PRODUCTION OF 50 MTPA POLYHYDROXYBUTYRATE FROM JATROPHA OIL

7/25/2019 PRODUCTION OF 50 MTPA POLYHYDROXYBUTYRATE FROM JATROPHA OIL

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PRODUCTION OF 50 MTPA POLYHYDROXYBUTYRATE FROMJATROPHA OIL

by

NURUL AIN BT IBRAHIM KE12004

QASTALANI BT GHAZALI KE11004

SHOBANA A/P SINNIAH KE11058

NUR FATIN NADIAH BT FAUZI KE11042

A design project submitted to the Faculty of Chemical and Natural ResourcesEngineering in partial fulfillment of the requirements for the degree of

Bachelor of Chemical Engineering (Biotechnology)

Faculty of Chemical and Natural Resources EngineeringUniversiti Malaysia Pahang

DECEMBER 2014


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Universiti Malaysia Pahang

Faculty of Chemical and Natural Resources Engineering

The undersigned certify that they have read, and recommend to the Faculty ofChemical and Natural Resources Engineering for acceptance, a design projectentitled Production of 50 MTPA Polyhydroxybutyrate from Jatropha Oilsubmitted by

NURUL AIN BT IBRAHIM KE12004

QASTALANI BT GHAZALI KE11004

SHOBANA A/P SINNIAH KE11058NUR FATIN NADIAH BT FAUZI KE11042

in partial fulfillment of the requirements for the degree of Bachelor ofChemical Engineering in Biotechnology.

Dr. Nur Hidayah Bt Mat Yasin

Date:


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ACKNOWLEDGEMENT

First and foremost, we would like to express our greatest gratitude and

sincere appreciation to our plant design supervisor, Dr. Nur Hidayah Binti Mat

Yassin for his exemplary guidance, monitoring and constant encouragement

throughout the process to complete this Plant Design Project. We appreciated

all efforts of supervisor in advising and be available at right time besides

providing valuable insights leading to the successful completion of our plant

design project. Without her guidance and help of overseeing the whole

progress of the team works until the end, we would not be able to accomplishthis design project successfully.

Besides, we would like to take this opportunity to express a deep sense

of gratitude to Dr Mior Ahmad Khusairi Bin Mohd Zahari and Mr. Rozaimi

Abu Samah for their cordial support, valuable information and guidance,

which helped us in completing this task through various stages especially in

simulation.

Furthermore, we are obliged to thank all panels during 3 stages of plant

design presentations for the valuable comments and information provided by

them in their respective fields. All these useful comments help a lot in

improving our plant design project. In addition, sincere thankful is also

extended to our lecturers who had provided us with assistance and

encouragements at any occasions. In addition, we would like to thank our

parents for their unconditional love in giving us support and motivation which

enable us to be determined and without giving up in completing the plant

design project.

Last but not least, to our beloved course mates and acquaintance,

constant encouragement and exchange of knowledge throughout our struggles

in completing this design project. May this report will benefits all readers not

only us in designing new plant for production of Polyhydroxybutyrate (PHB)

for overall stages.


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EXECUTIVE SUMMARY

Litter is a problem with a very negative social and environmental

impact. One way to tackle this problem is to use biodegradable plastics as an

environmentally-friendly solution for things such as plastic

bags. Biodegradable plastics are plastics that can be broken down by

microorganisms (bacteria or fungi) into water, carbon dioxide (CO2) and some

bio-material. Polyhydroxybutyrate is a polymer belonging to the polyesters

class that are of interest as bio-derived and biodegradable plastics.

Therefore the objective of this plant and process design is to develop a

new (PHB) plant using Jatropha oil as the main carbon source and

Cupriavidus necatorH16 as the biomass or the microorganism. Urea is

selected as nitrogen source as it could produce high PHB content. According

to a new study by World Bioplastics from The Freedonia Group. Inc, it stated

that global demand for biodegradable and bio-plastics will be more than triple

to more than 1.1 million tons in 2015, valued at $2.9 billion. Demand for

biodegradable polyesters is said to be growing by about 27.9% for a five years.

This is due to customer demand for more environmentally-sustainable

products, development of bio-based feed stocks for commodity plastic resins,

increasing restrictions on the use of non-degradable plastic products and high

rise of crude oil and natural gas prices. Frost & Sullivan have examined

current and future of the bioplastics market in Southeast Asia for the period

2004 to 2014. It stated that the bioplastics market is at a developing stage. The

total market for engineering plastics in Southeast Asia in 2007 was 12 tons.

These units are forecast to grow at a rate of about 129.8 percent per year andreach about 4063 tons by 2014 (Sullivan, 2008). Malaysias first fully

automated PHA Bioplastics Pilot Plant was launched and scaled-up to 2,000 L,

the bioreactor facilities and integrated manufacturing process of the plant are

able to produce various options of PHA materials from crude palm kernel oil

and palm oil mill effluent. Bioplastics based on PHA in 2013 has been

projected to reach 0.5 billion kg. According to observation of market survey, it

is proposed to produce 50 metric ton PHB per annum. The location is decidedto produce 50 MTPA of PHB which is at Sungai Bako area Kuching, Sarawak.


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Plant layout which consists of administration building, operational building,

waste treatment plant, laboratory and research center, and other ancillary

buildings has been sketched.

Based on the calculation, the total power usage in plant is 719.008

kWh. Average industrial tariff for electricity from Sarawak is 33.70 sen /kWh.

By applying the industrial tariff of electricity 33.70 sen /kWh, the total

electricity cost per year is equal to RM 4797652.781/year with operation hours

of 8000 per year. All the calculation is based on CEPCI 2014. The total steam

usages for main equipment are 1,186.67kg/h. Based on calculation using the

standard steam charges, the total steam cost is about RM 9140191.175 /year

with operation hours of 8000 per year. By conversion, the total steam cost is

RM 9140191.175 /year. The total water consumption for bioreactors and seed

fermenters is 12,849.51 kg/batch. Through calculation the total cost of water is

RM 7454515.37/year with operation hours 8000 per year. RM

7454515.37/year is needed for water cost. By addition of total cost by

electricity, steam and water cost, the total cost of utilities is RM 4797652.781

+ RM 9140191.175 + RM 7454515.37 = RM 21392359.33 /year.

This plant consists of five major equipment. There are seed fermenter,

fermenter, blending storage, disk stack centrifuge and spray dryer. Each of the

equipment has their own hazard. Hazard identification procedure is used to

identify the types of adverse health effects that can be caused by exposure to

some agent in question, and to characterize the quality and weight of evidence

supporting this identification. Risk assessment includes determination of the

events that can produce an accident, the probability of those events, and

consequences that could include human injury or loss of life, damage to the

environment, or loss of production and capital equipment. Hazard

identification can be performed independent of risk assessment, but it would

obtain best result if they are done together.

Economic and profitability analysis in the form of discounted cash

flow will be evaluated in this report as an effort to estimate profit or loss of

this PHB plant. Grass root capital (GRC) is the cost of equipment installation

in a plant and it costs major portion of total fixed capital cost. From


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calculation, it is determined that GRC for this PHB plant is approximately

RM3, 570,000.00. While as for the total capital investment (TCI) for this PHB

plant is approximately RM5, 378, 000.00. Profitability analysis will be

determined in this report by evaluating operating margin. Operating margin is

the ratio of operating profit to sales and it indicates how much of each

Malaysian Ringgit is left after operating expenses. A high operating margin

means that the plant has good cost control and that sales are increasing faster

than costs.


