the bioconversion of sludge to compost.
Besides that, the sludge can also become a food source for black soldier flies (BSF). The BSF will
utilize the sludge, and the by-product produced from the BSF can be used as fertilizer. The other
potential is that the BSF can be dried and used as feed for chicken and fish, and it promotes the
circular economy.
e) Final Discharge Wastewater – Recycle
Treated final discharge wastewater can be reused again. The overall wastewater generated from
the insulin manufacturing industry is 280m3. The wastewater can be recycled using ultrafiltration,
reverse osmosis, and ultraviolet light for disinfection. The treated wastewater can be used as
91
service water for cleaning and watering the plant. This could save water and promote the reuse
waste management hierarchy and prevent of using treated water from the water treatment plant or
municipal water supply for general cleaning and watering the plant.
Fig 4.12: Waste management hierarchy
4.5 SUSTAINABLE DEVELOPMENT GOAL
a) SDG 6: Clean Water and Sanitization
Produced wastewater can be reused and recycled by using Ultrafiltration (UF) and Reverse Osmosis
(RO) to treat the wastewater. Thus, the treated wastewater can be used again for the service water such
as cleaning, gardening and other activities that do not relate the production. Through this initiative the
water discharge to the river can be treated and reduced by recycling it and this can reduce the
consumption of raw water which are one step ahead towards the saving of natural resource depletion.
92
Figure 4.13: SDG 6, Clean Water and Sanitation
b) SDG 7: Affordable and Clean Energy
Insulin manufacturing industry consuming large amount of energy such as sterilization and heating
process. However, the waste produced from the insulin manufacturing industry is rich in an organic
material. Thus, there is a huge consumption of fuels thus in an alternative way the biogas from the
insulin manufacturing industry can be used. The insulin manufacturing industry waste product is rich
in an organic content and by the application of anaerobic reactor which are Up flow Anaerobic Sludge
Blanket reactor (UASB) it can convert all the organic rich waste into biogas by few steps that is
hydrolysis, acidogenesis, acetogenesis and methanogenesis. The biogas produced from the anaerobic
reactor can be used as an alternative fuel to the boiler which are renewable energy.
Figure 4.14: SDG 7, Affordable and Clean Energy

93
c) SDG 12: Responsible Consumption and Production
Biogas, wastewater sludge, and are the main waste that is produced from the insulin manufacturing
industry. Currently most of the insulin manufacturing industry in the world is disposing of all the waste
produced to the landfill. In SDG 12 and on target 12.5 it has been mentioned that – “By 2030,
substantially reduce waste generation through prevention, reduction, recycling and reuse.” The raw
material usage and waste generated in the insulin manufacturing industry cannot be prevented or
reduced due to the formula to synthesize the insulin. However, the waste produced can be reuse for
other usage as an example the sludge from the wastewater can be utilized as a feed to the black soldier
flies (BSF). Instead of disposing the waste into the landfill the waste can be reused to produce other
products.
Figure 4.15: SDG 12, Responsible Consumption and Production
d) SDG 14: Life Below Water
The insulin manufacturing industry produces a large amount of wastewater. The produced wastewater
contains a huge amount nutrient which are measured in BOD, COD, Nitrogen and Phosphorus. If the
wastewater is not treated it can cause eutrophication and damage to the environment. The cause of
eutrophication will cause depletion of oxygen content in the river basin. This will harm the life below
the water. Thus, the wastewater must be treated prior discharge to the river or environment.
94
Figure 4.16: SDG 14, Life Below Water

95
CHAPTER V
CONCLUSION
Diabetes is a chronic condition brought on by either insufficient insulin production by the pancreas
or inefficient insulin utilization by the body. A hormone called insulin controls blood sugar levels.
8.5% of people 18 years of age and older had diabetes in 2014. A total of 1.5 million deaths were
directly related to diabetes in 2019, and 48% of these deaths occurred in those under the age of 70.
Type 1 diabetes (previously known as insulin-dependent, juvenile or childhood-onset) is

