1. Topic: Production of PHB using
Alcaligenes eutrophus
KB Group 2:
1. Sonia Dilip Patel A133115
2. Tan Yi Von A132788
3. Chin Lee Nee A132359
4. Judy Loh Ea Ea A132395

2. CHARACTERISTICS:
~Partially crystalline
thermoplastic
~Good material for producing
biodegradable and/or
biocompatible plastic
~ Stiff and brittle
~Water insoluble & relatively
INTRODUCTION: resistant to hydration
~First discovered by Lemoigne degradation
(1925) ~Good in ultra-violet resistance Alcaligenes eutrophus:
~Accumulated in intracellular but poor in resistance to acid ~ Gram - & non-spore
granules by Gram+ & - and bases. forming bacillus
microorganusm. ~Optimal growth at 30 C
~ Required the limitation of an ~Obligate
essential nutrient element in the aerobe, facultative
presence carbon source for chemolithoautotroph
efficient synthesis of PHB. ~ Up to 80% of the dry
weight of A.eutrophus can
PHB
be composed of PHB
inclusions

3. MEDICAL INDUSTRY PHAMACEUTICAL INDUSTRY PACKING INDUSTRY
Biodegradable sutures, surgical Drug delivery on the base of PHB normally used in food industry
mesh, screws and plates for bone films based on its biodegradable
fixation, periodontal membranes and = used as drug delivery matrix for characteristics / bioplastics.
wound dressing. sustaining the release of various used in food related applications
drugs such as DP. such as films for food wrapping and
Bioabsorbable surgical sutures = Via diffusion & degradation thermoplastics for food packing and
= Possess necessary strength for = release diffusion depends on its food container such as bowls, plates
healing of myofascial wounds. nature, thickness, weight ration & & cups.
= High tensile strength and molecular weight of PHB. Also used to produce container such
longer strength retention = Regulate the rate by changing the as shampoo bottles, laminated foils,
characteristics. MW. one way cup & agriculture foils.
= Lighter inflammation when
compare with other type material. Drug delivery on the base
= Changes in surface morphology can microsphere & microcapsule
be determine by SEM & AFM = release coefficient depend on
diameter of microspheres.
Biodegradable screw & plates = possible produce a system with
= Avoidance secondary removal of prolonged uniform drug release.
hardware
= Do not cause imaging or
radiotherapy interface or discomfort.

4. Table 2.1 World demand for bioplastics to exceed 1 million tons in 2015 Greener
Package
WORLD BIOPLASTICS DEMAND (thousand metric tons)
% Annual Growth
Item 2005 2010 2015 2005-2010 2010-2015
Bioplastic 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
Source: Mohan 2011
• Global demand for bioplastics that derived from plant-based sources, has
been estimated to 0.9 billion kg in 2013, valued at approximately RM7.6
billion.
(Freedonia Group 2012).
• Factors: customer demand for more environmentally-sustainable products,
development of bio-based feed stocks for commodity plastic resins,
increasing restrictions on the use of nondegradable plastic products and
high rise of crude oil and natural gas prices.

5. 3.5
Capacity (million tonnes per Other • Production of
3
Bio-based Monomers bioplastics based on
2.5
PHA PHA in 2013 has been
2 projected to reach 0.5
year)
Bio-based Ethylene
PLA
billion kg.
1.5
• Therefore,
Starch Plastics
1
Demand – Supply
0.5 = (0.9 – 0.5) billion kg
0 = 0.4 billion kg
2003 2007 2009 2013 2020 • 0.4 billion kg x 0.1%
(Projection) (Projection) (Projection)
= 400 000 kg per
Figure 2.1 Estimated Worldwide capacities of bio-based annum
plastics until 2020 based on company announcements.
Source: (Shen et.al 2009)
Malaysia Demand And Supply Of Bioplastic
• Malaysia’s 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.

6. T = 30°C, P = 1
Fed- bar, pH =7
batch
mode
Lower Homogenizer
surface blended with
tension chloroform
together with
enzymatic
To obtain more method
concentrated
product

7. Synthesis Mixing Evaporator
of PHB chamber
Cultivation Centrifuge
Disc-stack
centrifuge
Blending Spray
Homogenizer Extractor dryer
tank

8.  C6H12O6 + 2.5027O2 + 0.6689NH3
2.2676CH1.75O0.41 N0.25 + 0.2676C4H6O2 + 2.6620CO2 +
4.2164H2O
 0.1 % of 0.4 billion kg = 400 000 kg per year.
*1 batch = 62 hours
 133 batches/year= 8246 kg of PHB/year = Production
of 48.5 kg/hour
*21 days off production for maintenance etc.