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TABLE OF CONTENTS

ACKNOWLEDGEMENT .................................................................................. iEXECUTIVE SUMMARY ............................................................................... ii

TABLE OF CONTENTS ................................................................................... vLIST OF FIGURES ........................................................................................ viiiLIST OF TABLES ............................................................................................ ixCHAPTER 1 ...................................................................................................... 1INTRODUCTION ............................................................................................. 1

1.1 Background ......................................................................................... 11.1.1 Plastics ......................................................................................... 11.1.2 Biodegradable plastics ................................................................. 21.1.3 Poly-(3-hydroxybutyrate), PHB ................................................... 21.1.4 Physical and chemical properties of PHB .................................... 31.1.5 Biodegradability of PHB .............................................................. 4

1.1.6 Storage and Handling ................................................................... 51.2 Applications of PHB ........................................................................... 5

1.2.1 Medical ........................................................................................ 61.2.2 Aquaculture .................................................................................. 61.2.3 Pharmaceutical ............................................................................. 7

1.3 Market Survey ..................................................................................... 71.3.1 Global Market Demand ................................................................ 71.3.2 Asian market demand .................................................................. 81.3.3 Malaysia market demand ............................................................. 81.3.4 Global production ........................................................................ 91.3.5 Future Prospect of PHB ............................................................. 101.3.6 Prices of Products, Raw Materials and Chemicals .................... 111.3.7 Jatropha Oil ................................................................................ 12

1.4 Screening of Synthesis Routes .......................................................... 151.4.1 Synthesis routes for PHB production ......................................... 151.4.2 Selected synthesis route ............................................................. 241.4.3 Utilization of Jatropha oil .......................................................... 241.4.4 Type of Microbial Production Strain ......................................... 241.4.5 Feeding source of nutrient supply .............................................. 261.4.6 PHB synthesis ............................................................................ 261.4.7 Downstream Process .................................................................. 27

CHAPTER 2 .................................................................................................... 28PROCESS FLOW SHEETING ....................................................................... 28

2.1 Selection of raw material and impurities management ..................... 282.2 Input and Output Flow Sheeting ....................................................... 28

2.2.1 Mechanical Equipment Description ........................................... 332.3 Material and Energy Balances ........................................................... 35

2.3.1 Material Balance ........................................................................ 362.3.2 Energy Balance .......................................................................... 64

2.4 Economic Potential ........................................................................... 712.4.1 Economic Potential 2 Based On Input and Output Structure .... 712.4.2 Economic Potential 3 Based On Recycle Structure .................. 75

2.5 Comparison of Simulation (SuperPro) and Manual CalculationResults .......................................................................................................... 78


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CHAPTER 3 .................................................................................................. 79UTILITIES & HEAT INTERGRATION ........................................................ 79

3.1 Introduction ....................................................................................... 793.2 Utilities .............................................................................................. 79

3.2.1 Electricity ................................................................................... 79

3.2.2 Steam .......................................................................................... 803.3 Heat Integration ................................................................................. 813.4 Economic Potential Level 5: Heat Integration System ..................... 84

CHAPTER 4 .................................................................................................... 85EQUIPMENT SIZING .................................................................................... 85

4.1 Introduction ....................................................................................... 854.2 Heat Sterilizer (ST-101 & ST-102) ................................................... 854.3 Media Preparation Tank (P-09) ......................................................... 864.4 Splitter (FSP-101 & FSP-102) .......................................................... 864.5 Gas Compressor (G-101)................................................................... 874.6 Air Filter (AF-101 & AF-102) .......................................................... 87

4.7 Seed Fermenter (V-101) .................................................................... 874.8 Main Fermenter (V-103) ................................................................... 894.9 Storage Tank (V-104) ........................................................................ 904.10 Centrifuge (DS-101, DS-102 & DS-103) .......................................... 904.11 Pumps ................................................................................................ 914.12 Spray Dryer (SDR-101) .................................................................... 924.13 Economic Potential Level 4 (EP4): Separation System .................... 92

4.13.1 General Structure of the Separation System .............................. 92CHAPTER 5 .................................................................................................... 94PROCESS CONTROL & SAFETY ................................................................ 94

5.1 Introduction ....................................................................................... 945.2 Identification of Hazard..................................................................... 94

5.2.1 Material Safety Data Sheet ........................................................ 955.2.2 DOW Fire and Explosion Index ................................................ 975.2.2 Toxicity .................................................................................... 104

5.3 Hazard and Operability Studies (HAZOP) of Major Equipment .... 1065.4 Major Equipment Control ............................................................... 108

5.4.1 Objectives of Control System .................................................. 1095.4.2 Process Control of Major Equipment....................................... 110

5.5 Piping and Instrumentation Diagram .............................................. 113CHAPTER 6 .................................................................................................. 114

WASTE MANAGEMENT AND POLUTION CONTROL ......................... 1146.1 Introduction ..................................................................................... 1146.1.1 Higher Up the Hierarchy .......................................................... 1156.1.2 Waste Minimization ................................................................. 1166.1.3 Objective of Waste Minimization ............................................ 1176.1.4 Waste Sources and Effect to Human and Environment ........... 1176.1.5 Waste Management Option for Each Waste Produced ............ 118

6.2 JABATAN ALAM SEKITAR (JAS) Schedule B and EQAENVIRONMETAL QUALITY ACT, 1974 .............................................. 122

6.2.1 Gaseous Emission .................................................................... 1226.2.2 Sewage, Industrial Effluent and Leachate Discharge .............. 125

6.3 Waste Treatment Option ................................................................. 1286.3.1 Biological Method ................................................................... 128


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6.3.2 Chemical Method ..................................................................... 1296.3.3 Physical Method ....................................................................... 1306.3.4 Selection of Method ................................................................. 130

6.4 Process Description ......................................................................... 1316.5 Waste in Polyhydroxybutyrate (PHB) Plant ................................... 132

CHAPTER 7 .................................................................................................. 135SITE SELECTION AND PLANT LAYOUT ............................................... 1357.1 Introduction ......................................................................................... 135

7.2 General Consideration of Plant Location ........................................ 1357.3 Type of Industry Preferred and Location ........................................ 136

7.3.1 Availability of Raw Material ................................................... 1367.3.2 Utilities ..................................................................................... 1377.3.3 Water Supply ........................................................................... 1377.3.4 Electricity Supply ..................................................................... 1387.3.5 Land Selling Price and Area Still Available ............................ 1387.3.6 Transportation System ............................................................. 139

7.3.7 Availability of Manpower ........................................................ 1407.3.8 Research and Development Organization ................................ 1407.3.9 Geography, Climate and Environment .................................... 1407.3.10 Government Incentive .............................................................. 1417.3.11 Waste and Effluent Disposal Facilities .................................... 141

7.4 Site Selection analysis ..................................................................... 1417.5 Plant Layout .................................................................................... 142

7.5.1 Introduction .............................................................................. 1427.5.2 Definition ................................................................................. 1427.5.3 Objectives of Plant Layout ....................................................... 1437.5.4 Factors Affecting the Plant Layout .......................................... 144

CHAPTER 8 .................................................................................................. 150ECONOMIC ANALYSIS ............................................................................. 150

8.1 Introduction ..................................................................................... 1508.2 Grass Root Capital .......................................................................... 1508.3 Capital Investment ........................................................................... 1528.4 Manufacturing Cost ......................................................................... 1538.5 Cash Flow Analysis ......................................................................... 159

8.5.1 Payback Period Analysis .......................................................... 1598.6 Profitability Analysis ....................................................................... 1658.7 Conclusion ....................................................................................... 166

CHAPTER 9 .................................................................................................. 1679.1 Conclusion ....................................................................................... 1679.2 Recommendation ............................................................................. 168

REFERENCES ................................................................................................. viAPPENDICES .................................................................................................. vi


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LIST OF FIGURES

Figure 1. 1: Chemical Structure of Polyhydroxybutyrate .................................. 3Figure 1. 2: Global Production capacities of bioplastics in 2012 (by region) ... 9

Figure 1. 3: World Biodiesel Production, 2005-2017 (Millions of gallons) inIndonesia, Argentina, Brazil, U.S, and Europe. ............................................... 10Figure 1. 4: TEM of Cupriavidus necator showing PHB inclusion bodies ..... 14Figure 1. 5: Total acreage of jatropha oil plantations in selected countries(Extracted from: http://www.jatropha-alliance.com) ....................................... 20

Figure 2. 1: Block Flow Diagram of PHB Production ..................................... 30Figure 2. 2: Block Flow Diagram of Upstream Process .................................. 31Figure 2. 3: Block Flow Diagram from Downstream Process ......................... 32Figure 2. 4: Process Flow Diagram of PHB plant ........................................... 33

Figure 2. 5: Process Flow Diagram Simulation in SuperPro ........................... 34Figure 2. 6: Input-output structure of PHB production process ....................... 71Figure 2. 7: Graph of concentration versus conversion of Jatropha oil ........... 73Figure 2. 8: Diagram of Recycle ...................................................................... 75Figure 2. 9: Graph of product, biomass, recycled biomass, Jatropha oil, andurea concentration versus Jatropha oil conversion. ......................................... 76Figure 2. 10: Graph of economic potential at the second level (EP2), economic

potential at the third level with recycle and economic potential at the thirdlevel without recycle. ....................................................................................... 78