characterized by deficient insulin production and requires daily insulin administration. In 2017
there were 9 million people with type 1 diabetes, and the insulin manufacturing industry rose
rapidly to meet the consumer demand.
The insulin manufacturing industry generates a highly concentrated effluent with COD, BOD, TN
and TP. However, a chemical such as zinc chloride poses a heavy metal. The wastewater produced
by the insulin manufacturing industry must be treated before being released into the environment.
Untreated wastewater released into the environment can cause eutrophication due to the nigh
nutrients contents from the insulin manufacturing wastewater effluent.
The insulin manufacturing wastewater is designed with primary, secondary, and tertiary treatment.
The zinc chloride stream is isolated from the wastewater because this stream contains heavy metal,
which can contaminate the other wastewater treatment by-product.
The by-products produced from the wastewater treatment plant, such as primary and secondary
clarifier sludge, can be used to produce fertilizer and as animal feed through bioconversion.
Besides that, the biogas generated can be converted into energy through the waste-to-energy
approach, and the wastewater generated also can be recycled and reused again for general cleaning
services. The wastewater treatment plant design follows the circular economy and focuses on the
cradle-to-cradle concept, which aligns with the United Nations: Sustainable Development Goal
approaches.
96
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C., Taylor, S. I., & Yatvin, A. L. (2018). Insulin Access and Affordability Working Group:
Conclusions and Recommendations. Diabetes Care, 41(6), 1299–
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Harrison, R. G., Todd, P. W., Rudge, S. R., & Petrides, D. P. (2015). Bioseparations Science and
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PMC5607278.
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Market Share, and Spending on Long-Acting Insulins, 2006-2018. JAMA, 321(16), 1627.
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INDUSTRIAL EFFLUENT TREATMENT SYSTEM

(IETS) DESIGN PROJECT

JASVINDER SINGH A/L TARA SINGH

UNIVERSITI KEBANGSAAN MALAYSIA

JASVINDER SINGH A/L TARA SINGH

WASTE AUDIT PROJECT SUBMITTED IN FULFILMENT FOR THE COURSE OF

FACULTY SCIENCE AND TECHNOLOGY

28 January 2023 JASVINDER SINGH A/L

CHAPTER II PROCESS DESCRIPTION

CHAPTER III MASS BALANCE

3.3 Reaction Section 36

CHAPTER IV WASTEWATER DESIGN

4.2 Wastewater Generated 51

4.3 Wastewater Design 60

Table No. Page

Table 4.0 Table of Waste Generated for Primary Reaction 55

Table 4.1 Table of Waste Generated for Reaction Section 55

Table 4.2 Table of Waste Generated for Final Purification System 56

Table 4.3 Summary of Waste Generated 57

Table 4.4 Total Volume of Waste Generated 58

Table 4.5 Summary of Sump Pit Design 62

Table 4.6 Summary of Primary Clarifier Design 66

Table 4.7 Summary of Calamity Tank Design 68

Table 4.8 Summary of Equalization Tank Design 70

Table 4.9 Summary of UASB Tank Design 80

Table 4.10 CAS System Design 82

Table 4.11 Summary of Aeration Tank Design 84

Table 4.12 Summary of Final Clarifier Design 85

Table 4.13 Overall Efficiency of Wastewater 86

Figure No. Page

Figure 1.1 $ 20.4 billion of global insulin market share by

Figure 1.3 Biocon Manufactured Insulin in Malaysia 10

Figure 1.4 Forecast Sales of Insulin in Malaysia 11

Figure 1.5 Total Revenue of Insulin Manufacturing Industry 12

Figure 2.0 Human Insulin from proinsulin fusion protein 15

Figure 2.1 Insulin Production PFD 17

Figure 2.2 Insulin Fermentation Vessel 20

Figure 2.3 Fermentation Vessel at Insulin Production Industry 20

Figure 2.4 Centrifugal Machine 22

Figure 2.5 Centrifugal Machine at Insulin Manufacturing Industry 22

Figure 4.0 Sump Pit 60

Figure 4.2 Primary Clarifier Design 64

Figure 4.3 Equalization Tank 70

Figure 4.4 Conversion of Organic Pollutants to Biogas by Anaerobic 72

Figure 4.5 UASB Reactor 72

Figure 4.6 UASB Reactor with GLS Separator 73

Figure 4.7 Anaerobic Digestion Conversion Process of Organic Material 76

Figure 4.8 Anaerobic Granular Sludge 77

Figure 4.9 Conventional Activated Sludge System 82

Figure 4.10 Overall Design of WWTP 88

Figure 4.11 Cenviro, Prescribed Premises Approved by DOE 90

Figure 4.12 Waste Management Hierarchy 92

Figure 4.11 SDG 6, clean Water and Sanitation 93

Figure 4.12 SDG 7, Affordable and Clean Energy 93

Figure 4.13 SDG 12, Responsible Consumption and Production 94

Figure 4.15 SDG 14, Life Below Water 95

1.2 HISTORY OF INSULIN

1.3 INSULIN GLOBAL MARKET

Source: Statistica, 2021

1.3.1 Product Type Insight

1.3.2 Application Insight

1.3.3 Type Insight

1.3.4 Distribution Channel Insight

1.3.5 Regional Insight