9. Glucos NH O2 Dry PHB CO2 H20 Total
e 3 Biomass
Feed 380 24 0 0 0 0 2596 3000
O2 gas 0 0 168.74 0 0 0 0 168.7
4
 In stream Produ - - - - - - - -
ct
Off-gas - - - - - - - -
Total 380 24 168.74 0 0 0 2596
Gluco NH3 O2 Dry PHB CO2 H20 Total
se Biomass
Feed - - - - - - - -
 Out stream O2 gas - - - - - - - -
Product 3.6 - - 113.8 48.5 0 2756 2921.
9
Off-gas 0 0 0 0 0 247 0 247
Total 3.6 0 0 113.8 48.5 247 2756

10. For inlet of fermenter,
Inlet Enthalpy Mass flow Molar flow Total
Componen change, ΔHi rate, ṁi rate, Ni enthalpy
ts (J/mol) (kg/h) (mol/h) change,
ΔHiNi
(kJ/h)
Glucose 345 380 2111.11 728.33
Ammonia 4240.9 24 1411.76 5987.13
Oxygen 88.45 168.74 5273.13 466.41
Water 225.83 2596 144222.22 32569.70
For outlet of fermenter,
Σ 3169 39 751.57
Outlet Enthalpy Mass flow Molar flow Total
Componen change, rate, ṁo rate, N0 enthalpy
ts ΔHo (J/mol) (kg/h) (mol/h) change,
ΔH0N0
(kJ/h)
Glucose 345 3.6 20 6.9
PHB 33.65 48.5 563.95 18.98
Carbon 113.35 247 5613.64 636.31
dioxide
Water 225.83 2756 153 111.11 34577.08
Biomass 34.62 113.8 4779.50 165.47
Σ 3169 35404.74

12. •Sterilization refers to physical, chemical or mechanical process
that completely destroys or removes all form of viable microorganisms.
•Mode of sterilization methods : a) continuous b) batch
•Advantages of continuous sterilization are shown below: (Source : Lee
2001)
i. Running costs are less
ii. Ease in scaling-up of the process.
iii. Easier to automate the process and therefore less labor intensive.
iv. Requires less steam by recovering heat from the sterilized medium
and thus requires less cooling water. It can averagely save about
30% steam and 40% of cooling water

13. Figure 7.1 Comparison of a batch (A) with a continuous sterilization strategy (B) for the temperature profile of the medium sterilized
Source: Shuler & Kargi 2002

14. Sterilization
Heating
section Cooling
Holding section
section
Plate-and-frame heat exchanger Shell-and-tube exchanger

15.  There are three sections in the sterilization:
a)heating section b) holding section c) cooling
section
 Indirect heating in plate-and-frame heat
exchanger is chosen and it can be used for cooling
purpose.
 The heated medium need to pass through holding
section which is composed of long tubes as the
temperature is assumed to be constant .
 Therefore, the time needed for heating, holding
and cooling are 17.27s, 11.45s and 12.73s
respectively, the sum of time required is 41.45s
and equal to 0.012 h.

16. Four
Baffles
Sigma 298
silicon
antifoam

17.  Case that can be selected as the criteria of scale-up:
scale up based on constant power input (P0/V) implies
constant OTR.
Volume of bioreactor (l) 75 10,000
Diameter of the vessel (m) 0.36 1.85
Diameter of impeller (m) 0.12 0.61
Height of liquid media (m) 0.72 3.70
Table 7.3 Values of scale-up operations for 75l and 10,000l bioreactor

18. The impeller rotation number after scale-up is 0.34. The energy input can be
calculated as 133.33; the impeller diameter can be assumed as 5.33; pump rate of
impeller can be assumed as 45.33; pump rate of impeller over volume is 0.36;
maximum impeller speed is 1.81 and Reynolds number is assumed as 9.0.
There are some additional information that need to calculated as complete
reference in scale-up operations:(1)Aeration rate = 6.25 x 10-4 m3/s for 0.5vvm
(2)Gas superficial velocity = 43.48m/h (3)Power calculation Pg1 = 15.71 hp; PI =
579.42hp;(4) Rotational speed 350rpm for N1 while N2 = 118.98 rpm for constant
power input and N2 = 68.85 rpm for constant input velocity.
Scale-up criterion Small fermenter,80l Constant Po/V
Energy input 1.0 125
Energy input/volume 1.0 1.0
Impeller rotation number 1.0 0.34
Table 7.2
Interdependence of
Impeller diameter 1.0 5.0
scale-up parameters
Pump rate of impeller 1.0 42.5
Pump rate of 1.0 0.34 Source: Shuler &
impeller/volume Kargi 2002
Maximum impeller speed 1.0 1.7
(max.shearing rate)
Reynolds number 1.0 8.5