Figure 5. 1: Procedure of hazard identification and risk assessment. (Source:Guidelines for Hazards Evaluation Procedures: American Institute ofChemical Engineers, 1985) .............................................................................. 95Figure 5. 2: General steps in determining DOW Fire and Explosion Index .... 99Figure 5. 3: Form used in DOW Fire and Explosion Index ........................... 100Figure 5. 4: Section of P & ID of Seed Fermenter ......................................... 111Figure 5. 5: Section of P & ID of Main Fermenter ........................................ 112Figure 5. 6: Section of P & ID of Disc Stack Centrifuges ............................. 112Figure 5. 7: Section of P & ID of Spray Dryer .............................................. 113

Figure 6. 1: Waste management hierarchy .................................................... 114Figure 6. 2: Conceptual Flow Diagram for Activated Sludge WastewaterTreatment System .......................................................................................... 131

Figure 7. 1: Plant Layout of PHB plant ......................................................... 146

Figure 8. 1: Undiscounted Cash Flow ............................................................ 161Figure 8. 2: Discounted Cash Flow ................................................................ 163


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LIST OF TABLES

Table 1. 1: Chemical properties of PHB ............................................................ 4Table 1. 2: World Bioplastic Demand for 20052015 ..................................... 7

Table 1. 3: Media for the production of PHB .................................................. 11Table 1. 4: The prices of the material components .......................................... 11Table 1. 5: Properties of Jatropha oil from Bionas Sdn. Bhd. ......................... 13Table 1. 6: Characteristics of urea ................................................................... 14Table 1. 7: Raw materials and their prices for production of 50 MTPA PHBusing soybean oil as carbon source. ................................................................. 17Table 1. 8: Raw materials and their prices for production of 50 MTPA PHBusing crude palm kernel oil (CPKO) as carbon source. ................................... 18Table 1. 9: Raw materials and their prices for production of 50 MTPA PHBusing Jatropha oil as carbon source. ................................................................ 21Table 1. 10: Comparison between soybean oil, jatropha oil, and crude palm

kernel oil based on its availability of raw materials, yield of PHB, concerns,cost and operation mode. ................................................................................. 22Table 1. 11: comparison between promising microorganisms in PHBcultivation, an analysis from Choi and Lee (1997). ......................................... 25

Table 2. 1: Input and Output of Heat Sterilizer (ST-101&ST-102) ................. 36Table 2. 2: Density of Each Components ........................................................ 38Table 2. 3: Summary of materials used in blending tank................................. 42Table 2. 4: Amount of input of Seed Fermenter and Main Fermenter ............ 43Table 2. 5: Input and Output of Compressor (G-101) ..................................... 43

Table 2. 6: Input and Output of Air Filter (AF-101) ........................................ 44Table 2. 7: Summary of material used in seed fermenter ................................ 47Table 2. 8: Summary of materials used in main fermenter .............................. 48Table 2. 9: Summary amount of input into Seed Fermenter and MainFermenter ......................................................................................................... 54Table 2. 10: Summary amount of output from fermenter ................................ 55Table 2. 11: Overall material balance of Seed Fermenter................................ 55Table 2. 12: Summary of Overall Material Balance of main fermenter .......... 56Table 2. 13: Input and output of Air Filter (AF-102) ...................................... 57Table 2. 14: Input and Output of Flat Bottom Tank (V-104) .......................... 57Table 2. 15: Input and output of Centrifugal (C-101) ...................................... 58Table 2. 16: Input and Output of Blending Tank (C-101) ............................... 59Table 2. 17: Input and output streams of mixer (MX-101) .............................. 59Table 2. 18: Input and Output Stream of Disc-stack Centrifuge (P-13/DS-102).......................................................................................................................... 61Table 2. 19: Input and Output Stream of Blending Tank (P-14/V-103) .......... 62Table 2. 20: Input and output of Disc-stack Centrifugal (C-03) ...................... 62Table 2. 21: Summary Input Stream of Spray Dryer (P-16/SDR-101) ............ 63Table 2. 22: Summary Output of Spray Dryer (P-16/SDR-101) ..................... 63Table 2. 23: Summary of energy balance of each stream ................................ 65Table 2. 24: Heat of formation ......................................................................... 68

Table 2. 25: Heat duty for each equipment ...................................................... 70Table 2. 26: Values for EP2 calculation .......................................................... 74


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Table 2. 27: Graph of EP2 versus Jatropha oil conversion .............................. 74Table 2.27: Table 2. 28: Data for EP2 and EP3 at both with recycle andwithout recycle. ................................................................................................ 77Table 2. 29: Comparison between Simulation and Manual Balance ............... 78

Table 3. 1: Total Power consumption of equipment used in plant design ....... 79Table 3. 2: Total steam consumption of equipment used in plant design ........ 80Table 3. 3: Total water consumption of equipment used in plant design ........ 81

Table 4. 1: Sizing Summary of Heat Sterilizer ................................................ 85Table 4. 2: Sizing Summary of Media Preparation Tank ................................ 86Table 4. 3: Sizing Summary of Splitter............................................................ 86Table 4. 4: Sizing Summary of Gas Compressor ............................................. 87Table 4. 5: Sizing Summary of Air Filter ........................................................ 87

Table 4. 6: Bare Module Cost (CBM) for Centrifuges .................................... 93

Table 5. 1: Degree of Hazard based on DOW Fire and Explosion Index (FEI)........................................................................................................................ 103Table 5. 2: Toxicity level ............................................................................... 104Table 5. 3: Toxicity rating system ................................................................. 105Table 5. 4: General Guide Words for HAZOP procedures (Crowl and Louvar,2002) .............................................................................................................. 106

Table 6. 1: Source and Waste Generated in PHB plant ................................. 117

Table 6. 2: Waste Management Options by Our Company ........................... 118Table 6. 3: Malaysian Standard Guidelines for Air Gaseous Pollutants ........ 119Table 6. 4: Malaysia, Canada and USA Ambient Air Quality Guidelines .... 120Table 6. 5: Characterization of Waste Type According to MIDA ................. 121Table 6. 6: Comparison of Aerobic and Anaerobic Treatment (Mittal, 2011)........................................................................................................................ 128Table 6. 7: Total Gaseous Waste ................................................................... 133Table 6. 8: Total Waste Summary ................................................................. 133Table 6. 9: Costing for Waste Treatment Option Employed in Our Company........................................................................................................................ 134

Table 7. 1: Water Provider Based on Location .............................................. 137Table 7. 2: Electricity Provider Based On Location ...................................... 138Table 7. 3: Building and Location in the Plant Layout .................................. 147

Table 8. 1: Bare Module Cost of Equipment in PHB Plant ........................... 150Table 8. 2: Estimation of Grass Root Capital, GRC. ..................................... 152Table 8. 3: Fixed and Total Capital Investment ............................................. 153Table 8. 4: Estimation of Operating Labor Cost ............................................ 155Table 8. 5: Summary of Manufacturing Cost ................................................ 156

Table 8. 6: Cash Flow Analysis for Undiscounted Rate, I% ......................... 160Table 8. 7: Discounted Cash Flow Summary ................................................ 162


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Table 8. 8: Net Present Value for Discounted Rate ....................................... 164Table 8. 9: Discounted Cash Flow at DCFRR=28.35% ................................ 165


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CHAPTER 1

INTRODUCTION

1.1 Background

This chapter will provides overview of Polyhydroxybutyrate (PHB) as

well as other components involved in the plant. The demand and supply of

PHB also was discussed.

1.1.1 Plastics

Plastics are man- made long chain polymeric molecules similar in

many ways to natural resins found in trees and other plants (Scott, 1999). On

the other hand, plastics are uniquely flexible materials that have seen them

occupy a huge range of functions, from simple packing materials to complex

engineering components (Jim and Alexandre et al., 2013). The history of

plastic begins from 1862 by Alexander Parkes. The main raw material in

plastic production is petroleum. The properties of plastic which is high

molecular weight and tightly bonded together make the plastic not degradable,

their disposal become difficult and give negative impact on the environment

(Sharmila et al., 2011). During the 1980s, the solid waste problem emerged as

a potential crisis in many areas of the US because of increasing amounts of

municipal solid waste (MSW), shrinking landfill capacity, rising costs and

strong public opposition to new solid waste facility sittings (Regan et al.,

1990). In 1960 plastics made waste less than half a percent of US MSW

generation. By 2010 they made up to 12.4% and only 8.2% is recovered (US

EPA, 2011).


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1.1.2 Biodegradable plastics

Biodegradable plastics were introduced in the 1980s to find ways to

produce non-petroleum based plastics as well as to reduce the environmental

effects because of the increased landfill (Gironi and Piemonte, 2010).

According to European Bioplastics, a plastic material is defined as a bioplastic

if it is either biobased, biodegradable, or features both properties. The term

biobased means that the part of material or product is derived from biomass.

Meanwhile, biodegradation is a chemical process which could be degraded by

the microorganism in the environment when proper conditions such as the

sunlight, moisture, oxygen and so forth are available convert materials into

natural substances such as water, carbon dioxide, and composition (Abe and

Doi, 2002).

1.1.3 Poly-(3-hydroxybutyrate), PHB

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a

polymer belong to the polyesters class that was first isolated and characterized

in 1925 by French microbiologist Maurice Lemoigne. PHB is produced by

microorganisms (like Ralstonia eutropha or recombinant Escherichia coli)

apparently in response to conditions of physiological stress. The polymer is

primarily a product of carbon assimilation (from glucose or starch) and is

employed by microorganisms as a form of energy storage molecule to be

metabolized when other common energy sources are not available. Microbial

biosynthesis of PHB starts with the condensation of two molecules of acetyl-

CoA to give acetoacetyl-CoA which is subsequently reduced to

hydroxybutyryl-CoA. This latter compound is then used as a monomer to

polymerize PHB (Lemoigne, 2009).

Since 1925, PHB has been produced through bacterial fermentation

(Rosa, 2004), being synthesized under limited culture conditions, and it is

usually produced through the use of microorganisms that belong to genres

Alcaligenes, Azobacter, Bacillus, andPseudomonas(Ugur, 2002).


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The poly-3-hydroxybutyrate (P3HB) form of PHB is probably the most

common type of polyhydroxyalkanoate, but many other polymers of this

class are produced by a variety of organisms: these include poly-4-

hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate

(PHH), polyhydroxyoctanoate (PHO) and their copolymers (Lemoigne, 2009).

Poly-(3-hydroxybutyrate), PHB is one of the most important members

of PHAs. According to Li et al. (1999), PHB is an intracellular carbon and

energy storage material produced by many microorganisms under unfavorable

growth condition such as limitation of (NH4)2SO4, PO32-, Mg2+ and oxygen.

PHB is synthesized from acetyl-CoA using three enzymatic steps (Paramjit

and Nitika, 2011). It is a biodegradable thermoplastic polyester which can be

used in various ways like the conventional non-degradable plastics (Li et al.,

1999). The chemical structure of PHB is shown as in Figure 1.1.

Figure 1. 1: Chemical Structure of Polyhydroxybutyrate

1.1.4 Physical and chemical properties of PHB

The physical properties of PHB are elastomeric, insoluble in water,

nontoxic, biocompatible, and piezoelectric, with high degree of polymerization

(Samantary et al, 2011). Besides, PHB is also resistant to water and ultraviolet

radiation and impermeable to oxygen. In addition, PHB is a partially

crystalline material with high melting temperature and high degree of

crystallinity. PHB is stiff and brittle. PHB does not contain any residues of

catalyst and is perfectly isotactic and does not include any chain branching. It

is not water soluble but is 100% biodegradable. PHB has low permeable for

O2, H2O and CO2 (Samantary et al, 2011). Chemical properties of PHB is

summarized in Table 1.1.


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Table 1. 1: Chemical properties of PHB

Parameter Value

Melting point (oC)

Glass transition temperature (oC)

Crystallinity (%)

Density (g cm-3)

Molecular weight (g/mol)

Molecular weight distribution

Heat capacity (kJ/kg.K)

Tensile strength [MPa]

Extension to break [%]

UV resistance

Solvent resistance

Oxygen permeability [cm3m-2atm-1d-1]

Biodegradability

171-182

5-10

65-80

1.23 - 1.25

6600000

2.23

1.465

40

68

Good

Poor

45

Good

1.1.5 Biodegradability of PHB

Biodegradation of PHB is dependent upon a number of factors such as

the microbial activity of the environment and the exposed surface area. In

addition, temperature, pH, molecular weight and crystallinity are important

factors. Biodegradation starts when microorganisms begin growing on the

surface of the plastic and secrete enzymes that break down the polymer into its

molecular building blocks, called hydroxyacids. The hydroxyacids are then

taken up by the microorganisms and used as carbon sources for growth. In

aerobic environments the polymers are degraded to carbon dioxide and water.

The environmental degradation behavior of PHB-g-VAc films (Xg:

0%, 5% and 15%) before and after saponification assessed by the BOD

method in environmental water. Many kinds of PVA-utilizing micro-

organisms have been found in the water of major rivers (Matsumura et al.,

1994), and it was confirmed that PVA could be degraded in environmentalwater from the lake at the Takasaki Advanced Radiation Research Institute.


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Biodegradation of PHA has also been tested in various aquatic

environments. In one study in Lake Lugano, Switzerland, items were placed at

different depths of water as well as on the sediment surface. A life span of 5-

10 years was calculated for bottles under these conditions (assuming no

increase in surface area), while PHA films were completely degraded in the

top 20 cm of sediment within 254 days at temperatures not exceeding 6C.

1.1.6 Storage and Handling

PHB is non-toxic biopolymer. Therefore, it is biocompatible and hence

is suitable for medical applications. It is important to minimize prematurePHB degradation during fabrication and storage. This is because PHB are

biodegradable polymer and its biodegradation is dependent upon a number of

factors such as the microbial activity of the environment and the exposed

surface area. In addition temperature, pH, molecular weight and crystallinity

are also play an important role. In one report, the maximum biodegradation

rates were observed at moisture level of 55% and temperatures of around

60C. Therefore, it is well advised to packed PHB in airtight, aluminum-

backed, or plastic foil pouches and kept it in the refrigerator.

1.2 Applications of PHB

There are many applications of PHB besides it is been used in the

production of biodegradable plastic. PHB have been chosen as petroleum

derived plastic replacement because of its properties that possess high

durability and endurance similar like regular plastics but unlike regular

plastics, it can be decomposed to water and carbon dioxide aerobic

microorganisms existing from sewage, sea or soil without forming any toxic

products. PHB can be used as wrapping materials like bags, containers and

throwaway items such as cup, plates and diapers.


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1.2.1 Medical

Since biodegradability and biocompatibility are properties of PHB, the

combination of PHB with hydroxyapatite (HA) were used as scaffolding

material in tissue engineering (Brigham, 2012). For instance, the medical

practitioners use PHB scaffolding material to treat bone defects. While the

combinations of copolymer of polyglycolic acid (PGA) and PHB was used to

produce pulmonary valve leaflets and pulmonary artery scaffolds in sheep.

PHB also used in medical devices such as for dental and skin surgery

(Bonartsev, 2007). The efficiency of these devices in term of biocompatibility,

biodegradation and therapeutic is still in progress.

1.2.2 Aquaculture

PHB was used as a food add-on to aquaculture animals in order to

control the enormous deaths caused by pathogenic contaminations. Larvae of

the brine shrimp Artemia franciscana serve as important feed in fish and

shellfish larviculture however, they are subject to bacterial diseases that

devastate entire populations and consequently hinder their use in aquaculture.

It was found that PHB might shield the fish meal which is gnotobiotic brine

shrimp Artemia franciscana against pathogenic vibriosis (Schryver, 2010).

The release of the PHB monomer -hydroxybutyric acid was suggested to

inhibit the growth and/or the activity of the pathogens (Schryver, 2010). By

integrating the accumulation of PHB in bio-flocs, this technique can possibly

decrease the rate of death during larval and young stages of aquaculture

animals and can therefore become all the more cost effective. No adverse

effects were observed when the feed is introduced for about 10% to the diet of

the fish.


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1.2.3 Pharmaceutical

In pharmaceutical, PHB is applied into slow-released carrier for lasting

drug delivery due to their biocompatibility and biodegradability properties. It

is also used as cell and tablet packaging material. PHB, 3-hydroxyhexanoate

(PHBHHx), and polylactic acid (PLA) were used to study drug sustained

release. The results showed that over a period of at least 20 days for PHB and

PHBHHx nanoparticles, while PLA nanoparticles and free drug lasted only 15

days and a week, respectively (Xiong, 2010).

1.3 Market Survey

1.3.1 Global Market Demand

Polyhydroxybutyrate (PHB) and similar polymers have obtained

worldwide interest because of their biodegradability in addition to their

durability and plasticity. Industrial production of PHA and other

biodegradable plastics is shown in Table 1.2.