19. Cell disruption
Non-mechanical Mechanical
Lysis Dessication Solid shear Liquid shear
(bead mill, (homogenization,
Physical grinding, untrasonic, French
(osmotic Enzymatic Hughes press) press)
shock, thermolysis)
Chemical
(osmotic, solubilisatio
n, lipid
dissolution, alkali
treatment)
Figure 8.1 Hierarchy chart for cell disruption
methods

20. The release of intracellular bioplastic PHB granules from fed-batch
cultured gram-negative bacterium Alcaligenes eutrophus using
combinations of non-mechanical and mechanical methods to disrupt
the first and second layers of the cells.
Non-mechanical : enzymatic pretreatment of bacterium with
lytic enzyme from Cytophaga;
5 fold dilution of
Wash & suspend 5mM of EDTA
cell suspension
in phosphate- is used to
in 50mM Tris-
buffered saline, destabilize the
HCl buffer, pH
pH 7.4 outer membrane
7.3.
Mechnical : disruptive by using an APV-Gaulin
15M-8BA and 30CD high-pressure
homogenizer with a ceramic valve seat.
 three passes at 60-70 Mpa for complete disruption
 two-stage process: primary point break of the cell
envelope & further breakage of
the cell wall and degradation of
cellular debris.

21. Nonviable material used in medical device which is
intended to interact with biological systems
( Williams 1987).
Biological
Unit operation Product usage
response
• Cyclone • Medical • Bioabsorbable
column • Phamaceutical suture
bioreactor • packing • Drug delivery
system

22. iv. Temperature
Probe
v. DO Probe
iii. Baffles ~ Temperature
deviation by a ~ Polarographic DO
~ Prevent formation of vortex couple of degree Probe
around walls of vessel can dimishish ~ anodemade from
~ made from metal strip & dramatically the silver; cathode made
Stainless Steel Grade 316L growth and from gold
ii. Impeller biosynthesis
productivity vi. pH Probe
~ Downward pumping
hydrofoil & Rushton ~Stainless Steel Pt ~ Speed of a reaction & solubility
turbine 100 of compound
~ Stainless Steel Grade ~ made from glass tube & silver
316L chloride covered silver wire
located inside solution in glass
i. Body Construction tube
~ Stainless Steel Grade 316L
vii. Sealing
~Excellent in a range of
~ between top plate and
atmospheric environment &
Cyclone vessel
many corrosive media
~intermittent service to 870°C
Column ~glass&glass;glass & metal;
Bioreactor metal & metal
In continuous service to 925°C
~ fabric-nitryl or butyl rubber
~ Solution treatment: heat to
~ gasket, lipseal and ‘O’ ring
1038-1149°C then rapid quench
Example: Heat exchanger

23. Bioabsorbable surgical suture
• Biodegradable P3/4HB monofilament suture has better tissue
compatibility than nature and chemosynthesis biodegradable suture.
• The tissue response for P3/4HB is less serious than chromic catgut and
Vicryl.
• Inflammation process will reduced slowly by indicated by disappearing
of leucocytes.
Drug delivery system
• Suitability depend on its biodegradation properties and also
biocompatibility.
• Slight inflammation in capsule zone during implantation period
changed from the mostly neutrophils granulocytes to mostly
lymphocytes.
• Typical host reaction to foreign implant.
• PHB did not inhibit growth of the cells.

24. • Stoichiometry Calculation
• Material Balance Of Fermenter
• Energy Balance Of Fermenter
• Economic Aspect
* Script files attached in the submitted CD

25. • Waste Generation – waste water, carbon
dioxide, biomass
• Discharge limit for waste, carbon dioxide
• Relevant Environmental Act
• Safety Precautions – Production plant, personal

26.  The demand of PHB keeps increasing.
 Our production of 0.1% of 0.4 billion had a total
mass in and out of 3169 kg/h.
 The total time of sterilization required is 0.012h.
 Scale up – from 75l to 10 000l.
 The criteria of bioreactor for production was
calculated.
 Operating way of homogenizer was understood.
 Material of bioreactor was studied in deep
together with biological response of PHB.
 MATLAB coding comparison and SuperPro usage
was understood.

27.  Thank you for your attention 