Table 1. 2: World Bioplastic Demand for 20052015

WORLD BIOPLASTICS DEMAND

(thousand metric tons)

Item 2005 2010 2015 %Annual Growth

2005 - 2010 2010-2015

Bioplastics Demand 130 300 1025 18.2 27.9

North America 34 80 242 18.7 24.8

Western Europe 60 125 347 15.8 22.7

Asia/Pacific 33 83 320 20.3 31.0

Other Regions 3 12 116 32.0 57.4

According to a new study, World Bioplastics, from The Freedonia

Group, Inc; it stated that global demand for biodegradable and bio-plastics will


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more than triple to more than 1 million metric tons (1.1 million tons) in 2015,

valued at $2.9 billion. Demand for biodegradable polyesters is said to be

growing by about 27.9% for a five years, and North America is belatedly

catching up with other regions for about 24.8 % of annual growth for 2010 to

2015. This is due to customer demand for more environmentally-sustainable

products, development of bio-based feed stocks for commodity plastic resins,

increasing restrictions on the use of non-degradable plastic products and high

rise of crude oil and natural gas prices.

1.3.2 Asian market demand

Frost & Sullivan have examined the bioplastics markets in Southeast

Asia. The research service presents current and future of the bioplastics market

in Southeast Asia for the period 2004 to 2014. It stated that the bioplastics

market is at a developing stage. The total market for engineering plastics in

Southeast Asia in 2007 was 12 tons. These units are forecast to grow at a rate

of about 129.8 percent per year and reach about 4063 tons by 2014 (Sullivan,

2008).

1.3.3 Malaysia market demand

Malaysias first fully automated PHA Bioplastics Pilot Plant was

launched by Science, Technology and Innovation Minister Datuk Seri Dr.

Maximus Johnity Ongkili at Jalan Beremban. Scaled-up to 2,000 L, the

bioreactor facilities and integrated manufacturing process of the plant are able

to produce various options of PHA materials from crude palm kernel oil and

palm oil mill effluent. Bioplastics based on PHA in 2013 has been projected to

reach 0.5 billion kg (First-Of-Its-Kind Sirim Bioplastics Pilot Plant Launched

in 2011).


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1.3.4 Global production

Figure 1. 2: Global Production capacities of bioplastics in 2012 (by region)

Bioplastics production capacities are growing fastest outside of

Europe. In 2012 production capacities amounted to approximately 1.4 million

tons. Market data of European Bioplastics forecasts production capacities will

multiply by 2017to more than 6 million tons. Based on the figure above, it

has shown that Asia has dominated the production of bioplastics which is 36.2

percent. It is about 0.5 million tons per year (Bioplastics, 2014).


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Figure 1. 3: World Biodiesel Production, 2005-2017 (Millions of gallons)

in Indonesia, Argentina, Brazil, U.S, and Europe.

The market of around 1.2 million tons in 2011 may see a five-fold

increase in production volumes by 2016, to almost 6 million tons. The productexpected to contribute most to this growth is bio-based PET (for plastic

bottles), which already accounts for approximately 40% of the global

bioplastics production capacity. The current production volume is expected to

grow to more than 4.6 million tons by 2016 as a result of demand from large

manufacturers of carbonated drinks. Early in 2013 the nova-Institute predicted

that by 2020 bioplastics production could rise to 12 million tons, principally

due to drop-in polymers, particularly bio-PET13. With an expected total

polymer production of about 400 million tons in 2020, the bio-based share

should increase from 1.5% in 2011 to 3% in 2020 (Development, 2013).

1.3.5 Future Prospect of PHB

Polyhydroxybutyrate (PHB) is diverse and versatile class of materials

that has potential applications in many sectors of the economy. Currently,


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productions of PHB are still in the developmental stage, but important

applications are beginning to emerge in packaging, food production, and

medicine. We have reached a critical point in the development of PHBs for

many applications. It is, therefore, an opportune time for a comprehensive

report detailing promising new developments in this field. In brief, production

of PHB has good future prospect because:

1.

PHB is biodegradable

2. Production of PHB protects the fossil resources

3. PHBs have a positive eco-balance sheet

4. Good example for a sustainable development in the spirit of the

agenda of 21stcentury

5.

Carbon dioxide neutral

6.

The use of biodegradable material creates over 20, 000 new and

secure workplaces in Europe and many times over in the world

(social factor)

1.3.6 Prices of Products, Raw Materials and Chemicals

Types of raw materials and amount used for pre-cultures are shown in

Table 1.3 while the market prices for PHB, raw materials of culture medium in

PHB production are shown in Table 1.4:

Table 1. 3: Media for the production of PHB

Materials Amount (g/L)

Jatropha oil 20.00 g/L

Urea 1.00 g/L

Table 1. 4: The prices of the material components

Materials Prices (MYR/kg) Source

Jatropha oil 2.73 Bionas Malaysia Sdn Bhd


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Urea 1.70 Petronas Fertilizer Sdn Bhd

PHB 27.45 -

1.3.7 Jatropha Oil

Jatropha oil is a potential renewable resource because Jatropha

plantations yield large amounts of oil, are highly resistant to drought and pests

and the oil is relatively cheap and non-edible. Jatropha oil is derived from

Jatropha curcas seeds. This plant was originally found in the Caribbean area

but is now widespread throughout Africa, the Americas and much of Asia. The

plant also is known as hardy Jatropha due to its resistance to pest and

drought, and also its ability to grow almost anywhere. The oil yield of this

plant is almost four times that of soybean, and 10 times that of maize.

Recently, Jatropha oil has been evaluated as a source of high quality biodiesel

production. However, it has not been evaluated as a feedstock for PHA

production (Ko-Sin Ng, 2010).

The genus Jatropha belongs to the Euphorbiaceous family which cansynthesize several toxic compounds, including carcinogenic phorbol ester,

trypsin inhibitor, lectin and saponin. The toxins render the oil non-edible, but

should not affect its utility for bioplastics production. In view of the above, it

is advantageous to use Jatropha oil which is not food-grade oil as the sole

carbon source to produce PHA (Ko-Sin Ng, 2010).

It has three Malaysian entities and six overseas joint ventures which

are Bionas Murabahah Bhd, Bionas Sdn Bhd and Biofuel Bionas Sdn Bhd,

Bionas Philippines, Bionas Indonesia, Bionas Vietnam, Bionas Cambodia,

Bionas Thailand and Bionas Taiwan. Its assets portfolio consists of over

600,000 acres planted areas, 3.3 million acres land bank, 313 seedling

nurseries & harvest collection centers and 3 processing plants.

As a result, the company has monthly supply and production capacity

of 100,000 tons seeds, 90,000 tons seedlings, 33,000 MT Crude Jatropha Oil(CJO) and 65,000 MT seed cakes (bio-mass). Now the company is extending


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its global presence by expanding to Taiwan, Thailand, Cambodia, Bangladesh

and Cambodia. With Bionas diverse operational experience and an unrivaled

business heritage, it is now poised to take a leading position in the global

business arena.

In the year 2008 and 2009, Bionas has been actively promoting the

cultivation of Jatropha in Malaysia. The numbers of planters had risen from

28,983 in 2008 to 112,484 in 2009 respectively. Bionas has also increased the

numbers of nurseries from 98 in 2008 to 221 in 2009. The number of planters

had risen to 238,541 and the number of nurseries to 313 in 2010. The company

has also setup four pressing mills in 2010 as part of the companys capacity

building to cater its needs for the production Bionas Jatropha Additives. 2011

has been a productive period for Bionas as the company has invested into the

setting up of two processing, blending plant, and storage facilities for Bionas

Jatropha Additives at two main ports of Malaysia which are located in Prai

Port, Penang and Kuching Port, Sarawak.

Table 1. 5: Properties of Jatropha oil from Bionas Sdn. Bhd.

Criteria Properties

Climate type Tropical

Seed oil content 37%

Average annual yield/Acre (1st-3

r

year)3.6 MT

Lifespan 50 years

Harvest period Monthly after six months

Crude oil price (MYR/MT) 2736.00

By-products Seed cakes i.e Biomass Briquette

1.3.7.3 Biomass: Cupri avidus Necator


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Cupriavidus necatorwas formerly known asAlcaligenes eutrophusis a

motile, rod shaped, Gram negative, non-sporing bacterium and major strains is

H16 and JMP 134 (Larsen and Pogliano, 2007). Larsen and Pogliano (2011)

stated that its optimal temperature is 30C while optimal pH is 7 and it is a

non-halophilic, which cannot live in high salt concentration. It is able to

produce PHB inside the inclusion bodies under limited nitrogen source but

excessive carbon source (Ojumu et al.2004).

Figure 1. 4: TEM of Cupriavidus necator showing PHB inclusion bodies

1.3.7.4 Urea

Urea is a white crystalline substance with the chemical formula CO

(NH2)2. It is highly water soluble and contains 46% nitrogen. Urea is

considered an organic compound because it contains carbon. It was the first

organic compound ever synthesized by chemists; this was accomplished in the

early 1800s. Urea supplies more nitrogen per ton of product than any other dry

fertilizer. It contains 46% nitrogen; this means that each ton of urea supplies

920 lbs. of nitrogen.

Table 1. 6: Characteristics of urea


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Other Name Urea

Molecular Formula CH4N2O

Molecular weight 60.06

1.4 Screening of Synthesis Routes

PHB has a great potential as a biodegradable bio plastic. However, the

major drawback to the commercialization of PHB is their high cost of

production compared with conventional petrochemical based plastic materials.

The cost of carbon feed stocks or raw materials required can significantly

affect the PHB production cost in large production scale. Therefore, the

production cost can be considerably lowered when alternative cost-effective

carbon feedstock, type of microbial production strain, as well as nutrient

supply during the biosynthesis for the commercialization of bio plastics are

identified.

1.4.1 Synthesis routes for PHB production

Various substrates especially plant oils have been evaluated as an

excellent carbon source in PHA production. Examples given are soybean oil

(Kahar et al., 2004), palm oil (Kek et al., 2008) and Jatropha oil (Khan et al.,

2013). Kahar et al. (2008) reported that plant oils are desirable as they are also

inexpensive carbon sources. Additionally, due to their high carbon content,

plant oils yield almost two-fold higher than from glucose and they are

appealing feed stocks for industrial PHA production because metabolism of

these compounds can influence the monomer composition of the resulting

PHA (Akiyama et al., 2003).

1.4.1.1 Production of PHB from soybean oil by Cupr iavidus necatorH16


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According to Kahar et al. (2008), high yield production of

polyhydroxyalkanoates has been identified from using soybean oil by wild-

type strainRalstonia eutrophaor now known as Cupriavidus necator, one of

the best known bacteria among PHA-producing microorganisms. Soybean oil

has a high yield of PHA obtained ranging from 0.72 to 0.76 g PHA/ g soybean

oil used and the PHA productivity obtained here was roughly calculated to be

1.0 g/L.h (Kahar et al. 2008).

According to Global Agricultural Information Network (GAIN) in

2012, there is no commercial cultivation of soybeans in Malaysia despite it is

one of the largest producers of soy drinks in Southeast Asia, with exports

going to neighboring countries as well as Australia, Japan and Europe.

Malaysia has to import soybean oil from U.S, hence this will increase

production cost as shipping and handling cost have to be considered.

Furthermore, contrary to sugar that can be directly utilized by cells,

soybean oil needs to be hydrolyzed by lipase and fatty acids. This would

increase production cost to acquire lipase and fatty acids as well as equipment

such as hydrolyzer. Environmental wise, large scale production from soybean

oil is environmentally friendly as the carbon dioxide emission from soybean

oil are very low compared to the petrochemical polymers if high yield of PHA

is produced (Kahar et al.2008). However, the use of edible oils in production

of bio plastics may cause depletion of global food supply and sources. Using

soybean oil is considered as unethical as it is wasteful to convert food to bio

plastics. Additionally, Ng et al. (2010) also stated that the edible plant oils

price has increased drastically because of recent crisis of food shortage and

increase of food demand.

1.4.1.1.1 Economic Potential Level 1

As we produce 50,000 kg of PHB per year and the price is RM 1,

373,000 per year. Below is price for raw materials of this synthesis route;


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Table 1. 7: Raw materials and their prices for production of 50 MTPA

PHB using soybean oil as carbon source.

Raw Material Price

(RM/kg)

Price

(RM/year)

Source

Soybean oil 2.90 44, 900.00 MGT Group Bhd

Ammonium

sulphate

10.35 12.40 Greymont Agrochem Sdn Bhd

Economic potential level 1 of this synthesis route can be calculates as;

EP1: RM 1, 373,000/year(RM 44, 900.00/yearRM 12.40/year)

= RM 1, 330, 000.00 per year

1.4.1.2 Production of PHB from crude palm kernel oil (CPKO) by

Cupr iavidus necator

Since the C. necatorhas the limitation on soybean oil as this cells grow

well on palmitic acid, linoeic and oleic acid but cannot grow well on linolenic

acid, the use of palm oil containing less linolenic acid may be a good choice to

accumulate a high dry cell weight and also could increase the yield of PHA

more than those with soybean oil (Kahar et al. 2008).

Today, Malaysia is both major producer and exporter of palm oil in the

world. Palm oil is a versatile oil that is currently used as edible oils as well as

for the production of oleo chemicals. Malaysia is one of the largest contributor

of palm oil in the world, surpassing Nigeria as the main producer since 1971

(Yusoff, 2006). Malaysia is the worlds second-leading oil palm producer and

exporter after Indonesia, supplying about 12.6% of global consumption of

vegetable oils (GAIN, 2012). This is firmly would support the supply of feed

stock for the PHA and PHB production.


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However, there are issues requiring serious attention such as

deforestation, waste disposals from palm oil mill and energy expenditure when

PHA is to be produced in large scale. In order to fulfill the PHA market

demand solely by using CPKO to produce PHA, approximately 53,000 tons of

CPKO (which is approximately 2.8% of Malaysias total CPKO production) is

required as carbon feed stock for microbial fermentation. In other words, the

production of 52,000 tons of PHA per annum would involve a total of 111,520

hectares of oil palm plantation. As the demand for plant oils increase for PHA

production, it may result in the further expansion of plantations into forests.

Also, like soybean oil, there are concerns about merits of converting food-

grade oil for bio plastics production at the expense of dwindling the worlds

food supply as palm oil provides nearly 30% of the worlds edible vegetable

oil (Carter et al.2007), with a production volume of 43.12 million tons in year

2008 (MPOB 2008).



373,000 per year. Below is price for raw materials of this synthesis route;

Table 1. 8: Raw materials and their prices for production of 50 MTPA

PHB using crude palm kernel oil (CPKO) as carbon source.

Raw Material Price

(RM/kg)

Price

(RM/year)

Source

Palm kernel oil 1.25 19, 350.00 KL Kepong Oleomas Sdn

Bhd

Ammonia 8.00 10.20 Petronas Chemicals

Ammonia Sdn.Bhd


EP1: RM 1, 373, 000/year(RM19, 350.00/yearRM 10.20/year)


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= RM 1, 354, 600.00/year

1.4.1.3 Production of PHB from jatropha oil by Cupr iavidus necatorH16

As a result of evaluation of using edible plant oils, Jatropha oil, as a

non-edible one, would not affect the global food chain crisis and has potential

as a renewable resource. It can be the alternative substrate for bio plastic

production. Jatropha oil, derived from Jatropha curcas seed, also yield high

amounts of oil, yielding almost four times than the soybean and ten times from

the maize (Fitzgerald, 2006). It is also relatively cheap, costing less than

soybean oil as the fertilizer and pesticide requirement of Jatropha is lower

(Gui et al, 2008).

As the world is in a state of biofuels fever, many countries have started

planting Jatropha plant as this non-edible plant has promising future as

biofuels. Approximately 900,000 hectares of Jatropha have already been

planted throughout the world. Although the industry is in its early stages, it is

identified 242 Jatropha plantation projects, totaling approximately 900,000

hectares. More than 85% of the land cultivated is located in Asia. Africa

counts for approximately 120,000 hectares followed by Latin America with

approximately 20,000 hectares. Jatropha saw enormous growth: 5 million

hectares were expected by 2010. The number and size of Jatropha projects

currently being developed is increasing sharply. This is the case in almost all

regions of the world which are suitable for Jatropha cultivation. It is predicted

that each year for the next 5-7 years approximately 1.5 to 2 million hectares of

Jatropha will be planted. This will result in a total of approximately 5 million

hectares by 2010 and approximately 13 million hectares by 2015.


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Figure 1. 5: Total acreage of jatropha oil plantations in selected countries

(Extracted from: http://www.jatropha-alliance.com)

Jatropha has been commercially farmed in Malaysia specifically

Sarawak owned by Bio Oil National Group Malaysia (The Star, 2011). A few

local private companies also have engaged in Jatropha cultivation scaling from

400 ha to 1000 ha. It has been identified that total current acreage of Jatropha

plantation projects is 1,712 ha. Project owners state plans to increase the

cultivation scale to a total of 57,601 ha by 2015. The Ministry of Plantation of

Industries and Commodities is undertaking a Jatropha pilot research projectfor which 300 ha have been allocated.

The operational and maintenance costs for the Jatropha oil extraction

are very minimal, estimated at approximately 1015% of the capital cost per

year. In Ghana, for instance, in 2010, whilst the cost of Jatropha oil and

kerosene were estimated to be US$0.085/liter and US$1.23/liter respectively,

the cost of biodiesel from jatropha oil and petroleum diesel were also

estimated at US$0.99/liter and US$1.21/liter respectively (Ofori-Boateng and


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Teong, 2011). On the other hand, the seeds from the northern part of Malaysia

contain high lipid content of 60% of oil (Salimon and Abdullah, 2008). Using

jatropha as carbon source in PHB production can reduce the recovery cost

(Choi and Lee, 1997) as it yields high PHB content.



373,000 per year. Below is price of raw materials of this synthesis route;

Table 1. 9: Raw materials and their prices for production of 50 MTPAPHB using Jatropha oil as carbon source.

Raw MaterialsPrice

(RM/kg)

Price

(RM/year)Source

Jatropha oil 2.73 19, 503.00 Bionas Malaysia Sdn Bhd

Urea 1.70 2.75 Petronas Fertilizers Kedah


EP1: RM 1, 373, 000/year(RM 19, 503.00/yearRM2.75/year)

= RM 1, 353, 000/year


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Table 1. 10: Comparison between soybean oil, jatropha oil, and crude palm kernel oil based on its availability of raw materials, yield of

PHB, concerns, cost and operation mode.

Raw material Jatropha Oil Soy Bean Oil Palm Oil

Microbial strain

used Cupriavidus necatorH16

Availability of

raw materials in

Malaysia

Malaysia has potential for jatropha

plantations because this plant grows more

quickly and produces more seed in the

tropics (Openshaw, 2000).

Jatropha seed from northen part of

Malaysia has a high lipid content of 60

wt% of oil (Salimon and Abdullah, 2008).

There is no commercial

cultivation of soybeans in

Malaysia. Malaysia has to import

soybean oil from United States

(GAIN, 2012).

Malaysia is the second largest palm oil

producer and exporter in the world (Kek et

al. 2011).

Yield of PHB Yield high amounts of oil, yielding almost

four times than the soybean and ten times

from the maize (Fitzgerald, 2006)

Yield almost two-fold higher than

from glucose (Akiyama et al.,

2003)

68 wt% PHB content from crude palm

kernel oil (Kek et al. 2010)

Concerns Jatropha oil is a non-edible oil would not

affect the global food chain crisis and has

The use of edible oils in

production of bio plastics may

High demand for PHA production may

cause more deforestation for palm oil


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potential as a renewable resource can be

the alternative substrate for bioplastic

production.

cause depletion of global food

supply and sources.

plantation.

Cost Jatropha plants are estimated to cost less

than soybeans due to lower fertilizer and

pesticides requirements (Gui et al. 2008)

Edible plant oils price has

increased drastically because of

recent crisis of food shortage and

increase of food demand. (Ng et

al. 2010)

Palm oil can be obtained locally hence

reducing shipping and handling cost.

However, supply for PHA production has

to compete with demand of palm oil as

commercial edible oil.

Operation mode Fed-batch fermentation


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1.4.2 Selected synthesis route

Based on evaluation from three synthesis routes, jatropha oil is chosen

as the main carbon source for PHB production with C. necator H16 as this

route provides more advantage in term of cost reduction and high yield of

PHB.

1.4.3 Utilization of Jatropha oil

The operational and maintenance costs for the Jatropha oil extraction

are very minimal, estimated at approximately 1015% of the capital cost peryear. In Ghana, for instance, in 2010, whilst the cost of Jatropha oil and

kerosene were estimated to be US$0.085/liter and US$1.23/liter respectively,

the cost of biodiesel from Jatropha oil and petroleum diesel were also

estimated at US$0.99/liter and US$1.21/liter respectively (Ofori-Boateng and

Teong, 2011). On the other hand, the seeds from the northern part of Malaysia

contain high lipid content of 60% of oil (Salimon and Abdullah, 2008). Using

Jatropha as carbon source in PHB production can reduce the recovery cost

(Choi and Lee) as it yields high PHB content.

1.4.4 Type of Microbial Production Strain

Several bacteria strains have been studied in accumulation of PHB.

Researches are conducted and microorganisms such as Alcaligenes latus

(Yamane et al., 1996), Alcaligenes eutrophus now is known as Cupriavidus

necator (Kim et al., 1994), Azotobacter vinelandii (Page and Knosp, 1989),

Pseudomonas oleovorans (Brandl et al. 1988), and recombinant Escherichia

coli (Lee and Chang, 1994). Lee et al., 1994 have showed some promising

high yield of PHB production. E. coli strains are considered as impractical in

large scale production of PHB as it is expensive. They require expensive

Luria-Bertani (LB) medium, ampicillin and pure O2(Liu et al.,1998).E.coliis

also unable of producing PHAs, however it can utilise several carbon sources


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including some substrates that cannot be easily used by most of the

microorganisms, such as lactose (Lee et al., 1997). Table 1.11 shows

comparison between promising microorganisms in PHB cultivation, an

analysis from Choi and Lee (1997). It is shown that PHB concentration, PHB

productivity and PHB yield are higher in C. necator.

Table 1. 11: comparison between promising microorganisms in PHB

cultivation, an analysis from Choi and Lee (1997).

Bacterium C. necator A. latusRecombinant

E.coli

Carbon source Glucose Sucrose Glucose

Limiting nutrient Nitrogen None None

Fermentation method Glucose

concentration

control

pH-stat pH-stat

Culture time (h) 50 28.45 39

Cell concentration (g/L) 164 143 110

PHB concentration (g/L) 121 71.4 85

PHB content (%) 76 50 77.3

PHB productivity (g/L.h) 2.42 2.5 2.18

PHB yield (g PHB/ g

substrate)

0.3 0.17 0.29

kg substrate/kg PHB 3.33 5.88 3.5

Reference Kim et al.

1994

Yamane et al.

1996

Lee and Chang

1994

Cupriviadus necator use up palmitic acids, oleic acids and linoleic

acids contained in jatropha oil. Freitas et al. (2009) reported that C. necator

has been proven to accumulate PHB up to 80% of its cell dry weight. Whilst

Khan et al. (2013) reported that cell growth curve of C. necator H16 has a

classical pattern with an exponential phase up to 50 hours followed by

stationary phase that lasted until 65 hours. They also stated that the highest


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cell dry weight of 11.6 g/L was obtained at 55 hour followed by highest PHB

concentration of 8.6 g/L at 61.5 hour. Kadouri et al. (2005) reported that C.

necator can tolerate adverse stress conditions such as heat, osmotic pressure,

UV radiation and toxins such as ethanol and hydrogen peroxide. It should be

clear that C. necator is the most suitable microorganism to be used with

jatropha oil in PHB production.

1.4.5 Feeding source of nutrient supply

Lee et al. (2008) found that different nitrogen sources affected both

cell biomass and PHA biosynthesis. They also discovered that both urea and

sodium nitrate resulted in better biomass production compared to other

nutrient supply. Ng et al. (2010) stated that urea is the most suitable nitrogen

source to pair up with Jatropha oil as carbon source. Besides, urea costs much

lower price and has high productivity of PHB (Kek et al., 2008). Khanna and

Srivastava (2005) as well as Sabra and Abou Zeid (2008) discovered that urea

can yield high cell biomass and PHA production significantly. Ng et al. (2010)

also reported that based on their analysis both cell dry weight (CDW) and

PHB accumulation increased when the urea concentration increased. They also

stated that the optimal concentration of urea is 0.54 g/L as CDW remained

constant while PHB accumulation decreased significantly after 0.54 g/L of

urea.

1.4.6 PHB synthesis

There are four methods identified to cultivate PHAs which are; in

vitro, via PHA-polymerase catalyzed polymerization; and in vivo with batch,

fed-batch and continuous cultures (Zinn et al., 2001). However, the fed-batch

mode is the most used for PHAs production to achieve high cell density,

which often crucial for the high productivity and yield for the desired product.

Fed-batch mode is chosen as it has a lot of advantages in production of PHB.

Heuristically, fed-batch can maintain the carbon-source concentration at very


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low concentration, to maximize the biomass yield. Ng et al. (2010) presented

Jatropha oil support the cell growth and PHB production in fed-batch

fermentation and high yield of product per Jatropha oil was obtained.

The fermentation process is relatively simple with multi-staging from

the petri dish to a shaker flask to a small fermenter which is then used to

inoculate the production reactor. In fermentation process, cells were

maintained and pre-cultivated in 2 g/L yeast extract, 10 g/L meat extract and

10g/L peptone (Khan et al.,2013).

1.4.7 Downstream Process

For subsequent process, several methods have been developed for the

recovery of PHAs, mostly PHB from the cells. Solvents such as chloroform,

methylene chloride, propylene carbonate and dichloroethane have been used

for the extraction of PHB (Ramsay et al.,1994). However, it was difficult to

remove the cell residues due to viscosity of 5% (w/v) PHB from extracted

polymer solution (Choi and Lee, 1997). Hahn et al.1994 suggested that PHB

can be recovered using a dispersion of hypochlorite and surfactant solution.

PHB recovery by this method is more efficient with less polymer degradation.

During recovery of PHB, the harvested cell pellets are treated with

surfactant solution (1%, w/v) at 25C for 1 hour of mean residence time. The

treat mean is then followed by hypochlorite digestion in flow-through manner

and then PHB is separated from supernatant by centrifugation. PHB granules

are rinsed with water and were finally spray-dried. (Lee and Choi, 1997).


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CHAPTER 2

PROCESS FLOW SHEETING

2.1 Selection of raw material and impurities management

In order to produce targeted amount of 50 metric ton per year of PHB,

and based on 8000 operation hours per year, the raw materials are decided

based on their high yield of product and economy wise, gives advantages to

cost of production. Based on its market availability, crude Jatropha oil ischosen as the substrate. Urea is selected as nitrogen source as it could produce

high PHB content according to Khan et al. (2013). Meanwhile, the bacteria

strain that used is Cupriavidus necator H16 which previously known as

Ralstonia eutropha. It was obtained from National Collection of Industrial,

Food and Marine Bacteria.

Since we are using crude Jatropha oil with high purity, which also can

be obtained with affordable price locally, there is no need to manage impurity

of substrate. The oil, which contains high carbon source is used directly in

inoculation.

2.2 Input and Output Flow Sheeting

Input and output flow sheeting is illustrated in block flow diagram to

give clear view of the process. Figure 2.1 illustrates the flow of PHB

production while Figure 2.2 and Figure 2.3 shows upstream and downstream

process, respectively. Upstream process includes early cell isolation and

cultivation, to cell banking and culture expansion of the cells (fermentation)

until final harvest (termination of the culture and collection of the live cell

batch). Downstream process starts from harvesting from fermenter, and then

processed to meet purity and quality requirements.


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Figure 2. 1: Block Flow Diagram of PHB Production


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Figure 2. 2: Block Flow Diagram of Upstream Process


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Figure 2. 3: Block Flow Diagram from Downstream Process


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Figure 2. 4: Process Flow Diagram of PHB plant


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Figure 2. 5: Process Flow Diagram Simulation in SuperPro


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Figure 2.4 illustrates the process flow diagram based on block flow

diagram, fully labelled of streams and pumps. The upstream process started

with the sterilization of mineral medium and jatropha oil in ST-101 and ST-

102, respectively and mixed into mixing tank MX-101. The microbesCupriavidus necator H16 is inoculated in shake flask for 48 hours before

subjected to seed fermenter. Solution from mixing tank is splitted and 10%

will go to seed fermenter and the rest is fed into main fermenter. Air is

supplied using gas compressor (G-101) and filtered to avoid contamination in

fermenters using air filter AF-101. Vent from main fermenter is filtered using

air filter AF-102.

The downstream process utilizes the centrifugation process only. The

separation stage was based on methodology from Choi & Lee (1997) as well

as Choi & Lee (1999). After the end of the fermentation, the bacterial cells are

harvested via continuous centrifugation (DS-101) of the fermentation broth

which collected in V-103. Microbial cell lysis or disruption is carried out via

combined surfactant-hypochlorite digestion. A surfactant solution (Triton X-

100) of 1% (w/v) is added to the microbial biomass, charged in stream S-122

and mixed at 25C. This treatment followed by centrifugation in DS-102 to

separate PHB granules and aqueous solution and then added hypochlorite

digestion, charged in stream S-124 in a flow-through manner. This combined

step results in microbial lysis and separation of PHB from residual cell

material. The aqueous solution containing the residual cell material is

separated further by centrifugation in DS-103, where PHB is rinsed with water

beforehand V-105. Finally, PHB granules are purified via washing with water

and drying via a spray-drying step.

2.2.1 Mechanical Equipment Description

Based on preliminary flow diagram in Figure 2.1, there are thirteen

equipment used to produce high purity of 50 MTPA PHB. Follows are

description for each equipment used in this design.


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2.2.1.1 Heat sterilizer (ST-101 & ST-102)

Two heat sterilizers are used to heat the raw materials media

continuously in 121C temperature before being sent to seed fermenter (V-

102) and main fermenter (V-103). Jatropha oil must be autoclaved separately

from mineral media. Sterilization is essential in bioprocess plant to avoid any

contamination during main process. These heat sterilizers have diameter of

0.10m and 7.42 m length.

2.2.1.2 Blending tank (V-101, V-105, & V-106)

Blending tank V-101 is installed to blend the sterilized Jatropha oil

from S-104 and sterilized mineral media from S-103. V-105 is used to blend

surfactant (S-122) with supernatant (S-121) while V-106 is used to blend

water from stream S-128 and PHB for washing and rinsing of granules. A

stainless steel 20 m3 blending tank with Siemens motors is used in this

process.

2.2.1.3 Disk Stack Centrifuge (DS-101, DS-102, & DS-103)

Centrifuge DS-101 is required to harvest the cells of fermentation

broth collected from storage by continuous centrifugation. DS-102 is used to

separate PHB from aqueous solution containing dissolved non-PHA cellular

material (NPCM) while DS-103 is used to rinse PHB granules with water.

2.2.1.4 Spray Dryer (SDR-101)

This last piece of equipment in this process is used to produce dry

powder from slurry consists of PHB granules and water (S-132). The product

is collected at S-133 while waste is channeled to waste treatment plant through

S-134. The design of this spray dryer is estimated with 0.80 m diameter with

2.39 m in height.


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2.2.1.5 Fermenter (V-103)

Fermenter with fed-batch mode is chosen to produce PHB from

Jatropha oil by C. necator H16. Fermenter with volume 200 m3 is used in

order to produce 50,000 kg of PHB per year.

2.2.1.6 Gas Compressor (G-101)

Axial gas compressor is chosen to continuously pressurized air from

surrounding environment as this compressor has high efficiency and large

mass flow rate. The pressure change in this compressor is 2 bar.

2.2.1.7 Air filter (AF-101 & AF-102)

Air filter is one of the essential equipment in bioprocess plant. Air

filter AF-101 is connected after gas compressor function as to filter any

particulates coming from surrounding air. It is needed to filter to avoid any

contamination in the main fermenter that might alter the product. AF-102 is

installed to ensure no microorganism exit from fermenter as they might be

harmful and toxic to plant personnel and environment. The throughput is

estimated as 0.70 m3/s.

2.3 Material and Energy Balances

This section provides details of manual calculation of material and

energy balance, and simulation using Superpro software was conducted and

the results was compared accordingly with manual calculation.


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2.3.1 Material Balance

In this section, we will perform material balance for each process unit

involved in this PHB production. Material balance is calculated by using

Microsoft Excel and the spreadsheet is as attached in Appendix A.1. The

overall material balance for the whole process is performed using the general

equation shown below:

Input + GenerationOutputConsumption = Accumulation

Input = total mass entering system boundaries

Generation = total mass produced within system

Output = total mass leaving system boundaries

Consumption = total mass consumed within the system

Accumulation = total mass built up within the system

Here, some assumptions have been made to make the calculation more

easily. The design-based assumptions are:

1.

No leakage in the pipes and vessels in the system.

2. All the components in the system behave as ideal c

PRODUCTION OF 50 MTPA POLYHYDROXYBUTYRATE FROM JATROPHA OIL

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